Production Of Mesenchymal Stem Cells Research Articles

The study of cancer cell in stromal environment through induced pluripotent stem cell-derived mesenchymal stem cells.

The development of mesenchymal stem cells (MSCs) has gained reputation from its therapeutic potential in stem cell regeneration, anti-inflammation, tumor suppression, and drug delivery treatment. Previous studies have shown MSCs have both promoting and suppressing effects against cancer cells. While the limitation of obtaining a large quantity of homologous MSCs for studies and treatment remains a challenge, an alternative approach involving the production of MSCs derived from induced pluripotent stem cells (iPSCs; induced MSCs [iMSCs]) may be a promising prospect given its ability to undergo prolonged passage and with similar therapeutic profiles as that of their MSC counterparts. However, the influence of iMSC in the interaction of cancer cells remains to be explored as such studies are not well established. In this study, we aim to differentiate iPSCs into MSC-like cells as a potential substitute for adult MSCs and evaluate its effect on non-small-cell lung cancer (NSCLC). iMSCs were derived from iPSCs and validated with reference to the International Society of Cellular Therapy guidelines on MSC criteria. To create a stromal environment, the conditioned medium (CM) of iMSCs was harvested and applied for coculturing of NSCLC of H1975 at different concentrations. The H1975 was then harvested for RNA extraction and subjected to next-generation sequencing (NGS) for analysis. The morphology of iMSCs-CM-treated H1975 was different from an untreated H1975. Our NGS data suggest the occurrence of apoptotic events and the presence of cytokines from H1975's RNA that are treated with iMSCs-CM. Our results have shown that iMSCs may suppress the growth of H1975 by releasing proapoptotic cytokines into coculture media. Using iPSC-derived MSC models allows a deeper study of tumor cross talk between MSC and cancer cells that can be applied for potential future cancer therapy.

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions.

Mesenchymal stem cells (MSCs) are classified as advanced therapy medicinal products, a new category of GMP (good manufacturing practice)-compliant medicines for clinical use. We isolated MSCs from 5 bone marrow (BM) samples using human platelet lysate (HPL) instead of foetal bovine serum (FBS). We used a new method of HPL production consisting of treating platelet (PLTs) pools with Ca-Gluconate to form a gel clot, then mechanically squeezing to release growth factors. We compared the new HPL (HPL-S) with the standard (HPL-E) obtained by freezing/thawing cycles and by adding heparin. HPL-S had not PLTs and fibrinogen but the quantity of proteins and growth factors was comparable to HPL-E. Therefore, HPL-S needed fewer production steps to be in compliance with GMP conditions. The number of colonies forming unit-fibroblasts (CFU-F) and the maintenance of stem markers showed no significant differences between MSCs with HPL-E and HPL-S. The cumulative population doubling was higher in MSCs with HPL-E in the earlier passages, but we observed an inverted trend of cell growth at the fourth passage. Immunophenotypic analysis showed a significant lower expression of HLA-DR in the MSCs with HPL-S (1.30%) than HPL-E (14.10%). In conclusion, we demonstrated that HPL-S is an effective alternative for MSC production under GMP conditions.

P407 A Phase IB/IIA study of remestemcel-L, an allogeneic bone marrow derived mesenchymal stem cell product, for the treatment of medically refractory ulcerative colitis: An interim analysis

