Wharton's Jelly Mesenchymal Stem Cells Research Articles

The healing effect of Wharton's jelly stem cells seeded on biological scaffold in chronic skin ulcers: A randomized clinical trial.

Chronic wound or nonhealing ulcer is essentially a wound that does not progress normally through the wound healing process. This study assessed the healing effect of umbilical cord Wharton's jelly stem cells seeded on biological scaffold in chronic skin ulcers. In a randomized clinical trial, five patients between 30 and 60years with chronic diabetic wounds were enrolled. To cover the wounds, acellular amniotic membrane seeded with Wharton's jelly mesenchymal stem cells (WJSCs) was used for 9days, every 3days with a follow-up of 1month. The percentage and time of wound healing and the size of wound were recorded for each patient. In treated patients, the wound healing time and wound size significantly decreased, and after 6 and 9days, the wound size significantly declined (P<0.002). As WJSCs seeded on amniotic membrane could significantly accelerate the healing effect in chronic diabetic wounds, they can be an alternative source in tissue engineering and repair of chronic ulcers.

Mitochondrial Transfer of Wharton's Jelly Mesenchymal Stem Cells Eliminates Mutation Burden and Rescues Mitochondrial Bioenergetics in Rotenone-Stressed MELAS Fibroblasts.

Wharton's jelly mesenchymal stem cells (WJMSCs) transfer healthy mitochondria to cells harboring a mitochondrial DNA (mtDNA) defect. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major subgroups of mitochondrial diseases, caused by the mt.3243A>G point mutation in the mitochondrial tRNALeu(UUR) gene. The specific aim of the study is to investigate whether WJMSCs exert therapeutic effect for mitochondrial dysfunction in cells of MELAS patient through donating healthy mitochondria. We herein demonstrate that WJMSCs transfer healthy mitochondria into rotenone-stressed fibroblasts of a MELAS patient, thereby eliminating mutation burden and rescuing mitochondrial functions. In the coculture system in vitro study, WJMSCs transferred healthy mitochondria to rotenone-stressed MELAS fibroblasts. By inhibiting actin polymerization to block tunneling nanotubes (TNTs), the WJMSC-conducted mitochondrial transfer was abrogated. After mitochondrial transfer, the mt.3243A>G mutation burden of MELAS fibroblasts was reduced to an undetectable level, with long-term retention. Sequencing results confirmed that the transferred mitochondria were donated from WJMSCs. Furthermore, mitochondrial transfer of WJMSCs to MELAS fibroblasts improves mitochondrial functions and cellular performance, including protein translation of respiratory complexes, ROS overexpression, mitochondrial membrane potential, mitochondrial morphology and bioenergetics, cell proliferation, mitochondrion-dependent viability, and apoptotic resistance. This study demonstrates that WJMSCs exert bioenergetic therapeutic effects through mitochondrial transfer. This finding paves the way for the development of innovative treatments for MELAS and other mitochondrial diseases.

Microvesicles derived from human Wharton's Jelly mesenchymal stem cells ameliorate acute lung injury partly mediated by hepatocyte growth factor.

Several studies have highlighted the underlying role of mesenchymal stem cells microvesicles (MSC-MVs) in acute lung injury (ALI). Hepatocyte growth factor (HGF) derived from MSC-MVs is partly involved in their therapeutic effects; however, the detailed mechanism remains unclear. MVs were isolated from human Wharton's Jelly MSCs. The rat model of ALI was established by intratracheal instillation of bleomycin (BLM). A co-culture model of alveolar epithelial cells or pulmonary endothelial cells and MSC-MVs was utilized. Total protein content in bronchoalveolar lavage fluid (BALF) was determined by bicinchoninic acid method. White blood cell (WBC) and neutrophil in BALF were counted. ELISA was used for the determination of cytokines and HGF in BALF. Apoptosis was determined by TUNEL assay and Annexin V-FITC/PI staining as well as caspase-3 activity detection. HE and Masson staining of lung tissues was used for histopathology analysis. The expression of HGF and proteins involved in the PI3K/AKT/mTOR pathway were measured by quantitative Real-Time PCR (qRT-PCR) and western blotting. Treatment with MSC-MVs significantly inhibited BLM-induced apoptosis and fibrosis in lung tissues and PI3K/AKT/mTOR activation, which was reversed by HGF mRNA deficient MVs. Intriguingly, these effects were completely abrogated by PI3K inhibitor. The therapeutic effect of MSC-MVs in ALI was partly mediated through HGF mRNA.

Bioreactor produced wharton's jelly mesenchymal stem cell- derived exosomes prevent lung injury in a pre-clinical model of bronchopulmonary dysplasia.

