Stem Cell Clinic

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