Minimal criteria for reporting mesenchymal stem cells in veterinary regenerative medicine.

The widespread application of mesenchymal stem cells (MSCs) in veterinary regenerative medicine highlights their promising therapeutic potential. However, the lack of standardized characterization and reporting practices across studies poses a significant challenge, compromising the assessment of their safety and efficacy. While criteria established for human MSCs serve as a foundation, the unique characteristics of animal-derived MSCs warrant updated guidelines tailored to veterinary medicine. A recent position statement outlining minimal reporting criteria for MSCs in veterinary research reflects efforts to address this need, aiming to enhance research quality and reproducibility. Standardized reporting criteria ensure transparency, facilitate evidence synthesis, and promote best practices adoption in MSC isolation, characterization, and administration. Adherence to minimal reporting criteria is crucial for maintaining scientific rigor and advancing the field of veterinary regenerative medicine. Ongoing collaboration among stakeholders is essential for effective implementation and adherence to updated guidelines, fostering excellence and innovation in MSC-based therapies for animal patients.

GMP-Compliant Production of Human Placenta-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells are one of the most attractive sources for stem cell research and therapy. Their safety and efficacy have been demonstrated in many clinical trials. Because of their low immunogenicity and immunomodulatory properties, allogenic MSCs have been transplanted in different clinical studies. MSCs could be in different adult- and fetal-derived tissues including pregnancy products. Placenta-derived mesenchymal stem cells (PLMSCs) that can be harvested without using any invasive procedures from a discarding tissue are one of the important types of mesenchymal stem cells for therapeutic applications. Stem cell manufacturing for therapeutic applications should be in compliance with the basic principles of good manufacturing practice (GMP). Herein, the current chapter is to describe GMP-compliant production of human PLMSCs, which are suitable for clinical applications.

Mesenchymal Stem Cells as a Treatment Strategy for Coronavirus Disease 2019 (COVID-19): Need for Authority Regulations and Clinical Guidelines.

The cause of Coronavirus Disease 2019 (COVID-19) known as the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, formerly designated 2019-nCoV) was first discovered in December 2019 in Wuhan, China. It then spread rapidly worldwide. Investigation for the discovery of drugs to cure this disease continues. The currently accepted treatments are supportive, but there is no specific disease curing intervention found yet. Since mid-February, therapies involving Mesenchymal Stem/Stromal Cells (MSCs) have been proposed for the treatment of patients with COVID-19. In light of these recent developments, this review will focus on: i) the mechanism of SARS-CoV-2 action and the subsequent pathology in COVID-19, ii) the proposed mechanism( s) of outcome-improving action of MSCs or MSC-derived extracellular vesicles in COVID-19 pneumonia, iii) registered MSC-based clinical trials and interventions for the treatment of COVID-19, iv) published case studies/series/trials reporting the use of MSC-based treatments in COVID-19 cases, and finally v) the need for authority regulations and clinical guidelines for MSCbased treatment strategies for COVID-19.

Logistics of an advanced therapy medicinal product during COVID-19 pandemic in Italy: successful delivery of mesenchymal stromal cells in dry ice.

BACKGROUND: During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option. METHODS: We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics. RESULTS: Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes. CONCLUSIONS: This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

Therapeutic Effects of Mesenchymal Stem Cells Derived From Bone Marrow, Umbilical Cord Blood, and Pluripotent Stem Cells in a Mouse Model of Chemically Induced Inflammatory Bowel Disease.