Background & Aim Mesenchymal stem cells (MSCs) have been safely and effectively used to treat lung and heart diseases. However, cell product variability and limited survival post transplantation has led to varying degrees of clinical success. Recent evidence suggest that exosomes rather than stem cell engraftment and differentiation mediate MSC regenerative effects. Exosomes are nanosized extracellular vesicles that a play a key role in intercellular and interorgan communications. Therefore, exosome therapy has emerged as a new therapeutic alternative to MSCs. One of the challenges for the transition of exosomes from the bench to the bedside is the generation of high quantity GMP-compliant products. Thus, we aimed to develop a production methodology of Wharton's Jelly MSC (WJMSC) derived exosomes in a bioreactor and test the hypothesis that the exosome product would significantly reduce neonatal lung injury in a preclinical model of Bronchopulmonary Dysplasia. Methods, Results & Conclusion WJMSCs were isolated from Wharton's Jelly and expanded to passage 1. After harvest, 30 × 106 cells were loaded into the quantum bioreactor (Terumo BCT) and expanded for 1 week to achieve 4.05 × 108 cells. Serum-free aMEM conditioned media was collected every 24 hours for 96 hours for exosome precipitation by ultracentrifugation. NanoSight analysis of the final products revealed a production of 15.0 × 1012± 0.32 × 1012 total particles with an average mode size of 111±4.78 nm. Furthermore, protein analysis revealed positive expression of exosome markers CD9, CD63, and CD81. To test the regenerative potential of this product, we treated newborn Sprague-Dawley rats (n=9/group) exposed to normoxia or hyperoxia (85% O2) from postnatal day 1-14 at day 3 with 1 × 1010 total particles (EXO) or placebo (PL). The degree of lung vascular remodeling, alveolarization, and pulmonary hypertension was then assessed. Results confirmed a significant lung protective effect of the EXO treatment in the hyperoxia groups by the demonstration of decreased percentage of muscularized vessels (67 ± 9% in PL vs 14 ± 7% in EXO, P

Developing a human mesenchymal stem cell culture medium with high growth efficient, 3d culture supported, serum-free and xeno-free properties.

Background & Aim Human mesenchymal stem cell (MSC) therapies have been tested clinically for a variety of disorders, including Crohn's disease, multiple sclerosis, graft-versus-host disease (GVHD), bone fractures, sepsis, and cartilage defects. Despite of the remarkable clinical advancements in this field, most cell manufacturing processes still rely on the traditional culture medium containing fetal bovine serum (FBS). The highly inconsistence nature and xenogenic infection risks of the bovine serum remain a challenge for the standardization and massive production for clinics. Here, we successfully develop a medium, featured with serum-free, xeno-free components and high efficacy for MSC growth, named as AllPhase. Methods, Results & Conclusion The medium on human adipose tissue-derived mesenchymal stem cells (ADSC) and Wharton's jelly mesenchymal stem cells (WJ-MSC) enables competitive expansion performance, comparing to traditional culture medium and can maintains tri-lineage mesoderm differentiation potential after fifth passages. Furthermore, most commercial serum-free media need expansive ECM coating, like fibronectin or other commercial coating materials, to support cell adhesion. Especially, AllPhase can support ADSC expansion under 3D culture condition without microcarriers. We hope that our medium can support most bioreactors and cell industrial production for MSC clinical applications.

Bach impeller for suspension microcarrier cultures of mesenchymal stromal cells

Background & Aim Mesenchymal stromal cells (MSC) form the majority of very promising late stage clinical trials for indications like stroke, heart failure and osteoarthritis. As the industry seeks to meet the requirements for commercial scale offerings to large patient populace, production of these cell therapies will have to be met in scalable suspension culture vessels. We evaluated the Bach impeller that has an exponential spiral design which operates by creating fluid-fluid motion to culture MSC at 1 and 5 litre scale compared to the conventional axial flow impeller and paddle blade impeller. Simulations indicated that suspension of microcarriers would be rapid (approx. 5 mins) at 100rpm and the Bach would be able to provide 3-4 times the oxygen transfer capability of the axial flow impeller at the equivalent power per unit volume input. Methods, Results & Conclusion Culture of Wharton Jelly MSC at 1 litre with Solohill microcarriers showed 25% improved cell densities of the Bach impeller over the axial flow and paddle impellers: 4 × 10e5 cells/ml vs. 3 × 10e5 cells/ml. This was primarily due to extensive clumping of cell-microcarrier aggregates seen in the conventional impellers from day 3 onwards; whereas the laminar flow prevented aggregation up to the final day of harvesting. Significantly, when MSC were cultured on Cytodex microcarriers at 1 litre, cell yields increased dramatically to 15 × 10e5 cells/ml; viability and identity were equivalent to axial flow or spinner flask cultures. Furthermore, when MSC cultures were scaled further to 5 litre, the Bach yielded 13 × 10e5 cells/ml, and high viability. Thus, the Bach impeller is suitable for microcarrier suspension cultures of both polystyrene as well as hydrogel based microcarriers, and is a promising new impeller to add to the field of regenerative medicine for cell therapy manufacturing.

