MSC transplantation on the development of PAH | JIR – Dove Medical Press

Fengjin Shao,1 3 Rui Liu,1 Xun Tan,1 4 Qiaoyan Zhang,1 3 Lujie Ye,1 3 Bingxuan Yan,1 3 Ying Zhuang,1,2,4 Jiaxue Xu1,2,4

1Department of Veterinary Medicine, Zhejiang University, Hangzhou, Zhejiang Province, Peoples Republic of China; 2Veterinary Medical Center, Zhejiang University, Hangzhou, Zhejiang Province, Peoples Republic of China; 3Institute of Preventive Veterinary Sciences, Zhejiang University, Hangzhou, Zhejiang Province, Peoples Republic of China; 4Hainan Institute of Zhejiang University, Sanya, Hainan Province, Peoples Republic of China

Correspondence: Xun Tan, Department of Veterinary Medicine, Zhejiang University, Yuhangtang Road 866, Hangzhou, 310058, Peoples Republic of China, Tel +86 571 8898 2393, Fax +86 571 8898 2310, Email [emailprotected]

Purpose: Pulmonary arterial hypertension (PAH) is a progressive and fatal pulmonary vascular disease initiated by endothelial dysfunction. Mesenchymal stromal cells (MSCs) have been shown to ameliorate PAH in various rodent models; however, these models do not recapitulate all the histopathological alterations observed in human PAH. Broiler chickens (Gallus gallus) can develop PAH spontaneously with neointimal and plexogenic arteriopathy strikingly similar to that in human patients. Herein, we examined the protective effects of MSC transplantation on the development of PAH in this avian model. Methods: Mixed-sex broilers at 15 d of age were received 2 106 MSCs or PBS intravenously. One day later, birds were exposed to cool temperature with excessive salt in their drinking water to induce PAH. Cumulative morbidity from PAH and right-to-left ventricle ratio were recorded. Lung histologic features were evaluated for the presence of endothelial damage, endothelial proliferation and plexiform lesions. Expression of proinflammatory mediators and angiogenic factors in the lung was detected. Matrigel tube formation assay was performed to determine the angiogenic potential of endogenous MSCs. Results: MSC administration reduced cumulative PAH morbidity and attenuated endothelial damage, plexiform lesions and production of inflammatory mediators in the lungs. No significant difference in the expression of paracrine angiogenic factors including VEGF-A and TGF- was determined between groups, suggesting that they are not essential for the beneficial effect of MSC transplantation. Interestingly, the endogenous MSCs from birds receiving MSC transplantation demonstrated endothelial differentiatial capacity in vitro whereas those from the mock birds did not. Conclusion: Our results support the therapeutic use of MSC transplantation for PAH treatment and suggest that exogenous MSCs produce beneficial effects through modulating inflammation and endogenous MSC-mediated vascular repair. Graphical Abstract:

Keywords: plexiform lesion, right ventricular hypertrophy, VEGF-A, TGF-, angiogenesis, bone marrow

Pulmonary arterial hypertension (PAH) is a devastating disease in humans characterized by sustained elevations in pulmonary vascular pressure, resulting in right heart failure and death. The histological hallmark of PAH is the structural and functional remodeling of small pulmonary arteries.1,2 Disordered angiogenesis causes the formation of glomeruloid structures called plexiform lesions, resulting in complete obliteration of pulmonary arteries.35 Although the detailed mechanisms underlying vascular pathology in PAH remain to be elucidated, accumulating evidence suggests that endothelial dysfunction acts as first trigger in driving this process.6

Despite major advances in pharmacological treatments, PAH remains a fatal disease.7 Over the last decades, stem cell-based therapies have attracted great interest in the field of PAH. One of the cell types currently undergoing preclinical trials is the mesenchymal stem cells (MSCs, also known as mesenchymal stromal cells). MSCs are one of the most well-characterized stem cells that can be obtained from various tissues involving bone marrow, peripheral blood and adipose tissue, are easily cultivated, expand extensively in vitro, have intrinsic differentiation potentials, and produce an abundance of bioactive factors mediating beneficial angiogenic effects as well as immunosuppression.811 Numerous preclinical studies demonstrated promising therapeutic potential of MSC therapy for PAH; however, the majority of the studies was conducted by using the monocrotaline (MCT) model.12 Notably, this model does not recapitulate all the histopathological alterations observed in human PAH, such as neointimal and plexogenic arteriopathy.13,14 Furthermore, many drugs that have demonstrated efficacy in the MCT models have failed to show clinical benefit in human trial.13,15 Thus, further preclinical data from more clinically-relevant models are needed.

Fast-growing meat-type broiler chickens (Gallus gallus) can spontaneously develop PAH (also known as ascites or pulmonary hypertension syndrome), with an estimated incidence of 3% in all broiler chickens reared under conditions that promote maximal growth. Increased oxygen demand (cold stress), hypobaric hypoxia (high altitude)16 or sodium chloride toxicity17 predisposes the birds to develop PAH. Broilers with PAH exhibit histological features closely resembling that of human idiopathic PAH, including medial hypertrophy, intimal hyperplasia and plexiform lesions in lung vasculature.1823 We have recently proposed a concept that the formation of plexiform lesions is associated with local immune/inflammatory response resulting from hemodynamic stress.21,24 It is now well established that, like that in humans, pulmonary artery endothelial cell damage plays a major role in the pathogenesis of PAH in broilers.2527 Thus, broilers offer a particularly useful model for the study of PAH pathogenesis and development of new therapies for PAH.

The aim of the present study was to provide additional information regarding the application of MSCs for PAH treatment by administrating MSCs to broiler chickens with PAH induced by cold stress combined with sodium chloride toxicity. Here, we used the chicken model to investigate the effects of MSC transplantation on PAH. We confirmed that intravenous-infused MSCs reduced PAH incidence and attenuated endothelial damage, plexogenic arteriopathy and inflammation. We demonstrated, for the first time, that MSC transplantation activated endogenous MSCs to differentiation to endothelial cells, which may thus contribute to the beneficial effects produced by MSC transplantation.

The animal experiments followed the guidelines for the ethical review of laboratory animal welfare in Zhejiang University and were approved by the Ethics Committee of the Zhejiang University (Approved No. ZJU2015-445-12).

MSCs were isolated from the bone marrow of healthy broilers (Ross 308) at 1-week old. In brief, the birds were killed by cervical dislocation and femurs and tibia bones were removed, cleaned of all soft tissues. The bones were soaked in 70% (v/v) ethanol for 10 minutes. Bone marrow cells were extracted as previously described.28 Mononuclear cell (MNC) fraction was enriched by density gradient centrifugation using Ficoll-Paque, density of 1.078g/mL (Haoyang Biological Manufacture Co., Ltd, Tianjin, China) and cultured in DMEM supplemented with 10% FBS, 100 IU/mL penicillin and 100 g/mL streptomycin at 39 C with 5% CO2. After 48 hours, hematopoietic cells and other non-adherent cells were removed. Medium was replaced every 23 days. The remaining cells were further expanded in culture until 80% confluence. Afterward, the cells were detached with 0.25% trypsin-EDTA (Sigma-Aldrich, Shanghai, China) and replated in other flasks at 1:6 ratios. Cultures up to passage 2 were used for the experiments.

At the end of passage 2, cells were harvested for evaluating mRNA expression of cell surface markers CD44, CD90, CD105, CD31, CD34 and CD45 by using reverse transcription (RT)-PCR. Briefly, total RNA was extracted from harvested cells with TRIzol reagent (TaKaRa, Dalian, China), and cDNA was synthesized from 1 g of total RNA using PrimeScript RT Reagent Kit (TaKara, Dalian, China). The cDNA was subjected to PCR amplification as previously described.29The oligonucleotide primers are presented in Table S1. PCR products were separated by electrophoresis with 1.2% agarose gel and visualized with GoldView (Yeasen Biotechnology, Shanghai, China).

The phenotypic expression of -SMA and CD133 in MSCs was examined by immunofluorescence. Cells were fixed in ice-cold methanol and stained as previously described.30 Briefly, cells were incubated with either monoclonal mouse anti-human -SMA (Bostar Bio, Wuhan, China) or polyclonal mouse anti-chicken CD133 (self-prepared) overnight at 4C, followed by incubation with a FITC-labeled secondary antibody for 60 min at 37C in dark. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).

Oil Red O stain was performed to determine adipocyte differentiation using our previously described protocol.31 In brief, cells in 6-well plates were fixed in 10% formalin and washed in 60% isopropanol. Lipid droplets were then visualized by fresh oil red O solution. To characterize osteogenic differentiation, cells were stained by alizarin red S as previously described.31 Orange/red calcific deposits were observed under a microscope.

Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, cells at passage 2 were seeded in 96-well plates at an initial density of 100 or 1000 cell per well. At the indicated time points, 20 L of MTT (5 mg/mL; Sigma-Aldrich, USA) solution was added into each well, followed by incubation of the plates at 39C in 5% CO2 for 4 h. The supernatant was then discarded and 150 L of dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan precipitation. The optical density of each well was measured at 570 nm using a microplate reader.

Mixed-sex 1-day-old broilers (Ross 308) were purchased from a local commercial hatchery. Birds were fed a commercial corn-soybean meal diet formulated to meet or exceed the NRC (1994) standards for all ingredients. Thermoneutral temperatures were applied to the birds until 15 d of age when the birds were randomly divided into 2 groups (body weight 300400 g) and received either 2106 MSCs suspended in 200 L PBS (MSC group) or PBS alone (mock group) through the wing vein. On d 16, birds were exposed to cool environmental temperatures in combination with excess salt in their drinking water to induce PAH as we have previously described.25 Briefly, when starting on d 16, the brooding temperature was gradually decreased by 1C per day until a final temperature of 17 C was reached. Along with the exposure to cold stress, sodium chloride (0.3%, w/v) was given in the drinking water to further accelerate the development of PAH.23 Birds had free access to full feed and water. Bird mortality was recorded daily and necropsies were performed to identify PAH-related death from d 16 onward. Birds that died with a right-to-total ventricle ratio (RV/TV) above 0.25 were included in the PAH mortality.32 Six birds were randomly selected from each group at weekly intervals for 4 weeks after MSC implantation and subjected to the following experiments (Figure 1).

Figure 1 Experimental design.

Abbreviation: MSCs, mesenchymal stem cells.

The birds were humanly killed and the lungs were perfused immediately with PBS through right ventricle. Thereafter, the right lungs were removed and cut in the transverse plane at the major rib indentations (costal sulci). One inter-rib division was collected and fixed in 4% (w/v) formaldehyde solution for 24 h. The paraffin-embedded tissues were subsequently serially cut in the transverse plane at 5 to 7 m thickness and stained with hematoxylineosin. Morphological changes of the pulmonary arterioles were observed under an optical microscope. To evaluate endothelial damage, at least 20 arterioles with an external diameter of <50 m in each slide were photographed, and the histological alterations of vascular endothelium were assessed by an expert. The percentage of the arterioles with normal endothelium was calculated. Plexiform lesions in each slide were also counted and lesion density (number of lesions per section/cm2 per section) was calculated as described previously.24,33

Hearts were removed and carefully dissected. Weights of the free-wall of the right ventricle (RV) and the total ventricle (TV) were measured, and the ratio of RV/TV was calculated as the RV hypertrophy index.34

Total RNA was extracted from frozen lung tissue by using RNA-Quick Purification Kit according to the manufacturers instructions (Yishan, Shanghai, China). First-strand cDNA was synthesized from 1000 ng total RNA using HiScript II Q RT SuperMix for qPCR with genomic DNA wiper (Vazyme, Nanjing, China). Gene expression was quantified by qPCR using Roche LightCycler 480 II system (Roche Diagnostics GmbH, Mannheim, Germany) with SYBR Green Realtime PCR Master Mix Plus (Vazyme, Nanjing, China). The primer sets used and the amplification program have been described previously.24 The relative expression of the target genes was corrected to reference genes B2M and RPL19 using efficiency corrected method (Pfaffl).

Lung tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer (Fudebio, Hangzhou, China) containing protease inhibitors (PMSF, Beyotime Technology, Nanjing, China). Protein concentration was quantified by using a bicinchoninic acid (BCA) method (Beyotime Technology, Nanjing, China). For Western blot, the samples were separated by sodium dodecyl sulfate (SDS)-12% polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were subsequently blocked in 5% non-fat milk in TBST and probed with primary antibodies against IL-1 (Abclonal, Wuhan, China), IL-6 (Huabio, Hangzhou, China) and TNF- (Santa Cruz, Texas, USA) at a final dilution of 1:1000 overnight at 4C, then immunoblotted with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Fudebio, Hangzhou, China). The immunoreactive bands were visualized by electrochemiluminescent (ECL).

Tubular formation assay was conducted as previously described.21 In brief, Matrigel matrix (BD Biosciences, San Jose, CA, USA) diluted in DMEM (1:1, v/v) was added into 96-well plates and allowed to solidify at 39 C for 30 min. MSCs suspended in DMEM were plated onto the surface of the matrix at 5104 cells/well with triplicate. After 4 h cells were visualized by a phase-contrast microscope (Nikon, Japan) and representative fields (20 magnification) were photographed.

Data are presented as mean SEM. PAH morbidity was analyzed using independent chisquared test. Normality was assessed for other data sets by ShapiroWilk test. RV/TV ratio, plexiform lesion density and the mRNA level of angiogenic factors were analyzed by using non-parametric MannWhitney U-test due to non-normal distribution or small sample size (n < 5). Other data were analyzed using an unpaired Students t-test. Significance was set at P < 0.05, and all P values are listed. Analysis was performed using the SPSS 22.0 (IBM Corp., Armonk, NY, USA).

In the present study, MNCs were isolated from chicken bone marrow and cultured in DMEM to obtain MSCs. The MSCs displayed spindle-shaped morphology, gradually grew into small colonies during the initial days of incubation (Figure 2A), and reached ~80% confluence by d 10 after plating. After passage, three subpopulations, ie, triangular or star-like cells, spindle-shaped cells, and large, flattened cells, could be observed (Figure 2B), in line with previous studies on human MSCs.35,36 Cells at Passage 2 were subjected to PCR for the determination of cell surface markers, which demonstrated that the cells were positive for MSC markers (CD44, CD90, and CD105) and negative for hematopoietic surface markers (CD34 and CD45) and hematopoietic/endothelial marker CD3137 (Figure 2C). Almost all of the cells expressed -SMA and CD133, a typical stem cell marker of mesenchymal origin (Figure 2D). The Alizarin Red and Oil Red O staining results confirmed the potential of the cells to differentiate into the osteogenic lineage and adipocytes (Figure 2E). Growth curves showed active proliferation of these cells (Figure 2F). All these results are inductive of the MSC phenotype of the cultured cells.

Figure 2 Characterization and differentiation of chicken bone marrow-derived MSCs. (A and B) Morphology of MSCs. The cells exhibit spindle-like morphology during the early growth in DMEM ((A), passage 0, day 3). In later passages, the cells display various morphologies including star-like (arrow), spindle-shaped (arrowhead), and flattened morphology (*) ((B), passage 1, d 2). (C) Reverse transcription (RT) PCR products of surface markers of cells at passage 2. actin was used as reference gene. Fragments were fractioned on a 1.3% agarose gel and stained with Goldview reagent. Note the lack of CD34, CD45 and CD31 expression. Images are representative of three independent experiments. (D) Representative immunofluorescence staining showing the expression of -SMA (upper) and CD133 (down) of cells at passage 2. The cell nuclei were labeled with DAPI. Images are representative of three independent experiments. (E) Representative photomicrographs of Oil Red O stain (left) showing intracytoplasmic lipid droplets (red) and of Alizarin Red S stain (right) showing calcium deposits (orange) of cells at passage 2. (F) The s-shape cell growth curves show that the chicken bone marrow-derived MSCs exhibit the feature of rapid proliferation. Cell growth reaches logarithmic growth phase even earlier at lower initial density of 1102 per well compared to 1103 per well.

Abbreviations: MSCs, mesenchymal stem cells; -SMA, -smooth muscle actin.

To identify MSCs in the lung after transplantation, MSCs were pre-labeled with 4,6-diamidino-2-phenylindole (DAPI). Approximately 90% of MSCs showed blue fluorescence after labeling (Figure 3A). One week after intravenous injection, DAPI-positive MSCs were sparsely present in the lung, which were mainly clustered in the parenchyma near to blood vessels (Figure 3B).

Figure 3 Detection of transplanted MSCs in the lung. (A) Representative image showing 4,6-diamidino-2-phenylindole (DAPI)-labelled MSCs. (B) DAPI-labelled MSCs (2106) (upper panel) were injected via the wing vein of broiler chickens. The unlabeled MSCs (lower panel) were served as a control. At d 6 post implantation, lung was collected and embedded in optimal cutting temperature (OCT) compound to obtain 15-m-thick sections for determination of DAPI-positive cells (arrow). Images were acquired with fluorescence microscopy. Bright field and DAPI-fluorescence are shown.

Abbreviation: MSCs, mesenchymal stem cells.

In order to determine whether MSC transplantation has a preventive effect on PAH, we delivered MSCs through wing vein injection to birds 1 d before PAH induction and measured PAH-related morbidity and RV/TV ratio as an indicator of increased pulmonary arterial pressure. Cases of PAH found at processing and cumulative PAH incidence are shown in Table 1. MSC transplantation markedly reduced the cumulative PAH morbidity as compared to mock treatment, coincident with reduced RV/TV ratio at weeks 2 and 3 post transplantation (Figure 4) when a peak incidence in the mock group was observed (Table 1). These results indicate that MSC transplantation prevents the development of PAH.

Table 1 Cases of PAH Found at Processing and Cumulative PAH Incidence

Figure 4 Right-to-total ventricle ratio (RV/TV) of birds treated with MSCs (MSC group) or PBS (mock group) at weeks 14 after MSC implantation. Data are expressed as mean SEM of 6 birds.

Abbreviation: MSCs, mesenchymal stem cells.

MSC treatment has been shown to attenuate inflammatory response in a MCT-induced PAH rat model.12 To determine if the protective effect of MSCs on the development of PAH is associated with attenuated inflammation in our avian model, production of major inflammatory mediators in the lung tissue was measured by Western blot. As shown in Figure 5, there were no significant differences in TNF- and IL-6 production between groups during the first 2 weeks post transplantation. However, MSC group had lower TNF- and IL-6 production than mock group at week 3. MSC transplantation led to a consistent reduction in IL-1 production as compared to mock treatment and a significant difference was observed at week 2. Taken together, the data suggest that MSC transplantation attenuates PAH-induced inflammatory response in the lung.

Figure 5 Regulatory effect of MSC transplantation on the production of inflammatory mediators in the lung. Lung samples of birds treated with MSCs (MSC group) or PBS (mock group) were collected at weeks 1, 2 and 3 post transplantation, homogenized and subjected to Western blot analysis with anti-tumor necrosis factor (TNF)- (A), anti-interleukin (IL)-6 (B) and anti- IL-1 (C). Tubulin was used as the equal loading control. Results are expressed as mean SEM of at least 5 birds. The data are representative of 2 separate experiments.

Abbreviation: MSCs, mesenchymal stem cells.

Endothelial cell damage is thought to be the first trigger of PAH.6 Since a peak incidence of PAH in mock group was observed between weeks 2 and 3, lung samples collected at weeks 3 were subjected to histological analyses of endothelial damage. While detachment of endothelial cells from the underlying basement membrane (Figure 6A), intimal thickening (Figure 6B), endothelial cell proliferation (Figure 6C), and plexiform lesions with perivascular inflammatory infiltrates (Figure 6D and E) were frequently observed in the lung of birds in mock group, the majority of the pulmonary vessels in MSC group displayed intact endothelium (Figure 6F and G). Particularly, the density of plexiform lesions in MSC group was significantly lower than that in mock group (Figure 6H).

Figure 6 Transplantation of MSCs alleviated the histologic features of PAH. (AE) Representative photographs of small pulmonary arteries from birds treated with PBS (mock group) showing (A) loss of endothelial cells, (B) eccentric intimal thickening (arrow), (C) endothelial proliferation, (D) an immature lesion, and (E) a mature glomeruloid-like plexiform lesion (arrowhead) with perivascular inflammatory infiltrates (arrow). (F) A small pulmonary artery from a bird treated with MSCs (MSC group) showing intact vascular endothelium. (G) Percentage of arterioles with normal endothelium. At least 20 arterioles with an outer diameter < 50 m were randomly selected in each slide for analysis. Data are expressed as mean SEM of six birds. (H) Plexiform lesion density in the lung. Data are expressed as mean SEM of at least three birds.

Abbreviations: MSCs, mesenchymal stem cells; PAH, pulmonary arterial hypertension.

Dysregulation of angiogenic factors has been implicated in the development and progression of PAH in both humans38 and broilers.20 To determine if MSCs exert protective effects by modulating angiogenesis in our avian model of PAH, we next assessed by qPCR the mRNA levels of vascular endothelial growth factor (VEGF), transforming growth factor (TGF-), hepatocyte growth factor (HGF), angiopoietin (Ang)-1 and angiopoietin receptor Tie-2 in the lung tissues. As shown in Figure 7, there were no significant differences in the mRNA levels of these factors between groups at weeks 3 post transplantation. However, the birds treated with MSCs exhibited a 2.95-fold increase in TGF- level in respect to their counterparts. In contrast, and in line with a previous study,39 MSC transplantation led to a downregulation in VEGF mRNA expression (0.44-fold relative to mock control). It is worthy to note that the expression levels of the genes investigated varied extremely between individuals.

