Patients accept therapy using embryonic stem cells for Parkinson’s … – BMC Medical Ethics

Discrete choice experiment

Preferences of patients with PD for potential cell-based therapies to treat PD were assessed by a Discrete Choice Experiment (DCE) in Swedish patients with PD. The DCE is a cross-sectional survey method to investigate individuals preferences and can be used to determine the relative importance of different characteristics of an intervention and predict uptake of different interventions [15]. Respondents of a DCE are faced with a set of hypothetical choice questions with two or more alternatives, characterized by different characteristics (i.e., attributes) with varying levels. The DCE method also allows for the calculation of attribute trade-offs [16].

We performed a scoping literature review to identify attributes of treatments for PD that potentially were of importance for patients with PD when choosing treatment. Qualitative and quantitative papers investigating preferences of patients with PD related to treatment for PD were included. All literature searches were performed in PubMed and the keywords used were Parkinson disease, patient preferences, preferences, treatment, medication, and attributes. We identified 193 papers, including 29 papers that were relevant for this project, of which 20 papers remained after excluding duplicates. After reading the full text papers, 209 potential attributes were identified. Out of the 209 attributes identified in the scoping literature review, 115 attributes were unique. These attributes were condensed down to 45 by merging similar concepts. The identified attributes were discussed in a group consisting of a representative patient of a Parkinson patient organization, neurologists, a research coordinator, a nurse working with patients with PD, and researchers knowledgeable in DCE methodology. Based on the discussions in this group, 11 attributes remained. We let 17 patients with PD rank the 11 attributes from most to least important, for their decision about PD treatment. Based on the mean ranks of the attributes and discussions with clinicians, eight attributes remained. These were re-categorized into the five attributes that were assigned relevant levels to be assessed by the DCE: (i) type of treatment, (ii) aim of treatment, (iii) available knowledge of the different types of treatments, (iv) effect on symptoms, and (v) risk for severe side effects (Table1).

We followed methodological guidelines to estimate the sample size needed to identify preferences of patients with PD and differences within those preferences [17]. We considered the number of attributes in the DCE (Table1) and the number of choice questions for each respondent (n=9). Based on the sample size requirements for a DCE and accounting for subgroup analysis, we aimed for a sample size of 500 respondents.

Patients with PD were recruited from neurology clinics at two university hospitals in Sweden. This study was approved by the Swedish Ethical Review Authority (Dnr 201906539). Information about the study was sent out by mail to all potential respondents fulfilling the inclusion criteria: patients diagnosed with PD, 18 years or older, able to read and understand Swedish. Patients with a known dementia diagnosis were excluded. Information about the study was sent out to 1266 patients. Patients who had not responded within two weeks were sent a reminder by mail. All respondents provided their informed consent before entering the survey. Two patients formally declined participation, and five patients were unable to participate due to technical or health-related restrictions. In total, 498 patients participated in the study (i.e., 39% response rate).

This survey was administered as a web-based survey that included three parts: (i) information about the attributes and levels, (ii) the DCE with hypothetical choice scenarios, and (iii) demographic and attitude questions (see supplementary file for survey). The survey was created for this study and administered using Sawtooth Software (Sawtooth Software Inc.). Each respondent was faced with nine hypothetical choice scenarios that each included three alternatives. The respondents were asked to select the alternative that they most preferred out of the three presented to them. The first two alternatives were experimentally designed to assess preferences for potential treatment alternatives for PD and the third was a fixed profile (i.e., nonexperimental) to represented standard care (drugs) for patients with PD (Fig.1). We used a Bayesian D-efficient design to construct the choice scenarios for the DCE using the NGene program (version1.2.1; ChoiceMetrics 2012). Prior information on the attribute importance was gathered from a pilot test (n=142) in patients with PD. The design used 500 Halton draws and 1000 repetitions. Using the pilot data, a multinomial logit (MNL) model was fitted, and the beta estimates was used as priors for the final experimental DCE design.

Some conditions were posted on the design: if the aim of treatment was to repair damage caused by disease, the treatment could not consist of electric stimulation or drug. If the aim of the treatment was to slow down disease progression, the treatment could not consist of electric stimulation. The final discrete choice survey consisted of 36 unique choice scenarios divided into four blocks; each respondent was randomized into one block and answered nine choice scenarios. The choice questions also included a hover function with further explanations of the attributes and the levels (see Table1 for full description of the attribute levels).

Example of a choice scenario

The demographical and attitude questions included background questions (e.g., age, gender, and education) and disease-related questions (e.g., disease duration, treatment, and side effects). Moreover, the respondents attitudes were gauged with a ranking exercise with eight statements that they were asked to place in the order they found most important. The attitude questions asked respondents about their moral stands on the status of an embryo, and a ranking exercise to prioritize eight statements.

The respondents were asked about their views on how to regard the products left over after IVF procedures, which may be used for hESC isolation, that is, the blastocyst. Whether this material was regarded as a lump of cells or something more was used to dichotomize the answers. Questions to assess respondents health literacy [18] and health numeracy [19] were also included to define the sample.

The statistical analyses, in particular the estimation of the latent class model were performed using R 4.0.2 (R Core Team, 2018), the mlogit (version 1.1-1; Yves Croissant, 2009) and the gmnl (version 1.13.3; Mauricio Sarrias, 2017) [20].

Demographics describing the populations age, gender, country of birth, occupational situation, education, health numeracy, health literacy, drug frequency, disease duration, number of experienced side effects, and experience of advanced treatment were presented in mean, median, and percentages. The overall level of health literacy and numeracy was calculated for each respondent. Individuals who responded strongly disagree or disagree to one of the items were categorized as having low health literacy. Individuals who responded with neither agree nor disagree with one of the items were categorized as having medium health literacy. Individuals responding agree or strongly agree to all the items were categorized as having high health literacy, and likewise for numeracy.