Mesenchymal Stem Cells from the Wharton's Jelly of the Human Umbilical Cord: Biological Properties and Therapeutic Potential.

Wharton’s jelly mesenchymal stem cells (WJ-MSCs) are a class of stem cells with high differentiative potential, an immuno-privileged status and easy access for collection, which raise no legal or ethical issues. WJ-MSCs exhibit several features of embryonic stem cells, both in the phenotypic and genetic aspects, with only a few differences, such as a shorter doubling time and a more extensive ex vivo expansion capacity. WJ-MSCs have immunomodulatory properties, involving both innate and adaptive immune responses. This review focuses on the role of WJ-MSCs in the management of graft-versus-host disease (GvHD), a life-threatening complication of the allogenic transplantation of hematopoietic stem cells. Different studies documented the beneficial effect of the infusion of WJ-MSCs, even when not fully HLA identical, in patients with severe GvHD, refractory to standard treatment. Finally, we summarized current ongoing clinical trials with WJ-MSCs and their potential in regenerative medicine.

Cultivation, screening, identification and transplantation of Muse cell from human umbilical cord-derived for spinal cord injury in rats

To investigate multilineage-differentiating stress-enduring (Muse) by immunomagnetic bead screening from Wharton's jelly mesenchymal stromal cells(WJ-MSCs), and explore transplantation of Muse cell for safety and effectivensess of sub acute cord injury in rats. Donated Wharton's Jelly-mesenchymal stromal cells (WJ-MSCs) were successfully derived from a human umbilical cord by a series of procedures namely physical isolation of Wharton's Jelly from cord membrane, collagenase and trypsin treatment and density gradient centrifugation. Magnetic activated cell sorting was performed to specifically select SSEA3+ Muse cells, and flow cytometry and immunocytochemistry were used to identify further. In vivo, spinal cord contusion injury model in rats was induced by NYU-III impactor, and were randomly divided and equally into four groups, namely group A (sham), group B (control), group C (Non-Muse cells transplantation) and group D (Muse cells transplantation). Laminectomy was conducted in group A but no spinal cord contusion injury. Laminectomy and cord injury were performed in group B, C and D, 10 g trip rod was freely falling down from 12.5 mm. Two weeks later, group B, C and D were received PBS injection, Non-Muse cells transplantation and Muse cells transplantation respectively, four-point injection were performed in each cord with totally 4×10⁵ cells. BBB scores were evaluated on 1 day, 1, 2, 3, 4, 5 and 6 week after injury. Four weeks after cell transplantation, the rats were sacrificed, and immunohistochemistry were carried out to observe survival, migration and differentiation of the injected cells. The expression of CD105, CD90 and CD73 were over 99.5% in the derived WJ-MSCs population, but CD45 and CD14 were lower than 0.5%, positive rate of SSEA3+ was 1.46% under flow cytometer, However, after MACS sorting, the percentage of 92.0% Muse cells expressed SSEA3 and CD105, and immunohistochemistry results of SSEA3 showed typically membrane morphology with special processes. In vivo, BBB scores was 21 in group A at different time points. One-way ANOVA and LSD analysis showed that BBB scores in group C and D were significantly higher than that in group B (P=0.004, 0.002), but there was no significantly difference between group C and D. Further intra-group paired t test showed that BBB score was significantly higher at 4 weeks than that 3 weeks in group C (P=0.005). However, in group D, BBB scores were significantly higher at 4 and 6 week than those at 3 and 5 weeks, P values were 0.005 and 0.016 respectively. Immunohistochemistry results showed that both Muse cells and Non-Muse cells could survive for 4 weeks in rats and they migrated from the four-point injection to injury site. But there showed more Muse cells survival than Non-Muse cells in the cord. Immunomagnetic bead screening is efficient to select large number of purified SSEA3+ Muse cells. Muse cells could survive and target-migrate in injured cord to improve BBB scores continuously. Muse cells are a novel kind of seed cells in the spinal cord injury treatment.

The Influence of Mesenchymal Stem Cell Wharton Jelly toward Prostaglandin E2 Gene Expression on Synoviocyte Cell Osteoarthritis.