Figure 7 Expression of angiogenic factors in the lung. Lung samples of birds treated with MSCs (MSC group) or PBS (mock group) at weeks 3 post transplantation were subjected to qPCR analysis for measuring the mRNA levels of transforming growth factor (TGF)-, vascular endothelial growth factor (VEGF)-A, hepatocyte growth factor (HGF) and angiopoietin (Ang)-1 and its receptor Tie-2. Results are expressed as mean SEM of 3 birds. The data are representative of 2 separate experiments.

MSCs are capable of differentiating into endothelial lineage cells upon the stimulation of proangiogenic factors, forming capillary-like network structures on Matrigel matrix.40 We thus performed a Matrigel tube formation assay to evaluate the endothelial differentiation of peripheral blood-derived MSCs from normal, mock- and MSC-treated birds at weeks 3 post PAH induction. While the peripheral MSCs from normal birds exhibited individual round cells on the Matrigel surface with the formation of few cords (Figure 8A), these from mock-treated animals gave rise to clusters (Figure 8B). By contrast, MSCs from birds that received exogenous MSCs developed well-organized tube-like network structures (Figure 8C). It is noteworthy that the cells were not treated with proangiogenic factors. The data suggest that MSC transplantation improves the endothelial differentiation of endogenous MSCs.

Figure 8 Transplantation of MSCs enhanced endothelial differentiation of endogenous MSCs. Blood samples were collected from normal (A), PBS- (B) and MSC-treated (C) broiler chickens at 3 weeks post transplantation. Cells at Passage 1 were suspended in DEME and seeded in 96-well plates pre-coated with Matrigel matrix at 5 104/well with triplicate. Tubular-like structures were observed udder phase-contrast microscopy within 4 h of incubation.

Abbreviation: MSCs, mesenchymal stem cells.

In the present study, intravenous administration of MSCs efficiently prevented the development of cold temperature/salt-induced PAH in birds, as evidenced by markedly reduced morbidity and right ventricular hypertrophy. This effect was associated with attenuated endothelial damage, plexogenic arteriopathy and local inflammation. In vitro angiogenic assay provided clear evidence that MSC transplantation potentiates the differentiation of endogenous MSCs into endothelial lineage cells, suggesting that endogenous MSCs are actively involved in endothelial repair or regeneration after MSC transplantation. To the best of our knowledge, functional modulation of transplanted MSCs on endogenous MSCs hasnever been investigated before.

MSCs are first discovered in mammalian bone marrow,41 which is currently the most studied source of MSCs for both experimental and clinical studies. MSCs from chicken bone marrow have also been previously characterized.42 In the present study, the chicken bone marrow-derived MSCs exhibited features consistent with those of mammalian MSCs in terms of morphology, rapid proliferation, adherence to plastic, multilineage differentiation, and immunophenotype.43,44 In this regard, we believed that the bioactive of avian MSCs would be comparable to that of mammals.

PAH in broilers can be induced by either cold temperature22 or excessive salt in drinking water.23 In this work, we challenged the birds with a combination of cold temperature and excessive salt in drinking water. Under this condition, 50% (15/30) of birds in the mock group developed PAH as assessed by RV/TV ratio. In contrast, and as expected, MSC transplantation reduced PAH morbidity by ~44% and delayed the occurrence of PAH by 2 weeks as compared to mock treatment. This finding is in line with a previous study where MSCs have been shown to confer a protective role in both chronic hypoxia- and Sugen5416/hypoxia/normoxia-exposed PAH in rat.45 Since birds with PAH were usually found dead or quickly died in response to capture stress during the experiment, our attempt to conduct MSC transplantation in PAH birds was hampered. Thus, the beneficial effect of MSCs on established PAH was not evaluated.

Accumulating evidence suggests that the efficacy of MSC therapies is mainly attributed to paracrine action.11 MSCs are found to produce a plenty of paracrine factors including angiogenic cytokines such as VEGF-A, HGF, Ang-1, and TGF-.46 However, it still remains uncertain whether the angiogenic factors play a role in the therapeutic mechanisms of MSCs for PAH, as conflicting results have been reported regarding the production of angiogenic cytokines in the lungs of MCT models after MSC therapies.39,47 After determining the protective effect of MSC transplantation on the progression of PAH in our avian model, we analyzed if the angiogenic factors are involved. Unexpectedly, we did not determine a remarkable difference in the expression of the tested angiogenic factors in the lung tissues between MSC and mock groups. Indeed, the expression of VEGF-A, which is known to play a predominant role in angiogenesis,48 tended to be downregulated in MSC group, which is in line with a previous study demonstrating decreased VEGF-A production in MCT model after MSC therapy.39 Thus, it is likely that the paracrine angiogenic factors are not essential for the protective and therapeutic effects of MSC transplantation. Indeed, it is suggested that the paracrine activity of MSCs for tissue repair is mainly ascribed to their immunomodulatory properties.49 In this work, MSC transplantation reduced the production of proinflammatory factors TNF-, IL-6 and IL-1 as compared to the mock treatment at different time points, coincident with a moderate increase in the expression of anti-inflammatory cytokine TGF-. Taken together, our observations suggest that the immunosuppressive cytokines released by the transplanted MSCs might play a more important role than paracrine angiogenic factors in pulmonary repair.

It is generally accepted that endothelial injury resulting in endothelial dysfunction and proliferation plays a central role in the initiation and progression of vascular pathology in PAH including vascular remodeling, inflammation and the formation of plexiform lesions.6,5052 MSCs have been shown to improve MCT-induced pulmonary endothelial injury.53 Similarly, the present study revealed that MSC transplantation significantly attenuated PAH-induced endothelial abnormalities seen in the mock group, ie, endothelial cell detachment and proliferation. Thus, our findings together with the observations in MCT models suggest that the implanted MSCs induce functional repair or regeneration of endothelial cells.

A growing body of literature suggests that differentiation into vascular cells in the arterial wall is not involved in the endothelial repair mechanisms of the transplanted MSCs, although they were found to be trapped in the lung after intravenous injection or intratracheal administration.45,47,53,54 Indeed, despite extensive research, the origin of the newly developed endothelial cells involved in vascular repair is still controversial and has been a subject of continued interest.55 Considering the fact that MSC population is selectively mobilized from bone marrow in response to hypoxia and endothelial injury,56,57 and that those cells are capable of differentiating into endothelial cells,58 we next determined whether MSC transplantation affect the angiogenic potential of endogenous MSCs. As a result, MSC transplantation significantly potentiated the endothelial differentiation of endogenous MSCs, as indicated by a robust enhancement of in vitro tubular formation of peripheral blood-derived MSCs in birds from MSC group. Thus, our results allow us to argue that the observed effects of MSC transplantation in our animal model are associated with endogenous MSC-mediated endothelial repair or regeneration. No previous studies have been conducted to address the proangiogenic effects of transplanted MSCs on endogenous MSCs. The mechanisms by which the transplanted MSCs regulate endothelial differentiation of endogenous MSCs remain unclear, but might be associated with their paracrine effects. It is also possible that the transplanted MSCs may directly contact and transfer cellular components to endogenous MSCs, thereby enhancing their angiogenic potential. Indeed, a contact-dependent mechanism of action by which MSCs ameliorate acute lung injury has been previously proposed.59 Further studies are warranted to determine the exact mechanisms underlying this process.

Some limitations of this work should be considered. Although PAH birds share all the reported histological features observed in human PAH, the plexiform lesions, which are considered as a hallmark of severe PAH in humans,14 can be found in birds without signs of PAH.20,21,33 Nevertheless, we recently provided clear evidence that the development of plexiform lesions in birds is associated with increased pulmonary arterial pressure.24 In addition, we evaluated the angiogenic potential of blood-derived MSCs after MSC transplantation. However, this population of MSCs might differ from the tissue-resident lung MSCs, as that observed between lung-derived and bone marrow-derived MSCs.60 Therefore, further studies should be carried out to determine the beneficial effects of exogenous MSCs on lung-resident MSCs. Finally, in this work, only a single dose of MSCs was performed. Whether two or more doses of MSCs yield enhanced endothelial repair during the development of PAH warrants further research.

In summary, this work demonstrates that MSC transplantation attenuates inflammation and prevents the progression of PAH. We show clear evidence that MSC transplantation potentiates the angiogenic potency of endogenous MSCs, providing a novel insight into the mechanisms accounting for the beneficial effects of MSC transplantation on PAH. Our findings encourage the development of MSC therapy for treating PAH.

All the operation was approved by the Ethics Committee of the Zhejiang University (Approved No. ZJU2015-445-12).

The study was supported by the National Natural Science Foundation of China (Project No. 31872444 to X.T.) and Zhejiang Provincial Natural Science Foundation of China (Project No. LR12C18001 to X.T.).

The authors report no conflicts of interest in this work.

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18. Kluess HA, Stafford J, Evanson KW, et al. Intrapulmonary arteries respond to serotonin and adenosine triphosphate in broiler chickens susceptible to idiopathic pulmonary arterial hypertension. Poult Sci. 2012;91(6):14321440. doi:10.3382/ps.2011-01919

19. Wideman RJ, Hamal KR. Idiopathic pulmonary arterial hypertension: an avian model for plexogenic arteriopathy and serotonergic vasoconstriction. J Pharmacol Toxicol Methods. 2011;63(3):283295. doi:10.1016/j.vascn.2011.01.002

20. Hamal KR, Erf GF, Anthony NB, Wideman RF. Immunohistochemical examination of plexiform-like complex vascular lesions in the lungs of broiler chickens selected for susceptibility to idiopathic pulmonary arterial hypertension. Avian Pathol. 2012;41(2):211219. doi:10.1080/03079457.2012.663077

21. Tan X, Juan FG, Shah AQ. Involvement of endothelial progenitor cells in the formation of plexiform lesions in broiler chickens: possible role of local immune/inflammatory response. J Zhejiang Univ Sci B. 2017;18(1):5969. doi:10.1631/jzus.B1600500

22. Tan X, Chai J, Bi SC, et al. Involvement of matrix metalloproteinase-2 in medial hypertrophy of pulmonary arterioles in broiler chickens with pulmonary arterial hypertension. Vet J. 2012;193(2):420425. doi:10.1016/j.tvjl.2012.01.017

23. Shao FJ, Ying YT, Tan X, Zhang QY, Liao WT. Metabonomics profiling reveals biochemical pathways associated with pulmonary arterial hypertension in broiler chickens. J Proteome Res. 2018;17(10):34453453. doi:10.1021/acs.jproteome.8b00316

24. Tan X, Shao FJ, Fan GJ, Ying YT. Expression of angiogenic factors and plexiform lesions in the lungs of broiler and layer chickens: a comparison. Poult Sci. 2018;97(5):15261535. doi:10.3382/ps/pey008

25. Tan X, Pan JQ, Li JC, et al. L-arginine inhibiting pulmonary vascular remodelling is associated with promotion of apoptosis in pulmonary arterioles smooth muscle cells in broilers. Res Vet Sci. 2005;79(3):203209. doi:10.1016/j.rvsc.2004.12.004

26. Pan JQ, Li JC, Tan X, et al. The injury effect of oxygen free radicals in vitro on cultured pulmonary artery endothelial cells from broilers. Res Vet Sci. 2007;82(3):382387. doi:10.1016/j.rvsc.2006.08.001

27. Bautista-Ortega J, Stallone JN, Ruiz-Feria CA. Effects of arginine and antioxidant vitamins on pulmonary artery reactivity to phenylephrine in the broiler chicken. Poult Sci. 2013;92(4):10621072. doi:10.3382/ps.2012-02472

28. Khatri M, OBrien TD, Sharma JM. Isolation and differentiation of chicken mesenchymal stem cells from bone marrow. Stem Cells Dev. 2009;18(10):14851492. doi:10.1089/scd.2008.0223

29. Bi S, Tan X, Ali SQ, Wei L. Isolation and characterization of peripheral blood-derived endothelial progenitor cells from broiler chickens. Vet J. 2014;202(2):396399. doi:10.1016/j.tvjl.2014.08.017

30. Karaoz E, Aksoy A, Ayhan S, et al. Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers. Histochem Cell Biol. 2009;132(5):533546. doi:10.1007/s00418-009-0629-6

31. Shah QA, Tan X, Bi S, Liu X, Hu S. Differential characteristics and in vitro angiogenesis of bone marrow- and peripheral blood-derived endothelial progenitor cells: evidence from avian species. Cell Prolif. 2014;47(4):290298. doi:10.1111/cpr.12111

32. Julian RJ, McMillan I, Quinton M. The effect of cold and dietary energy on right ventricular hypertrophy, right ventricular failure and ascites in meat-type chickens. Avian Pathol. 1989;18(4):675684. doi:10.1080/03079458908418641

33. Wideman RF, Hamal KR, Bayona MT, et al. Plexiform lesions in the lungs of domestic fowl selected for susceptibility to pulmonary arterial hypertension: incidence and histology. Anat Rec. 2011;294(5):739755. doi:10.1002/ar.21369

34. Tan X, Hu SH, Wang XL. The effect of dietary l-carnitine supplementation on pulmonary hypertension syndrome mortality in broilers exposed to low temperatures. J Anim Physiol Anim Nutr (Berl). 2008;92(2):203210. doi:10.1111/j.1439-0396.2007.00727.x

35. Nancarrow-Lei R, Mafi P, Mafi R, Khan W. A systemic review of adult mesenchymal stem cell sources and their multilineage differentiation potential relevant to musculoskeletal tissue repair and regeneration. Curr Stem Cell Res Ther. 2017;12(8):601610. doi:10.2174/1574888X12666170608124303

36. Haasters F, Prall WC, Anz D, et al. Morphological and immunocytochemical characteristics indicate the yield of early progenitors and represent a quality control for human mesenchymal stem cell culturing. J Anat. 2009;214(5):759767. doi:10.1111/j.1469-7580.2009.01065.x

37. Roubelakis MG, Tsaknakis G, Pappa KI, Anagnou NP, Watt SM. Spindle shaped human mesenchymal stem/stromal cells from amniotic fluid promote neovascularization. PLoS One. 2013;8(1):e54747. doi:10.1371/journal.pone.0054747

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39. de Mendonca L, Felix NS, Blanco NG, et al. Mesenchymal stromal cell therapy reduces lung inflammation and vascular remodeling and improves hemodynamics in experimental pulmonary arterial hypertension. Stem Cell Res Ther. 2017;8(1):220. doi:10.1186/s13287-017-0669-0

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The rest is here:
MSC transplantation on the development of PAH | JIR - Dove Medical Press

Stem Cell Mimicking Nanoencapsulation for Targeting Arthrit | IJN – Dove Medical Press

Introduction

Given the multi-lineage differentiation abilities of mesenchymal stem cells (MSCs) isolated from different tissues and organs, MSCs have been widely used in various medical fields, particularly regenerative medicine.13 The representative sources of MSCs are bone marrow, adipose, periodontal, muscle, and umbilical cord blood.410 Interestingly, slight differences have been reported in the characteristics of MSCs depending on the different sources, including their population in source tissues, immunosuppressive activities, proliferation, and resistance to cellular aging.11 Bone marrow-derived MSCs (BM-MSCs) are the most intensively studied and show clinically promising results for cartilage and bone regeneration.11 However, the isolation procedures for BM-MSCs are complicated because bone marrow contains a relatively small fraction of MSCs (0.0010.01% of the cells in bone marrow).12 Furthermore, bone marrow aspiration to harvest MSCs in human bones is a painful procedure and the slower proliferation rate of BM-MSCs is a clinical limitation.13 In comparison with BM-MSCs, adipose-derived MSCs (AD-MSCs) are relatively easy to collect and can produce up to 500 times the cell population of BM-MSCs.14 AD-MSCs showed a greater ability to regenerate damaged cartilage and bone tissues with increased immunosuppressive ability.14,15 Umbilical cord blood-derived MSCs (UC-MSCs) proliferate faster than BM-MSCs and are resistant to significant cellular aging.11

MSCs have been investigated and gained worldwide attention as potential therapeutic candidates for incurable diseases such as arthritis, spinal cord injury, and cardiac disease.3,1623 In particular, the inherent tropism of MSCs to inflammatory sites has been thoroughly studied.24 This inherent tropism, also known as homing ability, originates from the recognition of various chemokine sources in inflamed tissues, where profiled chemokines are continuously secreted and the MSCs migrate to the chemokines in a concentration-dependent manner.24 Rheumatoid arthritis (RA) is a representative inflammatory disease that primarily causes inflammation in the joints, and this long-term autoimmune disorder causes worsening pain and stiffness following rest. RA affects approximately 24.5 million people as of 2015, but only symptomatic treatments such as pain medications, steroids, and nonsteroidal anti-inflammatory drugs (NSAIDs), or slow-acting drugs that inhibit the rapid progression of RA, such as disease-modifying antirheumatic drugs (DMARDs) are currently available. However, RA drugs have adverse side effects, including hepatitis, osteoporosis, skeletal fracture, steroid-induced arthroplasty, Cushings syndrome, gastrointestinal (GI) intolerance, and bleeding.2527 Thus, MSCs are rapidly emerging as the next generation of arthritis treatment because they not only recognize and migrate toward chemokines secreted in the inflamed joints but also regulate inflammatory progress and repair damaged cells.28

However, MSCs are associated with many challenges that need to be overcome before they can be used in clinical settings.2931 One of the main challenges is the selective accumulation of systemically administered MSCs in the lungs and liver when they are administered intravenously, leading to insufficient concentrations of MSCs in the target tissues.32,33 In addition, most of the administered MSCs are typically initially captured by macrophages in the lungs, liver, and spleen.3234 Importantly, the viability and migration ability of MSCs injected in vivo differed from results previously reported as favorable therapeutic effects and migration efficiency in vitro.35

To improve the delivery of MSCs, researchers have focused on chemokines, which are responsible for MSCs ability to move.36 The chemokine receptors are the key proteins on MSCs that recognize chemokines, and genetic engineering of MSCs to overexpress the chemokine receptor can improve the homing ability, thus enhancing their therapeutic efficacy.37 Genetic engineering is a convenient tool for modifying native or non-native genes, and several technologies for genetic engineering exist, including genome editing, gene knockdown, and replacement with various vectors.38,39 However, safety issues that prevent clinical use persist, for example, genome integration, off-target effects, and induction of immune response.40 In this regard, MSC mimicking nanoencapsulations can be an alternative strategy for maintaining the homing ability of MSCs and overcoming the current safety issues.4143 Nanoencapsulation involves entrapping the core nanoparticles of solids or liquids within nanometer-sized capsules of secondary materials.44

MSC mimicking nanoencapsulation uses the MSC membrane fraction as the capsule and targeting molecules, that is chemokine receptors, with several types of nanoparticles, as the core.45,46 MSC mimicking nanoencapsulation consists of MSC membrane-coated nanoparticles, MSC-derived artificial ectosomes, and MSC membrane-fused liposomes. Nano drug delivery is an emerging field that has attracted significant interest due to its unique characteristics and paved the way for several unique applications that might solve many problems in medicine. In particular, the nanoscale size of nanoparticles (NPs) enhances cellular uptake and can optimize intracellular pathways due to their intrinsic physicochemical properties, and can therefore increase drug delivery to target tissues.47,48 However, the inherent targeting ability resulting from the physicochemical properties of NPs is not enough to target specific tissues or damaged tissues, and additional studies on additional ligands that can bind to surface receptors on target cells or tissues have been performed to improve the targeting ability of NPs.49 Likewise, nanoencapsulation with cell membranes with targeting molecules and encapsulation of the core NPs with cell membranes confer the targeting ability of the source cell to the NPs.50,51 Thus, MSC mimicking nanoencapsulation can mimic the superior targeting ability of MSCs and confer the advantages of each core NP. In addition, MSC mimicking nanoencapsulations have improved circulation time and camouflaging from phagocytes.52

This review discusses the mechanism of MSC migration to inflammatory sites, addresses the potential strategy for improving the tropism of MSCs using genetic engineering, and discusses the promising therapeutic agent, MSC mimicking nanoencapsulations.

The MSC migration mechanism can be exploited for diverse clinical applications.53 The MSC migration mechanism can be divided into five stages: rolling by selectin, activation of MSCs by chemokines, stopping cell rolling by integrin, transcellular migration, and migration to the damaged site (Figure 1).54,55 Chemokines are secreted naturally by various cells such as tumor cells, stromal cells, and inflammatory cells, maintaining high chemokine concentrations in target cells at the target tissue and inducing signal cascades.5658 Likewise, MSCs express a variety of chemokine receptors, allowing them to migrate and be used as new targeting vectors.5961 MSC migration accelerates depending on the concentration of chemokines, which are the most important factors in the stem cell homing mechanism.62,63 Chemokines consist of various cytokine subfamilies that are closely associated with the migration of immune cells. Chemokines are divided into four classes based on the locations of the two cysteine (C) residues: CC-chemokines, CXC-chemokine, C-chemokine, and CX3 Chemokine.64,65 Each chemokine binds to various MSC receptors and the binding induces a chemokine signaling cascade (Table 1).56,66

Table 1 Chemokine and Chemokine Receptors for Different Chemokine Families

Figure 1 Representation of stem cell homing mechanism.