Respondents attitude toward the moral status of a couple of days old human embryo was assessed using this question: The human is perceived to have a special moral position, in the sense of having rights just by being human. What moral position does a human embryo that is only a few days old have? The respondents had four statements from which to select: (1) The embryo is just a lump of cells; it is meaningless to discuss its moral status, (2) The embryo has a moral status that is in between being just a lump of cells and being a human being, (3) The embryo in its moral status is closer to being a human than just a lump of cells, and (4) The embryo has the same moral status as a human being. The variable was dichotomized based on the frequency of the data. Respondents answering The embryo is just a lump of cells; it is meaningless to discuss its moral status formed one group, and the rest another group. One-way analysis of variance and nonparametric measures were used to test the differences between the personal characteristics and the different perceptions of whether an embryo is more than a lump or cells or not.

The most important attitudinal statement was given a 1, the second most important the number 2 and so forth. The ranking exercise was illustrated with a boxplot by the median value of each statement, stratified on the different perceptions of whether an embryo is more than a lump of cells.

The latent class analysis was based on the a priori hypothesis that the authors thought would be associated with the willingness to accept a new treatment. Five variables were tested for class membership: (1) a summary of experience of different treatment, (2) experience of the summary of different side effects, (3) the perception of the moral status of the embryo, (4) experience of advanced treatment, and (5) the importance of religion. A sum of how many treatments each respondent had was calculated, and also how many side effects they had experienced. Advanced treatment was based on treatment experience with one or more of apomorphine subcutaneous injection, apomorphine subcutaneous infusion, deep brain stimulation, levodopa-carbidopa intestinal infusion, and levodopa-entacapone-carbidopa intestinal infusion. The variable the perception of the moral status of the embryo did not influence class membership and was therefore not included in the final class assignment model.

The statistical analyses of the preference data were based on a latent class model. A preference weight (i.e., coefficient) and a corresponding SE were estimated for all but one level of each attribute (i.e., reference attribute level) [21]. Dummy coding of the variables was selected for this analysis (i.e., corresponding to zero as the reference value). Each p-value is a measure of the statistical significance of the difference between the estimated preference weights for each level of the attribute compared to the reference attribute level. All results were considered statistically significant at p<0.05. Confidence intervals (95%) were also provided for each preference weight. The Akaike information criterion (AIC) and the log-likelihood values were considered when selecting the appropriate model.

The latent class model was used to identify hidden (latent) classes of respondents preferences [22]. In latent class analysis, unobserved preference heterogeneity among respondents preferences is modeled as classes with similar preference patterns but with different variances across classes. Once preference patterns have been stratified into classes, the model determines the extent to which demographic characteristics impact the likelihood of belonging to a certain class. The systematic utility component (V) describes the latent construct that participant r belonging to class c reported for alternative A, B or C in choice task t. The final utility functions were as follows:

Vr,t,A&B|c=1 * consist_hESCr,t,A&B|c+2 * consist_iPSr,t,A&B|c+3 * consist_electricr,t,A&B|c+4 * aim_slowr,t,A&B|c+5 * aim_repairer,t,A&B|c+6 * know_500r,t,A&B|c+7 * know_5000r,t,A&B|c+8 * effect_50r,t,A&B|c+9 * effect_80r,t,A&B|c+10 * sideeffects_0.001r,t,A&B|c+11 * sideeffects_0.01r,t,A&B|c+.

Vr,t,C|c=1 * consist_drugr,t,C|c+2 ** aim_reliefr,t,C|c+3 * know_5000r,t,C|c+4 * effect_50r,t,C|c+5 * sideeffects_0.01r,t,C|c+.

A class assignment model was fitted after the specified utility function. The variables: experience in treatment, side effects, advanced treatment therapy and religious beliefs were tested for their potential impact on class membership in the model. The final class assignment function was:

Vn|c=0+1* treatment_sum|c+2 * experience_sideeffects|c+3 * advanced_treatment|c+4 * Religion_dum|c+.

The relative importance of the attributes included in the DCE was calculated by estimating the difference in preference weights of the latent class model between the most preferred level of an attribute and the least preferred level of the same attribute [21]. The highest difference value was normalized to 1, which represents the most important value. The difference values were divided by the highest value to reveal the relative distance between all other attributes.

We calculated the predicted acceptance uptake for a potential treatment scenario using hESCs to treat patients with PD. Predicted acceptability can be understood as the probability that a participant will accept a described scenario. The scenario represents a hypothetical treatment scenario of treatments with hESCs based on the attributes assessment in the DCE. Attribute estimates assessed by the latent class model were used to calculate the predicted acceptability of attribute levels (treatment with hESCs, risk of severe side effects is 1 out of 1000 and 50 patients received treatment) in relevant future scenarios; (A) effect on symptoms is 2 out of 10, (B) effect on symptoms is 5 out of 10, and (C) effect on symptoms is 8 out of 10.

The predicted acceptability is presented as the percentage of 100 who would accept the presented scenario. The utility for the specific scenario was calculated by using the following equation:

VScenario 1=A+B+C.

The predicted acceptability, the probability of accepting a specific scenario, was then calculated by using the following equation:

Predicted acceptance uptake=1/(1+expVScenario 1).

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Patients accept therapy using embryonic stem cells for Parkinson's ... - BMC Medical Ethics

Automation is the key to future advances, says the Head of the … – National Health Executive

Below, Lee highlights how his passion for stem cell research brought him to the UKSCB, its role in the development of advanced therapeutics and discusses how automation and machine learning in the manufacturing process could mean faster patient access to stem cell therapies in the future.

My career has been hugely varied, taking me around the world across the public, private and academic sectors, but one thing has remained constant: my fascination with cell and gene therapies, and through research, bringing these closer to patients.

Ive been lucky enough to study cell and gene therapies in many different ways over the last twenty years, from the potential of stem cells as a therapy for Type I Diabetes and the role of genes in the cancer cell cycle, to perfecting techniques that differentiate stem cells into precursors for adult blood cells for transfusion therapies. But theres always more to discover.

One application that excites me the most is the use of stem cells for immuno-oncology, where immune cells are used to treat cancer. Were now able to take immune cells from donors and use gene modification so they can fight cancer in patients where they would normally be rejected.