Abstract Background Direct injection of mesenchymal stem cells (MSCs) have been used for the treatment of perianal Crohn’s fistulizing disease. However, no studies have looked at the direct injection of MSCs for luminal ulcerative colitis (UC). Methods Following screening and informed consent, patients with medically refractory UC were randomized in a, 2:1 fashion to receive remestemcel-L, an ex vivo expanded allogeneic bone marrow derived MSC product, at a dose of, 150 or, 300 million MSCs versus placebo delivered via direct injection using a, 23 G sclerotherapy needle at the time of colonoscopy. Patients were included if they had medically refractory UC defined as an established diagnosis for at least, 6 months prior to study enrollment and had lost response to at least one monoclonal antibody in the past. Patients were evaluated with validated clinical and endoscopic scoring systems at, 2 weeks, 6 weeks, and, 3 months post MSC delivery. Patients randomized to control crossed over at, 3 months to receive MSC treatment and followed the same schedule for post MSC evaluation. Results A total of six patients were enrolled and treated;, 4 were randomized to treatment and, 2 to placebo. All had been on prior anti-TNF or anti integrin-therapy. There were no adverse and serious adverse events related to investigational product. In the treatment group (n=4), the Mayo score and Mayo endoscopic severity score decreased in all patients by two weeks after MSC delivery. By six weeks post MSC treatment, all treated patients had achieved clinical and endoscopic remission as defined by the Mayo score and Mayo endoscopic severity score. Patient reported number of daily bowel movements decreased as did urgency and presence of blood in the stool. With regard to patient reported outcomes, at three months, based on the inflammatory bowel disease patient reported treatment impact (IBD-PRTI), all patients were extremely satisfied or satisfied with their MSC treatment. Treated response was described as excellent or good in all patients. In the control group (n=2), the Mayo score did not decrease. Patient reported number of daily bowel movements increased as did the number reporting blood in their stool; urgency remained unchanged. With regard to patient reported outcomes, at three months, based on the IBD-PRTI, one patient was neutral, and one patient was dissatisfied. Treated response was described as poor or unchanged in control patients. Conclusion MSCs offer a safe alternative therapeutic for the treatment of medically refractory UC. Early data suggests improved clinical and endoscopic scores by as early as two weeks following MSC delivery.

Nonfreezing Low Temperature Maintains the Viability of Menstrual Blood–Derived Endometrial Stem Cells Under Oxygen–Glucose Deprivation Through the Sustained Release of Autophagy-Produced Energy

Between the completion of the mesenchymal stem cell (MSCs) preparation and the transplantation into the patient, there is a time interval during which the quality control and transport of MSC products occur, which usually involves suspending the cells in normal saline in an oxygen–glucose deprivation (OGD) microenvironments. Thus, how to effectively maintain MSC viability during the abovementioned time interval is bound to play a significant role in the therapeutic effect of MSC-based therapies. Recently, menstrual blood–derived endometrial stem cells (MenSCs) have attracted extensive attention in regenerative medicine due to their superior biological characteristics, including noninvasive protocols for their collection, abundant source material, stable donation, and autotransplantation. Therefore, this study aimed to mainly determine the effect of storage temperature on the maintenance of MenSC viabilities in an OGD microenvironment, and to preliminarily explore its potential mechanism. Simultaneously, the effects of solvents commonly used in the clinic on MenSC viability were also examined to support the clinical application of MenSCs. Consequently, our results demonstrated that in the OGD microenvironment, a nonfreezing low temperature (4°C) was suitable and cost-effective for MenSC storage, and the maintenance of MenSC viability stored at 4°C was partly contributed by the sustained releases of autophagy-produced energy. Furthermore, the addition of human serum albumin effectively inhibited the cell sedimentations in the MenSC suspension. These results provide support and practical experience for the extensive application of MenSCs in the clinic.

IL-10 mRNA Engineered MSCs Demonstrate Enhanced Anti-Inflammation in an Acute GvHD Model.