The mechanisms underlying MSC and leukocyte migration are similar in terms of their migratory dynamics.55 P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are major proteins involved in leukocyte migration that interact with P-selectin and E-selectin present in vascular endothelial cells. However, these promoters are not present in MSCs (Figure 2).53,67

Figure 2 Differences in adhesion protein molecules between leukocytes and mesenchymal stem cells during rolling stages and rolling arrest stage of MSC. (A) The rolling stage of leukocytes starts with adhesion to endothelium with ESL-1 and PSGL-1 on leukocytes. (B) The rolling stage of MSC starts with the adhesion to endothelium with Galectin-1 and CD24 on MSC, and the rolling arrest stage was caused by chemokines that were encountered in the rolling stage and VLA-4 with a high affinity for VACM present in endothelial cells.

Abbreviations: ESL-1, E-selectin ligand-1; PSGL-1, P-selectin glycoprotein ligand-1 VLA-4, very late antigen-4; VCAM, vascular cell adhesion molecule-1.

The initial rolling is facilitated by selectins expressed on the surface of endothelial cells. Various glycoproteins on the surface of MSCs can bind to the selectins and continue the rolling process.68 However, the mechanism of binding of the glycoprotein on MSCs to the selectins is still unclear.69,70 P-selectins and E-selectins, major cell-cell adhesion molecules expressed by endothelial cells, adhere to migrated cells adjacent to endothelial cells and can trigger the rolling process.71 For leukocyte migration, P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) expressed on the membranes of leukocytes interact with P-selectins and E-selectins on the endothelial cells, initiating the process.72,73 As already mentioned, MSCs express neither PSGL-1 nor ESL-1. Instead, they express galectin-1 and CD24 on their surfaces, and these bind to E-selectin or P-selectin (Figure 2).7476

In the migratory activation step, MSC receptors are activated in response to inflammatory cytokines, including CXCL12, CXCL8, CXCL4, CCL2, and CCL7.77 The corresponding activation of chemokine receptors of MSCs in response to inflammatory cytokines results in an accumulation of MSCs.58,78 For example, inflamed tissues release inflammatory cytokines,79 and specifically, fibroblasts release CXCL12, which further induces the accumulation of MSCs through ligandreceptor interaction after exposure to hypoxia and cytokine-rich environments in the rat model of inflammation.7982 Previous studies have reported that overexpressing CXCR4, which is a receptor to recognize CXCL12, in MSCs improves the homing ability of MSCs toward inflamed sites.83,84 In short, cytokines are significantly involved in the homing mechanism of MSCs.53

The rolling arrest stage is facilitated by integrin 41 (VLA-4) on MSC.85 VLA-4 is expressed by MSCs which are first activated by CXCL-12 and TNF- chemokines, and activated VLA-4 binds to VCAM-1 expressed on endothelial cells to stop the rotational movement (Figure 2).86,87

Karp et al categorized the migration of MSCs as either systemic homing or non-systemic homing. Systemic homing refers to the process of migration through blood vessels and then across the vascular endothelium near the inflamed site.67,88 The process of migration after passing through the vessels or local injection is called non-systemic homing. In non-systemic migration, stem cells migrate through a chemokine concentration gradient (Figure 3).89 MSCs secrete matrix metalloproteinases (MMPs) during migration. The mechanism underlying MSC migration is currently undefined but MSC migration can be advanced by remodeling the matrix through the secretion of various enzymes.9093 The migration of MSCs to the damaged area is induced by chemokines released from the injured site, such as IL-8, TNF-, insulin-like growth factor (IGF-1), and platelet-derived growth factors (PDGF).9496 MSCs migrate toward the damaged area following a chemokine concentration gradient.87

Figure 3 Differences between systemic and non-systemic homing mechanisms. Both systemic and non-systemic homing to the extracellular matrix and stem cells to their destination, MSCs secrete MMPs and remodel the extracellular matrix.

Abbreviation: MMP, matrix metalloproteinase.

RA is a chronic inflammatory autoimmune disease characterized by distinct painful stiff joints and movement disorders.97 RA affects approximately 1% of the worlds population.98 RA is primarily induced by macrophages, which are involved in the innate immune response and are also involved in adaptive immune responses, together with B cells and T cells.99 Inflammatory diseases are caused by high levels of inflammatory cytokines and a hypoxic low-pH environment in the joints.100,101 Fibroblast-like synoviocytes (FLSs) and accumulated macrophages and neutrophils in the synovium of inflamed joints also express various chemokines.102,103 Chemokines from inflammatory reactions can induce migration of white blood cells and stem cells, which are involved in angiogenesis around joints.101,104,105 More than 50 chemokines are present in the rheumatoid synovial membrane (Table 2). Of the chemokines in the synovium, CXCL12, MIP1-a, CXCL8, and PDGF are the main ones that attract MSCs.106 In the RA environment, CXCL12, a ligand for CXCR4 on MSCs, had 10.71 times higher levels of chemokines than in the normal synovial cell environment. MIP-1a, a chemokine that gathers inflammatory cells, is a ligand for CCR1, which is normally expressed on MSC.107,108 CXCL8 is a ligand for CXCR1 and CXCR2 on MSCs and induces the migration of neutrophils and macrophages, leading to ROS in synovial cells.59 PDGF is a regulatory peptide that is upregulated in the synovial tissue of RA patients.109 PDGF induces greater MSC migration than CXCL12.110 Importantly, stem cells not only have the homing ability to inflamed joints but also have potential as cell therapy with the anti-apoptotic, anti-catabolic, and anti-fibrotic effect of MSC.111 In preclinical trials, MSC treatment has been extensively investigated in collagen-induced arthritis (CIA), a common autoimmune animal model used to study RA. In the RA model, MSCs downregulated inflammatory cytokines such as IFN-, TNF-, IL-4, IL-12, and IL1, and antibodies against collagen, while anti-inflammatory cytokines, such as tumor necrosis factor-inducible gene 6 protein (TSG-6), prostaglandin E2 (PGE2), transforming growth factor-beta (TGF-), IL-10, and IL-6, were upregulated.112116

Table 2 Rheumatoid Arthritis (RA) Chemokines Present in the Pathological Environment and Chemokine Receptors Present in Mesenchymal Stem Cells

Genetic engineering can improve the therapeutic potential of MSCs, including long-term survival, angiogenesis, differentiation into specific lineages, anti- and pro-inflammatory activity, and migratory properties (Figure 4).117,118 Although MSCs already have an intrinsic homing ability, the targeting ability of MSCs and their derivatives, such as membrane vesicles, which are utilized to produce MSC mimicking nanoencapsulation, can be enhanced.118 The therapeutic potential of MSCs can be magnified by reprogramming MSCs via upregulation or downregulation of their native genes, resulting in controlled production of the target protein, or by introducing foreign genes that enable MSCs to express native or non-native products, for example, non-native soluble tumor necrosis factor (TNF) receptor 2 can inhibit TNF-alpha signaling in RA therapies.28

Figure 4 Genetic engineering of mesenchymal stem cells to enhance therapeutic efficacy.

Abbreviations: Sfrp2, secreted frizzled-related protein 2; IGF1, insulin-like growth factor 1; IL-2, interleukin-2; IL-12, interleukin-12; IFN-, interferon-beta; CX3CL1, C-X3-C motif chemokine ligand 1; VEGF, vascular endothelial growth factor; HGF, human growth factor; FGF, fibroblast growth factor; IL-10, interleukin-10; IL-4, interleukin-4; IL18BP, interleukin-18-binding protein; IFN-, interferon-alpha; SDF1, stromal cell-derived factor 1; CXCR4, C-X-C motif chemokine receptor 4; CCR1, C-C motif chemokine receptor 1; BMP2, bone morphogenetic protein 2; mHCN2, mouse hyperpolarization-activated cyclic nucleotide-gated.

MSCs can be genetically engineered using different techniques, including by introducing particular genes into the nucleus of MSCs or editing the genome of MSCs (Figure 5).119 Foreign genes can be transferred into MSCs using liposomes (chemical method), electroporation (physical method), or viral delivery (biological method). Cationic liposomes, also known as lipoplexes, can stably compact negatively charged nucleic acids, leading to the formation of nanomeric vesicular structure.120 Cationic liposomes are commonly produced with a combination of a cationic lipid such as DOTAP, DOTMA, DOGS, DOSPA, and neutral lipids, such as DOPE and cholesterol.121 These liposomes are stable enough to protect their bound nucleic acids from degradation and are competent to enter cells via endocytosis.120 Electroporation briefly creates holes in the cell membrane using an electric field of 1020 kV/cm, and the holes are then rapidly closed by the cells membrane repair mechanism.122 Even though the electric shock induces irreversible cell damage and non-specific transport into the cytoplasm leads to cell death, electroporation ensures successful gene delivery regardless of the target cell or organism. Viral vectors, which are derived from adenovirus, adeno-associated virus (AAV), or lentivirus (LV), have been used to introduce specific genes into MSCs. Recombinant lentiviral vectors are the most widely used systems due to their high tropism to dividing and non-dividing cells, transduction efficiency, and stable expression of transgenes in MSCs, but the random genome integration of transgenes can be an obstacle in clinical applications.123 Adenovirus and AAV systems are appropriate alternative strategies because currently available strains do not have broad genome integration and a strong immune response, unlike LV, thus increasing success and safety in clinical trials.124 As a representative, the Oxford-AstraZeneca COVID-19 vaccine, which has been authorized in 71 countries as a vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread globally and led to the current pandemic, transfers the spike protein gene using an adenovirus-based viral vector.125 Furthermore, there are two AAV-based gene therapies: Luxturna for rare inherited retinal dystrophy and Zolgensma for spinal muscular atrophy.126

Figure 5 Genetic engineering techniques used in the production of bioengineered mesenchymal stem cells.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 were recently used for genome editing and modification because of their simpler design and higher efficiency for genome editing, however, there are safety issues such as off-target effects that induce mutations at sites other than the intended target site.127 The foreign gene is then commonly transferred into non-integrating forms such as plasmid DNA and messenger RNA (mRNA).128

The gene expression machinery can also be manipulated at the cytoplasmic level through RNA interference (RNAi) technology, inhibition of gene expression, or translation using neutralizing targeted mRNA molecules with sequence-specific small RNA molecules such as small interfering RNA (siRNA) or microRNA (miRNA).129 These small RNAs can form enzyme complexes that degrade mRNA molecules and thus decrease their activity by inhibiting translation. Moreover, the pre-transcriptional silencing mechanism of RNAi can induce DNA methylation at genomic positions complementary to siRNA or miRNA with enzyme complexes.

CXC chemokine receptor 4 (CXCR4) is one of the most potent chemokine receptors that is genetically engineered to enhance the migratory properties of MSCs.130 CXCR4 is a chemokine receptor specific for stromal-derived factor-1 (SDF-1), also known as CXC motif chemokine 12 (CXCL12), which is produced by damaged tissues, such as the area of inflammatory bone destruction.131 Several studies on engineering MSCs to increase the expression of the CXCR4 gene have reported a higher density of the CXCR4 receptor on their outer cell membrane and effectively increased the migration of MSCs toward SDF-1.83,132,133 CXC chemokine receptor 7 (CXCR7) also had a high affinity for SDF-1, thus the SDF-1/CXCR7 signaling axis was used to engineer the MSCs.134 CXCR7-overexpressing MSCs in a cerebral ischemia-reperfusion rat hippocampus model promoted migration based on an SDF-1 gradient, cooperating with the SDF-1/CXCR4 signaling axis (Figure 6).37

Figure 6 Engineered mesenchymal stem cells with enhanced migratory abilities.

Abbreviations: CXCR4, C-X-C motif chemokine receptor 4; CXCR7, C-X-C motif chemokine receptor 7; SDF1, stromal cell-derived factor 1; CXCR1, C-X-C motif chemokine receptor 1; IL-8, interleukin-8; Aqp1, aquaporin 1; FAK, focal adhesion kinase.

CXC chemokine receptor 1 (CXCR1) enhances MSC migratory properties.59 CXCR1 is a receptor for IL-8, which is the primary cytokine involved in the recruitment of neutrophils to the site of damage or infection.135 In particular, the IL-8/CXCR1 axis is a key factor for the migration of MSCs toward human glioma cell lines, such as U-87 MG, LN18, U138, and U251, and CXCR1-overexpressing MSCs showed a superior capacity to migrate toward glioma cells and tumors in mice bearing intracranial human gliomas.136

The migratory properties of MSCs were also controlled via aquaporin-1 (Aqp1), which is a water channel molecule that transports water across the cell membrane and regulates endothelial cell migration.137 Aqp1-overexpressing MSCs showed enhanced migration to fracture gap of a rat fracture model with upregulated focal adhesion kinase (FAK) and -catenin, which are important regulators of cell migration.138

Nur77, also known as nerve growth factor IB or NR4A1, and nuclear receptor-related 1 (Nurr1), can play a role in improving the migratory capabilities of MSCs.139,140 The migrating MSCs expressed higher levels of Nur77 and Nurr1 than the non-migrating MSCs, and overexpression of these two nuclear receptors functioning as transcription factors enhanced the migration of MSCs toward SDF-1. The migration of cells is closely related to the cell cycle, and normally, cells in the late S or G2/M phase do not migrate.141 The overexpression of Nur77 and Nurr1 increased the proportion of MSCs in the G0/G1-phase similar to the results of migrating MSCs had more cells in the G1-phase.

MSC mimicking nanoencapsulations are nanoparticles combined with MSC membrane vesicles and these NPs have the greatest advantages as drug delivery systems due to the sustained homing ability of MSCs as well as the advantages of NPs. Particles sized 10150 nm have great advantages in drug delivery systems because they can pass more freely through the cell membrane by the interaction with biomolecules, such as clathrin and caveolin, to facilitate uptake across the cell membrane compared with micron-sized materials.142,143 Various materials have been used to formulate NPs, including silica, polymers, metals, and lipids.144,145 NPs have an inherent ability, called passive targeting, to accumulate at specific sites based on their physicochemical properties such as size, surface charge, surface hydrophilicity, and geometry.146148 However, physicochemical properties are not enough to target specific tissues or damaged tissues, and thus active targeting is a clinically approved strategy involving the addition of ligands that can bind to surface receptors on target cells or tissues.149,150 MSC mimicking nanoencapsulation uses natural or genetically engineered MSC membranes to coat synthetic NPs, producing artificial ectosomes and fusing them with liposomes to increase their targeting ability (Figure 7).151 Especially, MSCs have been studied for targeting inflammation and regenerative drugs, and the mechanism and efficacy of migration toward inflamed tissues have been actively investigated.152 MSC mimicking nanoencapsulation can mimic the well-known migration ability of MSCs and can be equally utilized without safety issues from the direct application of using MSCs. Furthermore, cell membrane encapsulations have a wide range of functions, including prolonged blood circulation time and increased active targeting efficacy from the source cells.153,154 MSC mimicking encapsulations enter recipient cells using multiple pathways.155 MSC mimicking encapsulations can fuse directly with the plasma membrane and can also be taken up through phagocytosis, micropinocytosis, and endocytosis mediated by caveolin or clathrin.156 MSC mimicking encapsulations can be internalized in a highly cell type-specific manner that depends on the recognition of membrane surface molecules by the cell or tissue.157 For example, endothelial colony-forming cell (ECFC)-derived exosomes were shown CXCR4/SDF-1 interaction and enhanced delivery toward the ischemic kidney, and Tspan8-alpha4 complex on lymph node stroma derived extracellular vesicles induced selective uptake by endothelial cells or pancreatic cells with CD54, serving as a major ligand.158,159 Therefore, different source cells may contain protein signals that serve as ligands for other cells, and these receptorligand interactions maximized targeted delivery of NPs.160 This natural mechanism inspired the application of MSC membranes to confer active targeting to NPs.

Figure 7 Mesenchymal stem cell mimicking nanoencapsulation.

Cell membrane-coated NPs (CMCNPs) are biomimetic strategies developed to mimic the properties of cell membranes derived from natural cells such as erythrocytes, white blood cells, cancer cells, stem cells, platelets, or bacterial cells with an NP core.161 Core NPs made of polymer, silica, and metal have been evaluated in attempts to overcome the limitations of conventional drug delivery systems but there are also issues of toxicity and reduced biocompatibility associated with the surface properties of NPs.162,163 Therefore, only a small number of NPs have been approved for medical application by the FDA.164 Coating with cell membrane can enhance the biocompatibility of NPs by improving immune evasion, enhancing circulation time, reducing RES clearance, preventing serum protein adsorption by mimicking cell glycocalyx, which are chemical determinants of self at the surfaces of cells.151,165 Furthermore, the migratory properties of MSCs can also be transferred to NPs by coating them with the cell membrane.45 Coating NPs with MSC membranes not only enhances biocompatibility but also maximizes the therapeutic effect of NPs by mimicking the targeting ability of MSCs.166 Cell membrane-coated NPs are prepared in three steps: extraction of cell membrane vesicles from the source cells, synthesis of the core NPs, and fusion of the membrane vesicles and core NPs to produce cell membrane-coated NPs (Figure 8).167 Cell membrane vesicles, including extracellular vesicles (EVs), can be harvested through cell lysis, mechanical disruption, and centrifugation to isolate, purify the cell membrane vesicles, and remove intracellular components.168 All the processes must be conducted under cold conditions, with protease inhibitors to minimize the denaturation of integral membrane proteins. Cell lysis, which is classically performed using mechanical lysis, including homogenization, sonication, or extrusion followed by differential velocity centrifugation, is necessary to remove intracellular components. Cytochalasin B (CB), a drug that affects cytoskeletonmembrane interactions, induces secretion of membrane vesicles from source cells and has been used to extract the cell membrane.169 The membrane functions of the source cells are preserved in CB-induced vesicles, forming biologically active surface receptors and ion pumps.170 Furthermore, CB-induced vesicles can encapsulate drugs and NPs successfully, and the vesicles can be harvested by centrifugation without a purification step to remove nuclei and cytoplasm.171 Clinically translatable membrane vesicles require scalable production of high volumes of homogeneous vesicles within a short period. Although mechanical methods (eg, shear stress, ultrasonication, or extrusion) are utilized, CB-induced vesicles have shown potential for generating membrane encapsulation for nano-vectors.168 The advantages of CB-induced vesicles versus other methods are compared in Table 3.

Table 3 Comparison of Membrane Vesicle Production Methods

Figure 8 MSC membrane-coated nanoparticles.

Abbreviations: EVs, extracellular vesicles; NPs, nanoparticles.

After extracting cell membrane vesicles, synthesized core NPs are coated with cell membranes, including surface proteins.172 Polymer NPs and inorganic NPs are adopted as materials for the core NPs of CMCNPs, and generally, polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), chitosan, and gelatin are used. PLGA has been approved by FDA is the most common polymer of NPs.173 Biodegradable polymer NPs have gained considerable attention in nanomedicine due to their biocompatibility, nontoxic properties, and the ability to modify their surface as a drug carrier.174 Inorganic NPs are composed of gold, iron, copper, and silicon, which have hydrophilic, biocompatible, and highly stable properties compared with organic materials.175 Furthermore, some photosensitive inorganic NPs have the potential for use in photothermal therapy (PTT) and photodynamic therapy (PDT).176 The fusion of cell membrane vesicles and core NPs is primarily achieved via extrusion or sonication.165 Cell membrane coating of NPs using mechanical extrusion is based on a different-sized porous membrane where core NPs and vesicles are forced to generate vesicle-particle fusion.177 Ultrasonic waves are applied to induce the fusion of vesicles and NPs. However, ultrasonic frequencies need to be optimized to improve fusion efficiency and minimize drug loss and protein degradation.178

CMCNPs have extensively employed to target and treat cancer using the membranes obtained from red blood cell (RBC), platelet and cancer cell.165 In addition, membrane from MSC also utilized to target tumor and ischemia with various types of core NPs, such as MSC membrane coated PLGA NPs targeting liver tumors, MSC membrane coated gelatin nanogels targeting HeLa cell, MSC membrane coated silica NPs targeting HeLa cell, MSC membrane coated PLGA NPs targeting hindlimb ischemia, and MSC membrane coated iron oxide NPs for targeting the ischemic brain.179183 However, there are few studies on CMCNPs using stem cells for the treatment of arthritis. Increased targeting ability to arthritis was introduced using MSC-derived EVs and NPs.184,185 MSC membrane-coated NPs are proming strategy for clearing raised concerns from direct use of MSC (with or without NPs) in terms of toxicity, reduced biocompatibility, and poor targeting ability of NPs for the treatment of arthritis.