These stem cells can provide an unlimited supply of cancer-fighting T cells, available off-the-shelf to treat anyone quickly when referred to a clinic. This living drug has the potential to constantly adapt to cancer, combating resistance, and existing within patients to continue fighting the disease indefinitely.

In fact, this simplest form of cell can become almost any cell type for many applications in cell therapies, and the UKs national repository for all human embryonic stem cell lines derived within the UK, is here, at the MHRAs UK Stem Cell Bank.

Established 20 years ago to curate all human embryonic stem cells created in the UK and to regulate their use and provision for research and in the clinic, the UKSCB at the MHRAs South Mimms Laboratories is now recognised globally as a leading repository with over 180 different human embryonic stem cell lines.

During this time, weve received support from the Medical Research Council (MRC) and National Institute for Health Research (NIHR) to deliver our banking activities and, together with the lines available for research, weve banked over 30 lines now available for clinical application, making the UKSCB the largest source of clinical grade human embryonic stem cells in the world.

As part of the MHRA, the UKSCB plays an important role in providing regulatory guidance and workshops to the stem cell health sector across the UK, and weve established links with Harvard University to support international training programmes.

The UKSCB is a unique asset within the MHRA, sitting at the forefront of the advanced therapies landscape in the UK, and Im eager to build on its great history and legacy at a time where were only just starting to realise the full potential and application of advanced cell therapies.

As an aging population, we can expect to receive novel treatments for disease in the form of small molecules and prescription drugs but often these only serve to manage symptoms or limit disease progression. Using stem cells, we can now develop replacement joints, neurons for improved brain function and even entire hearts can be made in the lab without the need for human donors.

But what can we do to keep up with demand and ensure quality? The UKSCB has contributed to over 100 publications in international peer reviewed journals, establishing strong links with the World Health Organisation and international partners to improve standards and controls. Weve recently completed a successful international collaboration with Japanese partners, Sinfonia, to trial their automated cell processing robot and we aim to continue with these efforts towards the cost-effective scaling up of our manufacturing.

I believe that future improvements in customer service lies in automation. Reliance on scientists for the manufacture of stem cells is labour intensive, expensive and introduces human error. Automation will alleviate a lot of the manual aspects of cell culture, allowing us to scale-up manufacturing, drive down costs and ensure the highest quality and consistency, allowing stem cell-derived advanced therapies to be more accessible to patients and affordable for healthcare systems. By introducing automation into this process, we can free-up capacity for our leading experts to move away from labour-intensive manufacturing and instead work on improving and adapting novel advanced therapies.

When I look at my role as the third custodian of the UKSCB I hope to build on the significant past achievements of Glyn Stacey and Elsa Abranches that have established us as the leading stem cell institution in the UK today. I hope to move us forward with a sustainable business model that secures the long-term viability of the UKSCB. This means expanding our operations across various cell therapy platforms, supporting the development of specific cell types for use in human cell therapies, and developing new standards, enabling regulatory approval and eventual uptake by healthcare organisations.

I also want the UKSCB to continue making an impact around the world, supporting the development of stem cell therapies and reference materials for advanced therapies with the World Health Organisation. Over the last twenty years, weve delivered more than 370 cell line vials to 25 different countries for research, and in 2022, 54% of the stem cell lines requested have been of clinical grade, rising year on year. I want to see this trend continue as demand for these lines as starting materials for cell therapies grows further.

The future of the MHRAs UKSCB is diverse and exciting and, much like the cells we curate, there are endless possibilities for us to support research and clinical advances in the UK and around the world. I cannot wait to see where the next 20 years will take us.

Image credit: iStock

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Automation is the key to future advances, says the Head of the ... - National Health Executive

DNA aptamer finds novel application in regulating cell differentiation – Science Daily

Generating specific cell lineages from induced pluripotent stem cells and embryonic stem cells is the holy grail of regenerative medicine. Guiding iPSCs toward a target cell line has garnered much attention, but the process remains challenging. Now, researchers from Japan have discovered that an anti-nucleolin DNA aptamer, iSN04, can determine a cell's lineage during differentiation. By demonstrating the generation of cardiomyocytes from murine pluripotent stem cells, their concept shows promise as a regenerative therapy.

Self-renewal and pluripotency-the capacity to form any cell lineage-are inherent characteristics of induced pluripotent stem cells (iPSCs). Furthermore, they are highly prized in regenerative therapies targeting cardiovascular, neurological, and metabolic diseases as they are immunologically suitable for transplantation back into a donor. Unfortunately, regenerative medicine is not yet feasible outside a laboratory setting as available protocols to generate target cells are complicated and expensive. This raises a pertinent question: Can regulating the fate of stem cells in clinical settings and at scale be made more economical?

A team of researchers from Shinshu University, the National Institute of Advanced Industrial Science and Technology, and the University of Shizuoka in Japan set out to address this question by leveraging nucleic acid aptamers. Aptamers are single-stranded pieces of DNA that bind to target proteins and are able to modulate signaling cascades during cell differentiation when a stem cell commits to a specific functional role or phenotype. They hold promise in regenerative medicine as they are easily modified, can be synthesized economically, and are suitable for long-term storage.

The team, led by Associate Professor Tomohide Takaya from the Department of Agricultural and Life Sciences at Shinshu University, recently discovered that an anti-nucleolin aptamer, myogenetic oligodeoxynucleotide iSN04, induced myocardial differentiation in embryonic stem cells (ESCs). The study was led by Mina Ishioka, a graduate student in Dr. Takaya's laboratory, and published in The International Journal of Molecular Sciences on 21 September 2023.

"We had previously found that iSN04 promoted myogenic precursor cells (myoblasts) to differentiate into skeletal muscle cells and had hypothesized that the aptamer also enhanced differentiation of pluripotent stem cells. We were intrigued by the prospect of using iSN04 to promote iPSC differentiation into cardiomyocytes as this could lead to regenerating heart tissue," says Dr. Takaya, elaborating on the team's motivation to pursue the research.

