IntroductionAmong the plethora of cells under investigation to restore a functional myocardium, mesenchymal stromal cells (MSCs) have been granted considerable interest. However, whereas the beneficial effects of bone marrow MSCs (BM-MSCs) in the context of the diseased heart are widely reported, data are still scarce on MSCs from the umbilical cord matrix (UCM-MSCs). Herein we report on the effect of UCM-MSC transplantation to the infarcted murine heart, seconded by the dissection of the molecular mechanisms at play.MethodsHuman umbilical cord tissue-derived MSCs (UCX®), obtained by using a proprietary technology developed by ECBio, were delivered via intramyocardial injection to C57BL/6 females subjected to permanent ligation of the left descending coronary artery. Moreover, medium produced by cultured UCX® preconditioned under normoxia (CM) or hypoxia (CMH) was collected for subsequent in vitro assays.ResultsEvaluation of the effects upon intramyocardial transplantation shows that UCX® preserved cardiac function and attenuated cardiac remodeling subsequent to myocardial infarction (MI). UCX® further led to increased capillary density and decreased apoptosis in the injured tissue. In vitro, UCX®-conditioned medium displayed (a) proangiogenic activity by promoting the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), and (b) antiapoptotic activity in HL-1 cardiomyocytes subjected to hypoxia. Moreover, in adult murine cardiac Sca-1+ progenitor cells (CPCs), conditioned medium enhanced mitogenic activity while activating a gene program characteristic of cardiomyogenic differentiation.ConclusionsUCX® preserve cardiac function after intramyocardial transplantation in a MI murine model. The cardioprotective effects of UCX® were attributed to paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis, augment proliferation, and activate a pool of resident CPCs. Overall, these results suggest that UCX® should be considered an alternative cell source when designing new therapeutic approaches to treat MI.

BackgroundECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis.MethodsUCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time.ResultsUCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations.ConclusionThe results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1β, IL-8, LIF and TGFβ2.

Mesenchymal stromal cells (MSCs) play an important role in tissue regeneration mainly through the secretion of trophic factors that enhance the repair of damaged tissues. The main goal of this work was to study the paracrine mechanisms by which an umbilical cord tissue-derived MSC population (UCX(®)) promotes the migration capacity of human dermal fibroblasts and keratinocytes, which is highly relevant for skin regeneration. Furthermore, the differences between paracrine activities of MSCs from the umbilical cord tissue and the bone marrow (BM-MSCs) were also evaluated. In vitro scratch assays revealed that conditioned media (CM) obtained from both growing and stationary-phase UCX(®) cultures induced human dermal fibroblast (HDF) and keratinocyte (HaCaT) migration. These assays showed that the motogenic activity of UCX(®) CM to HaCaTs was significantly higher than to HDFs, in opposition to the effect seen with CM produced by BM-MSCs that preferentially induced HDF migration. Accordingly, a comparative quantification of key factors with vital importance in the consecutive stages of wound healing revealed very different secretome profiles between UCX(®) and BM-MSCs. The relatively higher UCX(®) expression of EGF, FGF-2, and KGF strongly supports early induction of keratinocyte migration and function, whereas the UCX(®)-specific expression of G-CSF suggested additional roles in mobilization of healing-related cells including CD34(-)/CD45(-) precursors (MSCs) known to be involved in tissue regeneration. Accordingly, in vitro chemotaxis assays and an in vivo transplantation model for chemoattraction confirmed that UCX(®) are chemotactic to CD34(-)/CD45(-) BM-MSCs via a cell-specific mobilization mechanism mediated by G-CSF. Overall, the results strongly suggest different paracrine activities between MSCs derived from different tissue sources, revealing the potential of UCX(®) to extend the regenerative capacity of the organism by complementing the role of endogenous BM-MSCs.

IntroductionStandardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).MethodsThe adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed.ResultsThe isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties.ConclusionsWe have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.