Acute inflammatory bowel disease (AIBD) is a wide clinical entity including severe gastrointestinal pathologies with common histopathological basis. Epidemiologically increasing diseases, such as necrotizing enterocolitis (NEC), gastrointestinal graft versus host disease (GVHD), and the primary acute phase of chronic inflammatory bowel disease (CIBD), exhibit a high necessity for new therapeutic strategies. Mesenchymal stem cell (MSC) cellular therapy represents a promising option for the treatment of these diseases. In our study, we comparatively assess the efficacy of human MSCs derived from bone marrow (BM), umbilical cord blood (UCB), human embryonic stem cells (ESCs), or human-induced pluripotent stem cells (iPSCs) in a mouse model of chemically induced acute enterocolitis. The laboratory animals were provided ad libitum potable dextrane sulfate sodium solution (DSS) in order to reproduce an AIBD model and then individually exposed intraperitoneally to MSCs derived from BM (BM-MSCs), UCB (UCB-MSCs), ESCs (ESC-MSCs), or iPSCs (iPSC-MSCs). The parameters used to evaluate the cellular treatment efficacy were the animal survival prolongation and the histopathological-macroscopic picture of bowel sections. Although all categories of mesenchymal stem cells led to statistically significant survival prolongation compared to the control group, significant clinical and histopathological improvement was observed only in mice receiving BM-MSCs and UCB-MSCs. Our results demonstrated that the in vivo anti-inflammatory effect of ESC-MSCs and iPSC-MSCs was inferior to that of UCB-MSCs and BM-MSCs. Further investigation will clarify the potential of ESCs and iPSC-derived MSCs in AIBD treatment.

Genetic Stability of Mesenchymal Stromal Cells for Regenerative Medicine Applications: A Fundamental Biosafety Aspect.

Mesenchymal stem/stromal cells (MSC) show widespread application for a variety of clinical conditions; therefore, their use necessitates continuous monitoring of their safety. The risk assessment of mesenchymal stem cell-based therapies cannot be separated from an accurate and deep knowledge of their biological properties and in vitro and in vivo behavior. One of the most relevant safety issues is represented by the genetic stability of MSCs, that can be altered during in vitro manipulation, frequently required before clinical application. MSC genetic stability has the potential to influence the transformation and the therapeutic effect of these cells. At present, karyotype evaluation represents the definitely prevailing assessment of MSC stability, but DNA alterations of smaller size should not be underestimated. This review will focus on current scientific knowledge about the genetic stability of mesenchymal stem cells. The techniques used and possible improvements together with regulatory aspects will also be discussed.

Improving the biocompatibility of biomaterial constructs and constructs delivering cells for the pelvic floor.

PURPOSE OF REVIEW: Interactions between biomaterials and biomaterial-delivering cells and the host tissues are complexly affected by the material itself, the ultrastructure of the overall construct and cells and other bioactive factors involved. The aim of this review is to review the current understanding on the definitions of biocompatibility and current advances in improving biocompatability of tissue-engineered constructs. RECENT FINDINGS: Some synthetic materials are associated with more foreign body reactions compared with natural materials; however, they allow fabrication of materials with a great diversity of physical and mechanical properties. Material design strategies can be tailored to mimic the natural extracellular matrix topography. There are also advancements in the pharmacological functionalization of materials with improved angiogenic potential that can lead to better tissue response. Stem cells are also used to improve the tissue response of tissue-engineered materials; however, the recent regulations on regenerative medicine products necessitate significant regulatory approval processes for these. SUMMARY: The biggest challenge faced in translation of tissue-engineered constructs into clinical practice relates to their engraftment and poor tissue integration into the challenging wound bed of the pelvic floor. Biocompatibility of tissue engineered constructs can theoretically be improved by the incorporation of bioactive agents, such as vitamins C or oestradiol.

Identification of miRNA Reference Genes in Extracellular Vesicles from Adipose Derived Mesenchymal Stem Cells for Studying Osteoarthritis.

