Umbilical Cord Wharton's Jelly Research Articles

The Immunosupressive Effect Of Wharton Jerry -Derived Mesenchymal stem Cells In vitro

BackgroundHuman mesenchymal stem cells (MSCs) can be isolated from several tissues, including bone marrow, umbilical cord, placenta, and adipose tissue. In recent reports, MSCs have the ability to migrate to inflammatory tissues and suppress adverse immune reactions. In fact, BM-derived MSCs are already applied for the patients with acute graft versus host disease (aGVHD) with promising efficacy. To avoid the potential risk of BM harvest, we intended to use umbilical cord Wharton's jelly (WJ) as an alternative source for MSCs. However, the mechanism by which WJ-MSCs exert their immunosuppressive effects is not completely understood. MethodsUmbilical cord was collected after informed consent from the mother. WJ-MSCs were isolated by explants method and in part, continued to culture for further expansion by passage 3 (P3). Otherwise, the remaining WJ-MSCs were cryopreserved in P1. Mixed lymphocyte reaction (MLR) was performed on WJ-MSCs or control. CFSE-labeled human peripheral blood (PB) or cord blood(CB) derived mononuclear cells (MNCs) as the responder cells were mixed with irradiated (50Gy) human CB-derived dendrite cells as stimulator cells at a 10:1 ratio, in the presence of low-dose anti-CD3 antibody. CFSE-labeled responder cells were analyzed by FACS. ResultsWJ-MSCs corresponded to the minimal criteria of MSCs defined by the International Society for Cell Therapy. Thus WJ-MSCs showed spindle-shaped plastic adherent with positive CD105, CD73, CD90, and negative for CD45, CD34, CD14, CD19, and HLA -DR. They also have the ability of differentiation into osteoblasts, adipocytes, chondroblasts in vitro. In MLR, WJ-MSCs efficiently inhibited the responder T cells derived from the same donor of WJ-MSCs, triggered by autologous or allogeneic dendritic cells (DC)(Autologous and allogeneic MLR). Moreover, 3rd party-derived WJ-MSCs also strongly suppressed allogeneic responder T cells proliferation triggered by allogeneic DC(3rd party MLR) (n=3). And WJ-MSCs could inhibit the proliferation of T cells upon PHA-L stimulation. In order to study whether the inhibitory effects of WJ-MSCs on MLR requires the cell to cell contact or not, we next performed MLR on separated WJ-MSCs using by transwell. We observed the inhibitory effect of WJ-MSCs on MLR, although the immunosuppressive effect of the separated WJ-MSCs was much less than that of cell to cell contact. The supernatants of WJ-MSCs, which were derived from 80% confluent WJ-MSCs indicated the mild inhibitory effects on MLR. These inhibitory effects of the WJ-MSCs on MLR were reversed by the addition of indoleamine 2,3-dioxygenase (IDO) inhibitor, 1-methyltryptophan (1-MT) , in a dose dependent manner as shown in Figure 1 (n=3). We also studied the influence of the passage of WJ-MSCs on MLR. The inhibitory effect was comparable among the early passage (∼P3) and long-term passage (P10), although the long-term passage did not show the osteogenic differentiation. DiscussionWe demonstrated WJ-MSCs have the immunosuppressive effect on the stimulated T cells in autologous, allogeneic and 3rd party MLR setting, indicating that immunosuppressive effect of WJ-MSCs is not restricted by MHC. Soluble IDO may play an important role in immunosuppressive effect of MLR, although we need to detect the additional factors especially in cell to cell contact between WJ-MSCs and the responder T cells. Conclusively, WJ-MSCs may be a feasible alternative non-invasive source of BM-MSCs for GVHD treatment and WJ-MSCs banking can be considered for the prompt clinical applications.▪ Disclosures:No relevant conflicts of interest to declare

Distinct Immunomodulatory and Migratory Mechanisms Underpin the Therapeutic Potential of Human Mesenchymal Stem Cells in Autoimmune Demyelination

