Jelly Mesenchymal Stromal Cells Research Articles

Implantation of a double allogeneic human engineered tissue graft on damaged heart: insights from the PERISCOPE phase I clinical trial

In preclinical studies, the use of double allogeneic grafts has shown promising results in promoting tissue revascularization, reducing infarct size, preventing adverse remodelling and fibrosis, and ultimately enhancing cardiac function. Building upon these findings, the safety of PeriCord, an engineered tissue graft consisting of a decellularised pericardial matrix and umbilical cord Wharton's jelly mesenchymal stromal cells, was evaluated in the PERISCOPE Phase I clinical trial (NCT03798353), marking its first application in human subjects. This was a double-blind, single-centre trial that enrolled patients with non-acute myocardial infarction eligible for surgical revascularization. Seven patients were implanted with PeriCord while five served as controls. Patients who received PeriCord showed no adverse effects during post-operative phase and one-year follow-up. No significant changes in secondary outcomes, such as quality of life or cardiac function, were found in patients who received PeriCord. However, PeriCord did modulate the kinetics of circulating monocytes involved in post-infarction myocardial repair towards non-classical inflammation-resolving macrophages, as well as levels of monocyte chemoattractants and the prognostic marker Meteorin-like in plasma following treatment. In summary, the PeriCord graft has exhibited a safe profile and notable immunomodulatory properties. Nevertheless, further research is required to fully unlock its potential as a platform for managing inflammatory-related pathologies. This work was supported in part by grants from MICINN (SAF2017-84324-C2-1-R); Instituto de Salud Carlos III (ICI19/00039 and Red RICORS-TERAV RD21/0017/0022, and CIBER Cardiovascular CB16/11/00403) as a part of the Plan Nacional de I+D+I, and co-funded by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) and AGAUR (2021-SGR-01437).

Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system

Background & aimsCell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. MethodsMSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. ResultsTaking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). ConclusionsTaken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.

Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model.

Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model. After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis. Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment. WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

Cell-free therapy based on conditioned medium derived from mesenchymal stromal cells (MSCs) has gained attention in the field of protective and regenerative medicine. However, the exact composition and properties of MSC-derived conditioned media can vary greatly depending on multiple parameters, which hamper standardization. In this study, we have optimized a procedure for preparation of conditioned medium starting from efficient isolation, propagation and characterization of MSCs from human umbilical cord, using a culture medium supplemented with human platelet lysate as an alternative source to fetal bovine serum. Our procedure successfully maximizes the yield of viable MSCs that maintain canonical key features. Importantly, under these conditions, the compositional profile and biological effects elicited by the conditioned medium preparations derived from these MSC populations do not depend on donor individuality. Moreover, approximately 120L of conditioned medium could be obtained from a single umbilical cord, which provides a suitable framework to produce industrial amounts of toxic-free conditioned medium with predictable composition.

Efficacy of Wharton Jelly Mesenchymal Stromal Cells infusions in moderate to severe SARS-Cov-2 related acute respiratory distress syndrome: a phase 2a double-blind randomized controlled trial.

The COVID-19 pandemic caused a wave of acute respiratory distress syndrome (ARDS) with a high in-hospital mortality, especially in patients requiring invasive mechanical ventilation. Wharton Jelly-derived Mesenchymal Stromal Cells (WJ-MSCs) may counteract the pulmonary damage induced by the SARS-CoV-2 infection through pro-angiogenic effects, lung epithelial cell protection, and immunomodulation. In this randomized, double-blind, placebo-controlled phase 2a trial, adult patients receiving invasive mechanical ventilation for SARS-CoV-2 induced moderate or severe ARDS were assigned to receive 1 intravenous infusion of 1 × 106 WJ-MSCs/kg or placebo within 48 h of invasive ventilation followed by 2 infusions of 0.5 × 106 WJ-MSCs/kg or placebo over 5 days. The primary endpoint was the percentage of patients with a PaO2/FiO2 > 200 on day 10. Thirty patients were included from November 2020 to May 2021, 15 in the WJ-MSC group and 15 in the placebo group. We did not find any significant difference in the PaO2/FiO2 ratio at day 10, with 18 and 15% of WJ-MSCs and placebo-treated patients reaching a ratio >200, respectively. Survival did not differ in the 2 groups with a 20% mortality rate at day 90. While we observed a higher number of ventilation-free days at 28 days in the WJ-MSC arm, this difference was not statistically significant (median of 11 (0-22) vs. 0 (0-18), p = 0.2). The infusions were well tolerated, with a low incidence of anti-HLA alloimmunization after 90 days. While treatment with WJ-MSCs appeared safe and feasible in patients with SARS-CoV2 moderate or severe ARDS in this phase 2a trial, the treatment was not associated with an increased percentage of patients with P/F > 200 at 10d, nor did 90 day mortality improve in the treated group. https://beta.clinicaltrials.gov/study/NCT04625738, identifier NCT04625738.