Osteoarthritis (OA) leads to chronic pain and disability, and traditional conservative treatments are not effective in the long term. The intra-articular injection of mesenchymal stem cells (MSCs) is considered a novel therapy for OA whose efficacy mainly relies on the adaptive release of paracrine molecules which are either soluble or extracellular vesicles (EVs) embedded. The correct quantification of EV-miRNAs using reliable reference genes (RGs) is a crucial step in optimizing this future therapeutic cell-free approach. The purpose of this study is to rate the stabilities of literature-selected proposed RGs for EV-miRNAs in adipose derived-MSCs (ASCs). EVs were isolated by ultracentrifugation from ASCs cultured with or without inflammatory priming mimicking OA synovial fluid condition. Expression of putative RGs (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA) was scored by using the algorithms geNorm, NormFinder, BestKeeper and ΔCt method. miR-16a-5p/miR-23a-3p yielded the most stable RGs, whereas let-7a-5p/miR-425-5p performed poorly. Outcomes were validated by qRT-PCR on miR-146a-5p, reported to be ASC-EVs enriched and involved in OA. Incorrect RG selection affected the evaluation of miR-146a-5p abundance and modulation by inflammation, with both values resulting strongly donor-dependent. Our findings demonstrated that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for ASC-EVs miRNAs evaluation. A correct approach would increase the accuracy of embedded molecule assessments aimed to develop therapeutic strategies for the treatment of OA based on EVs.

In vivo safety profile and biodistribution of GMP-manufactured human skin-derived ABCB5-positive mesenchymal stromal cells for use in clinical trials.

BACKGROUND AIMS: Human dermal ABCB5-expressing mesenchymal stromal cells (ABCB5+ MSCs) represent a promising candidate for stem cell-based therapy of various currently uncurable diseases in several fields of regenerative medicine. We have developed and validated a method to isolate, from human skin samples, and expand ABCB5+ MSCs that meet the guideline criteria of the International Society for Cellular Therapy. We are able to process these cells into a Good Manufacturing Practice-conforming, MSC-based advanced-therapy medicinal product. METHODS: To support the development of ABCB5+ MSCs for potential therapeutic topical, intramuscular and intravenous administration, we have tested our product in a series of Good Laboratory Practice-compliant nonclinical in-vivo studies addressing all relevant aspects of biosafety, including potential long-term persistence and proliferation, distribution to nontarget tissues, differentiation into undesired cell types, ectopic tissue formation, tumor formation and local tissue reaction. RESULTS: (i) Subcutaneous application of 1 × 107 ABCB5+ MSCs/animal and intravenous application of 2 × 106 ABCB5+ MSCs/animal, respectively, to immunocompromised mice did not result in safety-relevant biodistribution, persistence or proliferation of the cells; (ii) three monthly subcutaneous injections of ABCB5+ MSCs at doses ranging from 1 × 105 to 1 × 107 cells/animal and three biweekly intravenous injections of 2 × 106 ABCB5+ MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 106 ABCB5+ MSCs/animal to immunocompromised mice was locally well tolerated. DISCUSSION: The present preclinical in vivo data demonstrate the local and systemic safety and tolerability of a novel advanced-therapy medicinal product based on human skin-derived ABCB5+ MSCs.

Isolation, culturing and preparation for transplantation of amniotic mesenchymal stem cells Repetitive and reproducible laboratory, technical protocol.

To date, different methods of isolation of amniotic stem cells have been developed. Our previous studies have demonstrated that there are significant differences in viability and efficiency of the isolation and culture process depending on the enzyme and medium used. The aim of this study was to present efficient protocol, which can be used within good manufacturing practise conditions. Amniotic membranes were obtained from ten woman 31-39 years old who signed informed constent. GMP regulations are applicable. The described protocol aims to obtain a clinically significant cell yield (>1*108). The cells may be maintained in the growth phase even for 2 months. The mesenchymal cells constitute about 75-95% of the cells in primary culture. Supervisory authorities require repetitive and reproducible laboratory protocol for stem cells culture. Presented protocol allow achieving clinically significant cell yield (>1*108) in 4-5 weeks. Cells can be transplanted as suspension or cell sheet.

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property.

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-ß exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-ß- MSCs. Notably, PDGFR-ß+ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 106 GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-ß+ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92 ± 0.31 × 105 remained in wound by 7 days) and accelerated wound closure; meanwhile, PDGFR-ß- MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46 ± 0.26 × 105) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-ß+ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-ß+ MSCs migrated eightfold more than PDGFR-ß- MSCs toward PDGF-BB. Intriguingly, PDGFR-ß+ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-ß- MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-ß identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.

Safety Profile of Good Manufacturing Practice Manufactured Interferon γ-Primed Mesenchymal Stem/Stromal Cells for Clinical Trials.