Exosomes are natural NPs that range in size from 40 nm to 120 nm and are derived from the multivesicular body (MVB), which is an endosome defined by intraluminal vesicles (ILVs) that bud inward into the endosomal lumen, fuse with the cell surface, and are then released as exosomes.186 Because of their ability to express receptors on their surfaces, MSC-derived exosomes are also considered potential candidates for targeting.187 Exosomes are commonly referred to as intracellular communication molecules that transfer various compounds through physiological mechanisms such as immune response, neural communication, and antigen presentation in diseases such as cancer, cardiovascular disease, diabetes, and inflammation.188

However, there are several limitations to the application of exosomes as targeted therapeutic carriers. First, the limited reproducibility of exosomes is a major challenge. In this field, the standardized techniques for isolation and purification of exosomes are lacking, and conventional methods containing multi-step ultracentrifugation often lead to contamination of other types of EVs. Furthermore, exosomes extracted from cell cultures can vary and display inconsistent properties even when the same type of donor cells were used.189 Second, precise characterization studies of exosomes are needed. Unknown properties of exosomes can hinder therapeutic efficiencies, for example, when using exosomes as cancer therapeutics, the use of cancer cell-derived exosomes should be avoided because cancer cell-derived exosomes may contain oncogenic factors that may contribute to cancer progression.190 Finally, cost-effective methods for the large-scale production of exosomes are needed for clinical application. The yield of exosomes is much lower than EVs. Depending on the exosome secretion capacity of donor cells, the yield of exosomes is restricted, and large-scale cell culture technology for the production of exosomes is high difficulty and costly and isolation of exosomes is the time-consuming and low-efficient method.156

Ectosome is an EV generated by outward budding from the plasma membrane followed by pinching off and release to the extracellular parts. Recently, artificially produced ectosome utilized as an alternative to exosomes in targeted therapeutics due to stable productivity regardless of cell type compared with conventional exosome. Artificial ectosomes, containing modified cargo and targeting molecules have recently been introduced for specific purposes (Figure 9).191,192 Artificial ectosomes are typically prepared by breaking bigger cells or cell membrane fractions into smaller ectosomes, similar size to natural exosomes, containing modified cargo such as RNA molecules, which control specific genes, and chemical drugs such as anticancer drugs.193 Naturally secreted exosomes in conditioned media from modified source cells can be harvested by differential ultracentrifugation, density gradients, precipitation, filtration, and size exclusion chromatography for exosome separation.194 Even though there are several commercial kits for isolating exosomes simply and easily, challenges in compliant scalable production on a large scale, including purity, homogeneity, and reproducibility, have made it difficult to use naturally secreted exosomes in clinical settings.195 Therefore, artificially produced ectosomes are appropriate for use in clinical applications, with novel production methods that can meet clinical production criteria. Production of artificially produced ectosomes begins by breaking the cell membrane fraction of cultured cells and then using them to produce cell membrane vesicles to form ectosomes. As mentioned above, cell membrane vesicles are extracted from source cells in several ways, and cell membrane vesicles are extracted through polycarbonate membrane filters to reduce the mean size to a size similar to that of natural exosomes.196 Furthermore, specific microfluidic devices mounted on microblades (fabricated in silicon nitride) enable direct slicing of living cells as they flow through the hydrophilic microchannels of the device.197 The sliced cell fraction reassembles and forms ectosomes. There are several strategies for loading exogenous therapeutic cargos such as drugs, DNA, RNA, lipids, metabolites, and proteins, into exosomes or artificial ectosomes in vitro: electroporation, incubation for passive loading of cargo or active loading with membrane permeabilizer, freeze and thaw cycles, sonication, and extrusion.198 In addition, protein or RNA molecules can be loaded by co-expressing them in source cells via bio-engineering, and proteins designed to interact with the protein inside the cell membrane can be loaded actively into exosomes or artificial ectosomes.157 Targeting molecules at the surface of exosomes or artificial ectosomes can also be engineered in a manner similar to the genetic engineering of MSCs.

Figure 9 Mesenchymal stem cell-derived exosomes and artificial ectosomes. (A) Wound healing effect of MSC-derived exosomes and artificial ectosomes,231 (B) treatment of organ injuries by MSC-derived exosomes and artificial ectosomes,42,232234 (C) anti-cancer activity of MSC-derived exosomes and artificial ectosomes.200,202,235

Most of the exosomes derived from MSCs for drug delivery have employed miRNAs or siRNAs, inhibiting translation of specific mRNA, with anticancer activity, for example, miR-146b, miR-122, and miR-379, which are used for cancer targeting by membrane surface molecules on MSC-derived exosomes.199201 Drugs such as doxorubicin, paclitaxel, and curcumin were also loaded into MSC-derived exosomes to target cancer.202204 However, artificial ectosomes derived from MSCs as arthritis therapeutics remains largely unexplored area, while EVs, mixtures of natural ectosomes and exosomes, derived from MSCs have studied in the treatment of arthritis.184 Artificial ectosomes with intrinsic tropism from MSCs plus additional targeting ability with engineering increase the chances of ectosomes reaching target tissues with ligandreceptor interactions before being taken up by macrophages.205 Eventually, this will decrease off-target binding and side effects, leading to lower therapeutic dosages while maintaining therapeutic efficacy.206,207

Liposomes are spherical vesicles that are artificially synthesized through the hydration of dry phospholipids.208 The clinically available liposome is a lipid bilayer surrounding a hollow core with a diameter of 50150 nm. Therapeutic molecules, such as anticancer drugs (doxorubicin and daunorubicin citrate) or nucleic acids, can be loaded into this hollow core for delivery.209 Due to their amphipathic nature, liposomes can load both hydrophilic (polar) molecules in an aqueous interior and hydrophobic (nonpolar) molecules in the lipid membrane. They are well-established biomedical applications and are the most common nanostructures used in advanced drug delivery.210 Furthermore, liposomes have several advantages, including versatile structure, biocompatibility, low toxicity, non-immunogenicity, biodegradability, and synergy with drugs: targeted drug delivery, reduction of the toxic effect of drugs, protection against drug degradation, and enhanced circulation half-life.211 Moreover, surfaces can be modified by either coating them with a functionalized polymer or PEG chains to improve targeted delivery and increase their circulation time in biological systems.212 Liposomes have been investigated for use in a wide variety of therapeutic applications, including cancer diagnostics and therapy, vaccines, brain-targeted drug delivery, and anti-microbial therapy. A new approach was recently proposed for providing targeting features to liposomes by fusing them with cell membrane vesicles, generating molecules called membrane-fused liposomes (Figure 10).213 Cell membrane vesicles retain the surface membrane molecules from source cells, which are responsible for efficient tissue targeting and cellular uptake by target cells.214 However, the immunogenicity of cell membrane vesicles leads to their rapid clearance by macrophages in the body and their low drug loading efficiencies present challenges for their use as drug delivery systems.156 However, membrane-fused liposomes have advantages of stability, long half-life in circulation, and low immunogenicity due to the liposome, and the targeting feature of cell membrane vesicles is completely transferred to the liposome.215 Furthermore, the encapsulation efficiencies of doxorubicin were similar when liposomes and membrane-fused liposomes were used, indicating that the relatively high drug encapsulation capacity of liposomes was maintained during the fusion process.216 Combining membrane-fused liposomes with macrophage-derived membrane vesicles showed differential targeting and cytotoxicity against normal and cancerous cells.217 Although only a few studies have been conducted, these results corroborate that membrane-fused liposomes are a potentially promising future drug delivery system with increased targeting ability. MSCs show intrinsic tropism toward arthritis, and further engineering and modification to enhance their targeting ability make them attractive candidates for the development of drug delivery systems. Fusing MSC exosomes with liposomes, taking advantage of both membrane vesicles and liposomes, is a promising technique for future drug delivery systems.

Figure 10 Mesenchymal stem cell membrane-fused liposomes.

MSCs have great potential as targeted therapies due to their greater ability to home to targeted pathophysiological sites. The intrinsic ability to home to wounds or to the tumor microenvironment secreting inflammatory mediators make MSCs and their derivatives targeting strategies for cancer and inflammatory disease.218,219 Contrary to the well-known homing mechanisms of various blood cells, it is still not clear how homing occurs in MSCs. So far, the mechanism of MSC tethering, which connects long, thin cell membrane cylinders called tethers to the adherent area for migration, has not been clarified. Recent studies have shown that galectin-1, VCAM-1, and ICAM are associated with MSC tethering,53,220 but more research is needed to accurately elucidate the tethering mechanism of MSCs. MSC chemotaxis is well defined and there is strong evidence relating it to the homing ability of MSCs.53 Chemotaxis involves recognizing chemokines through chemokine receptors on MSCs and migrating to chemokines in a gradient-dependent manner.221 RA, a representative inflammatory disease, is associated with well-profiled chemokines such as CXCR1, CXCR4, and CXCR7, which are recognized by chemokine receptors on MSCs. In addition, damaged joints in RA continuously secrete cytokines until they are treated, giving MSCs an advantage as future therapeutic agents for RA.222 However, there are several obstacles to utilizing MSCs as RA therapeutics. In clinical settings, the functional capability of MSCs is significantly affected by the health status of the donor patient.223 MSC yield is significantly reduced in patients undergoing steroid-based treatment and the quality of MSCs is dependent on the donors age and environment.35 In addition, when MSCs are used clinically, cryopreservation and defrosting are necessary, but these procedures shorten the life span of MSCs.224 Therefore, NPs mimicking MSCs are an alternative strategy for overcoming the limitations of MSCs. Additionally, further engineering and modification of MSCs can enhance the therapeutic effect by changing the targeting molecules and loaded drugs. In particular, upregulation of receptors associated with chemotaxis through genetic engineering can confer the additional ability of MSCs to home to specific sites, while the increase in engraftment maximizes the therapeutic effect of MSCs.36,225

Furthermore, there are several methods that can be used to exploit the targeting ability of MSCs as drug delivery systems. MSCs mimicking nanoencapsulation, which consists of MSC membrane-coated NPs, MSC-derived artificial ectosomes, and MSC membrane-fused liposomes, can mimic the targeting ability of MSCs while retaining the advantages of NPs. MSC-membrane-coated NPs are synthesized using inorganic or polymer NPs and membranes from MSCs to coat inner nanosized structures. Because they mimic the biological characteristics of MSC membranes, MSC-membrane-coated NPs can not only escape from immune surveillance but also effectively improve targeting ability, with combined functions of the unique properties of core NPs and MSC membranes.226 Exosomes are also an appropriate candidate for use in MSC membranes, utilizing these targeting abilities. However, natural exosomes lack reproducibility and stable productivity, thus artificial ectosomes with targeting ability produced via synthetic routes can increase the local concentration of ectosomes at the targeted site, thereby reducing toxicity and side effects and maximizing therapeutic efficacy.156 MSC membrane-fused liposomes, a novel system, can also transfer the targeting molecules on the surface of MSCs to liposomes; thus, the advantages of liposomes are retained, but with targeting ability. With advancements in nanotechnology of drug delivery systems, the research in cell-mimicking nanoencapsulation will be very useful. Efficient drug delivery systems fundamentally improve the quality of life of patients with a low dose of medication, low side effects, and subsequent treatment of diseases.227 However, research on cell-mimicking nanoencapsulation is at an early stage, and several problems need to be addressed. To predict the nanotoxicity of artificially synthesized MSC mimicking nanoencapsulations, interactions between lipids and drugs, drug release mechanisms near the targeted site, in vivo compatibility, and immunological physiological studies must be conducted before clinical application.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2019M3A9H1103690), by the Gachon University Gil Medical Center (FRD2021-03), and by the Gachon University research fund of 2020 (GGU-202008430004).

The authors report no conflicts of interest in this work.

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More:
Stem Cell Mimicking Nanoencapsulation for Targeting Arthrit | IJN - Dove Medical Press

Autologous Adult Stem Cells in the Treatment of Stroke | SCCAA – Dove Medical Press

1Regenerative Medicine Centre, Arabian Gulf University, Manama, Bahrain; 2Department of Molecular Medicine, College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain

Introduction: Stroke is a leading cause of death and disability worldwide. The disease is caused by reduced blood flow into the brain resulting in the sudden death of neurons. Limited spontaneous recovery might occur after stroke or brain injury, stem cell-based therapies have been used to promote these processes as there are no drugs currently on the market to promote brain recovery or neurogenesis. Adult stem cells (ASCs) have shown the ability of differentiation and regeneration and are well studied in literature. ASCs have also demonstrated safety in clinical application and, therefore, are currently being investigated as a promising alternative intervention for the treatment of stroke. Methods: Eleven studies have been systematically selected and reviewed to determine if autologous adult stem cells are effective in the treatment of stroke. Collectively, 368 patients were enrolled across the 11 trials, out of which 195 received stem cell transplantation and 173 served as control. Using data collected from the clinical outcomes, a broad comparison and a meta-analysis were conducted by comparing studies that followed a similar study design. Results: Improvement in patients clinical outcomes was observed. However, the overall results showed no clinical significance in patients transplanted with stem cells than the control population. Conclusion: Most of the trials were early phase studies that focused on safety rather than efficacy. Stem cells have demonstrated breakthrough results in the field of regenerative medicine. Therefore, study design could be improved in the future by enrolling a larger patient population and focusing more on localized delivery rather than intravenous transplantation. Trials should also introduce a more standardized method of analyzing and reporting clinical outcomes to achieve a better comparable outcome and possibly recognize the full potential that these cells have to offer.

Keywords: adult stem cells, autologous, neurogenesis, inflammation, clinical application, stroke, stroke recovery, systematic review, meta-analysis

Stroke is the second leading cause of death worldwide and one of the leading causes of disability.1 The blockade or the rupture of a blood vessel to the brain leads to either ischemic or hemorrhagic stroke, respectively.2,3 The extent and the location of the damaged brain tissue may be associated with irreversible cognitive impairment or decline in speech, comprehension, memory, and partial or total physical paralysis.4

Four chronological phases, namely hyperacute, acute, subacute, and chronic, describe the strokes cellular manifestations.5 The hyperacute phase is immediate and associated with glutamate-mediated excitotoxicity and a progressive neuronal death that can last a few hours.6 The glutamate, a potent excitatory neurotransmitter, is also an inducer of neurodegeneration following stroke.7 The acute phase, which could last over a week after the stroke, is associated with the delayed and progressive neuronal death and the infiltration of immune cells.5 The following subacute phase can extend up to three months after the stroke and is mainly associated with reduced inflammation and increased plasticity of neurons, astrocytes, microglia, and endothelial cells, allowing spontaneous recovery.8 In the chronic phase that follows, the plasticity of cells is reduced and only permits rehabilitation-induced recovery.5

The immediate treatments differ for ischemic and hemorrhagic strokes. Immediate intervention is required to restore the blood flow to the brain following an ischemic stroke. Thrombolytic agents, such as activase (Alteplase), a recombinant tissue plasminogen activator (tPA), are commonly given intravenously to dissolve the blood clots. Other more invasive approaches, such as a thrombectomy, use stents or catheters to remove the blood clot.9 Antiplatelet agents like Aspirin, anticoagulants, blood pressure medicines, or statins are generally given to reduce the risk of recurrence. Some ischemic strokes are caused by the narrowing of the carotid artery due to the accumulation of fatty plaques; a carotid endarterectomy is performed to correct the constriction.

The treatment of a hemorrhagic stroke requires a different approach. An emergency craniotomy is usually performed to remove the blood accumulating in the brain and repair the damaged blood vessels. Accumulation of cerebrospinal fluid in brain ventricles (hydrocephalus) is also a frequent complication following a hemorrhagic stroke, which requires surgery to drain the fluid. Medications to lower blood pressure are given before surgery and to prevent further seizures.10

These immediate treatments are critical to minimize the long-term consequence of the stroke but do not address the post-stroke symptoms caused by neurodegeneration. New therapeutic approaches adapted to the physiology of each phase of the stroke are currently developed. A promising therapy has been the use of stem cells.11 In this review, different clinical trials involving the use of various stem cells for the treatment of stroke are presented and compared using a meta-analysis of the published results.

To narrow down the relevant literature, a search strategy focused on original literature and reporting the clinical application of stem cells in stroke was established. An NCBI PubMed word search for stroke, stem cells, and adult stem cells yielded 146 clinical studies between 2010 and 2021. Finally, 11 studies, using autologous adult stem cells in the treatment of stroke, were considered. A PRISMA flow diagram detailing an overview of the study selection procedure and the inclusion and exclusion of papers is included in Appendix I. The inclusion criteria comprise the injection of autologous adult stem cells at any stroke stages (hyperacute, acute, sub-acute, chronic), and clinical trials whose results have been published in the last 11 years. The exclusion criteria include studies published more than 11 years ago, studies not published in English, all preclinical studies, other diseases related to stroke (ex. cardiovascular diseases), embryonic or induced pluripotent stem cells, allogeneic stem cells, and other cell therapies. Two independent researchers reviewed and filtered the 146 studies by reading the titles and abstracts. All three authors approved the final selected studies.

Stem cells are undifferentiated and unspecialized cells characterized by their ability to self-renew and their potential to differentiate into specialized cell types.12 Ischemic stroke causes severe damage to the brain cells by destroying the heterogeneous cell population and neuronal connections along with vascular systems. The regenerative potential of several types of stem cells like embryonic stem cells, neural stem cells, adult stem cells (mesenchymal stem cells), and induced pluripotent stem cells have been assessed for treating stroke.

Adult stem cells exhibit multipotency and the ability to self-renew and differentiate into specialized cell types. They have been widely used in clinical trials and a safe option thus far in treating various diseases.12,13,14 The plasticity of these cells allow their differentiation across tissue lineages when exposed to defined cell culture conditions.15 There are multiple easily accessible sources of adult stem cells, mainly the bone marrow, blood, and adipose tissue. In clinical settings, both autologous and HLA-matched allogeneic cells have been transplanted and are deemed to be safe.

Adult stem cells can secrete a variety of bioactive substances into the injured brain following a stroke in the form of paracrine signals.1618 The paracrine signals include growth factors, trophic factors, and extracellular vesicles, which may be associated with enhanced neurogenesis, angiogenesis, and synaptogenesis (Figure 1). Also, mesenchymal stem cells (MSCs) are thought to contribute to the resolution of the stroke by attenuating inflammation,19 reducing scar thickness, enhancing autophagy, normalizing microenvironmental and metabolic profiles and possibly replacing damaged cells.20

Figure 1 Schematic depicting the clinical application of different cells in stroke patients. The cells were delivered in one of three ways, intravenously, intra-arterially, or via stereotactic injections. Once administered, the cells play a role in providing paracrine signals and growth factors to facilitate angiogenesis and cell regeneration, immunomodulatory effects that serve to protect the neurons from further damage caused by inflammation, and finally, trans-differentiation of stem cells. Data from Dabrowska S, Andrzejewska A, Lukomska B, Janowski M.19 Created with BioRender.com.

A few routes of administration have been used to deliver the stem cells to the patients. The most common is through intravenous injection. Intra-arterial delivery is also performed; but this mode can be extremely painful to patients compared to an intravenous transfusion. The third approach is via stereotactic injections. This is an invasive surgery that involves injecting the cells directly into the site of affected in the brain.

Also known as mesenchymal stromal cells or medicinal signaling cells, MSCs can be derived from different sources including bone marrow, peripheral blood, lungs, heart, skeletal muscle, adipose tissue, dental pulp, dermis, umbilical cord, placenta, amniotic fluid membrane and many more.21 MSCs are characterized by positive cell surface markers, including Stro-1, CD19, CD44, CD90, CD105, CD106, CD146, and CD166. The cells are also CD14, CD34, and CD45 negative.22,23 The cells are thought to provide a niche to stem cells in normal tissue and releases paracrine factors that promote neurogenesis (Figure 2).19,20,24 During the acute and subacute stage of stroke, MSCs may inhibit inflammation, thus, reducing the incidence of debilitating damage and symptoms that may occur post-stroke.

Figure 2 Schematic describing the role of mesenchymal stem cells in stroke. The cells release different growth factors, signals, and cytokines that serve to facilitate various functions. Through the release of cytokines, they can modulate inflammation and block apoptosis. The growth factors aid in promoting angiogenesis and neurogenesis. Data from Maleki M, Ghanbarvand F, Behvarz MR, Ejtemaei M, Ghadirkhomi E.23 Created with BioRender.com.

Derived from the bone marrow, mononuclear cells contain several types of stem cells, including mesenchymal stem cells and hematopoietic progenitor cells that give rise to hematopoietic stem cells and various other differentiated cells. They can produce and secrete multiple growth factors and cytokines. They are also attracted to the lesion or damage site where they can accelerate angiogenesis and promote repair endogenously through the proliferation of the hosts neural stem cells. Mononuclear cells have also demonstrated the ability to decrease neurodegeneration, modulate inflammation, and prevent apoptosis in animal models.25,26

Blood stem cells are a small number of bone marrow stem cells that have been mobilized into the blood by hematopoietic growth factors, which regulate the differentiation and proliferation of cells. They are increasingly used in cell therapies, most recently for the regeneration of non-hematopoietic tissue, including neurons. Recombinant human granulocyte colony-stimulating factor (G-CSF) has been used as a stimulator of hematopoiesis, which in turn amplifies the yield of peripheral blood stem cells.27

The literature review considered 11 clinical trials that satisfied the inclusion criteria. A total of 368 patients were enrolled including 179 patients treated with various types of adult stem cells. The clinical trial number 7 contained a historical control of 59 patients included in the data analysis (Figure 3). The analysis was done on the published clinical and functional outcomes of various tests such as mRS, and mBI. The analysis compared the patients clinical outcomes post stem cell therapy to the baseline clinical results. The variance in the patient population should be noted.

Figure 3 Schematic representing an overview of the total number of patients enrolled in all 11 clinical trials and the number of patients administered with each type of adult stem cell.