Mesenchymal stem cells (MSCs) are used in various studies to induce immunomodulatory effects in clinical conditions associated with immune dysregulation such as graft versus host disease (GvHD). However, most of these clinical trials failed to go beyond early phase 2 studies because of limited efficacy. Various methods have been assessed to increase the potency of MSCs. IL-10 is an anti-inflammatory cytokine that is known to modulate immune responses in GvHD. In this study, we evaluated the feasibility of transfecting IL-10 mRNA to enhance MSC therapeutic potential. IL-10 mRNA engineered MSCs (eMSCs-IL10) maintained high levels of IL-10 expression even after freezing and thawing. IL-10 mRNA transfection did not appear to alter MSC intrinsic characteristics. eMSCs-IL10 significantly suppressed T cell proliferation relative to naïve MSCs in vitro. In a mouse model for GvHD, eMSCs-IL10 induced a decrease in plasma level of potent pro-inflammatory cytokines and inhibited CD4+ and CD8+ T cell proliferation in the spleen. In summary, our studies demonstrate the feasibility of potentiating MSCs to enhance their immunomodulatory effects by IL-10 mRNA transfection. The use of non-viral transfection may generate a safe and potent MSC product for treatment of clinical conditions associated with immune dysregulation such as GvHD.

Novel Methods to Mobilize, Isolate, and Expand Mesenchymal Stem Cells.

Numerous studies demonstrate the essential role of mesenchymal stem cells (MSCs) in the treatment of metabolic and inflammatory diseases, as these cells are known to modulate humoral and cellular immune responses. In this manuscript, we efficiently present two novel approaches to obtain MSCs from equine or human sources. In our first approach, we used electro-acupuncture as previously described by our group to mobilize MSCs into the peripheral blood of horses. For equine MSC collection, culture, and expansion, we used the Miltenyi Biotec CliniMACS Prodigy system of automated cell manufacturing. Using this system, we were able to generate appoximately 100 MSC colonies that exhibit surface marker expression of CD105 (92%), CD90 (85%), and CD73 (88%) within seven days of blood collection. Our second approach utilized the iPSC embryoid bodies from healthy or diabetic subjects where the iPSCs were cultured in standard media (endothelial + mesoderm basal media). After 21 days, the cells were FACS sorted and exhibited surface marker expression of CD105, CD90, and CD73. Both the equine cells and the human iPSC-derived MSCs were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Both methods described simple and highly efficient methods to produce cells with surface markers phenotypically considered as MSCs and may, in the future, facilitate rapid production of MSCs with therapeutic potential.

Biological Characteristics and Optical Reflectance Spectroscopy of Human Placenta Derived Mesenchymal Stem Cells for Application in Regenerative Medicine.

Introduction: The efficiency of stem cell isolation, culture, and biological characterization techniques for treatment is facing serious challenges. The purpose of this study was to provide a protocol for isolation and culture of three types of mesenchymal stem cells (MSCs) derived from the human placenta, amniotic membrane, and umbilical cord with high efficiency used for cell therapy. Methods: During this experimental laboratory study, 10 complete placenta samples were prepared from cesarean section mothers. The protocol for isolation and culture of mesenchymal cells from the placenta tissue, umbilical cord, and amniotic membrane was enzymatically optimized. The morphological features of mesenchymal cells were investigated using an inverted microscope and their biological features were measured using flow cytometry. The differentiation potential of the cells was evaluated by measuring their differentiation capacity into osteocytes and adipocytes. The absorption and reflectance features of the cells were recorded by optical spectroscopy. Finally, the data were statistically analyzed. Results: The expression of CD44, CD73, CD90 and CD29 markers in human placenta tissue-derived cells was significant. CD14, CD34 and CD45 markers were not expressed or were slightly expressed. These cells were highly viable and successfully differentiated into osteocytes and adipocytes. MSCs absorbed more light than visible light by showing light absorption peaks at wavelengths of about 435 and 550 nm. Conclusion: The protocol used in this study for isolation and culture of human placenta tissue-derived MSCs had significant efficiency for the production of MSCs for use in cell therapy and tissue engineering.

Mesenchymal Stem Cells as a Cornerstone in a Galaxy of Intercellular Signals: Basis for a New Era of Medicine.