Mesenchymal stem cells (MSCs) are efficacious in a variety of intractable diseases. While bone marrow (BM)-derived MSCs (BM-MSCs) have been widely investigated, MSCs from other tissue sources have also been shown to be effective in several autoimmune and inflammatory disorders. In the present study, we simultaneously assessed the therapeutic efficacy of human BM-MSCs, as well as MSCs isolated from adipose tissue (Ad-MSCs) and umbilical cord Wharton's jelly (UC-MSCs), in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Prior to in vivo experiments, we characterized the phenotype and function of all three MSC types. We show that BM-MSCs were more efficient at suppressing the in vitro proliferation of mitogen or antigen-stimulated T-cell responses compared to Ad-MSCs and UC-MSCs. Notably BM-MSCs induced the differential expression of cytokines from normal and stimulated T-cells. Paradoxically, intravenous transplantation of BM-MSCs into C57Bl/6 mice with chronic progressive EAE had a negligible effect on the disease course, even when multiple MSC injections were administered over a number of time points. In contrast, Ad-MSCs had the most significant impact on clinical and pathological disease outcomes in chronic progressive and relapsing-remitting EAE models. In vivo tracking studies revealed that Ad-MSCs were able to migrate to the central nervous system (CNS), a property that most likely correlated with their broader expression of homing molecules, while BM-MSCs were not detected in this anatomic region. Collectively, this comparative investigation demonstrates that transplanted Ad-MSCs play a significant role in tissue repair processes by virtue of their ability to suppress inflammation coupled with their enhanced ability to home to the injured CNS. Given the access and relatively ease for harvesting adipose tissue, these data further implicate Ad-MSCs as a cell therapeutic that may be used to treat MS patients.

Wharton's Jelly Stem Cells: A Novel Cell Source for Oral Mucosa and Skin Epithelia Regeneration

Perinatal stem cells such as human umbilical cord Wharton's jelly stem cells (HWJSCs) are excellent candidates for tissue engineering because of their proliferation and differentiation capabilities. However, their differentiation potential into epithelial cells at in vitro and in vivo levels has not yet been reported. In this work we have studied the capability of HWJSCs to differentiate in vitro and in vivo to oral mucosa and skin epithelial cells using a bioactive three-dimensional model that mimics the native epithelial-mesenchymal interaction. To achieve this, primary cell cultures of HWJSCs, oral mucosa, and skin fibroblasts were obtained in order to generate a three-dimensional heterotypical model of artificial oral mucosa and skin based on fibrin-agarose biomaterials. Our results showed that the cells were unable to fully differentiate to epithelial cells in vitro. Nevertheless, in vivo grafting of the bioactive three-dimensional models demonstrated that HWJSCs were able to stratify and to express typical markers of epithelial differentiation, such as cytokeratins 1, 4, 8, and 13, plakoglobin, filaggrin, and involucrin, showing specific surface patterns. Electron microscopy analysis confirmed the presence of epithelial cell-like layers and well-formed cell-cell junctions. These results suggest that HWJSCs have the potential to differentiate to oral mucosa and skin epithelial cells in vivo and could be an appropriate novel cell source for the development of human oral mucosa and skin in tissue engineering protocols.

Immunological Properties of Extraembryonic Human Mesenchymal Stromal Cells Derived from Gestational Tissue

Mesenchymal stromal cells (MSCs) have been isolated from many tissues, including gestational tissue. To date, a study comparing the properties and suitability of these cells in cell-based therapies is lacking. In this study, we compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory capabilities of human MSCs derived from umbilical cord lining (CL-MSCs), umbilical cord blood (CB-MSCs), placenta (P-MSCs), and Wharton's jelly (WJ-MSCs). Differences were noted in differentiation, proliferation, and migration, with CL-MSCs showing the highest proliferation and migration rates resulting in prolonged survival in immunodeficient mice. Moreover, CL-MSCs showed a prolongation in survival in xenogeneic BALB/c mice, which was attributed to their ability to dampen TH1 and TH2 responses. Weaker human cellular immune responses were detected against CL-MSCs and P-MSCs, which were correlated with their lower HLA I expression. Furthermore, HLA II was upregulated less substantially by CL-MSCs and CB-MSCs after IFN-γ stimulation. MSC types did not differ in indolamine 2,3-dioxygenase (IDO) expression after IFN-γ stimulation. Despite their lower IDO, HLA-G, and TGF-β1 expression, only CL-MSCs were able to reduce the release of IFN-γ by lymphocytes in a mixed lymphocyte reaction. In summary, CL-MSCs showed the best characteristics for cell-based strategies, as they are hypo-immunogenic and show high proliferation and migration rates. In addition, these studies show for the first time that although immunomodulatory molecules HLA-G, HLA-E, and TGF-β play an important role in MSC immune evasion, basal and induced HLA expression seems to be decisive in determining the immunogenicity of MSCs.