Abbreviations: MSC, mesenchymal stem cells; PBSC, peripheral blood stem cells; MNC, mononuclear stem cells; ADSVF, adipose derived stromal vascular fraction; ALD401, aldehyde dehydrogenase-bright stem cells.

Meta-analyses were conducted using modified Rankin scale (mRS) and Barthel Index (BI) scores. In the clinical trials, mRS and BI scores are commonly used scales to assess functional outcome in stroke patients. The BI score was developed to measures the patients performance in 10 activities of daily life from self-care to mobility. An mRS score follows a similar outcome but measures the patients independence in daily tasks rather than performance. OpenMeta[Analyst], an open-source meta-analysis software, was used to produce random-effects meta-analyses and create the forest plots. The number of patients, mean, and standard deviation (SD) of the scores were calculated to determine the study weights and create the forest plots.

All 11 clinical trials were compared based on their clinical and functional outcomes (Table 1; Figure 4). The data shows that stem cell therapy is relatively safe and viable in the treatment of stroke, indicating an improvement in patients overall health. However, when compared to the control, the improvement is not significant as patients in the control group also exhibited an improved clinical and functional outcome. Across trials that assigned a control group, the patients either received a placebo, or alternative form of treatment including physiotherapy. Variance in functional and clinical tests used to assess patients, and the number of patients enrolled in each trial results in a discrepancy in reporting. Most of the trials failed to report whether the patients suffered from an acute, subacute or chronic stroke which also affects the results of the treatments, with acute and subacute being the optimal periods to receive treatment due to cell plasticity and inhibiting unwarranted inflammation.39 The deaths in both the treatment and control population were attributed to the progression of the disease and are likely not the result of the treatment. Albeit, it has been noted down as they had occurred during the follow-up period.

Table 1 Overview of Selected Clinical Trials

Figure 4 Overview of clinical outcomes of the 11 clinical trials (N=368). (A) The chart shows the percentages of patients who have either improved, remained stable, deteriorated, or deceased. Some clinical trials are without a control arm. (B) The plot shows the overall percentage of patients that have improved after receiving either the stem cell treatment versus the standard of care. (C) The plot shows the overall percentage of patients that have remained stable and showed no clinical or functional improvement in the follow up period. (D) The plot shows the overall percentage of the patients whose condition has deteriorated in the follow up period.

A meta-analysis was conducted using modified Rankin scale (mRS) and Barthel Index (BI) scores. The results of the mRS scores were analyzed (Figure 5A; Table 2). In terms of study weights, CT6 is the highest (40.07%) as shown in Table 2. The combined results of the mRS functional test from CT1, CT5, CT6, and CT11 show a non-significant statistical heterogeneity in the studies (p-value 0.113). In conjunction, BI scores were analyzed and a meta-analysis was conducted using four comparable trials (Figure 5B; Table 3). In terms of study weights, CT3 is the highest (32.384%) as shown in Table 3. The combined results of BI scores from CT5, CT3, CT10, and CT11 show a statistical heterogeneity in the results of the studies (p-value 0.004) thus, precision of results is uncertain. More comparable studies are needed to have a better outcome. Therefore, standardized testing in trails should be considered in future trials.

Table 2 Clinical Outcomes of mRS Test

Table 3 Clinical Outcomes of BI Test

Figure 5 Meta-analysis conducted using three comparable trials. (A) Meta-analysis conducted using four comparable trials (CT1, CT5, CT6, CT11) for the mRS test. (B) Meta-analysis conducted using four comparable trials (CT3, CT5, CT10, and CT11) for the BI test.

Across all trials, patients injected with the MSCs, and other cell types did not trigger a degradation of the patient conditions demonstrating the safety of the procedures. However, the efficacy of the use of adult stem cells is less clear when compared to patients in the control group. This discrepancy could, however, exhibit improvement in patients receiving the treatment compared to the baseline clinical outcomes. However, when therapy results are compared to the patients in the control population that either received a placebo, physiotherapy, or prescribed medication, the efficacy of the use of adult stem cells is less clear.

Although multiple adult stem cell types have been used, mesenchymal stem cells have been widely used in many clinical trials. Albeit there is a consensus that the therapeutic and clinical outcomes of mesenchymal stem cell treatments are not yet significantly effective compared to the control treatment. Some trials have shown patient improvements, such as CT6 and CT8, where the investigators used PBSCs or BMMNSC, respectively. Although subjectively, the cells appear to be therapeutic, objectively, there are many limitations to the study designs included in this review. Not all the trials enrolled a control arm for a better comparison as some were only testing safety rather than efficacy. Therefore, we cannot conclude whether autologous adult stem cells are an effective therapeutic stroke treatment. Only autologous cells were included in this review as they are non-immunogenic.

Another factor to consider is the evident discrepancy in the number of patients enrolled in each trial. The trials included in this review are in Phase I and II trials, which primarily focus on safety rather than efficacy. Intravenous injection was the most used method of cell delivery due to its convenience and safety. However, it is commonly considered that this approach is not the most effective way of delivery, as the majority of the transplanted cells get absorbed by non-targeted organs, and the remaining cells find difficulty passing the blood-brain barrier. Due to this dilemma, the most obvious approach would be to inject the cells directly into the brain. However, a stereotactic procedure is invasive and will require general anesthesia, which may compromise patients health, especially ones suffering from acute ischemic stroke.40 Thus, an intra-arterial delivery seems feasible to accomplish the task as it is less invasive and might be more effective than an intravenous treatment such as the cases observed in CT3 and CT8. In CT11, the patients demonstrated a visible fmRI recovery as well as recovery of motor function in patients that have received a stem cell treatment. However, the analysis and test scores show no significance between the treatment group and the control group.

Only a few studies were comparable using a similar evaluation approach. Considering these factors, better study designs enrolling a higher number of patients in randomized clinical trial against the standard of care are needed. Moreover, a better grouping of the patients based on the type and stage of stroke may provide more relevant information for the safety and efficacy of adult stem cells for the recovery and prevention of recurrence of stroke patients.

ADSVF, Adipose-derived stromal vascular fraction; ASCs, Adult stem cells; ALD-401, Aldehyde dehydrogenase 401; BI, Barthel Index; BM-MNC, Bone marrow-derived mononuclear cells; FLAIR, Fluid attenuated inversion recovery; fMRI, Functional magnetic resonance imaging; G-CSF, Granulocyte colony-stimulating factor; MRI, Magnetic resonance imaging; MSCs, Mesenchymal stem cells; mRS, modified Rankin Scale; NIHSS, National Institute of Health Stroke Scale; PBSC, Peripheral blood stem cells; SD, Standard deviation; tPA, tissue plasminogen activator.

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

There is no funding to report.

We declare there is no conflict of interest.

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Autologous Adult Stem Cells in the Treatment of Stroke | SCCAA - Dove Medical Press

James Shapiro, MD: Insulin Production In T1D Patients After Stem Cell Therapy – MD Magazine

Recently, data from studies developing novel cell replacement therapies to address significant unmet needs in severe disease, including type 1 diabetes (T1D).

The study in question is an ongoing, first-in-human Phase study that reported that its stem-cell therapy produced insulin in people with severe T1D. A total of 17 patients were implanted with the ViaCyte PEC-Direct device at 6 different centers, with the device comprising pancreatic cells (PEC-01) contained within pouches for subcutaneous placement.

In an interview with HCPLive, James Shapiro MD PhD, Canada Research Chair and Director of the Islet Transplant Program at the University of Alberta and lead author of the Cell Reports Medicine report, discussed the findings of the study and what they ultimately represent.

It was a very successful trial in terms of demonstrating the safety, it was absolutely safe for patients, while they were, you know, many different potential side effects on the anti rejection drugs and the minor surgeries that the patients went through, they tolerated the placement and the removal of the devices exceedingly well, Shapiro said.

The trial results indicated 34% of patients had evidence of C-peptide production, while 63% of patients had evidence of surviving insulin producing cells at different time points when the devices were taken out and examined under a microscope.

Shapiro went on to describe the next wave of trials using gene-edited products that will not require anti-rejection drugs, called PEC-QT. He noted the difference between a treatment and a cure is the limitless source of cells and lack of need for rejection drugs.

I think if that happened, then we really would have a therapy that could be given to children just diagnosed with diabetes, they could be given to patients with all forms of diabetes, not just patients with T1D, he said. So, I think this does herald a big step forward for for stem cell based therapists in the cure potential curative treatment for all forms of diabetes.

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James Shapiro, MD: Insulin Production In T1D Patients After Stem Cell Therapy - MD Magazine

Global Automated and Closed Cell Therapy Processing Systems – GlobeNewswire

New York, Dec. 27, 2021 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Global Automated and Closed Cell Therapy Processing Systems Market By type, By workflow, By scale, By Regional Outlook, Industry Analysis Report and Forecast, 2021 - 2027" - https://www.reportlinker.com/p06193295/?utm_source=GNW Cell therapy is a technology that is based on replacing any dysfunctional or diseased cell with active & functional cells. Stem cells have the capability to differentiate into certain cells required for repairing damaged or defective tissues or cells, which is the reason why they are utilized for these advanced therapies.

Cell therapy technologies are very important in the medicine and cell therapy sector, which has emerged as a crucial aspect of medical practice. In addition, these cell therapy technologies have common functionality as drug delivery, gene therapy, cancer vaccines, tissue engineering, and regenerative medicine. The process of cell therapy products delivery can vary from injections to surgical implantation by using some specific equipment.

The growth of the market is driven by the increasing popularity of regenerative medicines & cell therapies along with various advantages provided by automation technologies for the development of these therapies. Additionally, the market growth is further driven by the increasing combination of software technologies and sophisticated therapy development procedures.

COVID-19 Impact Analysis

The outbreak of the COVID-19 pandemic has positively impacted the growth of the automated and closed cell therapy processing systems market. It is due to the growing focus of the companies and governments on automation technologies. Along with that, companies have highly invested in the development of advanced therapies and regenerative medicines to fight against the COVID-19 virus. This would support the growth of the market in the coming years.

There are numerous key players that are also focusing on the development of new therapies like exosomes, natural killer cell therapy, stem cell therapy, and others, which would augment the growth of the market in the upcoming years. In addition, governments across the world have also provided their support to the companies for the development of advanced therapies for the coronavirus, thereby created lucrative growth opportunities for the market.

Market Growth Factors:

Growing cases of chronic diseases

Chronic disease is the term used for a group of diseases including cardiovascular diseases, cancer, and diabetes among others. There is a rise in the number of chronic diseases among the population across the globe. It is majorly due to the sedentary lifestyle, unhealthy diet, and consumption of tobacco. As per the US Centers for Disease Control and Prevention (CDC), chronic disease is a condition that lasts for more than one year and needs immediate medical attention or restricts daily activities or both and involves heart disease, cancer, diabetes, and many more.

The rising popularity of regenerative medicines & cell therapies

Regenerative medicine refers to a group of medicine, which makes different methods to repair, regrow or replace diseased or damaged cells, organs or tissues. In addition, regenerative medicine consists of the generation and usage of therapeutic stem cells, tissue development, and the making of artificial organs. Due to the high accuracy and effectiveness, regenerative medicines and cell therapies are estimated to witness a surge in demand, thereby bolstering the growth of the market.

Market Restraining Factor:

Lack of skilled professionals

As cell therapies are gaining more popularity, the automated processing systems market needs more skilled professionals to carry out these therapies and operate automated systems. However, the dearth of skilled professionals is estimated to hinder the growth of the automated and closed cell therapy processing systems market over the forecast period. In addition, the usage of technologically advanced and highly complicated flow cytometers and spectrophotometers for generating a huge amount of data outputs require knowledge for interpreting and reviewing would hinder the market growth.

Type Outlook

Based on type, the market is segmented into stem cell therapy, and non-stem cell therapy. The non-stem cell therapy segment acquired the highest revenue share of the market in 2020 and is estimated to display the fastest growth rate over the forecast period. This growth is attributed to the rising number of product launches for various non-stem cell therapy applications.

Workflow Outlook

Based on workflow, the market is segmented into separation, expansion, apheresis, fill-finish, cryopreservation and others. Among all, the expansion segment dominated the market with the highest revenue share in 2020. It is due to the rise in adoption of strategies like partnerships among the key market players for the application and adoption of systems.

Scale Outlook

Based on scale, the market is segmented into Pre-commercial/R&D Scale and Commercial Scale. Among these, the pre-commercial/R&D scale segment procured the maximum revenue share of the market in 2020. In the current scenario, the market is in its initial phase owing to the restricted number of products. In addition, many key companies are launching their products only for research objectives.

Regional Outlook

Based on Regions, the market is segmented into North America, Europe, Asia Pacific, and Latin America, Middle East & Africa. In 2020, North America emerged as the leading region in the market with the highest revenue share and is estimated to witness a significant growth rate over the forecast period due to the high demand for regenerative medicines across the regional healthcare sector.

The major strategies followed by the market participants are Partnerships. Based on the Analysis presented in the Cardinal matrix; Thermo Fisher Scientific, Inc. and Cytiva (Danaher Corp.) are the forerunners in the Automated And Closed Cell Therapy Processing Systems Market. Companies such as Terumo Corporation, Lonza Group AG, Miltenyi Biotec are some of the key innovators in Automated and Closed Cell Therapy Processing Systems Market.

The market research report covers the analysis of key stake holders of the market. Key companies profiled in the report include Lonza Group AG, Terumo Corporation, Cytiva (Danaher Corporation), Thermo Fisher Scientific, Inc., Miltenyi Biotec B.V. & Co. KG, Thermogenesis Holdings, Inc., Cellares Inc., Biospherix ltd., Sartorius AG, and Fresenius Kabi AG.

Recent Strategies Deployed in Automated and Closed Cell Therapy Processing Systems Market

Partnerships, Collaborations and Agreements:

Oct-2021: Terumo joined hands with BioCentriq, a clinical manufacturing facility for cell and gene therapies. This collaboration aimed to boost the adoption of automated manufacturing to provide novel cell and gene therapies (CGT) to patients more rapidly and cost-effectively.

Oct-2021: Terumo collaborated with BioCentriq laboratories, a clinical manufacturing facility for cell and gene therapies. This collaboration aimed to bring together the companies respective automation and CDMO knowledge, products, skills, and services to assist meet users where they are in their product development pathway and allow a scalable strategy for the future.

Jul-2021: Cellares Corporation signed an agreement with Poseida Therapeutics, a clinical-stage biopharmaceutical company. Under this agreement, Poseida joined Cellaress Early Access Partnership Program (EAPP).

Jun-2021: Lonza teamed up with CellPoint, a private, clinical-stage Biopharmaceutical Company. This collaboration aimed to swiftly develop numerous T-cell-based therapies and use the Cocoon Platform for clinical point-of-care manufacturing. The utilization of the Cocoon Platform, along with the range of CellPoints therapies & technologies, and Lonzas manufacturing capabilities, would assist to boost the path to the clinic and offer a smoother path to commercial approval.

May-2021: Cytiva collaborated with Multiply Labs, a leader in developing robotic systems for pharmaceutical manufacturing. This collaboration aimed to make a robotic manufacturing system, which would automate the manual portions of the cell therapy manufacturing workflow.

Apr-2021: Fresenius Kabi entered into a distribution agreement with Corvida Medical, provider of a smarter Closed System Transfer Device for Chemotherapy. In this agreement, Fresenius Kabi would be the exclusive U.S. distributor for the HALO Closed System Drug-Transfer Device (CSTD).

Jan-2021: Sartorius joined hands with RoosterBio, a biotechnology company. The collaboration aimed to advance the scale-up of hMSC manufacturing for regenerative medicine by using the top-class solutions of the companies to substantially decrease process development efforts, industrialize the supply chain and boost the development & commercialization of groundbreaking cell-based regenerative cures.

Aug-2020: Lonza came into collaboration with IsoPlexis, a life science technology company. This collaboration aimed at the evolution of cell therapy manufacturing.

Jun-2020: ThermoGenesis entered into an agreement with Corning Incorporateds Life Sciences Division. Under this agreement, ThermoGenesiss X-SERIES products would be distributed under the Corning brand.

Jun-2020: BioSpherix Medical teamed up with Sexton Biotechnologies, a provider of novel manufacturing solutions for the cell and gene therapy (CGT) industry. This collaboration aimed to identify the requirement for cost-efficient & flexible automation solutions during cell and gene therapy process development.

Acquisitions and Mergers:

Jul-2021: Sartorius Stedim Biotech, a division of Sartorius acquired Xell, an innovative partner for the biotech and pharmaceutical industry. This acquisition aimed to expand its current media offering, particularly by specialized media for manufacturing viral vectors and, along with the area of media analytics.

Jan-2020: Fresenius Kabi formed a joint venture with Wilson Wolf and Bio-Techne, namely, ScaleReady. This joint venture aimed to offer the manufacturing technologies & processes required to develop and commercialize the latest cell and gene therapies via individual company products and expertise.

Product Launches and Product Expansions:

Dec-2020: Thermo Fisher Scientific released its Gibco CTS Rotea Counterflow Centrifugation System. This system allows cost-effective, scalable cell therapy development and manufacturing. This CTS Rotea system is the Gibco instrument for cell therapy processing applications as well as streamlines workflows from research via GMP clinical development & commercial manufacturing.

Jul-2020: Miltenyi Biotec introduced the latest CliniMACS Prodigy Adherent Cell Culture System. This system allows the automated, scalable, and closed manufacturing of numerous adherent cell types that include stem cells and their derivatives. Tested procedures involve, for example, GMP-compliant expansion of human mesenchymal stromal cells, and pluripotent stem cells, and the differentiation of the latter into dopaminergic progenitors.

Scope of the Study

Market Segments covered in the Report:

By Type

Separation

Expansion

Apheresis

Fill-Finish

Cryopreservation

By Workflow

Stem Cell Therapy

Non-Stem Cell Therapy

By Scale

Pre-commercial/R&D Scale

Commercial Scale

By Geography

North America

o US

o Canada

o Mexico

o Rest of North America

Europe

o Germany

o UK

o France

o Russia

o Spain

o Italy

o Rest of Europe

Asia Pacific

o China

o Japan

o India

o South Korea

o Singapore

o Malaysia

o Rest of Asia Pacific

LAMEA

o Brazil

o Argentina

o UAE

o Saudi Arabia

o South Africa

o Nigeria

o Rest of LAMEA

Companies Profiled

Lonza Group AG

Terumo Corporation

Cytiva (Danaher Corporation)

Thermo Fisher Scientific, Inc.

Miltenyi Biotec B.V. & Co. KG

Thermogenesis Holdings, Inc.

Cellares Inc.

Biospherix ltd.

Sartorius AG

Fresenius Kabi AG

Unique Offerings

Exhaustive coverage

Highest number of market tables and figures

Original post:
Global Automated and Closed Cell Therapy Processing Systems - GlobeNewswire

Cellular Therapies Fill Unmet Needs in R/R Multiple Myeloma – Targeted Oncology

Innovative approaches in multiple myeloma that focus on cellular therapies offer hope to patients with multiple myeloma.

Current approaches for multiple myeloma are stratified by patient fitness and age. For patients who can tolerate them, 3- or 4-drug combinations, with or without an autologous stem cell transplant (ASCT), can result in a complete remission, ideally with no residual disease. For patients who are elderly or fragile, 2-drug or 3-drug regimens are the standard.

For the standard-risk patient, a regimen of bortezomib (Velcade), lenalidomide (Revlimid), and dexamethasone (VRd) plus a CD38 monoclonal antibody such as daratumumab (Darzalex) or isatuximab (Sarclisa) is the norm. As a whole, these combinatorial approaches are needed because multiple myeloma is a heterogenous disease whose optimal treatment takes advantage of multiple mechanisms of action. These regimens can result in first remissions that range from 4 to 5 years.

Although these outcomes are promising, there is still an unmet need for patients with relapsed or refractory disease. Innovative approaches in multiple myeloma that focus on cellular therapies offer hope to these patients.

In a presentation during the 39th Annual CFS Innovative Cancer Therapy for Tomorrow, Shambavi Richard, MD, an assistant professor in medicine, hematology, and medical oncology at The Mount Sinai Hospital in New York, New York, addressed the emerging therapeutic frontiers in multiple myeloma with a focus on chimeric antigen receptor (CAR) approaches and bispecific antibodies.1 Richard explored updated results from the KarMMa trial (NCT03361748), which enrolled 149 patients with relapsed/refractory multiple myeloma (RRMM) and who were previously exposed to immunomodulatory agents, proteasome inhibitors (PIs), and CD38 antibodies (mAbs) and reported poor outcomes. Evaluable patients received idecabtagene vicleucel (ide-cel; n = 128).2,3

At a median follow-up of 15.4 months, the objective response rate (ORR) was 73% and median progression-free survival (PFS) was 8.8 months for all treated patients (TABLE3 ). Investigators reported that at the highest targeted dose of 450 106 CAR T cells, the overall response rate (ORR) was 81%, the complete response (CR) rate was 39%, and the median PFS increased by 12.2 months with longer follow-up. In a subgroup analysis of difficult-to-treat patients, the ORR for patients with extramedullary disease was 70%; patients with high-tumor burden, 71%; and patients with R-ISS stage III disease, 48%.