BACKGROUND:Pharmacological therapy in the management of OA causes many new health problems due to side effects caused by long-term use of drugs, such as long-term use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) will cause gastric ulcers and impaired kidney function. In OA pathogenesis, PGE2 gene is involved in the inflammation process.AIM:This study aims to identify the influence of Wharton Jelly Mesenchymal Stem Cell (MSC-WJ) on PGE2 expression gene in synoviocyte by in vitro.MATERIAL AND METHODS:The method used in this study is the co-culture method of primary cells and stem cells in the appropriate media. This research is pure experimental research. The sample used came from synovial tissue of osteoarthritis patients who underwent Total Knee Replacement (TKR) surgery. This study was divided into 6 groups treated with 4 replications. The expression analysis of the Prostaglandin E2 gene was done using qPCR (Real-Time Polymerase Chain Reaction). The expression analysis of the Prostaglandin E2 gene was carried out before and after the co-culture with Wharton’s Jelly and continued with the analysis of statistical data processing using the SPSS.15 program. PGE2 gene expression data were processed using the Kruskal-Wallis test and continued with the Mann-Whitney test with a 95% confidence level.RESULTS:The results showed that Mesenchymal Stem Cells Wharton Jelly could reduce the expression of Prostaglandin E2 gene after co-culture for 24 hours and 48 hours in synoviocyte cells osteoarthritis significantly compared with the control group. The administration of Mesenchymal Stem Cells for 24 hours reduced the expression level of PGE2 gene by 0.61 times compared to the control group (p < 0.05) and the administration of Mesenchymal Stem Cells for 48 hours decreased the expression level of PGE2 gene by 0, 47 times compared to the control group (p < 0.05).CONCLUSION:This study concluded that MSC-WJ in OA synoviocyte significantly reduced the expression of the PGE2 gene (p < 0.05).

Molecular and Functional Verification of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) Pluripotency.

The properties of mesenchymal stem cells (MSCs), especially their self-renewal and ability to differentiate into different cell lines, are widely discussed. Considering the fact that MSCs isolated from perinatal tissues reveal higher differentiation capacity than most adult MSCs, we examined mesenchymal stem cells isolated from Wharton’s jelly of umbilical cord (WJ-MSCs) in terms of pluripotency markers expression. Our studies showed that WJ-MSCs express some pluripotency markers—such as NANOG, OCT-4, and SSEA-4—but in comparison to iPS cells expression level is significantly lower. The level of expression can be raised under hypoxic conditions. Despite their high proliferation potential and ability to differentiate into different cells type, WJ-MSCs do not form tumors in vivo, the major caveat of iPS cells. Owing to their biological properties, high plasticity, proliferation capacity, and ease of isolation and culture, WJ-MSCs are turning out to be a promising tool of modern regenerative medicine.

Differentiation potential of different regions-derived same donor human Wharton's jelly mesenchymal stem cells into functional smooth muscle-like cells.

The present study evaluates the transdifferentiation potential of different region-derived same donor Wharton's jelly MSCs (WJMSCs) into functional smooth muscle-like cells (SMLCs). All regions showed baseline expression for early smooth muscle cell (SMC) markers (αSMA and SM22-α) whereas mid marker CALPONIN gradually reduced during in vitro culture expansion and late marker myosin heavy chain type-11 (MHY-11) was completely absent. Furthermore, WJMSCs were induced to SMLCs using DMEM containing 10% FBS supplemented with different concentrations/combinations of TGF-β1 and PDGF-BB under normoxia (20% O2) condition. Three treatment groups namely group A: 2.5ng/ml TGF-β1, group B: 5ng/ml PDGF-BB and group C: 2.5ng/ml TGF-β1 + 5ng/ml PDGF-BB were used for the induction of WJMSCs into SMLCs. Cells were evaluated for SMC-specific marker expression at different time intervals. Finally, selection of the SMC-specific highly potent region along with the most suitable treatment group was done on the basis of highest outcome in terms of SMC-specific marker expression and functional competence of transdifferentiated cells. Among all regions, baby region-derived WJMSCs (B-WJMSCs) exhibited highest SMC marker expression and functional ability. To mimic the in vivo physiological conditions, hypoxic conditions (3% O2) were used to evaluate the effect of low oxygen on the SMLC differentiation potential of selected WJMSCs using previously used same parameters. Annexin-V assay was performed to check the effect of cytokines and different oxygen concentrations, which revealed no significant differences. It was concluded that different induction conditions have different but positive effects on the functional SMLC differentiation ability of WJMSCs.