Around 40% of the population will suffer at some point in their life a disease involving tissue loss or an inflammatory or autoimmune process that cannot be satisfactorily controlled with current therapies. An alternative for these processes is represented by stem cells and, especially, mesenchymal stem cells (MSC). Numerous preclinical studies have shown MSC to have therapeutic effects in different clinical conditions, probably due to their mesodermal origin. Thereby, MSC appear to play a central role in the control of a galaxy of intercellular signals of anti-inflammatory, regenerative, angiogenic, anti-fibrotic, anti-oxidative stress effects of anti-apoptotic, anti-tumor, or anti-microbial type. This concept forces us to return to the origin of natural physiological processes as a starting point to understand the evolution of MSC therapy in the field of regenerative medicine. These biological effects, demonstrated in countless preclinical studies, justify their first clinical applications, and draw a horizon of new therapeutic strategies. However, several limitations of MSC as cell therapy are recognized, such as safety issues, handling difficulties for therapeutic purposes, and high economic cost. For these reasons, there is an ongoing tendency to consider the use of MSC-derived secretome products as a therapeutic tool, since they reproduce the effects of their parent cells. However, it will be necessary to resolve key aspects, such as the choice of the ideal type of MSC according to their origin for each therapeutic indication and the implementation of new standardized production strategies. Therefore, stem cell science based on an intelligently designed production of MSC and or their derivative products will be able to advance towards an innovative and more personalized medical biotechnology.

Enhancement of anti-inflammatory and immunomodulatory effects of adipose-derived human mesenchymal stem cells by making uniform spheroid on the new nano-patterned plates

Human mesenchymal stem cells (MSCs) are known to have anti-inflammatory and immunomodulatory functions; thus, several MSC products have been applied as cell therapy in clinical trials worldwide. Recent studies have demonstrated that MSC spheroids have superior anti-inflammatory and immunomodulatory functions to a single cell suspension. Current methods to prepare MSC spheroids include hanging drop, concave microwell aggregation, spinner flask, and gravity circulation. However, all these methods have limitations such as low scalability, easy cell clumping, low viability, and irregular size distribution. Here, we present a nano-patterned culture plasticware named PAMcell™ 3D plate to overcome these limitations. Nano-sized silica particles (700 nm) coated with RGD peptide were arrayed into fusiform onto the PLGA film. This uniform array enabled the seeded MSCs to grow only on the silica particles, forming uniform-sized semi-spheroids within 48 h. These MSC spheroids have been shown to have enhanced stemness, anti-inflammatory, and immunomodulatory functions, as revealed by the increased expression of stem cell markers (Oct4, Sox2, and Nanog), anti-inflammatory (IL-10, TSG6, and IDO), and immunomodulatory molecules (HGF, VEGF, CXCR4) both at mRNA and protein expression levels. Furthermore, these MSC spheroids demonstrated an increased palliative effect on glycemic control in a multiple low-dose streptozotocin-induced diabetes model compared with the same number of MSC single cell suspensions. Taken together, this study presents a new method to produce uniform-sized MSC spheroids with enhanced anti-inflammatory and immunomodulatory functions in vitro and in vivo.

Multiomics analyses of cytokines, genes, miRNA, and regulatory networks in human mesenchymal stem cells expanded in stirred microcarrier-spinner cultures

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.

Wogonin Strengthens the Therapeutic Effects of Mesenchymal Stem Cells in DSS-Induced Colitis via Promoting IL-10 Production.

Inflammatory bowel diseases (IBD) are prevalent and debilitating diseases; their clinical remedy is desperately unmet. Mesenchymal stem cells (MSCs) are pluripotent stem cells with multiple immunomodulatory effects, which are attributed to their efficacy in the IBD rodent model. Optimization of MSC regimes in IBD is a crucial step for their further clinical application. Wogonin is a flavonoid-like compound, which showed extensive immunomodulatory and adjuvant effects. This research is aimed at investigating whether and how Wogonin boosted the therapeutic efficiency of MSCs on DSS-induced colitis. Our results showed that the MSC treatment with Wogonin significantly alleviated the intestinal inflammation in IBD mice by increased IL-10 expression. In vitro experiments, Wogonin obviously raised the IL-10 production and ROS levels of MSCs in a dose-dependent manner. Meanwhile, western blot data suggested Wogonin improves the IL-10 production by inducing transcript factor HIF-1α expression via AKT/GSK3β signal pathway. Finally, the favorable effects of Wogonin on MSCs were confirmed by IL-10 blockade experiment in vivo. Together, our results suggested that Wogonin significantly increased the IL-10 production and enhanced the therapeutic effects of MSCs in DSS-induced colitis. This work suggested Wogonin as a novel optimal strategy for MSC clinical application.