Using various assays like RNA sequencing, cell staining and imaging, and molecular interaction and pathway analysis, the researchers investigated iSN04's effect on murine ESCs and iPSCs. iSN04 treatment under differentiating conditions inhibited stem cell commitment to the cardiac lineage. However, when these pluripotent stem cells were treated after experiencing differentiating conditions for five days, specific marker genes were upregulated, and the cells committed to forming beating cardiomyocytes.

"Ours is the first report to confirm a DNA aptamer that allows cardiomyocytes to develop from iPSCs," explains Dr. Takaya when asked about the significance of the work. "We uncovered two mechanisms of nucleolin interference with iSN04 at play whereby early treatment inhibits cardiomyogenesis, while treatment at a later stage enhances the generation of cardiac progenitors. First, iSN04 governs the translocation of nucleolin protein between the cytoplasm, plasma membrane, and nucleus. Second, it results in the modulation of the Wnt signaling pathway that governs cell differentiation."

The immunostaining experiments revealed that nucleolin was retained in the nucleoli following iSN04 treatment. Nucleolar nucleolin has a role in chromatin remodeling and gene transcription, and interestingly enough, Wnt pathway genes were differentially expressed in the RNA-seq data following iSN04 suppression. The team postulates that the iSN04-anchored nucleolin alters gene expression and Wnt signaling. Ultimately, terminal cell differentiation commits to the cardiomyocyte lineage.

And how could these findings impact regenerative medicine and patients' lives in the long term? Dr. Takaya provides insights into the broader implications of their work. "We believe there is a strong case to be made for further studies evaluating DNA aptamers in regenerative medicine. Aptamers are cost-effective and open up the possibility of producing specific cells from the patient's stem cells. But it doesn't end there! Since the aptamers can regulate stem cell fate, they can serve as therapeutic agents for many conditions linked to stem cell dysfunction," he concludes.

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DNA aptamer finds novel application in regulating cell differentiation - Science Daily

A comprehensive review of human trophoblast fusion models: recent … – Nature.com

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A comprehensive review of human trophoblast fusion models: recent ... - Nature.com

Cell atlases of the human brain – Science Daily

In two parallel projects, researchers at Karolinska Institutet have been involved in creating the most comprehensive atlases of human brain cells to date. The two studies, which are published in Science, provide clues on different brain diseases and give hope for medical advancements in the future, such as new cancer drugs.

Knowing what cells constitute the healthy brain, where different cell types are located and how the brain develops from the embryo stage is fundamental to the ability to compare and better understand how diseases arise. There are at present advanced atlases of the mouse brain, but not for the human brain. Until now.

A brain-cell census

"We've created the most detailed cell atlases of the adult human brain and of brain development during the first months of pregnancy," says Sten Linnarsson, professor of molecular system biology at the Department of Medical Biochemistry and Biophysics at Karolinska Institutet in Sweden. "You could say that we've taken a kind of brain-cell census."

The first project was led by Kimberly Siletti from Linnarsson's group. It was conducted in close collaboration with Ed Lein at the Allen Institute for Brain Science in Seattle, USA, as part of the international Human Cell Atlas initiative, and based on three donated human brains from adults. The researchers analysed more than three million individual cell nuclei using the technique of RNA sequencing, which reveals each cell's genetic identity. All in all, the researchers studied cells from just over a hundred brain regions and found over 3,000 cell types, some 80 per cent of which were neurons, the remainder being different kinds of glial cells.

"A lot of research has focused on the cerebral cortex, but the greatest diversity of neurons we found in the brainstem," says Professor Linnarsson. "We think that some of these cells control innate behaviours, such as pain reflexes, fear, aggression and sexuality."

Groundwork for medical advances

The researchers could also see that the cells' identity reflects the place in the brain where they first developed in the fetus, which links to the second project. Here, Emelie Braun and Miri Danan-Gotthold from Sten Linnarsson's group collaborated with the Swedish consortium for the Human Developmental Cell Atlas to analyse over a million individual cell nuclei from 27 embryos at different stages of development (between 5 and 14 weeks of fertilisation). The study enabled the researchers to show how the entire brain develops and is organised over time.

Even though the results are examples of molecular biological basic research, the new knowledge generated can also lay the groundwork for medical advances. Professor Linnarsson's research group has used similar methods to examine different kinds of brain tumours, one of which was a glioblastoma -- a cancer with a poor prognosis.

"The tumour cells resemble immature stem cells and it looks like they're trying to form a brain, but in a totally disorganised way," he explains. "What we observed was that these cancer cells activated hundreds of genes that are specific to them, and it might be interesting to dig into whether there is any potential for finding new therapeutic targets."

Freely available brain atlases

The brain atlases will be freely available to researchers around the world so that they can compare the brain diseases they are researching with what a normally developed brain looks like.

The studies are part of a larger package of articles published simultaneously in the scientific journal Science. The study on the adult brain was supported by a grant from the National Institutes of Health, while the embryo study was financed by the Knut and Alice Wallenberg and Erling-Persson foundations.

Sten Linnarsson is a scientific advisor at Moleculent, Combigene and Oslo University Center of Excellence in Immunotherapy. He and co-authors Alejandro Mossi Albiach and Lars E. Borm also hold shares in EEL Transcriptomics AB, which owns the intellectual property rights to the EEL method ("Spatial RNA localization"). Co-authors aneta Andrusivov and Joakim Lundeberg are scientific consultants for 10x Genomics, which holds the intellectual property rights to the Spatial Transcriptomics (Visium) technique.

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World Arthritis Day sheds light on innovative treatments – Omnia Health Insights

In the UAE, arthritis affects one in five people and is the top cause of disability globally. Arthritis includes over 100 autoimmune and rheumatic musculoskeletal conditions, and while it is commonly associated with ageing, younger patients are also affected.

World Arthritis Day, held on October 12 annually, emphasises the importance of recognising the symptoms of this painful disease, which can help lead to early intervention. Renowned organisations such as the Middle East Arthritis Foundation (MEAF) host focused events to further raise awareness and assist those who suffer from this debilitating disease.

While traditional treatments such as medications, physiotherapy, and surgery can alleviate symptoms, as World Arthritis Day is observed, we examine some alternatives and advancements that may offer hope for an enhanced quality of life.