Downregulation of the Netrin-1 Receptor UNC5b Underlies Increased Placental Angiogenesis in Human Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a common metabolic disorder, defined by high blood glucose levels during pregnancy, which affects foetal and post-natal development. However, the cellular and molecular mechanisms of this detrimental condition are still poorly understood. A dysregulation in circulating angiogenic trophic factors, due to a dysfunction of the feto-placental unit, has been proposed to underlie GDM. But even the detailed study of canonical pro-angiogenic factors like vascular endothelial growth factor (VEGF) or basic Fibroblast Growth Factor (bFGF) has not been able to fully explain this detrimental condition during pregnancy. Netrins are non-canonical angiogenic ligands produced by the stroma have shown to be important in placental angiogenesis. In order to address the potential role of Netrin signalling in GDM, we tested the effect of Netrin-1, the most investigated member of the family, produced by Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSC), on Human Umbilical Vein Endothelial Cells (HUVEC) angiogenesis. WJ-MSC and HUVEC primary cell cultures from either healthy or GDM pregnancies were exposed to physiological (5 mM) or high (25 mM) d-glucose. Our results reveal that Netrin-1 is secreted by WJ-MSC from healthy and GDM and both expression and secretion of the ligand do not change with distinct experimental glucose conditions. Noteworthy, the expression of its anti-angiogenic receptor UNC5b is reduced in GDM HUVEC compared with its expression in healthy HUVEC, accounting for an increased Netrin-1 signalling in these cells. Consistently, in healthy HUVEC, UNC5b overexpression induces cell retraction of the sprouting phenotype.

The Influence of Wharton Jelly Mesenchymal Stem Cell toward Matrix Metalloproteinase-13 and RELA Synoviocyte Gene Expression on Osteoarthritis.

BACKGROUND:Therapy for osteoarthritis (OA) with satisfactory results has not been found to date. In OA pathogenesis, RELA gene involved in cartilage degradation and MMP-13 in degrade cartilage, as a member family of NF-κβ genes, RELA serves to modulate inflammatory responses and activates pro-inflammatory cytokines.AIM:This study aims to identify the influence of Wharton Jelly Mesenchymal Stem Cell (MSC-WJ) on MMP-13 and RELA expression gene in synoviocyte by in vitro.MATERIAL AND METHODS:This research is pure experimental research. The sample used derived from synovial tissue of OA patients who underwent Total Knee Replacement (TKR) surgery. This study was divided into six groups treated with 4 replications. Group I and II (control groups) were synoviocyte of OA incubated for 24 and 48 hours, respectively. Group III and IV were MSC-WJ incubated for 24 and 48 hours, respectively. Group V and VI were Synoviocyte-MSC-WJ co-culture group incubated for 24 and 48 hours, respectively. Identification of MMP-13 and RELA gene expression in each group was performed by using qPCR.RESULT:The results showed that MSC-WJ reduced MMP-13 gene expression after co-culture for 24 and 48 hours in OA synoviocyte. The highest gene expression of MMP-13 was in Group I and II (1.00 ng/μl), followed by Group III (0.41 ng/μl), Group IV (0.24 ng/μl), Group V (0.13 ng/μl), and Group VI (0.04 ng/μl). MSC-WJ administration also decreased RELA gene expression. The highest gene expression of RELA gene was in Group I and II (1.00 ng/μl), Group V (0.67 ng/μl), Group III (0.58 ng/μl), Group IV (0.16 ng/μl), and Group VI (0.16 ng/μl).CONCLUSION:This study concluded that MSC-WJ in OA synoviocyte significantly reduced the expression of MMP-13 and RELA gene (p <0.05).

Cycloxygenase-2 inhibition potentiates trans-differentiation of Wharton's jelly–mesenchymal stromal cells into endothelial cells: Transplantation enhances neovascularization-mediated wound repair

BackgroundNeo-vascularization, an indispensible phenomenon for tissue regeneration, facilitates repair and remodeling of wound tissues. This process is impaired in chronic wounds due to reduced number and recruitment of endothelial cells (ECs), thereby necessitating development of newer strategies to enhance the EC repertoire as a therapeutic approach. MethodsWe explored the ‘plasticity’ of Wharton's jelly derived–mesenchymal stromal cells (WJ-MSCs) using an anti-inflammatory drug-mediated enhanced trans-differentiation into ECs, based on our observation of temporal decrease in COX-2 expression during trans-differentiation of MSCs into ECs at day 7 and 14 along with mature ECs. ResultsAt a physiological level, an increased DiI-labeled acetylated-low density lipoprotein (DiI-Ac-LDL) uptake, proliferation, migration and chick chorio allantoic membrane (CAM)-vasculogenesis occurred while at a molecular level significant up-regulation in messenger RNA (mRNA) and protein expression of endothelial-specific markers, Vegfr2, Pecam, eNOS, VE-Cadh and Tie-2, along with an activated p-VEGFR2 and its downstream mediators were observed in celecoxib-preconditioned ECs as compared with WJ-MSCs. Green fluorescent protein (GFP)-expressing stable WJ-MSCs and trans-differentiated EC-D14 in the absence/presence of celecoxib were generated using antibiotic selection for intradermal transplantation at the wound site on a murine ‘excisional splinting wound’ model. Engraftment of transplanted human cells in immunosuppressant-treated mice was confirmed by a significant increase in the expression levels of human gene-specific endothelial markers at the regenerated wound sites. Morphometrically, increased vascularity and percent wound closure were observed in regenerated wounds of mice transplanted with celecoxib-preconditioned-EC-D14. ConclusionCox-2 inhibition led to an enhanced trans-differentiation of WJ-MSCs into ECs that, when transplanted, accelerated the skin regeneration by engraftment and neo-vascularization at the wound bed, suggesting a plausible new therapeutic role of celecoxib.