A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton's jelly mesenchymal stromal cells for cell therapy and tissue engineering.

Purpose: Obtaining sufficient numbers of cells in a short time is a major goal of cell culturing in cell therapy and tissue engineering. However, current bidimensional (2D) culture methods are associated to several limitations, including low efficiency and the loss of key cell differentiation markers on cultured cells. Methods: In the present work, we have designed a novel biofabrication method based on a three-dimensional (3D) culture system (FIBRIAGAR-3D). Human Wharton's jelly mesenchymal stromal cells (HWJSC) were cultured in 3D using 100%, 75%, 50%, and 25% concentrations of fibrin-agarose biomaterials (FA100, FA75, FA50 and FA25 group) and compared with control cells cultured using classical 2D systems (CTR-2D). Results: Our results showed a significant increase in the number of cells generated after 7days of culture, with cells displaying numerous expansions towards the biomaterial, and a significant overexpression of the cell proliferation marker KI67 was found for the FA75 and FA100 groups. TUNEL and qRT-PCR analyses demonstrated that the use of FIBRIAGAR-3D was not associated with an induction of apoptosis by cultured cells. Instead, the 3D system retained the expression of typical phenotypic markers of HWJSC, including CD73, CD90, CD105, NANOG and OCT4, and biosynthesis markers such as types-I and IV collagens, with significant increase of some of these markers, especially in the FA100 group. Finally, our analysis of 8 cell signaling molecules revealed a significant decrease of GM-CSF, IFN-g, IL2, IL4, IL6, IL8, and TNFα, suggesting that the 3D culture system did not induce the expression of pro-inflammatory molecules. Conclusion: These results confirm the usefulness of FIBRIAGAR-3D culture systems to increase cell proliferation without altering cell phenotype of immunogenicity and opens the door to the possibility of using this novel biofabrication method in cell therapy and tissue engineering of the human cornea, oral mucosa, skin, urethra, among other structures.

Antibacterial PVA/Chitosan/alginate/Meropenem-based hydrogel as a potential therapeutic strategy for chronic ulcers infections

One of the most critical challenges when treating chronic wounds is proper infection control. Traditionally, antibiotics are administered in high doses to inhibit bacterial growth, but they usually adversely affect the patient’s health. To overcome this problem, the use of local release systems, such as hydrogels, allows the release of therapeutic concentrations of antimicrobial agents in situ. Here we developed a polyvinyl alcohol/chitosan/alginate hydrogel (PVA/CS/ALG) loaded with the antibiotic Meropenem (MRP). The physicochemical properties of four different formulations and the biological responses of Wharton’s Jelly Mesenchymal Stromal Cells (hWJ-MSCs) cultured in vitro with them allowed the selection of the best formulation to incorporate the antibiotic. Subsequently, the selected delivery system was evaluated for biocompatibility through cytotoxicity and cell viability assays. After, antibacterial activity was studied in Gram-positive and Gram-negative bacteria. The PVA/CS/ALG 2:1:1 formulation was selected for MRP incorporation (HA-MRP) because it showed a lower swelling degree (140.68% ± 10.52%), degradation percentage (52.21% ± 5.84%), and a significantly higher Young’s Modulus compared to the other formulations. The results obtained with the HA-MRP release system showed that it is biocompatible, sustainably releases MRP, and has a potent broad-spectrum antibacterial effect. Overall, these results indicate that the HA-MRP delivery system could be a promising dressing with potential application for effectively treating chronic wound infections.