Microvesicles Derived from Human Umbilical Cord Wharton’s Jelly Mesenchymal Stem Cells Attenuate Bladder Tumor Cell Growth In Vitro and In Vivo

Several studies suggest that mesenchymal stem cells (MSCs) possess antitumor properties; however, the exact mechanisms remain unclear. Recently, microvesicles (MVs) are considered as a novel avenue intercellular communication, which may be a mediator in MSCs-related antitumor effect. In the present study, we evaluated whether MVs derived from human umbilical cord Wharton’s jelly mesenchymal stem cells (hWJMSCs) may inhibit bladder tumor T24 cells growth using cell culture and the BALB/c nu/nu mice xenograft model. CCK-8 assay and Ki-67 immunostaining were performed to estimate cell proliferation in vitro and in vivo. Flow cytometry and TUNEL assay were used to assess cell cycle and apoptosis. To study the conceivable mechanism by which hWJMSC-MVs attenuate bladder tumor T24 cells, we estimated the expression of Akt/p-Akt, p-p53, p21 and cleaved Caspase 3 by Western blot technique after exposing T24 cells to hWJMSC-MVs for 24, 48 and 72h. Our data indicated that hWJMSC-MVs can inhibit T24 cells proliferative viability via cell cycle arrest and induce apoptosis in T24 cells in vitro and in vivo. This study showed that hWJMSC-MVs down-regulated phosphorylation of Akt protein kinase and up-regulated cleaved Caspase 3 during the process of anti-proliferation and pro-apoptosis in T24 cells. These results demonstrate that hWJMSC-MVs play a vital role in hWJMSC-induced antitumor effect and may be a novel tool for cancer therapy as a new mechanism of cell-to-cell communication.

Multipotent to Pluripotent Properties of Adult Stem Cells

Stem cells have captured the attention of both researchers and the public alike because of the promise of tissue regeneration, drug screening, and organogenesis. Stem cells are broadly classified as embryonic or adult with differentiation capacities ranging from pluripotent and multipotent to unipotent. In general, pluripotent stem cells are ascribed to cells derived from the inner cell mass of blastocyst stage embryos or to those generated experimentally using reprogramming factors. Adult stem or progenitor cells are generally tissue-restricted but reside in most organs. While the use of pluripotent stem cells has been limited to a few clinical trials due to safety concerns, adult stem cells have shown evidence of safety and are broadly employed in both clinical trials (http://www.clinicaltrial.gov/) and clinical practice. Transplantation of adult bone marrow hematopoietic stem cells, for example, represents a standard method of clinical care for autoimmune diseases and hematological disorders. While hematopoietic stem cells cannot be expanded in vitro, other stem cells such as mesenchymal stem cells (MSCs) can be easily expanded from autologous and allogeneic sources for clinical testing. Moreover, adult-derived testicular spermatogonial stem cells (SSCs) have been reported to be the only cells in the body that can be dedifferentiated/reprogrammed to a pluripotent state in vitro and grown into ES-like colonies. It has been advocated that adult stem cells have remarkable plasticity, transdetermination, and transdifferentiation ability. These properties have raised hope that adult stem cells could become a universal source of stem cells for tissue/organ repair in lieu of embryonic stem cells, which can easily form tumors. MSCs, for example, can be isolated from various tissues like bone marrow, gut, lungs, liver, blood, adipose tissue, umbilical cord Wharton's jelly, dental pulp, amniotic fluid, and so forth and have the ability to form bone, cartilage, adipose tissue, skeletal muscle, liver, neurons, skin, pancreatic islets, endothelial cells, intestine, renal, epithelial, and germ cells. More importantly, MSCs can be given across allogeneic barriers. Nevertheless, many trials with MSCs have shown only marginal benefits and some have proposed that the effects may be more paracrine in nature than being regenerative. This special issue, comprised of 10 articles, focuses on adult stem cells that examine the following claims: Why are MSCs ubiquitous? Are they indeed stem cells or just stromal cells which constitute the somatic niche for tissue specific stem cells in various body organs? Have we looked carefully at MSCs growing in a culture dish? Are ES-like colonies observed only from testicular biopsy but also from ovarian and endometrial biopsies? The article by D. Bhartiya addresses these issues and reviews the available literature on the presence of novel pluripotent very small ES-like stem cells (VSELs) as a subgroup among MSCs. It also highlights that Oct-4A transcripts need to be studied to conclude pluripotent state rather than Oct-4. An article by Dr. P. Rameshwar's group studied the safety issue of MSCs since these stem cells have been shown to support tumor growth. Other papers have discussed various sources of adult stem cells including dental pulp stem cells (S. Arrifin et al.), satellite stem cells in skeletal muscles (S. Fujimaki et al.), and bone marrow stem cells (I. Catacchio et al.) and their multipotent properties and transdifferentiation potential. Further characterization of these stem cells and their functional potential is essential. The role of cannabinoid receptor type I in differentiation and survival of MSCs is discussed by A. Gowran et al. M. Akita et al. show that CD133 positive cells have the capacity to form endothelial capillary tubes in 3D culture. Cultures of cardiac explants result in the formation of cardiospheres which contain both stem and progenitor cells implicated in cardiac regeneration (L. Barile et al.). Y. Togo et al. have reviewed the literature on the use of MSCs as a nonviral gene carrier for gene transfer in vitro. Finally, N. Sakayori et al. discuss how neural stem/progenitor cells function is regulated by lipids. The diverse areas of the manuscripts in this issue underscore the wide field of adult stem cell biology. The papers indicate that more in-depth research and functional studies are required to take adult stem cells safely to the clinic for regenerative medicine. Deepa Bhartiya Kenneth R. Boheler Pranela Rameshwar