Regarding safety, 97% of patients had cytopenia and 89% had grade 3/4 neutropenia; 52% experienced thrombocytopenia and 60% developed anemia. Cytokine release syndrome (CRS) had a median onset of 1 day, with a median duration of 5 days. CRS was seen in 84% of patients but grade 3/4 was observed in only 6% of patients. Neurologic toxicity was observed in 18% of patients and 4% were grade 3/4.

Updated results from the CARTITUDE-1 trial (NCT03548207)4 showed that ciltacabtagene autoleucel (cilta-cel) yielded early, deep, and durable responses in heavily pretreated patients with multiple myeloma, with a manageable safety profile at the recommended phase 2 dose.

In the study, 97 patients with a median of 6 prior lines received cilta-cel. The overall response rate per independent review committee (primary end point) was 97% (95% CI, 91%-99%), with 67% of patients achieving stringent CR (sCR). The median time to first response was 1 month (range, 1-9), and median time to CR or better was 2 months (range, 1-15). Responses deepened over time, and the median duration of response was not reached. Of 57 patients evaluable for minimal residual disease (MRD) assessment, 93% were MRD-negative at 10-5. The 12-month PFS and overall survival (OS) rates (95% CI) were 77% (66%-84%) and 89% (80%-94%), respectively; the median PFS was not reached.

In terms of adverse events, neutropenia was 94.8% grade 3/4, and 60.8% of patients had grade 3/4 anemia, said Richard. CRS was almost universal, with any-grade CRS seen in 94.8% of patients. This was a little different compared with ido-cel in terms of time of onset, which was 7 days with this product vs 1 day with the ido-cell product, she said. In both of these trials, early death within the first 2 to 3 months was 2% or less.

When comparing ide-cel to conventional treatment, according to findings presented by Shah et al,5 the investigators observed that ide-cel was associated with a significantly higher ORR compared with conventional treatment (OR, 5.11; 95% CI, 2.92-8.94; P < .001). Similarly, ide-cel significantly extended PFS (HR, 0.55; 95% CI, 0.42-0.73; P < .001) and OS (HR, 0.36; 95% CI, 0.24-0.54; P < .001) vs conventional treatment. Richard said this analysis aimed to compare efficacy outcomes observed with ide-cel treatment in KarMMa and conventional treatment in the Monoclonal Antibodies in Multiple Myeloma: Outcomes After Therapy Failure (MAMMOTH) study.6 Investigators analyzed outcomes of 275 patients with multiple myeloma with disease refractory to CD38 monoclonal antibodies at 14 academic centers.

Turning to the challenge of resistance to therapies in multiple myeloma, Richard noted that there are 3 main strategies in play: multiple myelomacell directed, T-cell directed; and CAR construct.

Possible strategies employed that use multiple myeloma celldirected treatments involve pooling CAR T products with different antigens; using dual CAR products that are constructed using 2 antigen specifi cities, such as B-cell maturation antigen (BCMA)/CD19; or taking a tandem CAR approach. Investigators also can focus on alternate antigens including SLAMF7, CD138, or integrin beta7.

Strategies that are T-cell directed can focus on those that are enriched for central or stem cell memory T cells or use combination approaches with checkpoint inhibitors or immunomodulatory imide drugs and cereblon E3 ligase modulators (CelMoD).

Efforts that tweak the CAR construct are also undergoing evaluation. These include FasTCAR, in which manufacturing takes 24 to 36 hours; next-generation CARs, which are armored CAR T cells that prevent T-cell exhaustion; CARs that use a safety switch to mitigate adverse effects; and allogeneic CARS.

Richard highlighted results from a study evaluating teclistamab, a bispecific antibody that binds to BCMA and CD3 to redirect T cells to attack multiple myeloma cells.

Findings from MajesTEC-1 (NCT03145181) demonstrated that the ORR in response-evaluable patients treated at the recommended phase 2 dose (n = 40) was 65% (95% CI, 48%-79%); 58% achieved a very good partial response or better.7 At the recommended phase 2 dose, the median duration of response was not reached. After 7.1 months median follow-up, 22 (85%) of 26 responders were alive and continuing treatment. During the 2021 American Society of Clinical Oncology Annual Meeting, Krishnan et al presented updated findings showing 58% of evaluable patients had achieved a very good partial response or better and 30% had achieved a CR or better; the median time to first confirmed response was 1.0 month (range, 0.2-3.1).8

Another bispecific antibody, talquetamab, has continued to show promising clinical activity in patients with RRMM. Updated findings from a phase 1 trial (NCT03399799)9 showed the ORR at the recommended phase 2 dose (RP2D) in response-evaluable patients (n = 24) was 63%, with 50% reaching very good partial response or better; 9/17 (53%) evaluable patients with triple-class refractory disease and 3/3 (100%) patients who were penta-refractory had a response. Median time to first confirmed response at the RP2D was 1.0 month (range, 0.2-3.8). Overall, responses were durable and deepened over time (median follow-up, 6.2 months [range, 2.7-9.7+] for responders at the RP2D).

When comparing CAR T-cell therapy to bispecific antibodies, Richard noted that patients undergo CAR T-cell therapy once with no further therapy indicated. Additionally, patients can enjoy a long chemotherapy holiday, whereas bispecific antibodies require more frequent doses. Toxicities are similar for the 2 approaches, although Richard said that CRS can be slightly more profound and at a somewhat higher grade with the CAR T-cell approach compared with that of bispecific antibodies.

In conclusion, Richard also noted that the costs associated with both these approaches will have an impact, especially in high up-front costs. Bispecific c antibodies, however, due to their chronic recurrent administration, may also come with a long-term financial burden.

REFERENCES:

1. Richard S. New therapeutic frontiers for RRMM: CAR T and bispecifi c antibodies. Presented at: 39th Annual CFS. Chemotherapy Foundation Symposium. Innovative Cancer Therapy for Tomorrow. November 3-5, 2021; New York, NY.

2. Munshi NC, Anderson LD Jr, Shah N, et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med. 2021;384(8):705-716. doi:10.1056/NEJMoa2024850

3. Anderson LD, Munshi NC, Shah N, et al. Idecabtagene vicleucel (ide-cel, bb2121), a BCMA-directed CAR T cell therapy, in relapsed and refractory multiple myeloma: Updated KarMMa results. J Clin Oncol. 2021;39(suppl 15):8016-8016. doi: 10.1200/JCO.2021.39.15_suppl.8016

4. Usmani SZ, Berdeja JG, Madduri D, et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy, in relapsed/refractory multiple myeloma: updated results from CARTITUDE-1. J Clin Oncol. 2021;39(suppl 15; abstr 8005). doi: 10.1200/JCO.2021.39.15_suppl.8005

5. Shah N, Ayers D, Davies FE, et al. A matching-adjusted indirect comparison of efficacy outcomes for idecabtagene vicleucel (ide-cel, bb2121), a BCMA-directed CAR T cell therapy versus conventional care in triple-class-exposed relapsed and refractory multiple myeloma. Presented at: 62nd American Society of Hematology Meeting and Exposition, December 5-8, 2020. Abstract 1653. https://bit.ly/3nQb458

6. Gandhi UH, Cornell RF, Lakshman A, et al. Outcomes of patients with multiple myeloma refractory to CD38-targeted monoclonal antibody therapy. Leukemia. 2019;33(9):2266-2275. doi:10.1038/ s41375-019-0435-7

7. Usmani SZ, Garfall AL, van de Donk NWCJ, et al. Teclistamab, a B-cell maturation antigen CD3 bispecific antibody, in patients with relapsed or refractory multiple myeloma (MajesTEC-1): a multicentre, open-label, single-arm, phase 1 study. Lancet. 2021;398(10301):665- 674. doi:10.1016/S0140-6736(21)01338-6

8. Krishnan AY, Garfall Al, Mateos M-V, et al. J Clinical Oncol. 2021;39(suppl 15):8007-8007. doi: 10.1200/JCO.2021.39.15_suppl.8007

9. Berdeja JG, Krishnan AY, Oriol A, et al. Updated results of a phase 1, first-in-human study of talquetamab, a G protein-coupled receptor family C group 5 member D (GPRC5D) CD3 bispecific antibody, in relapsed/refractory multiple myeloma (MM). J Clin Oncol. 2021;39(suppl 15):8008. doi: 10.1200/JCO.2021.39.15_suppl.8008

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Cellular Therapies Fill Unmet Needs in R/R Multiple Myeloma - Targeted Oncology

COVID-19 Takes a Toll on People with Blood Cancers and Disorders – Cancer Health Treatment News

As the COVID-19 pandemic continues to evolve, five studies presented during the 63rd American Society of Hematology (ASH) Annual Meeting and Exposition shed light on the persisting burden that COVID-19 has had on people with underlying blood disorders.

We take care of the patients at the highest risk for COVID-19 illness and those who are among the least likely to respond to the vaccine; these and other studies underscore the dual vulnerability facing many of our patients, said press briefing moderator,Laura Michaelis, MD, of the Medical College of Wisconsin. Hematologists have continued to play a unique role in contributing to the emerging science of COVID-19, especially given our expertise in clotting, and ASH has continued to provide leadership in an uncertain time with vetted resources and timely guidance for how best to manage our patients amid the pandemic.

Two studies analyze data from theASH Research Collaborative (ASH RC) COVID-19 Registry for Hematology, which started in the early days of the pandemic to provide real-time observational data summaries to clinicians on the front lines of the fight against COVID-19, as well as researchers and providers around the world.

In September 2021, the Centers for Disease Control and Prevention (CDC) awarded the ASH RC funding to identify the overall burden of COVID-19, the effects of health disparities and outcomes, and the areas where future resources should be focused for treatment for people living with hematologic malignancies. Specifically, CDC funding, in part, supports additional data submissions to the ASH RC COVID-19 Registry, real-time public data summaries, and research activities. As the Registry dataset has grown, researchers have identified potential drivers of severe illness, hospitalization, and mortality. The data also suggest that aggressive supportive treatment of COVID-19 can improve outcomes for many patients and should be offered.

A third study conducted among individuals living with sickle cell disease suggests COVID-19 infection can cause occlusive events, resulting in pain episodes, but these patients seem to respond to COVID-19 treatments and also were quick to adopt precautions and shift to virtual appointments as needed.

The final two studies look at antibody response following vaccination among people with various hematologic malignancies, helping give clues into which groups of patients may still be at high risk of COVID-19 after getting the vaccines.

A number of studies have shown that people with blood cancers have less than optimal responses to vaccination, and there is a need to continue to push for mitigation strategies, said Dr. Michaelis.

Abstract 3040: Risks for Hospitalization and Death Among Patients with Blood Disorders from the ASH RC COVID-19 Registry for Hematology

Patients with blood cancers, particularly those with more advanced disease, are especially vulnerable to serious COVID-19 outcomes, including an elevated chance of severe illness and death from COVID-19, according to an analysis of more than 1,000 patients in the ASH RC COVID-19 Registry for Hematology. Based on the report, 17% of patients with blood cancers who developed COVID-19 died from COVID-related illness, a strikingly higher mortality rate than what was seen in the general population, according to researchers. Older age, male sex, poor cancer prognosis, and electing to defer intensive care when it was recommended were all independently associated with a heightened chance of dying.

In our analysis, having a poor prognosis for underlying disease prior to COVID-19 and deciding to forgo ICU-level care for that disease were the most powerful predictors of mortality among patients with blood cancer and COVID-19and the two may very well be related, saidLisa K. Hicks, MD, MSc, of St. Michaels Hospital in Toronto, Canada. If someone is sick enough to require ICU-level care and their preference is not to receive this type of care, we would expect that decision to have a major impact on their survival.

According to the data, patients whose physician had estimated that they had less than six months to live due to their cancer before getting COVID-19 had six-fold higher odds of dying and these odds nearly doubled among people who decided to forgo more intensive care due to COVID-19. However, these groups represented a small proportion of the overall sample with only 7% estimated to have a pre-COVID-19 prognosis of under six months, and 9% deferring ICU care.

Of particular interest to the field was whether blood cancer treatment would affect COVID-19 mortality. Most patients included in the dataset (71%) received cancer treatment during the previous year; others were either in remission or had not yet needed treatment. In addition, receiving cancer treatment in the year prior to COVID-19 infection did not significantly increase the risk of death as some had feared; however, it was linked to an increased risk of hospitalization if infected by COVID-19. Older age, being male, having active cancer, and having other health conditions were also associated with an increased risk of hospitalization from COVID-19 among patients with blood cancers.

In the early days of the pandemic, there was a lot of uncertainty about whether we should withhold or modify blood cancer treatments in regions with high levels of COVID-19, said Dr. Hicks. The data are somewhat reassuring in that, while recent cancer treatment was linked to a higher risk of hospitalization among those with blood cancer and COVID-19, it wasnt independently associated with a statistically greater likelihood of dying. The type of blood cancer was also not associated with a higher risk of COVID-19 mortality. These findings suggest that patients who need treatment for their hematologic malignancy should likely proceed with that treatment.

Data were collected between April 1, 2020, and July 2, 2021, as part of the ASH RCs COVID-19 Registry for Hematology, which is a public-facing, volunteer registry reporting outcomes of COVID-19 infection in patients with underlying blood disorders. A total of 1,029 patients from around the globe were included in this analysis. Of these, 41% were female. The median age was 50 to 59 years of age, and patients ranged from five to more than 90 years of age; 27% had at least one co-existing condition such as heart disease, hypertension, respiratory disease, or diabetes. Researchers sought to identify factors associated with a higher likelihood of hospitalization and death from COVID-19.

Of people included in the analysis, 354 (34%) had acute leukemia or myelodysplastic syndromes (MDS), 255 (25%) had lymphoma, 206 (20%) had plasma cell dyscrasia (myeloma/amyloid/POEMS), 116 (11%) had chronic lymphocytic leukemia (CLL), and 98 (10%) had myeloproliferative neoplasm (MPN).

Patients with MPN and plasma cell dyscrasia had less severe COVID-19 illness overall compared to patients with CLL, leukemia, MDS, or lymphoma, which Dr. Hicks said is not surprising as patients with MPN typically live with their disease for many years, are generally in better health, and may not require immunosuppressive treatment.

The data from the ASH RC COVID-19 Registry has limitations and findings should generally be regarded as hypothesis generating, Dr. Hicks said. Nonetheless, the data do suggest that patients with blood cancers are at substantial risk from COVID-19; this finding has implications for our patients, how we manage our clinics amid COVID-19 and the changing variants, and how vaccines, boosters, and antibody treatments are distributed.

In this analysis, 17% of those with blood cancers died of COVID-19; the mortality rate among those infected with SARS-CoV-2 in the general U.S. population has been reported to be between 1.6% and 6.2% at various times during the pandemic, Dr. Hicks added.

The ASH RC Registry is a public voluntary registry that continues to accrue cases and provide the information on a public dashboard to help keep the hematology community apprised on changing trends. Dr. Hicks said the team will also be looking at how the risks of hospitalization and death changed as vaccines and COVID-19 treatments became more widely available.

Abstract 2800: Clinical Predictors of Outcome in Adult Patients with Acute Leukemias and Myelodysplastic Syndrome and COVID-19 Infection: Report from the American Society of Hematology Research Collaborative (ASH RC) Data Hub

In separate analyses of 257 patients with acute leukemia or MDS who developed COVID-19 and are part of the ASH RC COVID-19 Registry for Hematology, both neutropenia (a type of low white blood cell count) and having active MDS or leukemia (versus being in remission) were found to strongly and independently predict severe COVID-19 illness.Once hospitalized, active disease by itself whether someone was newly diagnosed or had relapsed was not tied to a greater odds of dying from COVID-19, nor was receiving ongoing cancer treatment.

For this retrospective analysis, which included data from 135 patients with acute myeloid leukemia (AML), 82 with acute lymphocytic leukemia (ALL) and 40 with MDS who were diagnosed with COVID-19 from 2019 to present, researchers sought to identify characteristics that put patients at higher risk of severe illness or death from COVID-19. At the time of COVID-19 diagnosis, 46% were in remission and 44% had active disease.

COVID-19 severity was defined as mild (no hospitalization required), moderate (hospitalization required), or severe (ICU admission required). After adjusting for several risk factors, active disease and neutropenia at the time of COVID-19 diagnosis were also associated with severe COVID-19 illness that necessitated ICU-level care.

Overall, one out of five (21%) patients died from COVID-19, which was higher than the mortality rate reported for the registry as a whole (17%) or what was seen in the general public during the same period of time, researchers reported. Mortality among hospitalized patients with COVID-related illness was 34%, and mortality among patients once admitted in the ICU was 68%. The two factors most strongly associated with a higher likelihood of dying among these patients were: 1) how long someone was perceived to live from the underlying MDS or leukemia before getting COVID-19, as defined as a physicians estimated prognosis of less than six months survival, and 2) whether or not they decided to go to the ICU if it was recommended. Older age, male sex, and neutropenia at diagnosis were also associated with COVID-19 mortality though less strongly.

This is a particularly vulnerable population and we suspected they may do worse because they are immunocompromised and, as it is, the average survival for acute blood cancers if untreated is three to six months, so if COVID-19 comes together with that diagnosis, its very concerning, saidPinkal Desai, MD, MPH, of Weill Cornell Medical College, New York. Our data suggest these patients can survive COVID-19 and their underlying disease itself was not associated with worse mortality, which means that if these patients are given appropriate and aggressive treatment, we can help them recover. But if there are decisions that are made after they get to the hospital (for example, whether to go to the ICU) that clearly plays a role.

In fact, patients for whom ICU-level care was recommended and declined had five times higher odds of dying compared with patients who opted to go to the ICU.

Patients who went to the ICU did better regardless of disease status, said Dr. Desai. Just having acute leukemia or MDS puts these patients at high risk of severe COVID-19, and they need to be hospitalized and receive treatments, but decisions about the ICU should be individualized, a patients prognosis should be discussed, and if a patient wants aggressive care for COVID-19 that should be offered.

Patients were more likely to forgo ICU care if they were older, male, smokers, or if they had active disease or an estimated pre-COVID-19 survival of less than six months. Forgoing ICU care was associated with a higher COVID-19 mortality in all patients.

Our data show that these patients do survive COVID-19 after receiving care in the ICU and underscore that cancer treatments should not be withheld as inferior treatment would quickly put many of these patients into the category of a prognosis of less than six months, said Dr. Desai. COVID-19 vaccination is also critically important.

The data are limited in that they were collected before COVID-19 vaccines were widely available; future data should inform about mortality rates among vaccinated patients.

Patient Vigilance and Virtual Visits Credited for Reducing Exposure, Illness, and Death Due to COVID-19 in Cohort With Sickle Cell Disease

Abstract 3105: COVID-19 Infection and Outcomes at a Comprehensive Sickle Cell Center

The Georgia Comprehensive Sickle Cell Center at Grady Hospital in Atlanta the nations largest treatment center for adults living with sickle cell disease (SCD) quickly switched to offering virtual visits for routine follow-up care of its more than 1,300 patients as the COVID-19 pandemic emerged. People living with SCD, an inherited disorder characterized by crescent- or sickle-shaped red blood cells, are immunocompromised and thus at high risk for COVID-19. The center established a database to track all COVID-19 cases among its patients.

The first report from that database the largest single-center study to date on COVID-19 in people with SCD now shows that between March 2020 and March 2021, just 55 (4%) of the centers 1,343 patients contracted COVID-19, of whom 16 (29%) were hospitalized and two ultimately died from complications of infection with the virus. Eleven patients (20%) required neither hospitalization nor emergency-room treatment for complications of either COVID-19 or SCD during the one-year follow-up period.

Our findings show that when supported by virtual visits, most of our patients successfully reduced their exposure to and complications from COVID-19, said study authorFuad El Rassi, MD, of Emory University and director of research at the Grady Comprehensive Sickle Cell Center. They understood the risks and followed recommendations to stay at home and avoid interacting with other people.

The 55 patients who contracted COVID-19 were aged 28 on average and 51% were female. Of those who visited an emergency room or were hospitalized during the year of follow-up, 27 (49%) sought care for a painful episode of SCD and 15 (27%) for complications of COVID-19. Among those who sought care for COVID-19 symptoms, 32 (58%) had pain as their primary symptom, followed by cough and fever (40%) and shortness of breath (31%); 25% had chest X-ray evidence of pneumonia. Sixteen patients received treatment, with nine receiving the antibody treatment remdesivir, eight receiving the steroid drug dexamethasone, and seven receiving red-blood-cell products to treat pain.

Twenty cases of COVID-19 were diagnosed between March and September of 2020. The two patient deaths from COVID-19 occurred in June and July of 2020. Among the 35 cases diagnosed between October 2020 and March 2021, no patients died and the number of hospitalizations decreased as better treatments for COVID-19 became available.

One of the patient deaths was due to a blood clot in the lungs, Dr. El Rassi said. This unfortunately occurred before it became the standard of care to treat hospitalized COVID-19 patients with blood thinners, he said.

Despite the second peak in COVID-19 cases in the winter of 2021, there were no reported deaths among our patients who developed the disease, Dr. El Rassi added. This suggests that the patients vigilance in staying home may have been crucial to reducing illness and death, and having the option for virtual visits was also key. Patients who needed blood tests or to obtain medication refills were sent to satellite centers.

Patient adherence to COVID-19 precautions was measured based on their responses to physician questions at intake and during virtual follow-up visits.