Culturing at Low Cell Density Delays Cellular Senescence of Human Bone Marrow-Derived Mesenchymal Stem Cells in Long-Term Cultures.

Background and ObjectivesMesenchymal stem cells (MSCs) have immense therapeutic potential for treating intractable and immune diseases. They also have applications in regenerative medicine in which distinct treatments do not exist. Thus, MSCs are gaining attention as important raw materials in the field of cell therapy. Importantly, the number of MSCs in the bone marrow is limited and they are present only in small quantities. Therefore, mass production of MSCs through long-term culture is necessary to use them in cell therapy. However, MSCs undergo cellular senescence through repeated passages during mass production. In this study, we explored methods to prolong the limited lifetime of MSCs by culturing them with different seeding densities.Methods and ResultsWe observed that in long-term cultures, low-density (LD, 50 cells/cm2) MSCs showed higher population doubling level, leading to greater fold increase, than high-density (HD, 4,000 cells/cm2) MSCs. LD-MSCs suppressed the expression of aging-related genes. We also showed that reactive oxygen species (ROS) were decreased in LD-MSCs compared to that in HD-MSCs. Further, proliferation potential increased when ROS were inhibited in HD-MSCs.ConclusionsThe results in this study suggest that MSC senescence can be delayed and that life span can be extended by controlling cell density in vitro. These results can be used as important data for the mass production of stem cell therapeutic products.

Bioglass enhances the production of exosomes and improves their capability of promoting vascularization

Recently, exosomes have been extensively applied in tissue regeneration. However, their practical applications are severely restricted by the limited exosome secretion capability of cells. Therefore, developing strategies to enhance the production of exosomes and improve their biological function attracts great interest. Studies have shown that biomaterials can significantly enhance the paracrine effects of cells and exosomes are the main signal carriers of intercellular paracrine communication, thus biomaterials are considered to affect the exosome secretion of cells and their biological function. In this study, a widely recognized biomaterial, 45S5 Bioglass® (BG), is used to create a mild and cell-friendly microenvironment for mesenchymal stem cells (MSCs) with its ion products. Results showed that BG ion products can significantly improve exosome production of MSCs by upregulating the expression of neutral sphingomyelinase-2 (nSMase2) and Rab27a which enhanced the nSMases and Rab GTPases pathways, respectively. Besides, microRNA analysis indicates that BG ion products can modulate the cargoes of MSCs-derived exosomes by decreasing microRNA-342-5p level while increasing microRNA-1290 level. Subsequently, the function of exosomes is modified as their capabilities of promoting the vascularization of endothelial cells and facilitating the intradermal angiogenesis are enhanced. Taken together, BG ion products are confirmed to enhance exosome production and simultaneously improve exosome function, suggesting a feasible approach to improve the practical application of exosomes in regenerative medicine.