In vitro and in vivo evaluation of electrospun poly (ε-caprolactone)/collagen scaffolds and Wharton's jelly mesenchymal stromal cells (hWJ-MSCs) constructs as potential alternative for skin tissue engineering

Dermal substitutes bear a high clinical demand because of their ability to promote the healing process of cutaneous wounds by reducing the healing time the appearance and improving the functionality of the repaired tissue. Despite the increasing development of dermal substitutes, most of them are only composed of biological or biosynthetic matrices. This demonstrates the need for new developments focused on using scaffolds with cells (tissue construct) that promote the production of factors for biological signaling, wound coverage, and general support of the tissue repair process. Here, we fabricate by electrospinning two scaffolds: poly(ε-caprolactone) (PCL) as a control and poly(ε-caprolactone)/collagen type I (PCol) in a ratio lower collagen than previously reported, 19:1, respectively. Then, characterize their physicochemical and mechanical properties. As we bear in mind the creation of a biologically functional construct, we characterize and assess in vitro the implications of seeding human Wharton's jelly mesenchymal stromal cells (hWJ-MSCs) on both scaffolds. Finally, to determine the potential functionality of the constructs in vivo, their efficiency was evaluated in a porcine biomodel. Our findings demonstrated that collagen incorporation in the scaffolds produces fibers with similar diameters to those in the human native extracellular matrix, increases wettability, and enhances the presence of nitrogen on the scaffold surface, improving cell adhesion and proliferation. These synthetic scaffolds improved the secretion of factors by hWJ-MSCs involved in skin repair processes such as b-FGF and Angiopoietin I and induced its differentiation towards epithelial lineage, as shown by the increased expression of Involucrin and JUP. In vivo experiments confirmed that lesions treated with the PCol/hWJ-MSCs constructs might reproduce a morphological organization that seems relatively equivalent to normal skin. These results suggest that the PCol/hWJ-MSCs construct is a promising alternative for skin lesions repair in the clinic.

PSEN1 E280A Cholinergic-like Neurons and Cerebral Spheroids Derived from Mesenchymal Stromal Cells and from Induced Pluripotent Stem Cells Are Neuropathologically Equivalent.

Alzheimer's disease (AD) is a chronic neurological condition characterized by the severe loss of cholinergic neurons. Currently, the incomplete understanding of the loss of neurons has prevented curative treatments for familial AD (FAD). Therefore, modeling FAD in vitro is essential for studying cholinergic vulnerability. Moreover, to expedite the discovery of disease-modifying therapies that delay the onset and slow the progression of AD, we depend on trustworthy disease models. Although highly informative, induced pluripotent stem cell (iPSCs)-derived cholinergic neurons (ChNs) are time-consuming, not cost-effective, and labor-intensive. Other sources for AD modeling are urgently needed. Wild-type and presenilin (PSEN)1 p.E280A fibroblast-derived iPSCs, menstrual blood-derived menstrual stromal cells (MenSCs), and umbilical cord-derived Wharton Jelly's mesenchymal stromal cells (WJ-MSCs) were cultured in Cholinergic-N-Run and Fast-N-Spheres V2 medium to obtain WT and PSEN 1 E280A cholinergic-like neurons (ChLNs, 2D) and cerebroid spheroids (CSs, 3D), respectively, and to evaluate whether ChLNs/CSs can reproduce FAD pathology. We found that irrespective of tissue source, ChLNs/CSs successfully recapitulated the AD phenotype. PSEN 1 E280A ChLNs/CSs show accumulation of iAPPβ fragments, produce eAβ42, present TAU phosphorylation, display OS markers (e.g., oxDJ-1, p-JUN), show loss of ΔΨm, exhibit cell death markers (e.g., TP53, PUMA, CASP3), and demonstrate dysfunctional Ca2+ influx response to ACh stimuli. However, PSEN 1 E280A 2D and 3D cells derived from MenSCs and WJ-MSCs can reproduce FAD neuropathology more efficiently and faster (11 days) than ChLNs derived from mutant iPSCs (35 days). Mechanistically, MenSCs and WJ-MSCs are equivalent cell types to iPSCs for reproducing FAD in vitro.

Production cell analysis and compound-based boosting of small extracellular vesicle secretion using a generic and scalable production platform.