Mesenchymal stem/stromal cells (MSCs) are widely studied by both academia and industry for a broad array of clinical indications. The collective body of data provides compelling evidence of the clinical safety of MSC therapy. However, generally accepted proof of therapeutic efficacy has not yet been reported. In an effort to generate a more effective therapeutic cell product, investigators are focused on modifying MSC processing protocols to enhance the intrinsic biologic activity. Here, we report a Good Manufacturing Practice-compliant two-step MSC manufacturing protocol to generate MSCs or interferon γ (IFNγ) primed MSCs which allows freshly expanded cells to be infused in patients on a predetermined schedule. This protocol eliminates the need to infuse cryopreserved, just thawed cells which may reduce the immune modulatory activity. Moreover, using (IFNγ) as a prototypic cytokine, we demonstrate the feasibility of priming the cells with any biologic agent. We then characterized MSCs and IFNγ primed MSCs prepared with our protocol, by karyotype, in vitro potential for malignant transformation, biodistribution, effect on engraftment of transplanted hematopoietic cells, and in vivo toxicity in immune deficient mice including a complete post-mortem examination. We found no evidence of toxicity attributable to the MSC or IFNγ primed MSCs. Our data suggest that the clinical risk of infusing MSCs or IFNγ primed MSCs produced by our two-step protocol is not greater than MSCs currently in practice. While actual proof of safety requires phase I clinical trials, our data support the use of either cell product in new clinical studies. Stem Cells Translational Medicine 2017;6:1868-1879.

Mesenchymal Stromal Cells as Anti-Inflammatory and Regenerative Mediators for Donor Kidneys During Normothermic Machine Perfusion.

There is great demand for transplant kidneys for the treatment of end-stage kidney disease patients. To expand the donor pool, organs from older and comorbid brain death donors, so-called expanded criteria donors (ECD), as well as donation after circulatory death donors, are considered for transplantation. However, the quality of these organs may be inferior to standard donor organs. A major issue affecting graft function and survival is ischemia/reperfusion injury, which particularly affects kidneys from deceased donors. The development of hypothermic machine perfusion has been introduced in kidney transplantation as a preservation technique and has improved outcomes in ECD and marginal organs compared to static cold storage. Normothermic machine perfusion (NMP) is the most recent evolution of perfusion technology and allows assessment of the donor organ before transplantation. The possibility to control the content of the perfusion fluid offers opportunities for damage control and reparative therapies during machine perfusion. Mesenchymal stromal cells (MSC) have been demonstrated to possess potent regenerative properties via the release of paracrine effectors. The combination of NMP and MSC administration at the same time is a promising procedure in the field of transplantation. Therefore, the MePEP consortium has been created to study this novel modality of treatment in preparation for human trials. MePEP aims to assess the therapeutic effects of MSC administered ex vivo by NMP in the mechanisms of injury and repair in a porcine kidney autotransplantation model.

Comparative Efficacy of Multiple Variables of Mesenchymal Stem Cell Transplantation for the Treatment of Neuropathic Pain in Rats.

OBJECTIVES: The current treatment options for neuropathic pain due to nerve injuries are limited and largely unsatisfactory. Mesenchymal stem cell transplantation (MSC) has shown promise as an emerging therapy for neuropathic pain. However, a number of critical parameters, including the sources of cells, the number of cells, and routes of transplantation, need to be elucidated before it can be tested clinically. METHODS: MSCs were isolated from rat bone marrow (rBM-MSCs) and adipose tissue (rAD-MSCs) and characterized by flow cytometry and functional differentiation. Rats with chronic constriction injury of the sciatic nerve were transplanted either intravenously or intrathecally with rBM-MSCs or rAD-MSCs in two different doses. The effects were evaluated by using paw withdrawal thresholds in response to noxious stimulation. The MSCs labeled with Dil dye were traced. A total of 75 Sprague-Dawley rats were used for these experiments. RESULTS: Both intravenous and intrathecal transplantation of MSCs significantly attenuated neuropathic pain. Comparable results were achieved by either rBM-MSCs or rAD-MSCs. No differences were noted between the two doses of cell transplantation. MSCs were found on the surface of the spinal cord and dorsal root ganglia. The animals did not show any signs of toxicity throughout the whole course of the experiments. CONCLUSIONS: Both intravenous and intrathecal MSC transplantations were safe and efficacious and both rBM-MSCs and rAD-MSCs are suitable for transplantation.