Differential distribution of cathepsin B in human umbilical cord tissues.

The extracellular matrix components are differentially distributed among various structures of the umbilical cord. Wharton's jelly is especially rich in collagens and growth factors. Cathepsin B is a major cysteine protease involved in collagen degradation, as well as in the activation of precursor forms of other collagenolytic enzymes and growth factors. We assessed the activity and expression of cathepsin B in the umbilical cord arteries, veins and Wharton's jelly. Extracts of separated umbilical cord components were subjected to an activity assay with the use of specific fluorogenic substrate. The expression of cathepsin B protein was qualitatively evaluated by Western immunoblotting and quantitatively determined with an immunoenzymatic method. The total cathepsin B activity and content calculated per gram of DNA were higher in Wharton's jelly than in the umbilical cord vessels, and the latter parameter was the lowest in the umbilical cord arteries. Moreover, the expression and the activity of latent cathepsin B (following activation by pepsin digestion) calculated per gram of DNA were the highest in Wharton's jelly and the lowest in the umbilical cord arteries. High expression and activity of latent, pepsin-activatable cathepsin B related to DNA content in Wharton's jelly seem to reflect the stimulation of its cells by high amounts of collagen I and growth factors.

Restricted Myogenic Potential of Mesenchymal Stromal Cells Isolated from Umbilical Cord

Nonhematopoietic cord blood cells and mesenchymal cells of umbilical cord Wharton's jelly have been shown to be able to differentiate into various cell types. Thus, as they are readily available and do not raise any ethical issues, these cells are considered to be a potential source of material that can be used in regenerative medicine. In our previous study, we tested the potential of whole mononucleated fraction of human umbilical cord blood cells and showed that they are able to participate in the regeneration of injured mouse skeletal muscle. In the current study, we focused at the umbilical cord mesenchymal stromal cells isolated from Wharton's jelly. We documented that limited fraction of these cells express markers of pluripotent and myogenic cells. Moreover, they are able to undergo myogenic differentiation in vitro, as proved by coculture with C2C12 myoblasts. They also colonize injured skeletal muscle and, with low frequency, participate in the formation of new muscle fibers. Pretreatment of Wharton's jelly mesenchymal stromal cells with SDF-1 has no impact on their incorporation into regenerating muscle fibers but significantly increased muscle mass. As a result, transplantation of mesenchymal stromal cells enhances the skeletal muscle regeneration.

Recent Patents and Advances on Isolation and Cellular Therapy Applications of Mesenchymal Stem Cells from Human Umbilical Cord Wharton's Jelly

Recent Patents and Advances on Isolation and Cellular Therapy Applications of Mesenchymal Stem Cells from Human Umbilical Cord Wharton's Jelly

Effect of substrate stiffness on differentiation of umbilical cord stem cells.