Intrathecal transplantation of Wharton’s jelly mesenchymal stem cells suppresses the NLRP1 inflammasome in the rat model of spinal cord injury

After spinal cord injury (SCI) local inflammation is induced following secretion of interleukin-1beta (IL-1β) and IL-18. It has been described that the secretion of IL-1β and IL-18 is mediated by a cytoplasmic multiprotein complex, termed inflammasome. Mesenchymal stem cells (MSCs) have been extensively used for treating inflammatory diseases in which they showed immunomodulation characteristics. We utilized the anti-inflammatory potential of Wharton’s jelly mesenchymal stem cells (WJ-MSCs) to target inflammasome complex in rat SCI model. Real time-polymerase chain reaction, western blotting, and ELISA assay were done one week after SCI to measure the expression of the inflammasome components including NLRP1, ASC, and active caspase-1 as well as IL-1β, IL-18, and tumor necrosis factor-α (TNF-α). The histologic alteration and hind-limb locomotion were evaluated three weeks after injury by nissl staining and Basso, Beattie, Bresnahan (BBB), respectively. Our results showed that WJ-MSCs transplantation significantly decreased the SCI-induced expression of the evaluated factors in both mRNA and protein levels. In addition, WJ-MSCs significantly increased the number of normal-appearance neurons in the ventral horn of spinal cord. Noteworthy, these effects resulted in a significant improvement of motor function recovery. We conclude that inflammasome inhibition may be one of the mechanisms for the anti-inflammatory effect of MSCs in the SCI.

Improved Bioactivity of 3D Printed Porous Titanium Alloy Scaffold with Chitosan/Magnesium-Calcium Silicate Composite for Orthopaedic Applications.

Recently, cases of bone defects have been increasing incrementally. Thus, repair or replacement of bone defects is gradually becoming a huge problem for orthopaedic surgeons. Three-dimensional (3D) scaffolds have since emerged as a potential candidate for bone replacement, of which titanium (Ti) alloys are one of the most promising candidates among the metal alloys due to their low cytotoxicity and mechanical properties. However, bioactivity remains a problem for metal alloys, which can be enhanced using simple immersion techniques to coat bioactive compounds onto the surface of Ti–6Al–4V scaffolds. In our study, we fabricated magnesium-calcium silicate (Mg–CS) and chitosan (CH) compounds onto Ti–6Al–4V scaffolds. Characterization of these surface-modified scaffolds involved an assessment of physicochemical properties as well as mechanical testing. Adhesion, proliferation, and growth of human Wharton’s Jelly mesenchymal stem cells (WJMSCs) were assessed in vitro. In addition, the cell attachment morphology was examined using scanning electron microscopy to assess adhesion qualities. Osteogenic and mineralization assays were conducted to assess osteogenic expression. In conclusion, the Mg–CS/CH coated Ti–6Al–4V scaffolds were able to exhibit and retain pore sizes and their original morphologies and architectures, which significantly affected subsequent hard tissue regeneration. In addition, the surface was shown to be hydrophilic after modification and showed mechanical strength comparable to natural bone. Not only were our modified scaffolds able to match the mechanical properties of natural bone, it was also found that such modifications enhanced cellular behavior such as adhesion, proliferation, and differentiation, which led to enhanced osteogenesis and mineralization downstream. In vivo results indicated that Mg–CS/CH coated Ti–6Al–4V enhances the bone regeneration and ingrowth at the critical size bone defects of rabbits. These results indicated that the proposed Mg–CS/CH coated Ti–6Al–4V scaffolds exhibited a favorable, inducive micro-environment that could serve as a promising modification for future bone tissue engineering scaffolds.