Related:Understanding Systemic Juvenile Idiopathic Arthritis and gut health

The use of remote monitoring offers a promising way to decrease hospital visits for arthritic patients through a combination of self-management and telemedicine. With the potential to replace labour-intensive outpatient clinic visits, this could positively impact healthcare utilisation while keeping disease activity low.

Regenerative medicine in arthritis could utilise cell therapy, bioengineering and gene therapy to stimulate the body's natural healing response. A key development is Platelet-Rich Plasma (PRP), derived from a patient's blood, PRP can be used to treat pain, damaged tendons, hair loss and ageing skin. While it can relieve symptoms and boost healing, its effectiveness may vary based on preparation methods and patient factors.

Similar to fat-based PRP, Autologous Micro-fragmented Adipose Tissue (AMAT) involves liposuction to extract fat, which is then injected into areas needing treatment. Studies show improvements in osteoarthritis pain and function, but consistency in AMAT quality remains a challenge.

Stem cell therapy holds great potential for tissue repair and regeneration. The various stem cell types include: embryonic, adult, induced pluripotent, and very small embryonic-like stem cells. Clinical trials are ongoing and the FDA cautions against unproven therapies from for-profit clinics. It emphasises the need for standardised guidelines and further research to discover the full potential of stem cells in arthritis care.

Related:Gen AI may power the next generation of immunotherapies

Surgical options such as Osteochondral Autograft Transplantation Surgery (OATS) and Matrix-Induced Autologous Chondrocyte Implantation (MACI) use a patient's own or donor tissue to repair localised cartilage damage, preventing arthritis progression. Researchers are exploring gene editing tools like CRISPR-Cas9 to create custom-designed cells and gene therapies that target inflammatory proteins in osteoarthritis.

While these treatments in regenerative medicine demonstrate substantial potential, continuous research, protocol refinement, and establishing standardised methodologies are imperative to fully discover their benefits in the field of arthritis care.

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World Arthritis Day sheds light on innovative treatments - Omnia Health Insights

Adipose stromal cells bioproducts as cell-free therapies … – Journal of Translational Medicine

Cell culture

ASC from lipoaspirates from three donors were processed by the group of Prof. Karen Bieback (University of Heidelberg, Heidelberg, Germany) after informed consent. The Mannheim Ethics Commission II approved the study (vote 2011-215N-MA). The ASC were cultured using MEM- media, Gibco, ThermoFisher Scientific, 2,561,029) and 10% Fetal Bovine Serum (FBS, 10270-106, Gibco, MA, USA) at 37 with 5% CO2 and controlled humidity. These three ASC batches (referred to a N=3 biological replicates in the figure legends) were shipped as cryo-aliquots to the other two centers to be cultured under identical harmonized culture conditions from passage 46 as detailed previously [29]. Bioproducts were derived from the conditioned medium of either 3D cultured ASC, processed by size exclusion chromatography to yield (1) EV-SEC or the (2) protein-rich fraction, or 2D-cultured cells, processed by ultracentrifugation to yield (3) EV-UC or after concentration to yield (4) the conditioned medium (CM) or (5) the respective wash-off (CM-WO) (Fig.1).

At 80% confluence, ASC were passaged and seeded in a hollow-fibber bioreactor at a concentration of 14106 cells/cartridge (20kDa MWCO, 450cm2, C2025D, FiberCell System-KD Bio, France). Prior to injecting the cells, a pre-culture step was carried out to initiate and activate the bioreactor, first Dulbeccos phosphate-buffered saline (PBS) for 24h, followed by fibronectin coating over-night. After the pre-culture process, ASC were seeded in serum-free MEM- in the extra-capillary space, at 37 with 5% CO2 and controlled humidity for 7days without harvesting the supernatant, with continuous monitoring of glucose levels. Serum-containing medium was used as circulating medium, given that EVs and high molecular weight proteins cannot cross the 20 kD MWCO filter fiber and thus do not contaminate the cell-derived EVs harvested from the extra-capillary-space (according to the Hollow Fiber Bioreactor Protocol for Mesenchymal Stem Cells by fibercellsystems.com) ASC were cultured for 4weeks in the bioreactor and during this period, the supernatant was collected daily. Following centrifugation to remove cell debris (5min at 420g), the supernatant was stored at 80 until EV isolation by size exclusion chromatography (see below) was performed. Cells were harvested and counted to calculate the bioproduct per producer cell concentration.

The secretome obtained in vitro, also named conditioned media (CM), was generated from ASC at passage 4 to 6. Upon reaching 80% confluence, cells were washed with PBS and incubated for 24h in serum-free MEM- medium. The supernatant was collected and centrifuged for 5min at 400g to remove cell debris before being placed in centrifugal concentrator units of 3KDa molecular weight cut-off (UFC9003, Merck Millipore, USA). The CM was centrifuged for 90min at 3,000g, 4 using an Eppendorf 5810 R Centrifuge to achieve tenfold concentration. The flow-through resulted from the concentration step (thereafter named wash-off, CM-WO) was kept and used as a control. Concentrated conditioned media samples were stored at 80 until further use. Cells were harvested and counted to calculate the bioproduct per producer cell concentration.

When the cells reached 80% confluence, they were starved for 1624h in serum-free medium. The supernatant was collected and centrifuged for 20min at 3000g to remove cell debris and apoptotic cells. The supernatant was then ultracentrifuged for 2h at 100,000g, 4 using Beckman Coulter Optima L-100K Ultracentrifuge (Beckman Coulter, CA, USA) with the rotor type 70Ti. The EV pellet was resuspended in PBS supplemented with 1% DMSO. The suspension of EVs (EV-UC) was then stored at80 until further use. EVs were collected from ASC at 4-6th passage. Cells were harvested and counted to calculate the cell equivalents used for cell treatments.