Abstract 487: Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy

Abstract Background: Mesenchymal stem cells (MSCs) have the potential as a drug delivery tool in cancer immunotherapy. We previously demonstrated that MSCs can be used to deliver BCG immunotherapy to the hypoxic core of tumors (#AACR-Abstract-3903). However, lack of clinical grade MSCs hamper the use of MSCs in clinical practice. The production of clinical grade human derived MSCs in accordance with current Good Manufacture Practice (cGMP) is an unmet need in the cancer immunotherapy. However, the selection criterion for characterizing the safety of MSC-based products is not well cited yet. In an effort to generate a potent therapeutic cell based product, we design a series of in-vitro safety assays for the production of high quality tissue specific human MSCs (hMSCs) as a final products before releasing from cGMP. Method: Here, we have isolated MSCs from Bone marrow (BM), Adipose tissue (AD) and Wharton Jelly (WJ) and expanded in serum free media. The in-vitro proliferation and phenotypic profiling of stem cell markers performed to check purity of the ex-vivo cultured hMSCs. To determine the clinical safety, we have done stemness marker analysis and cellular senescence capacity. To determine the functional aspects, tri-lineage differentiation and immunopotency of hMSCs were performed. Additionally, to assure the in vitro anti-tumorigenic properties of characterized hMSCs subjected to 2D colony forming unit (CFU) assay, 3D clonogenic assay as 3D organoid culture system and karyotyping. Results: Here, we proposed bioprocess design that allowed successful isolation of MSCs from BM, AD and WJ separately. Growth profiles of all isolated MSCs (n = 3) under serum free condition media with respect to serum containing media shows an exponential growth phase. The decline phase started after Day 7. Results also shows significant variation in population doubling time (PDT) of all isolated hMSCs at Passage 3 (n = 3) and Passage 5((n = 3) in comparison between serum free condition media & serum containing media. We found all hMSCs cultured in serum free media shows positive for CD105-APC (>80%), CD73-PE (>80%), CD90- PerCP (>80%), CD29- FITC (>80%) and HLAI (>80%), and negative HLAII- FITC(<2.20 %) and CD34/45- FITC (<2.20 %) expressions at Passage 3 & passage 5, indicating a stem cell phenotype (n = 3). Isolated hMSCs showed osteocyte, adipocytes and chondrocytes differentiation and high capacity to inhibit the proliferation of activated lymphocytes. Additionally, hMSCs positively expressed Nanog and Sox-2 stemness gene and cellular senescence from passage 7. In 2D CFU assay these cells showed colony forming capacity and in 3D organoid culture system in soft agar based matrix, we found that these cells are non-tumorigenic and didn't showed any karyotypic alteration. Conclusion: The collective result provides compelling evidence of the clinical safety of cGMP-compliant BM-MSCs, AD-MSCs and WJ-MSCs potentially suitable for future therapeutic application. Citation Format: Sorra Sandhya, Sonali Rawat, Suchi Gupta, Harshita Sharma, Bikul Das, Sujata Mohanty. Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 487.

Antimicrobial activity of mesenchymal stem cells against Staphylococcus aureus

IntroductionThere have been limited advances in the treatment of bone and joint infections, which currently involves a combination of surgery and antibiotic administration. There is a timely need in orthopedics to develop more effective and less invasive forms of antimicrobial prophylaxis and treatment. The antibacterial effect of adult tissue-derived mesenchymal stem cells (MSCs) has recently been investigated against Escherichia coli and Staphylococcus aureus. The main mechanism of action is postulated to be via MSC production of the cationic antimicrobial peptide, LL-37.MethodsThis study examines the antimicrobial activity of adipose-derived human MSCs (ASCs) on S. aureus, specifically examining the role of LL-37 and regulation of its expression. Bacteria colony-forming unit (CFU) assay was used to assess antimicrobial activity.ResultsOur results showed that the ASC-conditioned medium significantly inhibited the growth of S. aureus under standard culture conditions with or without the continued presence of ASCs. Also, the treatment of ASCs with 1,25-dihydroxy vitamin D3 elevated LL-37 expression and enhanced their antimicrobial activity. In support, treatment with the vitamin D receptor inhibitor, GW0742, blocked the antimicrobial activity of ASCs.ConclusionOur findings clearly demonstrate the antimicrobial activity of adult ASCs against S. aureus and implicate a key regulatory role for vitamin D. Further testing in in vivo models is being pursued to assess the potential application of ASCs as a biocompatible, adjunct treatment for musculoskeletal infections.