Dr. El Rassi and his colleagues plan to conduct further studies to evaluate the impact of the delta variant on diagnosis, illness, and death from COVID-19 among the sickle cell centers patients.

Some People With Blood Disorders May Continue to Face High Risk of COVID-19 After Vaccination

Abstract 218: Antibody Response to Vaccination with BNT162b2, mRNA-1273, and ChADOx1 in Patients with Myeloid and Lymphoid Neoplasms

According to a new study, about 15% of people with blood cancers and other blood disorders had no vaccination-related antibodies after receiving a COVID-19 vaccine. While researchers say it is encouraging that 85% of study participants did show an antibody response, the findings suggest that additional precautions may be warranted to prevent COVID-19 infection among people with blood disorders.

The study examined antibody levels after COVID-19 vaccination in people with blood cancers such as lymphoid and myeloid neoplasms, autoimmune disorders, and non-cancerous disorders of blood or immune cells. The results suggest that patients with lymphoma and those currently receiving treatment are the least likely to build antibodies in response to a COVID-19 vaccine.

Some patients with hematologic diseases do not have an adequate antibody response and might, therefore, not have sufficient protection from vaccination, saidSusanne Saussele, MD, of III. Medizinische Klinik, Medizinische Fakultt Mannheim, Universitt Heidelberg, Germany. This study can help guide vaccination strategies for these patients. In addition, our study suggests that when it is possible to delay beginning treatment for their underlying disorder, it may be best to wait so that a patient can receive a vaccine or booster first.

People with blood disorders face a high risk of hospitalization and death if they become infected with COVID-19, especially if they are older or have received therapies that reduce B-cells, a type of immune cell. Since the majority of participants in the study did respond to COVID-19 vaccines, the results underscore the role of vaccination as an important strategy for preventing severe disease, researchers said. However, the findings also suggest vaccination should be complemented with other precautions. We should recommend ongoing protective measures such as masks, social distancing, and screenings, as well as prioritizing vaccination for family members and caregivers to protect the patients, Dr. Saussele said.

For the study, the researchers recruited 373 patients treated for blood disorders at University hospital Mannheim in Germany and measured vaccine-related antibodies in their blood a median of 12 weeks after final vaccination. More than 90% of participants had blood cancer, while 9% had either autoimmune disease or a non-malignant blood disorder. Most patients had received the Pfizer-BioNTech [BNT162b2]vaccine; 10% received the Moderna vaccine [mRNA-1273], 7% received the AstraZeneca vaccine [ChADOx1], and 6% received one dose from each of the two vaccine types.

Overall, 85% of participants tested positive for vaccine-related antibodies and 15% tested negative. The rate of negative antibody results was highest among those with lymphoid neoplasms, a group of diseases that include lymphoma, myeloma, and lymphoid leukemia. Among these patients, 36% tested negative for vaccine-related antibodies. Patients with indolent non-Hodgkin lymphoma, a slow-growing type of lymphoma, had the weakest response to vaccination overall.

Being on active therapy was associated with a reduced antibody response. Overall, 61% of study participants were on active therapy. Of those who tested negative for vaccine-related antibodies, most (71%) were on active therapy. Therapies correlated with a negative response were rituximab, ibrutinib/acalabrutinib, and ruxolitinib.

Our study suggests that most people with blood malignancies not only those who are currently under treatment should monitor their antibody levels and work closely with their care team to determine how to continue to protect themselves from COVID-19, Dr. Saussele said. Antibody measurements offer a hint of who has responded to the vaccine and can perhaps ease up on precautions a bit.

Dr. Saussele noted that the results are limited in that the study did not examine participants T-cell response to vaccination, meaning that some patients level of protection may have been underestimated. The researchers plan to continue to measure antibody levels for at least a year and to assess participants rates of breakthrough infections and response to vaccine boosters.

Strong Antibody Response Seen in Patients With AML and MDS After Second Dose of mRNA COVID-19 Vaccine

Abstract 217: Responses to SARS-Cov-2 Vaccines in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia

In one of the largest studies to date of the antibody response to vaccination against COVID-19 in people who had been treated for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), patients responded well to two doses of the Moderna mRNA vaccine and saw a particularly dramatic increase in levels of antibodies against the virus after receiving their second vaccine dose.

We observed a strong antibody response to the vaccine in a group of patients at high risk for severe COVID-19, including among patients who were on active treatment for AML or MDS, said Jeffrey Lancet, MD, of the H. Lee Moffitt Cancer Center and Research Institute in Florida. The fact that antibody levels increased so dramatically after the second vaccine dose suggests potential utility in additional dosing, even for patients who initially respond poorly to the vaccine.

Previous studies had shown that patients with other types of blood cancer specifically, B-cell lymphomas or chronic lymphocytic leukemia often have a poor antibody response to vaccination with one of the COVID-19 mRNA vaccines. Treatment of these cancers suppresses the ability of the immune system to produce white blood cells such as B cells and T cells to fight off infection.

The treatment of myeloid cancers such as AML and MDS, including allogeneic transplantation, also suppresses white blood cells and leaves patients vulnerable to infection, said Dr. Lancet. We conducted this study to find out whether patients with these cancers would also have a suppressed or absent immune response to COVID-19 vaccination.

The study involved 46 patients who either had previously or were currently undergoing treatment for AML or MDS. The patients median age was 68 years; 59% were male and 96% were white. On average, they were about two years out from the diagnosis of their cancer. Fifteen patients (33%) were receiving treatment for their cancer at the time they were vaccinated. Thirty-two patients (70%) had undergone a transplant of blood-forming stem cells from a healthy donor as part of their cancer treatment. Forty patients (87%) were in remission when they were vaccinated. (Note that some patients are counted twice e.g., if they had undergone a stem cell transplant and were in remission, they would be counted in both categories. For this reason, the percentages add up to more than 100%.)

All patients received a first dose of the Moderna mRNA vaccine (this vaccine type was being given at the clinic) in late January 2021 and a second dose four weeks later. The investigators collected blood specimens from each patient before each vaccine dose was administered and again at four weeks after the second dose. The primary aims of the study were to describe the immune response and assess the safety profile of the vaccine in a cohort of patients with AML or MDS.

Blood test results at 29 days after the first vaccine dose showed that 70% of patients had an antibody response; at 57 days following the second dose 97% had an antibody response. Antibody levels were significantly higher after the second dose compared with after the first dose. Patients antibody response was not significantly affected by age, gender, race, disease status (i.e., active or in remission), time from disease diagnosis to vaccination, number of treatments patients had undergone for their cancer, whether patients had received a stem cell transplant, or whether they were on active treatment at the time of vaccination.

The most common adverse events following vaccination were the typical ones reported after vaccination with a COVID-19 mRNA vaccine, such as fatigue, headache, arm swelling, and mild pain at the injection site.

The study results should be confirmed in a larger group of patients, Dr. Lancet said. However, based on these data, we feel comfortable advising patients with AML or MDS that they should get vaccinated against COVID-19. Due to their vulnerability to COVID-19, they stand to benefit from the vaccine more than most.

This is an observational study without an identified control, or comparator, group, Dr. Lancet cautioned. Another limitation is that because the participants were overwhelmingly white, it is not known whether patients of other races or ethnicities would show a similar antibody response. In addition, the actual protective effect of the vaccine and the T-cell responses to it in this patient population are not yet known; the researchers are currently gathering these data.

The investigators are now following the same cohort of patients to determine whether a third dose of the vaccine can achieve even higher antibody levels than were seen after the second dose.

This press release was published by the American Society of Hematology on December 11, 2022.

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Bristol Myers Squibb exec on the companys growth in Seattle and beating cancer with immune cells – GeekWire

BMS inherited Junos headquarters in Seattles South Lake Union neighborhood. (BMS Photo)

Pharmaceutical giant Bristol Myers Squibb has been quietly growing in the Seattle area.

Since acquiring Celgene and its Seattle operations two years ago, BMS now has more than 1,240 employees in the region, hundreds more than when the deal was announced.

BMS brings big pharma clout and a chunk of its $9.2 billion annual R&D budget to Seattles prospering biotech ecosystem, where global drug anchor companies have been as rare as bigfoot since Amgen shut down its Seattle hub five years ago.

Only Bothell, Wash.-based Seagen, with more than 2,500 employees worldwide, may top BMS in size among biopharma companies in the Seattle area. BMS has more than 150 open positions in the region, jostling with Sana Biotechnology, Umoja Biopharma and other cell and gene therapy biotech companies for workers.

BMS Seattle-area outpost, devoted to cell therapy and immuno-oncology, is one of about a dozen BMS R&D centers worldwide. Seattle BMS scientists are developing new ways to attack tumors by harnessing cells of the immune system and they are improving on two CAR T cellular therapies approved for certain blood cancers, Breyanzi and Abecma.

Breyanzi emerged from research at Seattles flagship cell therapy biotech company, Juno Therapeutics, which Celgene acquired in 2018 in a multi-billion dollar deal. BMS continues partnerships that Juno forged with Fred Hutch and Seattle Childrens Research Institute when Juno spun out of these institutions, and it is building new biotech collaborations to develop the next generation of therapies.

Leading BMS Seattle effort is Teresa Foy, who previously helmed Seattles Celgene operations and rose up through the ranks as an executive at two small Seattle biotech startups, VLST Corp and Oncofactor.

Our presence here is strong, Foy told GeekWire in an interview. Were hiring and were growing.

BMS footprint in the region includes a 266,000 square foot Seattle R&D facility built by Juno, and a manufacturing facility in Bothell, where the company manufactures Breyanzi.

The Seattle operation oversees clinical trials for Breyanzi and other cell therapies. BMS, for instance, aims to expand the eligible patient group for Breyanzi, which is currently approved for adults with certain types of lymphoma who have relapsed or do not respond after two front-line therapies. BMS recently released data in support of expanding the therapy to patients at an earlier stage of treatment.

BMS aims to reduce the steep cost of manufacturing CAR T cells, which involves engineering a patients own cells to attack their tumor. One option is to instead enable off-the-shelf therapies, derived from healthy donor cells or even stem cells.

The next generation of cellular therapies are also being built in Seattle. The company is engineering CAR T cells to overcome a hostile tumor environment and to recognize more than one molecular target. Such research aims to counteract the development of resistance to treatment and expand the therapy to solid tumors. BMS is also advancing TCR-engineered T cells which can target molecules inside tumor cells, not just on the cell surface as with CAR T cells.

Meanwhile, BMS is looking beyond cellular therapies at immune cell engagers. These are agents that interact with immune cells and direct them to recognize and attack cancer cells. BMS is testing such agents in phase 1 clinical trials for blood cancers and solid tumors.

Other cell and immune therapy companies pursue similar aims, but BMS brings multiple research strategies under one roof, bolstered by its strong clinical and manufacturing capabilities and web of academic and industry collaborations.

We talked with Foy about the companys growth and its vision for treating cancer with the immune system.

The interview with Foy below has been edited for clarity and brevity.

GeekWire: What do you think has kept BMS in the Seattle region?

Bristol Myers Squibb executive Teresa Foy: With the acquisition, BMS hired Celgene president Rupert Vessey as its president of research and early development. They really liked his research model, and BMS needed a kind of refresh on their research strategy. Vessey had helped build a distributed research model, with different innovation hubs, and each of those hubs has a different area of focus.

Part of what BMS recognized in the acquisition was that this was an important concept to keep intact, not just for Seattle, but for the other hubs as well. That was not traditionally how BMS operated their research. They were very centrally located in New Jersey, but now theyve sort of embraced this model. It leverages different locations for hiring and also allows you to tap into the ecosystems of local regions for academic expertise, other small companies to partner with, as well as the talent.

BMS wants to maintain the core expertise thats here and the critical mass of people to do cell therapy development. It takes a while to build that expertise and depth of experience. Being able to retain that and grow that here in Seattle is a real strength for us.

How does the cell therapy ecosystem in Seattle bolster your work, and do regional companies benefit as potential collaborators?

Foy: We certainly have a lot of academic history here in Seattle with Seattle Childrens, with Fred Hutch, and we still maintain those relationships. The talent pool has been shared across the region, and thats great for the ecosystem in Seattle.

We have partnerships and equity investments in some of the local companies [BMS investments include Presage Biosciences, Zymeworks, Silverback Therapeutics, and Lyell Immunopharma, which are based in the Seattle area or have operations there]. But we dont currently have any large collaborations with any current [local] companies. Part of the reason is some of our programs are competitive with each other. The technology and innovation cycle is continuing and we continue to have discussions with Sana, Lyell and other new companies.

We have partnerships with people all over the country and all over the world. But Seattle is a kind of center of excellence for cell therapies. Certainly, people around the country recognize that there was a strong foundation built here with Juno that expanded, and that has now seeded a bunch of new companies. I think thats an advantage for all of us because it brings talent here. It brings great scientific discussions and opportunity for collaboration.

Youre working in a field at the cutting edge of cancer research. What are you excited about for the future?

Foy: I do think that the progress will be exponential in the next five to 10 years because theres been so much innovation in technology and in bioinformatics, machine learning and artificial intelligence, which can help inform all the data that we gather from our patients. We can learn so much to feed back into improving the first generations of cell therapies. The technology is advancing things at kind of this frantic pace.

Im excited for us to be able to expand what weve learned in hematology, lymphoma and myeloma into solid tumors. And then well have to also apply what we learned in the immuno-oncology space with checkpoint inhibitors [immune modulating cancer drugs like BMS nivolumab]. What resistance mechanisms prevent checkpoint inhibitors from having longer effects or what prevents some patients from responding? Some of those same themes are holding true for cell therapies.

How are your research collaborations building the next generation of cellular therapies?

Foy: We have a partnership with Arsenal Bio, which is developing logic gates for solid tumors [enabling activation of therapeutic cells only within a tumor or under other conditions]. Obsidian therapeutics does regulated expression of proteins were adding particular proteins to our cell therapies that we can regulate to turn on just when we want them to, primarily for solid tumors to overcome tumor environment challenges. And then we have a partnership with Immatics, which has identified engineered T cell receptors to solid tumor targets, and were putting those in our in our cell therapies. So, theres lots going on with the next generation.

Were working on a couple of different approaches for off-the-shelf cell therapies. Allogeneic approaches [therapies from donor cells] are in the queue for both CD19 and BCMA [the targets of Breyanzi and Abecma]. And then a long ways off, were looking at other things like iPSC [stem-cell] derived therapies. We dont yet have a partnership there, but were working on exploring that.

Can you tell us about your other research efforts?

Foy: About 30% of our portfolio is focused on immune cell engagers. Those complement the cell therapy modality. They have some advantages in that they could be off the shelf, maybe give access to more patients. But obviously, you cant engineer as many things into those biologics as you can into a cell therapy. So its kind of a nice breadth for our portfolio to have optionality with both of them.

Any highlights from your clinical programs?

Foy: Both CD19 and BCMA CAR T cells [Breyanzi and Abecma] are in a next generation of manufacturing. And those essentially have a similar design but a different manufacturing process [that enables cells to persist longer in the body]. We entered a CAR T cell against a novel target, GPRC5D, for multiple myeloma, and that is in phase 1 now. Behind that we have an ROR1 CAR T cell well be enrolling patients early next year for that program, and that will be in chronic lymphocytic leukemia initially and then solid tumors subsequent to that.

What has it been like for you moving from small biotech companies to Celgene and then BMS?

Foy: I grew up as a bench scientist and gradually became a leader and then a chief scientific officer. I thought, Im not sure Im going to like the big company, but I found that doing good science is doing good science, regardless of whether youre in a small company or a big company. But being able to have resources to do it really well, and to actually see patients benefit from it, that was a huge, rewarding part.

Any final words?

Foy: Were really excited about segueing into solid tumors in the next five to 10 years and also looking at ways to make these cell therapies more affordable and off-the-shelf donor-derived. I think thats really the next generation of where the cell therapies will go.

Were also really proud of the community outreach weve done and our STEM efforts in Washington state. They are a really important part of our mission here [BMS is involved in outreach programs at the Pacific Science Center and other efforts]. Our staff is really excited to help mentor and educate the next generation of young scientists and hopefully keep the Seattle and Washington state ecosystem thriving with scientists of the future.

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Bristol Myers Squibb exec on the companys growth in Seattle and beating cancer with immune cells - GeekWire

Upregulated expression of actin-like 6A is a risk factor | CMAR – Dove Medical Press

Introduction

Pancreatic cancer (PC) with high aggressiveness and malignancy has become an enormously common cancer of the digestive system during 10 years. Globally, the 5year overall survival (OS) rate of patients with PC is less than 9%, and the mortality rate is predicted to peak by 2030.1 Due to insidious symptoms, only less than 10% of PC is initially diagnosed with a local stage, and the prognosis of PC is extremely poor.2 Therefore, further investigation into novel cancer-related genes is required and meaningful for the improvement of prognosis.

SWI/SNF complexes are evolutionarily conserved multi-subunit molecular machines that mediate transcriptional regulation3 and are linked to a poor prognosis across several cancer types.46 Among them, Actin-like 6A (ACTL6A), encoded by Actl6a, acts as a chromatin-remodeling factor and regulates the function of progenitor and stem cell transcriptionally.7,8 In addition, ACTL6A expression is associated with prognosis in many types of cancer, such as hepatocellular carcinoma,9 colon cancer,10 and esophageal squamous cell carcinoma.11 Recently, research revealed that epithelial-to-mesenchymal transition (EMT) was also regulated by ACTL6A.9,12 In addition, the study showed that ACTL6A overexpression could lead to increased repair of cisplatin-DNA adducts and cisplatin resistance.13 However, the role of ACTL6A in tumorigenicity and clinical prognosis of PC remains unclear so far.

In this connection, we analyzed the differences of ACTL6A expression in PC tissues and normal tissues, and we investigated the prognostic effect of ACTL6A on PC based on cases in public databases and confirmed it in our center.

Differential expression of Actl6a mRNA between pancreatic tumor and normal tissues was analyzed using the Gene Expression Profiling Interactive Analysis website (GEPIA; http://gepia.cancerpku.cn/). Data for 179 patients with PC and 171 normal tissue samples analyzed on the GEPIA website were obtained from TCGA and normal tissue samples from Genotype-Tissue Expression (GTEx).1416 The gene expression, determined as transcripts per million (TPM), was calculated by log2 (TPM + 1) for comparison. Based on the expression levels of Actl6a mRNA, the overall survival (OS) of patients was also analyzed.

A total of 60 patients with PC confirmed by histopathology from January 2013 to June 2020 at Zhongda Hospital, Medical School of Southeast University were selected for the study. Sixty paired pancreatic tumor and normal tissues from patients who did not receive chemotherapy or radiotherapy were obtained to detect ACTL6A expression. Any patients with incomplete epidemiological and clinical information or lack of follow-up information were excluded. The results of serum tumor markers were collected from 60 healthy individuals who were admitted to the hospital for physical examination at the same time. All patients provided informed consent. Patients were followed up by telephone or at office visits every 3 months from the end date of surgery. The latest follow-up ended in July 2021. According to the eighth edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, pathological stages were validated. The study was conducted with approval from the ethics committee of Zhongda Hospital, Southeast University. The study protocol protected the private information of enrolled patients in accordance with the provisions of the Helsinki Declaration.

The paraffin-embedded pathological specimens were cut into 4-m-thick sections. After being dewaxed in xylene and rehydrated in grade alcohol, the paraffin sections were submerged in a pH 6.0 citric acid solution and heated at 95C for approximately 15 minutes for antigen retrieval. Next, the sections were incubated with rabbit ACTL6A antibody (Abcam Corp, USA, diluted 1:200) overnight at 4C and washed 3 times with phosphate buffer saline (PBS). The sections were then incubated with horseradish peroxidase-conjugated secondary antibody for 30 minutes at room temperature in the dark. After stained with freshly prepared 3,3-diaminobenzidine (DAB), they were counterstained with hematoxylin and differentiated with 1% hydrochloric acid. PBS was used to substitute the primary antibody as negative control. Finally, the sections were dehydrated with alcohol and sealed with neutral gum, and pictures were taken by microscope for positive cell calculation. Immunohistochemical staining analysis was performed independently by two pathologists according to the staining intensity and the percentage of positive cells. The staining intensities were 0 (negative), 1 (positive 1+), 2 (positive 2+), and 3 (positive 3+), respectively. The percentages of cells were 0 (negative), 1 (125%), 2 (2650%), 3 (5175%), and 4 (76100%), respectively.17,18 Total scores were calculated by multiplying the scores of staining intensity and percentage.

Statistical analyses and mapping were performed using SPSS software (version 18.0, IBM Corporation, Armonk, NY, USA), GraphPad Prism (Version 8.4.3, GraphPad Software, La Jolla, CA, USA), and R (version 3.4.1, http://www.r-project.org/) in the present study. Wilcoxon test was used to evaluate significant differences between pancreatic cancer and normal tissues, and the 2 test and continuity correction were used to explore the relationship between ACTL6A expression and clinicopathological features. The diagnostic efficiency of ACTL6A expression was analyzed through receiver operating characteristic (ROC) curves for PC. The sensitivity and specificity were evaluated at an optimal cutoff. The expression of ACTL6A was classified as high expression and low expression according to the cutoff. Survival analysis was analyzed using KaplanMeier curve, and difference among groups was assessed using Log rank test. Both univariable and multivariable analyses were used in survival analysis, respectively. The clinicopathological factors with significant associations (p < 0.1) in the aforementioned univariable analysis were subjected to multivariate analysis. p < 0.05 was considered to be statistically significant.