Extracellular vesicles (EVs) are a novel format of advanced therapeutical medicinal products (ATMPs). They can act regenerative or immune-modulatory as cell therapy substitutes or as a platform for designer exosomes. The biotechnological production of therapeutic EVs is still very much uncharted territory so standardized host cells, production setups, and isolation methods are not yet implemented. In this work, we present a tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) purification setup that is compatible for industry applications. Moreover, we evaluated a series of potential host cell lines regarding their EV productivity, characteristics, and biological functionality. It was found that telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) secrete high amounts of EVs per cell with regenerative capabilities. On the other hand, Cevec's amniocyte producer cells® (CAP®) and human embryonic kidney (HEK293) suspension cells are suitable platforms for designer EVs with high yields. Finally, we aimed to boost the EV secretion of HEK293 cells via chemical adjuvants and verified four compounds that heighten cellular EV secretion in a presumably cAMP-dependent manner. A combination of fenoterol, iodoacetamide, and dinitrophenol increased the EV yield in HEK293 cells threefold and cellular secretion rate fivefold.

Immunomodulatory effect of first-in-human PeriCord cardiac bioimplant: preliminary data of the PERISCOPE clinical trial

Abstract Background The PERISCOPE is a first-in-human, phase I, double blind, one centre clinical trial to test the safety of the PeriCord, an advanced therapy medicinal product (PEI18–140), for the treatment of patients with infarcted myocardial tissue. The PeriCord is a GMP-complying allogeneic engineered tissue graft consisting on a decellularised pericardial matrix colonised with umbilical cord Wharton's jelly mesenchymal stromal cells. The PeriCord implantation has shown to promote damaged tissue revascularisation, reduce infarct size, adverse remodelling and fibrosis, and ultimately improve cardiac function preclinically. Purpose To assess the safety and monitor the monocyte and cytokine response after PeriCord implantation in patients with infarcted myocardial tissue. Methods The PERISCOPE clinical trial has been approved by AEMPS and the Local Ethics Committee of our institution (Eudra-CT 2018–001964–49). Twelve patients with transmural myocardial infarction (>50% by MRI) who were candidates for surgical revascularisation were included: two roll-in and 10 randomised into a Control group (n=5; CABG) or Treatment group (n=5; n=7 in total; CABG and PeriCord implantation over the transmural myocardial scar). In conjunction with clinical follow-up, whole blood was sampled at baseline and 3-, 6-, and 9-days post-surgery for monocyte populations analysis by flow cytometry and plasma cytokine and monocyte chemoattractant chemokine levels by multiplex ELISA. Results The target patient enrolment has been met and no adverse effects related to PeriCord implantation were observed so far. Recruitment of CCR2+ activated monocytes in peripheral blood peaked on day 3 post-surgery in both groups (increase in the %CCR2+ monocytes of 6.1±5.7% in Control, p=0.38; and 6.5±1.8% in PeriCord Treated patients, p=0.0004), concomitant with a surge in the monocyte chemoattractant CCL2 plasma levels. At the same time, there was a decrease in the number of “non-classical” monocytes regardless of treatment (decrease in CD14-/CD16+ monocytes of 16.7±11.1% in Control, p<0.01; and 24.0±15.0% in PeriCord Treated patients, p<0.001) and a marked reduction in CX3CR1 expression in monocytes of PeriCord treated patients (decrease of 53.2±20.6% in mean fluorescence intensity at day 3, p<0.01), while plasma CX3CL1 levels were undetectable. Both populations recovered on day 9 only in treated patients (p<0.05). Conclusions Implantation of the PeriCord in myocardial infarction patients did not promote adverse reactions and modulated circulating monocyte subsets that have been associated with post-infarction myocardial tissue healing (CCR2 and CX3CR1). These results will be analysed for correlation with therapeutic benefits by the end of patient follow-up. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto Carlos III, Generalitat de Catalunya

Characteristics of Pooled Wharton's Jelly Mesenchymal Stromal Cells (WJ-MSCs) and their Potential Role in Rheumatoid Arthritis Treatment.