CRISPR/Cas9-Mediated Disruption of PD-L1 Reduces the T Cell Suppressive Effect of Wharton's Jelly Mesenchymal Stromal Cells and Their Extracellular Vesicles

BackgroundMesenchymal stem/stromal cells (MSCs) are a promising treatment for acute graft-versus-host disease and other inflammatory diseases. MSCs express programmed death-ligand 1 (PD-L1) that has been reported to contribute to MSC-mediated immune suppression through T-cell receptor (TCR)-mediated inhibitory signaling. MSCs actively secrete extracellular vesicles (EVs) that have immune modulatory properties resembling their cell of origin. Although the release of EVs by MSCs has been extensively reported, the presence and functional impact of immune inhibitory checkpoint molecules in MSC-derived EVs is still being determined. We sought to determine if PD-L1 and other immune inhibitory checkpoint molecules are enriched in MSC-derived EVs. In this study we report that PD-L1 is enriched in MSC-EVs, and that PD-L1(+) EVs and PD-L1(-) EVs differ in their T cell immune suppressive capacity.MethodsUmbilical cords and healthy donor blood samples were collected on separate IRB-approved protocols after written informed consent (HSC#1546/HSC#5929). Umbilical cord Wharton's jelly-MSCs (WJMSC) were isolated from umbilical cords under GMP conditions and characterized by morphology, multiparameter flow cytometry (MPFC), and differentiation capacity. Three clinical-grade WJMSC cell lines were cultured at low passage (2-5) in EV-depleted media. EVs were isolated from the media by differential ultracentrifugation and the 100K pellets enriched with exosomes were characterized by Western blot (WB), nanoparticle tracking analysis (NTA), and electron microscopy (EM). To further identify EV-enriched immune proteins, the WJMSC and EV proteomes were characterized by Tandem Mass Tag (TMT) labeling and LC MS/MS, using isobaric labels to compare protein quantities between samples. Gene disruption of PD-L1 was performed through site-specific CRISPR/Cas9 introducing a single cysteine nucleotide deletion in exon 3; the genetic mutation was confirmed by Sanger sequencing. The absence of PD-L1 transcript was confirmed by RT-PCR, and the absence of PD-L1 protein was confirmed by WB, immunofluorescence, MPFC, and IFNγ-response. PBMC were isolated from healthy donor blood samples; T cells were activated by CD3/CD28 beads and induced CD154 was measured on CD4(+) T cells by MPFC. Alternatively, CMV-experienced T cells were activated with CMV-pp65 peptide and induced IFNγ was measured on CD8(+) T cells by MPFC. Activated T cells were treated with PD-L1(+) and PD-L1(-) WJMSCs or their derived EVs. Phospho-Zap-70 (Tyr319)/Syk (Tyr352) MPFC was used to analyze T-cell receptor (TCR)-mediated signaling.ResultsWJMSCs were CD73(+), CD90(+), CD105(+), and CD34(-), CD45(-), CD11b(-), CD19(-) and HLA-DR(-); and inhibited proliferation in a mixed lymphocyte reaction. WJMSCs-derived EVs were cup-shaped 100-200nm nanoparticles by EM and were CD9(+), CD81(+), CD73(+), CD90(+), and CD105(+) by WB. Quantitative proteomic profiling revealed a total of 1,634 proteins across WJMSC-derived EV preparations. Proteins that were highly enriched in MSC-derived EVs and that were classified under “negative regulation of lymphocyte activation” (Gene Ontology Biological Processes; GOBP) included PDL1, CD276, DLG1, TNR6, ERBB2, TGFβ1, LYN, and HMGB3. Both WJMSCs and EVs suppressed CD4(+) T cell activation by CD3/CD28 beads and CD8(+) T cell activation by CMV peptide. CRISPR/Cas9 edited WJMSC exhibited normal morphology and differentiation characteristics. PD-L1 gene disruption efficiently reduced PD-L1 protein expression on WJMSC and their derived EVs, and reduced the ability of both to suppress T cell activation. Similarly, blocking PD-L1 with a specific internalization antibody decreased WJMSC and EV-mediated T cell activation.ConclusionProteomic analyses reveal that PD-L1 is enriched in WJMSC-derived EVs. CRISPR/Cas9-mediated disruption of PD-L1 in WJMSCs alone did not affect the release of EVs in vitro. Gene-edited WJMSCs stably generate PD-L1(-) EVs. The PD-L1(-) EVs generated from PD-L1(-) WJMSC did not suppress T cell activation in co-culture. These findings indicate that PD-L1 is enriched in WJMSC EVs and is biologically active. WJMSC-EVs can deliver membrane-bound PD-L1 to T cells, resulting in attenuated TCR signaling. DisclosuresMcGuirk:Fresenius Biotech: Research Funding; Bellicum Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Pluristem Ltd: Research Funding; Gamida Cell: Research Funding; Kite Pharma: Honoraria, Other: travel accommodations, expenses, speaker ; Novartis Pharmaceuticals Corporation: Honoraria, Other: speaker, Research Funding.