High content analysis reveals distinct morphological profiles of human MSCs under different culture conditions

Background & Aim The number of clinical trials employing Mesenchymal Stem Cells (MSC) for the treatment of human diseases has grown substantially in the last decade. In this context new characterisation tools for cell and media manufacturing process are required. With this in mind, we sought to develop an automated high-content imaging assay to characterize the morphological profile of MSC in response to different media formulations. Methods, Results & Conclusion High content analysis of high resolution images indicated that MSC growing in absence of serum displayed distinct morphological features compared to SC MSC. Specifically, in the absence of serum cells showed a smaller and more circular morphology, with less protrusions compared to SC MSC, which displayed an elongated shape, with numerous cellular protrusions. These phenotypic differences were expressed for instance by a significant higher circularity index and lower form factor by SF MSC, which measure respectively the overall roundness and spreading of a cell. Both automated workflows were able to successfully cluster the two experimental groups based on several morphological descriptors, allowing the generation of a morphological profile. Of note, the assay produced similar morphological profiles using MSC from two donors. The development of serum-free (SF) and chemically defined media (CDM) for the clinical production of human MSCs is a high priority within the cell therapy community. Small molecules as media supplements are an attractive tool as they are generally easily available, chemically defined and tunable, and chemical media design presents an attractive opportunity for improving MSC manufacturing. In this context, we are developing a novel analytical technology aimed at designing chemically defined media (CDM) tailored to the MSC production process. For this purpose, MSC Morphology Profile will be used as one of the read out assays to screen small molecules, with the goal of identifying molecules that sustain proliferation of MSC while retaining their potency. Taken together, we report the development of a high-content assay to readily capture and analyse the morphological profile of MSC grown in different culture conditions and quantitatively showed that serum influences MSC morphology. Importantly, the assay may represents a straightforward strategy to benchmark MSC preparation and media formulations during production processes for therapeutic application.

Effects of Mesenchymal Stem Cell Coculture on Human Lung Small Airway Epithelial Cells.

Mesenchymal stem cells (MSCs) and their secreted extracellular vesicles have been used effectively in different lung disease animal models and clinical trials. Their specific beneficial effects, the potential differences between MSCs derived from different organs, and interactions between MSC products and target cells still need to be studied further. Therefore, we investigated the effects of secreted products of human MSCs derived from the bone marrow and adipose tissue on human lung small airway epithelial (AE) cells in vitro. AE cells were cocultured with MSCs in inserts that allowed the free exchange of medium but did not allow direct cell-to-cell contact. We examined the effects on AE cell viability, proliferation, cell numbers, expression of AE cell-specific genes, and CD54 (intercellular adhesion molecule 1 (ICAM1)) surface positivity, as well as the secretion/uptake of growth factors relevant for AE cell. We found that coculture increased the viability of AE cells. The majority of AE cells expressed CD54 on their surface, but the percentage of cells being positive for CD54 did not increase in coculture. However, ICAM1 gene expression was increased in coculture. Also, we observed increased gene expression of mucin (MUC1), a lung-enriched cell surface glycoprotein. These observed effects were the same between bone marrow and adipose tissue MSCs. However, MSCs derived from adipose tissue reduced angiopoietin concentrations in coculture, whereas those from the bone marrow did not. Conclusively, MSCs influenced AE cells positively by increasing their viability and affecting gene expression, with some effects being specific for the tissue origin of MSCs.

Advances in regenerative therapy: A review of the literature and future directions.