Heterogeneity in Studies of Mesenchymal Stromal Cells to Treat or Prevent Graft-versus-Host Disease: A Scoping Review of the Evidence.

Effective treatments are lacking for the treatment of steroid-refractory graft-versus-host disease (GVHD), a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Mesenchymal stromal cells (MSCs) have demonstrated promise but there is uncertainty regarding their clinical effectiveness. A systematic scoping review of the literature was performed to characterize the heterogeneity of published studies and identify opportunities for standardization. Thirty studies were identified, including 19 studies (507 patients) addressing the treatment of acute or chronic GVHD and 11 prevention studies (277 patients). Significant heterogeneity was observed in the age and diagnoses of study subjects, intensity and specifics of the conditioning regimens, degree of HLA matching, and source of hematopoietic cells. MSCs were derived from bone marrow (83% of studies), cord blood (13%), or adipose tissue (3%) and were cryopreserved from third-party allogeneic donors in the majority of studies (91% of prevention studies and 63% of treatment studies). Culture conditions and media supplements were highly variable and characterization of MSCs did not conform to all International Society for Cellular Therapy criteria in any study. MSCs were harvested from cell culture at passage 1 to 7 and the dosage of MSCs ranged from 0.3 to 10 × 10(6)/kg, using varying schedules of administration. Treatment response criteria were not standardized and effectiveness in controlled treatment studies (5 studies) was unconvincing. Details of actively recruiting trials suggest heterogeneity still persists with only 53% of registered trials describing the use of standard GVHD response criteria and few detailing methods of MSC manufacturing. Future studies will need to make substantial coordinated efforts to reduce study heterogeneity and clarify the role of MSCs in GVHD.

Inflammatory response to the administration of mesenchymal stem cells in an equine experimental model: effect of autologous, and single and repeat doses of pooled allogeneic cells in healthy joints.

BACKGROUND: Mesenchymal stem cells (MSCs) transplantation has become a promising therapeutic choice for musculoskeletal injuries. Joint-related disorders are highly prevalent in horses. Therefore, these animals are considered as suitable models for testing MSC-based therapies for these diseases. The aim of this study was to investigate the clinical and inflammatory responses to intra-articular single and repeat dose administration of autologous or of pooled allogeneic MSCs in healthy equine healthy joints. Six horses were intra-articularly injected with a single autologous dose of bone marrow derived MSCs (BM-MSCs) and two separate doses of allogeneic BM-MSCs pooled from several donors. All contralateral joints were injected with Lactated Ringer's Solution (LRS) as the control vehicle. Signs of synovitis and lameness were evaluated at days 0, 1, 2, 3, 5 and 10 after injection. Total protein (TP), white blood cell count (WBC) and neutrophil count (NC) in synovial fluid were also measured at the same time-points. RESULTS: A mild synovial effusion without associated lameness was observed after all BM-MSCs injections. The second allogeneic injection caused the lowest signs of synovitis. Local temperature slightly increased after all BM-MSCs treatments compared to the controls. TP, WBC and NC in synovial fluids also increased during days 1 to 5 after all BM-MSCs injections. Both, clinical and synovial parameters were progressively normalized and by day 10 post-inoculation appeared indistinguishable from controls. CONCLUSIONS: Intra-articular administration of an allogeneic pool of BM-MSCs represents a safe therapeutic strategy to enhance MSCs availability. Importantly, the absence of hypersensitivity response to the second allogeneic BM-MSCs injection validates the use of repeat dose treatments to potentiate the therapeutic benefit of these cells. These results notably contribute to the development of stem cell based therapies for equine and human joint diseases.