Four types of human platelet lysate, including one virally inactivated by solvent-detergent, can be used to propagate Wharton jelly mesenchymal stromal cells

There is accumulating experimental evidence that human platelet lysate (HPL) made from platelet concentrates can replace fetal bovine serum (FBS) as a xeno-free clinical-grade supplement of growth media to expand mesenchymal stromal cells (MSCs). However, uncertainties exist in regard to impacts that various manufacturing methods of HPL can exert on the expansion and differentiation capacity of MSCs. In particular, there is a need to evaluate the possibility of implementing virus-inactivation treatment during HPL production to ensure optimal safety of industrial HPL pools. Expired human platelet concentrates from four different donors were pooled and subjected to freeze-thaw cycles (-80/+37 °C), followed or not by serum-conversion by calcium chloride, heat-treatment at 56 °C for 30 min, or solvent-detergent (S/D) virus inactivation. The concentrations of total proteins, growth factors and fibrinogen, and the chemical compositions of the HPLs were characterized. The impact of HPL supplementation on the cell morphology, doubling time, immunophenotype and trilineage differentiation capacity of Wharton jelly MSCs (WJMSCs) were compared over five passages, using FBS as a control and normalizing the protein content. Data showed that WJMSCs expanded equally well, exhibited a typical fibroblast morphology, had short doubling times, maintained their immunophenotypes, and differentiated into chondrocyte, osteocyte, and adipocyte lineages in all HPL-supplemented media, all of which were more effective than FBS. In conclusion, we found minimal detectable impact of the HPL manufacturing process, including S/D virus inactivation, on the suitability of expanding WJMSCs in vitro.

P53-NF-κB Crosstalk in Febrile Temperature-Treated Human Umbilical Cord-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are successful for their therapeutic application in immune and inflammatory contexts due to their anti-inflammatory, trophic, and immunomodulatory roles. However, though MSCs have the potential to provide regenerative treatment toward a wide range of devastating diseases, massive cell death of transplanted MSCs remains an obstacle to overcome. The relation between MSCs and inflammation is multifactorial and challenging to comprehend. Fever is a critical component of the inflamed microenvironment. Also, the choice of MSC source could be critical in determining the fate of transplanted cells under stress conditions. Here we investigated the thermosensitivity of Wharton's jelly MSCs (WJ-MSCs) to elevated temperature in the physiological fever range. We explored the effect of febrile range temperature on morphology, viability, proliferation kinetics, and cell cycle status of WJ-MSCs. WJ-MSCs adopted a flattened morphology at 40°C, and our data from proliferation kinetics study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays showed that WJ-MSCs had reduced proliferation and viability at 40°C compared with control cultures. There was also a G0/G1 cell cycle arrest, which was further confirmed by messenger RNA (mRNA) levels of genes specific for different stages of cell cycle. On evaluating p53 status, we observed an increase in p53 protein expression and its nuclear localization in WJ-MSCs exposed to 40°C. Its downstream effector p21 too was upregulated. Moreover, this temperature-induced p53 induction was inhibited on exposure to 40°C in the presence of NF-κB pathway inhibitor, pyrrolidinedithiocarbamate (PDTC) or endonuclease-prepared small interfering RNA (esiRNA) targeting p65. Febrile temperature exposure did not affect the senescence status of WJ-MSCs. The MSC-specific surface antigen profile at 40°C was similar to control WJ-MSCs. Our findings suggest that under febrile temperature stress conditions, WJ-MSCs exhibit G0/G1 cell cycle arrest and reduction in viable cell count, while retaining their basic characteristics, with an underlying interplay of p53 and NF-κB pathway.

Pancreatic endocrine-like cells differentiated from human umbilical cords Wharton's jelly mesenchymal stem cells using small molecules.

Following success of pancreatic islet transplantation in the treatment of Type I diabetes mellitus, there is a growing interest in using cell-based treatment approaches. However, severe shortage of donor islets-pancreas impeded the growth, and made researchers to search for an alternative treatment approaches. In this context, recently, stem cell-based therapy has gained more attention. The current study demonstrated that epigenetic modification improves the in vitro differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) into pancreatic endocrine-like cells. Here we used two histone deacetylase (HDAC) inhibitors namely trichostatin A (TSA) and TMP269. TSA inhibits both class I and II HDACs whereas TMP269 inhibits only class IIa HDACs. WJMSCs were differentiated using a multistep protocol in a serum-free condition with or without TSA pretreatment. A marginal improvement in differentiation was observed after TSA pretreatment though it was not significant. However, exposing endocrine precursor-like cells derived from WJMSCs to TMP269 alone has significantly improved the differentiation toward insulin-producing cells. Further, increase in the expression of paired box 4 (PAX4), insulin, somatostatin, glucose transporter 2 (GLUT2), MAF bZIP transcription factor A (MAFA), pancreatic duodenal homeobox 1 (PDX-1), and NKX6.1 was observed both at messenger RNA and protein levels. Nevertheless, TMP269-treated cells secreted higher insulin upon glucose challenge, and demonstrated increased dithizone staining. These findings suggest that TMP269 may improve the in vitro differentiation of WJMSCs into insulin-producing cells.