After thawing at 4, samples were centrifuged for 10min at 300g and 20min at 4000g. After, the supernatant was filtered through a 0.2m syringe filter and concentrated with a 100kD MWCO concentration filter to a final volume of 10mL. The qEV10-IZON column 35mm was initially washed with sterile PBS, and then 10mL of the sample was added to concentrate it to the final volume of 1.5mL (Vivaspin 20, 100,000 MWCO PE, Sartorius). Each EV sample (EV-SEC) and the resultant supernatant containing the protein fraction (Protein-Rich Fraction) were collected, concentrated (Vivaspin 20, 100,000 MWCO PE, Sartorius) and stored at 80 until further use.

ASC derived bioproducts were used at a ratio of 2:1 and 20:1 relative to recipient cells. To do so, we counted the number of ASC after harvesting and used it to relate the number of particles/volumes generated of EVs and CM respectively for each bioproduct.

After the isolation, the concentration of all the samples was measured (a) by Nanosight NS300 or (b) ZetaView.

After the isolation, the concentration of all the samples was measured (a) by Nanosight NS300 (Malvern Instruments Ltd., Malvern, UK) equipped with a 488nm laser module that utilizes Brownian motion and refraction index. The particle size scatters 10nm to 1000nm, although the optimized size range is 70300nm. It uses the scattered light to detect a particle and tracks its motion as a function of time. The particles scattered light was recorded with a light-sensitive camera under a 90 angle to the irradiation plane. This angle allows the Brownian motion of the EVs. Samples were diluted 1:100 in physiologic solution. For each sample, 3 videos of 60s at camera level 15 and threshold 5 were captured using a syringe pump 30. All the samples were characterized with NTA 3.2.16 Analytical software. The NTA settings were kept constant between samples.

After the isolation, the concentration of all the samples was measured b) by ZetaView (Particle Metrix GmbH, Germany). 1L of concentrated EVs was diluted in sterile-filtered PBS in a dilution 1:1,000 and visualized using the ZetaView (sensitivity 80%, shutter 100, 11 positions, 2 cycles; Particle Metrix, Germany).

Super-resolution microscopy pictures of EVs were obtained using a temperature-controlled Nanoimager S Mark II microscope from ONI (Oxford Nanoimaging, Oxford, UK) equipped with a 100 , 1.4NA oil immersion objective, an XYZ closed-loop piezo 736 stage, and 405nm/150mW, 473nm/1W, 560nm/1W, 640nm/1W lasers and triple emission channels split at 640/and 555nm. For sample preparation, we followed the manufacturers protocol using EV profiler Kit ONI (Alfatest, Rome, Italy). Before each imaging session, bead slide calibration was performed for aligning the channels, to achieve a channel mapping precision smaller than 12nm. Images were taken in dSTORM mode using 50% laser power for the 647nm channel, 30% laser power for the 488nm laser channel, and 30% for the 555 channel. Three-channels (2000 frames per channel) (647, 555 and 488) were acquired sequentially at 30Hz (Hertz) in total reflection fluorescence (TIRF) mode. Single-molecule data was filtered using NimOSsoftware (v.1.18.3, ONI) based on the point spread function shape, photon count and localization precision to minimize background noise and remove low-precision and non-specific colocalization. Data has been processed with the Collaborative Discovery (CODI) online analysis platform https://www.alto.codi.bio/ from ONI and the drift correction pipeline version 0.2.3 was used. Clustering analysis was performed on localizations and BD clustering-constrained parameters were defined (photon count 300-max, sigma 0200nm, p-value 01, localization precision 020nm). Colocalization was defined by a minimum number of localizations for each fluorophore/protein within a distance of 100nm or a distance used from the centroid position of a cluster.

MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) containing fluorescent labeled (FITC-PE) capture beads coupled to 37 exosomal surface epitopes and 2 isotope controls was used, following the manufacturers instructions (in detail: CD3, CD4, CD19, CD8, HLA-DR, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1, CD209, CD9, SSEA-4, HLA-ABC, CD63, CD40, CD62P, CD11c, CD81, MCSP, CD146, CD41b, CD42a, CD24, CD86, CD44, CD326, CD133-1, CD29, CD69, CD142, CD45, CD31, REA control, CD20, CD14, mIgG1 control). Briefly, 15L of beads were added to 120L of buffer or sample, including a total of 1109 EVs, and the complex was then incubated on a rotor overnight at 4. After the incubation and washing steps, a cocktail of APC fluorescent antibodies against tetraspanins (CD9, CD63 and CD81) was added (allowing the detection of beads bound EVs) and set on the rotor for 1h at room temperature. After washing, samples were detected using BD FACSCelestaTM Flow Cytometer (BD Bioscience, NJ, USA). Median background values of buffer control were subtracted, and samples were normalized to the median fluorescence intensity of tetraspanins.

Proteins extracted from Hela cells were used as cellular control, the pellet was resuspended in RIPA buffer (50mM TrisHCl, pH7.4, 150mM NaCl, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 0.1mM CaCl2, and 0.01mM MgCl2 supplemented with protease inhibitor cocktail (Thermo Fisher Scientific), incubate 30min in ice vortexing every 10min and centrifuge 20min at 20,000g. An equal volume of bioproducts (38L) was loaded and separated on 415% Mini-PROTEAN TGX Precast Gels (Bio-Rad, USA). Bioproducts and cell lysates were treated with protein loading dye (Laemmli sample buffer; Bio-Rad) with freshly added -mercaptoethanol 10%; v/v; Sigma, Germany) and boiled for 5min at 95 before SDS-PAGE. Proteins were subsequently blotted to a nitrocellulose blotting membrane (0.2m; 1,060,000; GE Healthcare, USA). Membranes were blocked in 5% BSA (Carl Roth, Germany) in 0.1% Tween in TBS (TBS-T). After blocking, blots were probed with the following primary antibodies diluted in 5% BSA/TBS-T: Calnexin (1:500 dilution, E-10, Santa Cruz Biotechnology). After overnight incubation at 4, membranes were washed 3 times with TBS-T and subsequently incubated with the secondary antibody dilution: Polyclonal Goat anti-mouse HRP (1:5000 dilution; P0447) for 1h at room temperature followed by washing. Blots were then developed using Western Bright ECL (541,004; Biozym Scientific, Germany) and protein bands were detected using the FusionCapt Advanced Solo 4 (Vilber, Germany).