To explore the potential role of ACTL6A in PC, the expression of Actl6a mRNA was analyzed with the publicly available GEPIA database. In clinical PC specimens (n = 178) and normal tissues (n = 171), Actl6a mRNA had significant differential expression between the two groups. What is more, Actl6a mRNA was upregulated in PC than normal tissues (p < 0.05, Figure 1A). Then, the protein expression of ACTL6A was validated and compared in PC samples (n = 60) and normal tissues (n = 60) with immunohistochemistry staining in our center. The typical immunohistochemical results of normal tissues and PC tissues are shown in Figure 1B, which demonstrated that ACTL6A was mainly observed in the nucleus of cells. By multiplying the staining intensity and percentage, the protein expression of ACTL6A was also overexpressed in pancreatic cancer (p < 0.001, Figure 1C). Table 1 shows the number of patients with different scores based on immunohistochemistry staining. The results above indicated that ACTL6A was upregulated in PC.

Table 1 The Number of Patients in Different Scores Based on Immunohistochemistry Staining

Figure 1 Expression of ACTL6A in PC and normal tissues. (A) Differential expression of Actl6a mRNA between pancreatic tumor and normal tissues. (B) Immunohistochemical results of typical normal tissues and PC tissues with different staining intensities. (C) Differential expression of ACTL6A between pancreatic tumor and normal tissues. (D) ACTL6A represented a moderate diagnostic value. The ROC of pancreatic cancer samples and normal tissues. (E) ROC for the diagnostic efficiency of ACTL6A, serum CEA, and serum CA199. *p<0.05.

Abbreviations: ACTL6A, actin like 6A; PC, pancreatic cancer; ROC, receiver operating characteristic curves; CA199, carbohydrate antigen 199; CEA, carcinoembryonic antigen.

To investigate the diagnostic value of ACTL6A expression for PC, we performed ROC analysis on total scores of pancreatic cancer and normal pancreatic tissue, as shown in Figure 1D and E, and the AUC value was 0.724, which was higher than that of carbohydrate antigen 199 (CA199) and carcinoembryonic antigen (CEA). These results represented a moderate diagnostic value for PC. The specificity and sensitivity of ACTL6A expression for PC diagnosis were 0.867 and 0.567, respectively. The cut-off value established for ACTL6A expression for the diagnosis of PC was 5.

To further understand the role of ACTL6A in PC, we analyzed the relationship between ACTL6A expression and the clinicopathological characteristics. Patients with PC were divided into ACTL6A low-expression group (score 05; n = 34) and ACTL6A high-expression group (score 612; n = 26) with the cut-off value of score 5. The relationship between ACTL6A expression and clinicopathological factors of pancreatic cancer is summarized in Table 2. Lymphovascular space invasion (LVSI) of PC was significantly associated with ACTL6A expression, which was more likely to occur in the ACTL6A high group. LVSI was present in 55.9% (19/34) of patients in the ACTL6A high group and 26.9% (7/26) in ACTL6A low group.

Table 2 Relationships Between the Expression Level of ACTL6A and the Clinicopathological Characteristics of PC Patients

The survival data of 178 PC patients was obtained from TCGA dataset. Patients are split into two groups according to the median value of Actl6a mRNA expression. One-half (89 patients) was defined as high Actl6a mRNA expression, and the other was defined as low Actl6a mRNA expression. Obviously, high Actl6a mRNA was associated with poor overall survival in patients with PC (p < 0.001, Figure 2A). Furthermore, based on data from our center, the KaplanMeier method was used to investigate the relationship between the expression of ACTL6A protein and OS of patients. The median OS in PC patients for the high and low expression of ACTL6A was 8.0 0.4 months and 13.0 1.6 months, respectively. Obviously, patients with low ACTL6A expression had significantly longer survival time than those with high ACTL6A expression (p < 0.001, Figure 2B).

Figure 2 (A) KaplanMeier curves of overall survival in PC patients with high and low Actl6a mRNA expression. (B) KaplanMeier curves of overall survival in PC patients with high and low ACTL6A expression.

Abbreviations: ACTL6A, actin like 6A; PC, pancreatic cancer.

Univariate and multivariate Cox analyses were performed to identify the prognostic factors on OS of patients with PC. The results demonstrated that ACTL6A overexpression (p = 0.032) and grade (p = 0.008) were risk factors for survival in patients with PC through univariate Cox analysis. Further multivariate Cox analysis showed that ACTL6A expression (p = 0.046) was an independent risk factor for poor prognosis of PC (Table 3). As shown in Figure 3A and B, the forest plot visualizes the specific HR of risk factors.

Table 3 Univariate and Multivariate Analysis of Clinicopathological Characteristics Affecting Prognosis of Patients with PC

Figure 3 Forest plot of univariate (A) and multivariate (B) cox regression.

Abbreviations: ACTL6A, actin like 6A; PC, pancreatic cancer; LVSI, lymphovascular space invasion.

Worldwide, PC has become a malignancy with a dismal prognosis and high mortality, which has a 5-year survival rate of less than 10%.19 There are two clinical features that are involved with the poor prognosis of PC. First, initial symptoms of PC are insidious, which leads to many challenges for early diagnosis. Second, PC has a significant potential for invasion and metastasis.20 In detail, the distant spread may occur in the early stages of PC, and more than 50% of patients with PC have no possibility to be treated with surgical resection.21 Scientific problems covering early diagnosis, the mechanisms of metastasis, and the risk factors of prognosis are necessary to be solved to improve survival of PC. In this study, we clarified that ACTL6A is highly expressed in PC, and it is a reliable marker for predicting the prognosis of PC patients.

ACTL6A is involved in a variety of cellular processes, including vesicle transport, spindle orientation, nuclear migration, and chromatin remodeling.7,22 Increasing evidence has suggested its involvement with tumorigenesis and development of cancer.7 ACTL6A has been reported to be overexpressed in a variety of malignancies, including hepatocellular carcinoma,9 ovarian cancer,18 cervical cancer,23 and esophageal squamous cell carcinoma,11 which is correlated with the prognosis of patients with malignancies. This evidence suggests that ACTL6A is a potential oncogene, and it is observed that ACTL6A expression is also upregulated in PC in our study, which is consistent with previous studies. Researchers have been constantly exploring diagnostic markers for PC. Jelski et al reported that the activity of alcohol dehydrogenase (ADH) class III isoenzyme in pancreatic cancer was significantly higher than that in normal tissues.24 And the total activity of ADH and class III isoenzyme was increased in the serum of patients with PC, which can be due to the release of this isoenzyme from PC cells.25 Nevertheless, it was not observed that other types of ADH isoenzymes (I, II, IV) had a significant change in either pancreatic tissue or serum. Further exploration revealed that ADH III had the diagnostic value for PC.26 Also, our evidence demonstrated a potential role for ACTL6A as a marker of PC.

ACTL6A plays a vital role in the invasion and metastasis of tumors by promoting EMT, leading to poor prognosis. ACTL6A expression is higher in fibroblasts and progenitor cells and inhibits the epithelial properties of epidermal tissues.27,28 Moreover, the functions of ACTL6A are similar to features of stem cells, including the inhibition of cell differentiation and the ability of self-renewal, which is closely related to the biological functions of EMT.28 In hepatocellular carcinoma, ACTL6A activated Notch1 signaling via SOX2, which regulated EMT to affect the biological function and clinical prognosis of hepatocellular carcinoma. Other studies also revealed ACTL6A as an EMT activator to promote metastasis in osteosarcoma29 and colon cancer,10 respectively. Some studies mentioned the potential role of ACTL6A involvement with tumors. Zhang et al found that ACTL6A was a glycolytic regulator by phosphoglycerate kinase 1(PGK1) in ovarian cancer and participated in FSH-induced tumorigenesis of ovarian cancer.18 And in triple negative breast cancer, ACTL6A promoted tumor cell proliferation by enhancing the stability of MYC oncogene.30 Additional evidence suggested that ACTL6A promoted the progression of cervical cancer and laryngeal squamous cell carcinoma through activation of yes-associated protein (YAP) signaling.23,31 Besides, ACTL6A could stabilize transcriptional regulators YAP and transcriptional coactivator with PDZ-binding motif (TAZ) to regulate the proliferation, migration, and invasion of glioma.32 Further studies revealed that the knockdown of ACTL6A gene resulted in the inhibition of protein kinase B (AKT) signaling pathway to suppress cell migration and increased sensitivity of glioma cells to temozolomide.33 Moreover, in vivo and in vitro, Shrestha et al revealed that p21Cip1, a tumor suppressor, was suppressed by ACTL6A in epidermal squamous cell carcinoma, leading to epidermal squamous cell carcinoma progression.34 More importantly, overexpressed ACTL6A was related to cisplatin-induced DNA damage and led to resistance to cisplatin.13 These studies have further confirmed the contribution of ACTL6A in the invasion, metastasis, and clinical prognosis of tumors.

In this research, we reveal a correlation between the expression of ACTL6A and the invasion and prognosis of PC. It was found that LVSI was more likely to occur in PC patients with high ACTL6A expression, which might be related to the high aggressiveness caused by ACTL6A. Univariate and multivariate Cox analysis suggested that ACTL6A expression and grade were independent risk factors for poor prognosis of PC. This study also confirmed ACTL6A as a valid prognostic biomarker and potential therapeutic target in PC. Given a follow-up and survival analysis of survival data of PC patients, patients with high ACTL6A expression had significantly poorer prognosis. It was suggested that ACTL6A expression in PC was a risk factor, which was consistent with the existing studies. And ACTL6A overexpression was associated with tumor progression. However, whether ACTL6A could induce PC cell proliferation, invasion, and metastasis in vitro, as well as the specific regulatory mechanisms, deserved further investigation.

In conclusion, it was found that levels of ACTL6A expression were elevated in PC tissues, which was associated with LVSI. Moreover, it was demonstrated that ACTL6A was an independent risk prognostic indicator for PC. ACTL6A could be used as a valuable biomarker to predict the prognosis of PC, assisting clinicians to develop preventative measures and better treatment strategies to improve mortality in patients with PC.

The authors are grateful to all the patients, researchers and institutions that participated in the TCGA and GTEx database.

The authors report no conflicts of interest in this work.

1. Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209249.

2. Zhang L, Sanagapalli S, Stoita A. Challenges in diagnosis of pancreatic cancer. World J Gastroenterol. 2018;24(19):20472060.

3. Mittal P, Roberts CWM. The SWI/SNF complex in cancer - biology, biomarkers and therapy. Nat Rev Clin Oncol. 2020;17(7):435448.

4. Naito T, Udagawa H, Umemura S, et al. Non-small cell lung cancer with loss of expression of the SWI/SNF complex is associated with aggressive clinicopathological features, PD-L1-positive status, and high tumor mutation burden. Lung Cancer. 2019;138:3542.

5. Cyrta J, Augspach A, De Filippo MR, et al. Role of specialized composition of SWI/SNF complexes in prostate cancer lineage plasticity. Nat Commun. 2020;11(1):5549.

6. Fukumoto T, Magno E, Zhang R. SWI/SNF complexes in ovarian cancer: mechanistic insights and therapeutic implications. Mol Cancer Res. 2018;16(12):18191825.

7. Krasteva V, Buscarlet M, Diaz-Tellez A, Bernard MA, Crabtree GR, Lessard JA. The BAF53a subunit of SWI/SNF-like BAF complexes is essential for hemopoietic stem cell function. Blood. 2012;120(24):47204732.

8. Panwalkar P, Pratt D, Chung C, et al. SWI/SNF complex heterogeneity is related to polyphenotypic differentiation, prognosis, and immune response in rhabdoid tumors. Neuro Oncol. 2020;22(6):785796.

9. Xiao S, Chang RM, Yang MY, et al. Actin-like 6A predicts poor prognosis of hepatocellular carcinoma and promotes metastasis and epithelial-mesenchymal transition. Hepatology. 2016;63(4):12561271.

10. Zeng Z, Yang H, Xiao S. ACTL6A expression promotes invasion, metastasis and epithelial mesenchymal transition of colon cancer. BMC Cancer. 2018;18(1):1020.

11. Li RZ, Li YY, Qin H, Li SS. ACTL6A promotes the proliferation of esophageal squamous cell carcinoma cells and correlates with poor clinical outcomes. Onco Targets Ther. 2021;14:199211.

12. Nieto MA, Huang RY, Jackson RA, Thiery JP. Emt: 2016. Cell. 2016;166(1):2145.

13. Xiao Y, Lin FT, Lin WC. ACTL6A promotes repair of cisplatin-induced DNA damage, a new mechanism of platinum resistance in cancer. Proc Natl Acad Sci U S A. 2021;118(3):e2015808118.

14. Cancer Genome Atlas Research N; Weinstein JN, Collisson EA, et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. 2013;45(10):11131120.

15. Consortium GT. Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science. 2015;348(6235):648660.

16. Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017;45(W1):W98W102.

17. Rao X, Wang J, Song HM, Deng B, Li JG. KRT15 overexpression predicts poor prognosis in colorectal cancer. Neoplasma. 2020;67(2):410414.

18. Zhang J, Zhang J, Wei Y, Li Q, Wang Q. ACTL6A regulates follicle-stimulating hormone-driven glycolysis in ovarian cancer cells via PGK1. Cell Death Dis. 2019;10(11):811.

19. Zhu H, Li T, Du Y, Li M. Pancreatic cancer: challenges and opportunities. BMC Med. 2018;16(1):214.

20. Ansari D, Tingstedt B, Andersson B, et al. Pancreatic cancer: yesterday, today and tomorrow. Future Oncol. 2016;12(16):19291946.

21. Lamb YN, Scott LJ. Liposomal irinotecan: a review in metastatic pancreatic adenocarcinoma. Drugs. 2017;77(7):785792.

22. Zhao K, Wang W, Rando OJ, et al. Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell. 1998;95(5):625636.

23. Zhao J, Li L, Yang T. MiR-216a-3p suppresses the proliferation and invasion of cervical cancer through downregulation of ACTL6A-mediated YAP signaling. J Cell Physiol. 2020;235(12):97189728.

24. Jelski W, Chrostek L, Szmitkowski M. The activity of class I, II, III, and IV of alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in pancreatic cancer. Pancreas. 2007;35(2):142146.

25. Jelski W, Zalewski B, Szmitkowski M. Alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) activity in the sera of patients with pancreatic cancer. Dig Dis Sci. 2008;53(8):22762280.

26. Jelski W, Kutylowska E, Laniewska-Dunaj M, Szmitkowski M. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) as candidates for tumor markers in patients with pancreatic cancer. J Gastrointestin Liver Dis. 2011;20(3):255259.

27. Bao X, Tang J, Lopez-Pajares V, et al. ACTL6a enforces the epidermal progenitor state by suppressing SWI/SNF-dependent induction of KLF4. Cell Stem Cell. 2013;12(2):193203.

28. Lu W, Fang L, Ouyang B, et al. Actl6a protects embryonic stem cells from differentiating into primitive endoderm. Stem Cells. 2015;33(6):17821793.

29. Sun W, Wang W, Lei J, Li H, Wu Y. Actin-like protein 6A is a novel prognostic indicator promoting invasion and metastasis in osteosarcoma. Oncol Rep. 2017;37(4):24052417.

30. Jian Y, Huang X, Fang L, et al. Actin-like protein 6A/MYC/CDK2 axis confers high proliferative activity in triple-negative breast cancer. J Exp Clin Cancer Res. 2021;40(1):56.

31. Dang Y, Zhang L, Wang X. Actin-like 6A enhances the proliferative and invasive capacities of laryngeal squamous cell carcinoma by potentiating the activation of YAP signaling. J Bioenerg Biomembr. 2020;52(6):453463.

32. Ji J, Xu R, Zhang X, et al. Actin like-6A promotes glioma progression through stabilization of transcriptional regulators YAP/TAZ. Cell Death Dis. 2018;9(5):517.

33. Chen X, Xiang Z, Li D, Zhu X, Peng X. ACTL6A knockdown inhibits cell migration by suppressing the AKT signaling pathway and enhances the sensitivity of glioma cells to temozolomide. Exp Ther Med. 2021;21(2):175.

34. Shrestha S, Adhikary G, Xu W, Kandasamy S, Eckert RL. ACTL6A suppresses p21(Cip1) expression to enhance the epidermal squamous cell carcinoma phenotype. Oncogene. 2020;39(36):58555866.

Link:
Upregulated expression of actin-like 6A is a risk factor | CMAR - Dove Medical Press

Syrian refugee is thriving five years after last-gasp gene therapy – STAT – STAT

In the summer of 2015, a 7-year-old named Hassan was admitted to the burn unit of the Ruhr University Childrens Hospital in Bochum, Germany, with red, oozing wounds from head to toe.

It wasnt a fire that took his skin. It was a bacterial infection, resulting from an incurable genetic disorder. Called junctional epidermolysis bullosa, the condition deprives the skin of a protein needed to hold its layers together and leads to large, painful lesions. For kids, its often fatal. And indeed, Hassans doctors told his parents, Syrian refugees who had fled to Germany, the young boy was dying.

The doctors tried one last thing to save him. They cut out a tiny, unblistered patch of skin from the childs groin and sent it to the laboratory of Michele de Luca, an Italian stem cell expert who heads the Center for Regenerative Medicine at the University of Modena and Reggio Emilia. De Lucas team used a viral vector to ferry into Hassans skin cells a functional version of the gene LAMB3, which codes for laminin, the protein that anchors the surface of the skin to the layers below.

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Then the scientists grew the modified cells into sheets big enough for Ruhr University plastic surgeons Tobias Hirsch and Maximilian Kueckelhaus to graft onto Hassans raw, bedridden body, which they did over the course of that October, November, and the following January.

It worked better than the boys doctors could have imagined. In 2017, de Luca, Hirsch, Kueckelhaus, and their colleagues reported that Hassan was doing well, living like a normal boy in his lab-grown skin. At the time though, there was still a big question on all their minds: How long would it last? Would the transgenic stem cells keep replenishing the skin or would they sputter out? Or worse could they trigger a cascade of cancer-causing reactions?

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Today, the same team is out with an update. Five years and five months after the initial intervention, Hassan is still, for the most part, thriving in fully functional skin that has grown with the now-teenager. He is attending school, and playing sports with his friends and siblings, though he avoids swimming due to blistering in the areas that werent replaced by the lab-grown skin. One of his favorite activities is a pedal-powered go kart. There are no signs his modified stem cells have lost their steam, and no traces of tumors to be found.

The encouraging follow-up data has been instrumental in moving forward a larger clinical trial of the approach, offering hope to the 500,000 epidermolysis bullosa patients worldwide currently living without treatment options.

We were astonished by the speedy recovery, Kueckelhaus, now at University Hospital Muenster, told STAT via email. But experience from skin transplantation in other settings made him and his colleagues wary of the grafts failing as the months and years wore on. Thankfully, wrote Kueckelhaus, those fears never materialized. We are very happy to be able to prove that none of these complications appeared and the genetically modified skin remains 100% stable. The chances are good that he will be able to live a relatively normal life.

Over the last five years, Hassans team of doctors and researchers has put his new skin through a battery of tests checking it for sensitivity to hot and cold, water retention, pigmentation and hemoglobin levels, and if it had developed all the structures youd expect healthy skin to have, including sweat glands and hair follicles. Across the board, the engineered skin appeared normal, without the need for moisturizers or medical ointments. The only flaw they found was that Hassans skin wasnt as sensitive to fine touch, especially in his lower right leg. This mild neuropathy they attributed not to the graft itself, but to how that limb was prepared doctors used a more aggressive technique that might have damaged the nerves there.

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The team also used molecular techniques to trace the cells theyd grown in the lab as they divided and expanded over Hassans body. They found that all the different kinds of cells composing the boys new skin were being generated by a small pool of self-renewing stem cells called holoclone-forming cells, carrying the Italian teams genetic correction.

This was quite an insight into the biology of the epidermis, said de Luca. Its an insight he expects will have huge consequences for any efforts to advance similar gene therapies for treating other diseases affecting the skin. You have to have the holoclone-forming cells in your culture if you want to have long-lasting epidermis, he said.

The approach pioneered by de Lucas team will soon be headed for its biggest clinical test yet, after nearly a decade of fits and starts. They expect to begin recruiting for a multi-center Phase 2/3 trial sometime next year.

De Luca first successfully treated a junctional EB patient in 2005. But then a change to European Union laws governing cell and gene therapies forced his team to stop work while they found ways to comply with the new rules. It took years of paperwork, building a manufacturing facility, and spinning out a small biotech company called Holostem to be ready to begin clinical research again. Hassan came along right as they were gearing up for a Phase 1 trial, but data from the boys case, which was granted approval under a compassionate use provision, convinced regulators that the cell grafts could move to larger, more pivotal trials, according to de Luca.

We didnt cure the disease, he told STAT. But the skin has been restored, basically permanently. We did not observe a single blister in five years. The wound healing is normal, the skin is robust. From this point of view, the quality of life is not even comparable to what it was before.

Science Writer

Megan Molteni is a science writer for STAT, covering genomic medicine, neuroscience, and reproductive tech.

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Syrian refugee is thriving five years after last-gasp gene therapy - STAT - STAT