Mesenchymal stromal cells (MSC) from Wharton's jelly of umbilical cord is primitive and serve as an inexhaustible source of stem cells with greater potential in clinics. The existence of heterogeneity among the donor MSCs makes it difficult to predict the properties and clinical outcome of WJ-MSCs. We developed a strategy to minimize the donor to donor heterogeneity and produce consistency in biological properties by pooling three individual donors WJ-MSCs. Further, evaluated the effectiveness of the pooled MSCs in regulating the disease severity of Rheumatoid arthritis (RA) in animal models. WJ-MSCs were isolated from umbilical cord obtained from different donors, characterised and pooled based on the gender of baby. The biological properties of the pooled WJ-MSCs were compared to the individual WJ-MSCs. Further, the pooled WJ-MSCs were analysed for their safety profile in both in vitro and in vivo settings. The efficiency of pooled WJ-MSCs in regulating RA pathogenesis was also analysed in mice models of Collagen induced arthritis (CIA). We identified differences in proliferation capacity, pro inflammatory gene expression levels among individual WJ-MSCs isolated from different donors and the variation is also attributed to gender difference. WJ-MSCs pooled and cultured from different donor's exhibit all the MSC characteristics and exhibited superior immunosuppressive capabilities. In the in vivo toxicity study, pooled MSCs are found to be safe, and further in the RA preclinical studies, they were found to decrease the disease severity in these animals. Pooled WJ-MSCs reduces heterogeneity of individual donors and have superior immunosuppressive property. It is also effective in reducing the disease severity in the experimental animal models of RA.

Improved Repopulation Efficacy of Decellularized Small Diameter Vascular Grafts Utilizing the Cord Blood Platelet Lysate.

Background: The development of functional bioengineered small-diameter vascular grafts (SDVGs), represents a major challenge of tissue engineering. This study aimed to evaluate the repopulation efficacy of biological vessels, utilizing the cord blood platelet lysate (CBPL). Methods: Human umbilical arteries (hUAs, n = 10) were submitted to decellularization. Then, an evaluation of decellularized hUAs, involving histological, biochemical and biomechanical analysis, was performed. Wharton’s Jelly (WJ) Mesenchymal Stromal Cells (MSCs) were isolated and characterized for their properties. Then, WJ-MSCs (1.5 × 106 cells) were seeded on decellularized hUAs (n = 5) and cultivated with (Group A) or without the presence of the CBPL, (Group B) for 30 days. Histological analysis involving immunohistochemistry (against Ki67, for determination of cell proliferation) and indirect immunofluorescence (against activated MAP kinase, additional marker for cell growth and proliferation) was performed. Results: The decellularized hUAs retained their initial vessel’s properties, in terms of key-specific proteins, the biochemical and biomechanical characteristics were preserved. The evaluation of the repopulation process indicated a more uniform distribution of WJ-MSCs in group A compared to group B. The repopulated vascular grafts of group B were characterized by greater Ki67 and MAP kinase expression compared to group A. Conclusion: The results of this study indicated that the CBPL may improve the repopulation efficacy, thus bringing the biological SDVGs one step closer to clinical application.

Wharton's Jelly Mesenchymal Stromal Cells and Derived Extracellular Vesicles as Post-Myocardial Infarction Therapeutic Toolkit: An Experienced View.

Outstanding progress has been achieved in developing therapeutic options for reasonably alleviating symptoms and prolonging the lifespan of patients suffering from myocardial infarction (MI). Current treatments, however, only partially address the functional recovery of post-infarcted myocardium, which is in fact the major goal for effective primary care. In this context, we largely investigated novel cell and TE tissue engineering therapeutic approaches for cardiac repair, particularly using multipotent mesenchymal stromal cells (MSC) and natural extracellular matrices, from pre-clinical studies to clinical application. A further step in this field is offered by MSC-derived extracellular vesicles (EV), which are naturally released nanosized lipid bilayer-delimited particles with a key role in cell-to-cell communication. Herein, in this review, we further describe and discuss the rationale, outcomes and challenges of our evidence-based therapy approaches using Wharton’s jelly MSC and derived EV in post-MI management.

Xeno-Free Human Wharton's Jelly Mesenchymal Stromal Cells Maintain Their Characteristic Properties after Long-Term Cryopreservation.