The capacity of ASC or their bioproducts to inhibit induced proliferation of peripheral blood mononuclear cells (PBMCs) was analyzed as described before [27]. PBMCs were isolated from leukapheresis samples from healthy donors, provided by the German Red Cross Blood Donor Service in Mannheim (Mannheim Ethics Commission; vote number 2018-594N-MA). To assess their proliferation, PBMCs were labelled with proliferation dye Cytotell Green (ATT Bioquest, 22,253) (1:500 dilution) and seeded at a 1:10 ASC/bioproduction:PBMCs ratio in RPMI, supplemented with 10% FBS, 2% l-glutamine (PAN Biotech, P04-80100), 1% Penicillin/Streptomycin (PAN Biotech, P06-07100), and 200U/mL IL-2 (Promokine, C61240). PBMC proliferation was stimulated with phytohemagglutinin-L (PHA, 4.8g/mL (Biochrom, Merck Millipore, M5030)). PBMCs cultured alone without ASC in the absence and presence of PHA served as negative and positive controls, respectively. After 5days, PBMC proliferation was measured based on the dilution of Cytotell Green dye using a FACS Canto II (BD Biosciences) and the data were analyzed with FlowJo Software.

THP-1 monocyte-like cell line (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 growth medium with l-Glutamine (Sigma-Aldrich Ireland Ltd. Wicklow, Ireland) supplemented with 10% FBS (Sigma-Aldrich), 1% penicillin G (100U/mL) and streptomycin (100g/mL) solution (Sigma-Aldrich). In vitro assessment of phagocytic activity was done as described before [30]. THP-1 cells were seeded at a density of 5104 cells/well in dark 96 well-plates (Perkin Elmer Ireland Ltd. Dublin, Ireland) and exposed to 1g/mL of para-methoxyamphetamine (PMA, Sigma-Aldrich, St. Louis, MO, USA) for 48h to induce a macrophage-like phenotype. Cultures were washed with DPBS and fed with growth media for 24h. Afterwards, cells were activated with 100ng/mL of lipopolysaccharide (LPS, Sigma-Aldrich) for 24h. To measure the phagocytic capacity, Zymosan A FITC BioParticles (Thermo Fisher Ltd.) were used. Particles were opsonized with human serum (2mg/mL per 2107 particles, Sigma-Aldrich) for 1h and added to the cells in experimental media containing ASC bioproducts and growth media for 4h. Then, cells were washed twice with DPBS, fixed with 4% PFA for 15min and stained with Hoechst 33,342 (Invitrogen, Thermo Fisher Ltd). Images were taken on the Cytation 1 Imaging Reader at 20X (BioTek, with Gen5 Version 3.04 software, Swindon, UK). Six replicates were undertaken per condition and particle analysis was done by counting particle opsonization in a minimum of 200 cells per well.

HUVEC were seeded in 48-well plates at 84,000 cells/cm2 and cultured overnight. Subsequently, a p200 tip was used to create a scratch in each monolayer. Cultures were washed with DPBS before adding EVs/CM as described before. Complete EndoGRO-LS medium was used as a positive control, while EndoGRO-LS without FBS and VEGF served as a negative control. Scratches were imaged immediately after the addition of CM (0h) and after 8- and 24-h incubation using the automated Cytation 1 Imaging Reader at 4X. Six replicates were undertaken, and the total area of each scratch was measured using Image J. The percentage of closure was calculated relative to time 0h.

20,000 ASC were seeded in a 96-well Essen ImageLock plate and cultured overnight. Then, a 96-pin WoundMaker was used to create precise and reproducible wounds in all the wells. After the wound, the cells were washed 2 times with DPBS and ASC bioproducts added in different concentrations. Plates were then cultured in an IncuCyte ZOOM incubator and every 3h were taken a picture with the software. The results were analyzed after 24h. Relative Wound Density algorithm was used to report data.

Human umbilical vascular endothelial cells (HUVEC) either from Lonza or prepared as described before [31] and cultured until the 6th passage in EndoGRO-LS Complete Culture Media Kit (SCME001, Sigma-Aldrich, St. Louis, MO, USA). In vitro formation of capillary-like structures was performed on growth factorreduced Matrigel (356,231, Corning, NY, USA, center 1 and 3) or geltrex (Geltrex LDEV-free reduced growth factor matrix; Thermo Fisher Scientific, United States, center 2) HUVEC cells were treated with EVs or CM as described before, seeded at a density of 10103cells/well on a 48-well plate. Positive control was full EndoGro-LS medium, negative control medium without VEGF and FBS (as used for all the conditions). Cells were periodically observed with a Nikon TE2000E inverted microscope (Nikon, Tokyo, Japan), and experimental results were recorded after 16h; 3 images were taken per well. Image analysis was performed with the ImageJ software v.1.53c, using the Angiogenesis Analyzer (center 1,3). The data from three independent experiments were expressed as the meanSD of tube length in arbitrary units per field. Center 2 used live cell imaging (Incucyte Zoom) to assess network formation as described before [32].

Presence of vascular endothelial growth factor (VEGF) on ASC bioproducts was determined by solid phase sandwich ELISA using the human VEGF DuoSet ELISA (R&D Systems, USA) according to manufacturers instructions. The samples were read immediately at 450nm with a wavelength correction at 570nm using a VICTOR X4 multilabel plate reader (Perkin Elmer, Waltham, Massachusetts, USA). Levels of cytokines were quantified against an eight-point standard curve using twofold serial dilutions in reagent diluent.

The Pierce BCA Protein Assay Kit (ThermoFisher Scientific, UK) was used to determine protein concentration. In order to quantify the total amount of protein, samples were first lysed with RIPA buffer 4:1 (ThermoFisher Scientific, UK) for 30min on ice. The assay was carried out as per manufacturers instructions. The absorbance values were read in a VICTOR X4 plate reader (Perkin Elmer) at a 550nm wavelength, and the protein concentrations of the samples were quantified against the standard curve.