Tissue formation and maintenance is regulated by various factors, including biological, physiological and physical signals transmitted between cells as well as originating from cell-substrate interactions. In our study, the osteogenic potential of mesenchymal stromal/stem cells isolated from umbilical cord Wharton's jelly (UC-MSCs) was investigated in relation to the substrate rigidity on polyacrylamide hydrogel (PAAM). Osteogenic differentiation of UC-MSCs was enhanced on stiff substrate compared to soft substrates, illustrating that the mechanical environment can play a role in differentiation of this type of cells. These results show that substrate stiffness can regulate UC-MSCs differentiation, and hence may have significant implications for design of biomaterials with appropriate mechanical properties for regenerative medicine.

Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

BackgroundThe possibility for isolating bovine mesenchymal multipotent cells (MSCs) from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D) environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM). The three-dimensional (3D) cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ) cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking.ResultsBovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when bovine-derived UC-WJ cells was included in the culture which demonstrated the immunossupression profile typically observed among isolated mesenchymal cells from other species. After classified the UC-WJ cells as mesenchymal stromal phenotype the in vitro 3D cultures was performed using the AlgiMatrix® protocol. Based on the size of spheroids (283,07 μm ± 43,10 μm) we found that three weeks of culture was the best period to growth the UC-WJ cells on 3D dimension. The initial cell density was measured and the best value was 1.5 × 106 cells/well.ConclusionsWe described for the first time the isolation and characterization of UC-WJ cells in a serum-free condition and maintenance of primitive mesenchymal phenotype. The culture was stable under 60 consecutive passages with no genetic abnormalities and proliferating ratios. Taken together all results, it was possible to demonstrate an easy way to isolate and culture of bovine-derived UC-WJ cells under 2D and 3D serum-free condition, from fetal adnexa with a great potential in cell therapy and biotechnology.

Inflammation and Toll-Like Receptor Ligation Differentially Affect the Osteogenic Potential of Human Mesenchymal Stromal Cells Depending on Their Tissue Origin

Mesenchymal stromal cells (MSCs) can be isolated not only from bone marrow (BM) but also from other tissues, including adipose tissue (AT) and umbilical cord Wharton's Jelly (WJ). Thanks to their ability to differentiate into various cell types, MSC are considered attractive candidates for cell-based regenerative therapy. In degenerative clinical settings, inflammation or infection is often involved. In the present work, we hypothesized that an inflammatory environment and/or Toll-like receptor (TLR) ligation could affect the MSC differentiation potential. MSC were isolated from BM, AT, and WJ. Inflammation was mimicked by a cytokine cocktail, and TLR activation was induced through TLR3 and TLR4 ligation. Osteogenesis was chosen as a model for differentiation. Osteogenic parameters were evaluated by measuring Ca2+ deposits and alkaline phosphatase (ALP) activity at day 7, 14, and 21 of the culture in an osteogenic medium. Our results show that WJ-MSC exhibit a much lower osteogenic potential than the other two MSC types. However, inflammation was able to strongly increase the osteogenic differentiation of WJ-MSC as calcification, and ALP activity appeared as early as day 7. However, this latter enzymatic activity remained much lower than that disclosed by BM-MSC. TLR3 or TLR4 triggering increased the osteogenesis in AT- and, to lesser extent, in BM-MSC. In conclusion, WJ-MSC constitutively disclose a lower osteogenic potential as compared with BM and AT-MSC, which is not affected by TLR triggering but is strongly increased by inflammation, then reaching the level of BM-MSC. These observations suggest that WJ-MSC could constitute an alternative of BM-MSC for bone regenerative applications, as WJ is an easy access source of large amounts of MSC that can effectively differentiate into osteoblasts in an inflammatory setting.