Objective The past decade has witnessed a rapid growth in harnessing the potential of adult stem cells for regenerative medicine. An investigational new drug (IND) or a regenerative medicine advanced therapy (RMAT) product must fulfil many requirements, such as stability studies, after cryopreservation. Such studies are important to ascertain the utility of off-the-shelf allogeneic cells for clinical applications. The present work describes a complete characterisation of xeno- free human Wharton’s Jelly mesenchymal stromal cells (hWJ-MSCs) before and up to 28 months post-cryopreservation. Materials and Methods In this experimental study, culture methods that involved plasma derived human serum and recombinant trypsin were used to develop clinical grade cells. Complete cell characterisation involved flow cytometry studies for viability, positive and negative markers, colony forming unit (CFU) potential, population doubling time (PDT), soft agar assay to evaluate in vitro tumourigenicity, karyotype analysis and differentiation studies which were performed before and at 6, 12, 18 and 28 months post-cryopreservation. Results Our data showed consistency in the flow cytometry, CFU assay, PDT, soft agar assay, karyotyping and differentiation studies. ConclusionUsing our protocols for extended xeno-free culture and cryopreservation of hWJ-MSCs, we could establish the shelf life of the cell-based product for up to 28 months.

Microvesicles derived from human umbilical cord mesenchyme promote M2 macrophage polarization and ameliorate renal fibrosis following partial nephrectomy via hepatocyte growth factor.

The intraoperative ischemia in partial nephrectomy (PN) often leads to postoperative renal function impairment and fibrosis, which can be regulated by macrophage polarization. We have previously demonstrated that microvesicles derived from human Wharton's Jelly mesenchymal stromal cells (hWJMSC-MVs) attenuated renal ischemia-induced renal fibrosis and contained a substantial quantity of hepatocyte growth factor (HGF). Herein, we investigated whether MSC-MVs regulate macrophage polarization and ameliorate renal fibrosis following ischemia-PN via transferring HGF. A rat model of ischemia-PN was established by 45min of left renal ischemia followed by removal of 1/3 upper left kidney. MSC-MVs were injected through the tail vein immediately after ischemia. Renal injury biomarkers were measured and histologic analysis was performed to analyze renal injury. A co-culture model of THP-1 macrophages and MSC-MVs was utilized. The expression of M1 markers and M2 markers were determined to evaluate macrophage polarization. MSC-MV administration significantly ameliorated renal inflammation, lesions, and fibrosis in ischemia-PN rats, and promoted M2 macrophage polarization both in rat remnant renal tissues and LPS-treated THP-1 cells. These effects of MSC-MVs were compromised when HGF expression was downregulated in MSC-MVs. Collectively, MSC-MVs promote M2 macrophage polarization and attenuate renal fibrosis following ischemia-PN via transferring HGF.

Muscle functional recovery is driven by extracellular vesicles combined with muscle extracellular matrix in a volumetric muscle loss murine model

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function.

Human Platelet Lysate Supports Efficient Expansion and Stability of Wharton's Jelly Mesenchymal Stromal Cells via Active Uptake and Release of Soluble Regenerative Factors.

Manufacturing of mesenchymal stromal cell (MSC)-based therapies for regenerative medicine requires the use of suitable supply of growth factors that enhance proliferation, cell stability and potency during cell expansion. Human blood derivatives such as human platelet lysate (hPL) have emerged as a feasible alternative for cell growth supplement. Nevertheless, composition and functional characterization of hPL in the context of cell manufacturing is still under investigation, particularly regarding the content and function of pro-survival and pro-regenerative factors. We performed comparative analyses of hPL, human serum (hS) and fetal bovine serum (FBS) stability and potency to support Wharton’s jelly (WJ) MSC production. We demonstrated that hPL displayed low inter-batch variation and unique secretome profile that was not present in hS and FBS. Importantly, hPL-derived factors including PDGF family, EGF, TGF-alpha, angiogenin and RANTES were actively taken up by WJ-MSC to support efficient expansion. Moreover, hPL but not hS or FBS induced secretion of osteoprotegerin, HGF, IL-6 and GRO-alpha by WJ-MSC during the expansion phase. Thus, hPL is a suitable source of factors supporting viability, stability and potency of WJ-MSC and therefore constitutes an essential raw material that in combination with WJ-MSC introduces a great opportunity for the generation of potent regenerative medicine products.