Statistical analysis was performed using GraphPad prism v9.4.2 (GraphPad software, USA). Data are expressed as meanstandard deviation (SD). N indicates biological replicates; n indicates technical replicates. Statistical differences among groups were calculated using ordinary two-way analysis of variance (ANOVA) and Tukeys post-hoc test when group distributions were normal (ShapiroWilks test) and variances of populations were equal (Bartletts test). When either or both assumptions were violated, non-parametric analysis was conducted; KruskalWallis test used to perform multiple comparison analysis and Dunns multiple comparison test for pairwise comparison. Results were considered statistically significant when p>0.05.

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Adipose stromal cells bioproducts as cell-free therapies ... - Journal of Translational Medicine

SIRT2 Works Against Cardiac Aging in Mice and Monkeys – Lifespan.io News

Working with non-human primates, scientists have discovered that the protein SIRT2, a member of the sirtuin family, might play an important role in slowing cardiac aging [1].

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The heart is arguably the hardest worker among the organs, constantly pumping enormous amounts of blood without ever skipping a beat (well, almost). This marvel of evolution works for decades before it begins to show its age. Heart aging happens due to all the usual culprits, including chronic inflammation, mitochondrial dysfunction, oxidative stress, and telomere damage [2].

In this study published in Nature Aging, the researchers used long-tailed macaques to elucidate the molecular aspects of cardiac aging using multi-omics analysis. Unlike short-lived mice and rats, non-human primates like these have hearts that closely resemble those of humans and, due to their relatively long lifespan, suffer from spontaneous heart conditions as well.

The researchers compared the hearts of eight young (4-6 years) and eight aged (18-21 years) monkeys, which roughly translates to 16 and 65 human years. In aged monkeys, hearts exhibited all the familiar signs of aging: they contained more senescent cells and more fibrotic areas, and their heart muscle cells were significantly enlarged with structural abnormalities. The levels of several inflammatory factors were elevated as well.

Using proteomic analysis, the researchers identified 126 upregulated and 43 downregulated aging-associated differentially expressed proteins (DEPs).Further analysis showed that the upregulated DEPs were mainly related toinflammation, blood clotting, and fibrosis, while protein synthesis, mitochondrial function, and lipid metabolism DEPs were downregulated.

The researchers then compared those DEPs to genes known to be involved in age-related cardiovascular diseases. SIRT2, a SIRT family protein that often pops up in studies of aging [3],was the only protein that was downregulated in aged monkey hearts and was also linked to all four types of cardiovascular diseases. It was also the only downregulated DEP that overlapped with the aging-related genes from the Aging Atlas database.

The scientists then generated human SIRT2-deficient cardiomyocytes from embryonic stem cells. The resulting cells resembled old cardiomyocytes, including hypertrophy and an increased percentage of senescent cells. They also showed signs of mitochondrial dysfunction.

Transcriptomic analysis showed that many genes were either upregulated or downregulated in SIRT2-deficient cardiomyocytes compared to young healthy cells. The researchers were able to identify the transcription factor (a gene that regulates expression of other genes) STAT3 as a major driver of those changes. STAT3 is also a well-known mediator of inflammation.

Notably, STAT3 was the only transcription factor that controlled changes in gene expression in both SIRT2-deficient human cardiomyocytes and in aged hearts of monkeys of both sexes. The researchers then confirmed via a technique called co-immunoprecipitation that STAT3 was one of the few transcription factors to interact with SIRT2.

SIRT2 acts by deacetylating proteins (removing an acetyl group from lysine residues, which alters the proteins function). Overexpression of SIRT2 led to decreased levels of acetylated STAT3, suggesting that SIRT2 recognizes STAT3 as a substrate. STAT3 levels did not react to overexpression of a SIRT2 mutant that lacked deacetylation ability. Aged monkey hearts had more acetylated STAT3 than young ones, showing impaired deacetylation.

Since transcription factors act by changing the expression of other genes, the researchers searched for downstream targets that would be relevant to the cardiac aging phenotype. They identified the gene CDKN2B, which encodes the senescence-related protein p15, as an important target. Apparently, acetylated STAT3 induces the transcription of CDKN2B, which, in turn, induces cellular senescence in cardiomyocytes. By deacetylating STAT3, SIRT2 intervenes in this process and ameliorates cardiac aging.

The researchers tested this hypothesis by injecting the hearts of aged mice with viral vectors containing SIRT2. Decreased ejection fraction and fractional shortening, two major markers of cardiac aging observed in old mice, were partially reversed by the treatment, as was the age-related enlargement of cardiomyocytes, indicating a possible cardioprotective role for SIRT2.

In this study, we systemically surveyed the multi-dimensional profiles of the NHP heart and unveiled a panel of critical biological pathways that shifted during primate heart aging. We identified SIRT2 as a key mediator of geroprotection in primate heart aging and showed that SIRT2-deficient human cardiomyocytes recaptured key senescence features of aged primate hearts. We also found that SIRT2 formed complexes with STAT3 and deacetylated it on Lys685, which, in turn, transcriptionally inactivated the senescence inducer CDKN2B. Thus, our results suggest a SIRT2STAT3CDKN2B axis, regardless of sex, in the regulation of primate cardiomyocyte senescence.

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[1] Ye, Y., Yang, K., Liu, H., Yu, Y., Song, M., Huang, D., & Liu, G. H. (2023). SIRT2 counteracts primate cardiac aging via deacetylation of STAT3 that silences CDKN2B. Nature Aging, 1-19.

[2] Li, H., Hastings, M. H., Rhee, J., Trager, L. E., Roh, J. D., & Rosenzweig, A. (2020). Targeting age-related pathways in heart failure. Circulation research, 126(4), 533-551.

[3] de Oliveira, R. M., Sarkander, J., Kazantsev, A. G., & Outeiro, T. F. (2012). SIRT2 as a therapeutic target for age-related disorders. Frontiers in pharmacology, 3, 82.

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SIRT2 Works Against Cardiac Aging in Mice and Monkeys - Lifespan.io News