There is enormous global anticipation for stem cell-based therapies that are safe and effective. Numerous pre-clinical studies present encouraging results on the therapeutic potential of different cell types including tissue derived stem cells. Emerging evidences in different fields of research suggest several cell types are safe, whereas their therapeutic application and effectiveness remain challenged. Multiple factors that influence treatment outcomes are proposed including immunocompatibility and potency, owing to variations in tissue origin, ex-vivo methodologies for preparation and handling of the cells. This communication gives an overview of literature data on the different types of cells that are potentially promising for regenerative therapy. As a case in point, the recent trends in research and development of the mesenchymal stem cells (MSCs) for cell therapy are considered in detail. MSCs can be isolated from a variety of tissues and organs in the human body including bone marrow, adipose, synovium, and perinatal tissues. However, MSC products from the different tissue sources exhibit unique or varied levels of regenerative abilities. The review finally focuses on adipose tissue-derived MSCs (ASCs), with the unique properties such as easier accessibility and abundance, excellent proliferation and differentiation capacities, low immunogenicity, immunomodulatory and many other trophic properties. The suitability and application of the ASCs, and strategies to improve the innate regenerative capacities of stem cells in general are highlighted among others.

Thawed Mesenchymal Stem Cell Product Shows Comparable Immunomodulatory Potency to Cultured Cells In Vitro and in Polymicrobial Septic Animals

Mesenchymal stem cells (MSCs) have been shown to exert immunomodulatory effects in both acute and chronic diseases. In acute inflammatory conditions like sepsis, cell therapy must be administered within hours of diagnosis, requiring “off-the-shelf” cryopreserved allogeneic cell products. However, their immunomodulatory potency, particularly in abilities to modulate innate immune cells, has not been well documented. Herein we compared the stabilities and functionalities of cultured versus thawed, donor-matched MSCs in modulating immune responses in vitro and in vivo. Cultured and thawed MSCs exhibited similar surface marker profiles and viabilities at 0 hr; however, thawed MSCs exhibited higher levels of apoptotic cells beyond 4 hrs. In vitro potency assays showed no significant difference between the abilities of both MSCs (donor-matched) to suppress proliferation of activated T cells, enhance phagocytosis of monocytes, and restore endothelial permeability after injury. Most importantly, in animals with polymicrobial sepsis, both MSCs significantly improved the phagocytic ability of peritoneal lavage cells, and reduced plasma levels of lactate and selected inflammatory cytokines without significant difference between groups. These results show comparable in vitro and in vivo immunomodulatory efficacy of thawed and fresh MSC products, providing further evidence for the utility of a cryopreserved MSC product for acute inflammatory diseases.

EXTH-49. THERAPEUTIC EFFICACY OF ENGINEERED, HYDROGEL ENCAPSULATED BIMODAL MSC IN GLIOBLASTOMA STRATIFIED ON CELL SURFACE RECEPTOR EXPRESSION

Abstract Engineered stem cell therapeutics present a promising therapeutic strategy for glioblastoma (GBM). However, their clinical translation in GBM patients is limited due to lack of mouse models that mimic clinical GBM scenario, dearth of stratification techniques for target cohorts and safety concerns. In this study, we have validated the potential of stratifying therapy responders based on a circulating tumor cell screen and developed a unique “off-the-shelf” cell surface receptor targeted engineered bi-functional mesenchymal stem cell (MSC) for resected GBMs. We created a dual promoter self-inactivating lentiviral vector plasmid encoding death receptor (DR4/5) ligand (DRL), the tumor specific therapeutic agent and the prodrug-converting enzyme, herpes simplex virus-thymidine kinase (HSV-TK) and engineered clinical grade human bone marrow derived MSC to express the bi-modal therapeutic. We show that hyaluronic acid (HA) hydrogel encapsulated bimodal MSC are retained in the tumor resection cavity and have significant therapeutic efficacy in vitro and in vivo in mice bearing patient derived GBM tumors following DR4/5 stratification by circulating tumor cells in the blood and GBM surgical resection. We also show that fate of engineered MSC can be tracked by PET imaging and activation of pro-drug, HSV-TK enhances the therapeutic efficacy of MSC and results in their elimination post-tumor treatment. Our study reveals that local delivery of hydrogel encapsulated engineered MSC has therapeutic benefits; circumvents concerns of bio-distribution, increases on-site retention and permits tumor-selective migration. Based on this data, we are preparing for a first-in-human study of a hydrogel encapsulated engineered allogeneic MSC product, in primary GBM patients.