Osteogenic potential of Wharton's jelly mesenchymal stromal cells cultured on a biomimetic scaffold

Background & Aim In regenerative medicine, Wharton's jelly mesenchymal stromal cells (WJ-MSCs) have a growing interest in its properties similar to those of bone marrow cells and the immunomodulatory potential. Tissue engineering combines the design of biomaterials and the potential of cells implanted in the scaffold that interacts with the added growth factors, and its main objective is to return morphology and function by providing a biological mechanically stable niche. This evidence has directed the approach of tissue regeneration towards the use of decellularized native cell matrices. The mature organs and tissues adequately decellularized, can be used as scaffolds, because the immunogenic determinants present in the cells are eliminated, conserving a native MEC, in which its structure, architecture and mechanical properties are preserved, along with the bioavailability of proteins and growth factors necessary for tissue regeneration. We aimed is provide a temporary bone biomimetic scaffold capable of inducing osteogenic differentiation of Wharton's jelly mesenchymal stromal cells. Methods, Results & Conclusion We decellularized, demineralized, and hydrolyzed the collagen protein of the bovine bone. Then we evaluate the structure and decellularization of the scaffolds. We performed scanning electron microscopy and chemical analysis by EDS. Raman Spectroscopy and FTIR techniques determined the chemical composition by identifying functional groups and the fingerprint of type I collagen protein. We aisled WJ-MSCs and characterize surface antigens profiles of WJ-MSCs; we kept the cells in culture for 3,7,14,21 and 28 days to evaluate the biocompatibility of the scaffolds and the cellular response in vitro. On histology sections, the scaffolds showed the conservation of the structure of the ECM and cell removal. We determined that the main component is collagen type I with different degrees of denaturation depending on the concentration of hydrochloric acid (HCl) by Raman and FTIR. We eliminate the mineral phase according to the guidelines established for the FDA for demineralized scaffolds. The scaffolds also showed excellently swelling and mechanical stability. In the in vitro cell culture, the scaffolds have no cytotoxic effect and mineralized matrix deposit after 28 days in culture in the absence of growth factors or inducers of mineralization. Future studies using animal subjects are required to evaluate the degree of mineralization and scaffold response.

Is HLA matching relevant for treating Spinal Cord Injury with intrathecal administration of expanded Wharton's Jelly Mesenchymal Stromal Cells?

Background & Aim Background Spinal cord injury (SCI) is a devastating pathology with no effective treatment. There are several treatment approaches under investigation and Mesenchymal Stromal Cells (MSC) are within one of the preferred cells under investigation. Autologous treatment requires previous patient's surgery resulting in long and expensive procedure but allogeneic cryopreserved Wharton jelly (WJ) MSC is a ready to use product with increasing interest. Methods, Results & Conclusion Methods Phase I/IIa, randomized, two-arm, double-blind, placebo-controlled trial in 10 patients affected with chronic traumatic SCI (T2-T11, ASIA A) (NCT03003364). Primary and secondary objectives were safety and efficacy, respectively. Patients were randomized to WJ-MSC/Placebo (PLC) or PLC/WJ-MSC and received the 1st infusion. At 6m all patients, except 1 (voluntarily withdrawn), received the 2nd infusion. At 12m, patients were asked to give consent to receive an additional infusion of WJ-MSC with a follow-up to month 24. Medication contained 10 × 106 WJ-MSC in a 4±1ml prefilled blinded syringe. Allelic profile was obtained from a sample of WJ-MSC of the donated umbilical cord units (UCu) and from all patients, but the compatibility was not a requirement to select the donor. Revision of results were made to identify whether the HLA compatibility resulted in a better response to treatment. The study was conducted between Nov 2017 and Jul 2019. Results 9 patients received 2 WJ-MSC and 1 PLC. Patient (P)#6 withdraw voluntarily after the 3rd infusion. 2 UCu were used to produce all dosages. Allelic profiles (UCu and patients) are detailed in table 1 and 2. No relevant safety issues were detected during the trial. Anti-HLA Ab and chimerism resulted all negative after the 1st active infusion, but patient #1 developed Abs to both UCu after the 2nd active infusion. No changes were detected in the ASIA classification or for the lower extremity motor subscores. For the rest of ISNCSCI parameters (table 3), P#1 and P#4 had the best response, while P#10 and P#2 had the worst one. Based on HLA compatibility one could expect a better response for P#2 and P#6, but this was not the case. Conclusions A better response has not been detected in patients with a greater degree of HLA compatibility with the treatment, so the patient's compatibility seems independent of the response to the treatment. There is the need to deeper investigate other or concomitant parameters that could explain the difference in the response to treatment.