Human umbilical cord wharton's jelly mesenchymal stem cells do not transform to tumor‐associated fibroblasts in the presence of breast and ovarian cancer cells unlike bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hBMMSCs) were shown to transform into tumor-associated fibroblasts (TAFs) when in the vicinity of breast cancer tumors and played an important role in tumor enhancement and metastasis. In early human development MSCs migrating from the yolk sac and aorta-gonad-mesonephros (AGM) via the umbilical cord to the placenta and back to the fetal bone marrow were shown to get trapped in the gelatinous Wharton's jelly of the umbilical cord. The common origin of the Wharton's jelly MSCs and the finally homed hBMMSCs prompted us to evaluate whether hWJSCs are also involved in TAF transformation. hWJSCs and hBMMSCs were grown in the presence of breast and ovarian cancer cell conditioned medium (MDA-TCM, TOV-TCM) for 30 days. No changes were observed in the hWJSCs but the hBMMSCs transformed from short to thin long fibroblasts, their proliferation rates increased and CD marker expression decreased. The transformed hBMMSCs showed positive staining for the tumor-associated markers FSP, VEGF, EGF, and Tn-C. Real-time PCR and multiplex luminex bead analysis showed upregulation of TAF-related genes (FSP, FAP, Tn-C, Tsp-1, EGF, bFGF, IL-6, α-SMA, VEGF, and TGF-β) for hBMMSCs with low expression for hWJSCs. The luciferase assay showed that hWJSCs previously exposed to MDA-TCM or TOV-TCM had no stimulatory growth effect on luciferase-tagged MDA or TOV cells unlike hBMMSCs. The results confirmed that hWJSCs do not transform to the TAF phenotype and may therefore not be associated with enhanced growth of solid tumors making them a safe MSC for cell based therapies.

Human umbilical cord wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Wharton's jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC-CM) and cell lysate (hWJSC-CL)] on breast adenocarcinoma (MDA-MB-231), ovarian carcinoma (TOV-112D), and osteosarcoma (MG-63) cells. The cells were treated with either hWJSC-CM (50%) or hWJSC-CL (15 µg/ml) for 48-72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC-CL and hWJSC-CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2-6% and 30-60%, while Transwell migration assay showed inhibition by 20-26% and 31-46% for hWJSC-CM and hWJSC-CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub-G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV-FITC and TUNEL positive cells seen in TOV-112D and MDA-MB-231 suggested that inhibition was via apoptosis while the presence of anti-BECLIN1 and anti-LC3B antibodies seen with MG-63 indicated autophagy. Upregulation of pro-apoptotic BAX and downregulation of anti-apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG-63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts.

Evaluation of the Cell Viability of Human Wharton's Jelly Stem Cells for Use in Cell Therapy

Human umbilical cord Wharton's jelly stem cells (HWJSCs) are gaining attention as a possible clinical source of mesenchymal stem cells for cell therapy and tissue engineering due to their high accessibility, expansion potential, and plasticity. We employed a combination of highly sensitive techniques to determine the average cell viability levels and proliferation capabilities of 10 consecutive cell passages of cultured HWJSCs and then used RNA microarrays to identify genes associated with changes in cell viability levels. We found an initial decrease in cell viability from the first to the third cell passage followed by an increase until the sixth passage and a final decrease from the sixth to tenth cell passages. The highest cell viability levels corresponded to the fifth and sixth passages. The intracellular ionic contents of potassium, sodium, and chlorine suggest that the lower cell viability levels at passages 2, 3, and 8-10 may be associated with apoptotic cell death. In fact, gene expression analysis revealed that the average cell viability was significantly associated with genes with a function in apoptotic cell death, especially pro-apoptotic FASTKD2, BNIP3L genes and anti-apoptotic TNFAIP8 and BCL2L2 genes. This correlation with both pro-apoptotic and anti-apoptotic genes suggests that there may be a complex live-death equilibrium in cultured HWJSCs kept in culture for multiple cell passages. In this study, the highest cell viability levels corresponded to the fifth and sixth HWJSC passages, suggesting that these passages should be preferentially employed in cell therapy or tissue engineering protocols using this cell type.

Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo

Bone marrow mesenchymal stromal cells (BMMSCs) have been used as feeder support for the ex vivo expansion of hematopoietic stem cells (HSCs) but have the limitations of painful harvest, morbidity, and risk of infection to the patient. This prompted us to explore the use of human umbilical cord Wharton's jelly MSCs (hWJSCs) and its conditioned medium (hWJSC-CM) for ex vivo expansion of HSCs in allogeneic and autologous settings because hWJSCs can be harvested in abundance painlessly, are proliferative, hypoimmunogenic, and secrete a variety of unique proteins. In the presence of hWJSCs and hWJSC-CM, HSCs put out pseudopodia-like outgrowths and became highly motile. Time lapse imaging showed that the outgrowths helped them to migrate towards and attach to the upper surfaces of hWJSCs and undergo proliferation. After 9 days of culture in the presence of hWJSCs and hWJSC-CM, MTT, and Trypan blue assays showed significant increases in HSC numbers, and FACS analysis generated significantly greater numbers of CD34(+) cells compared to controls. hWJSC-CM produced the highest number of colonies (CFU assay) and all six classifications of colony morphology typical of hematopoiesis were observed. Proteomic analysis of hWJSC-CM showed significantly greater levels of interleukins (IL-1a, IL-6, IL-7, and IL-8), SCF, HGF, and ICAM-1 compared to controls suggesting that they may be involved in the HSC multiplication. We propose that cord blood banks freeze autologous hWJSCs and umbilical cord blood (UCB) from the same umbilical cord at the same time for the patient for future ex vivo HSC expansion and cell-based therapies.

Wharton's Jelly Stem Cells as Agents for Cancer Therapy

Stem cell based therapy has significant potential to treat various diseases including primary and metastatic cancers. The umbilical cord matrix stem cells (UCMSC) derived from human umbilical cord Wharton's jelly (also termed WJMSC) have been shown to exhibit low immunogenicity, which potentially negates immune consequences after cytotherapy. The homing ability of human and rat UCMSC to inflammatory tissues, including cancer tissues, further confers upon these cells the potential for targeted cancer therapy. Our previous studies demonstrated that un-engineered human and rat UCMSC significantly attenuated the growth of multiple cancer cell lines in vivo and in vitro through multiple mechanisms. We have also demonstrated that these cells can be engineered to express cytotoxic cytokines before being delivered to the tumor and can be preloaded with nanoparticle payloads and attenuate tumors after homing to them. In this review, intrinsic stem cell-dependent regulation of cancer growth, potential mechanisms involved in this unique biological function, delivery of exogenous anti-cancer agents, and the potential for clinical applications will be discussed.

Human mesenchymal stem cells seeded on extracellular matrix‐scaffold: Viability and osteogenic potential

The development and the optimization of novel culture systems of mesenchymal osteoprogenitors are some of the most important challenges in the field of bone tissue engineering (TE). A new combination between cells and extracellular matrix (ECM)-scaffold, containing ECM has here been analyzed. As source for osteoprogenitors, mesenchymal stem cells obtained from human umbilical cord Wharton's Jelly (hWJMSCs), were used. As ECM-scaffold, a powder form of isolated and purified porcine urinary bladder matrix (pUBM), was employed. The goals of the current work were: (1) the characterization of the in vitro hWJMSCs behavior, in terms of viability, proliferation, and adhesion to ECM-scaffold; (2) the effectiveness of ECM-scaffold to induce/modulate the osteoblastic differentiation; and (3) the proposal for a possible application of cells/ECM-scaffold construct to the field of cell/TE. In this respect, the properties of the pUBM-scaffold in promoting and guiding the in vitro adhesion, proliferation, and three-dimensional colonization of hWJMSCs, without altering viability and morphological characteristics of the cells, are here described. Finally, we have also demonstrated that pUBM-scaffolds positively affect the expression of typical osteoblastic markers in hWJMSCs.

Promising new potential for mesenchymal stem cells derived from human umbilical cord Wharton's jelly: sweat gland cell-like differentiative capacity

Mesenchymal stem cells derived from Wharton's jelly of the human umbilical cord (hUC-MSCs) possess various advantageous properties, similar to bone marrow-derived mesenchymal stem cells (BM-MSCs), including self-renewal, extended proliferation potential and multilineage differentiation potential. In this study, we hoped to determine whether hUC-MSCs could be induced to differentiate into sweat gland cell-like cells, that would be potential in sweat glands restoration after injury. In this study, the results of flow cytometry analysis revealed that hUC-MSCs showed the typical antigen profile of MSCs and were positive for CD29, CD44, CD90, CD105 and Oct-4; they were negative for the antigens of CD34, CEA and CK14. Remarkably, hUC-MSCs maintained proper proliferation and differentiation ability. After culture in sweat gland cell-conditioned medium (induction group 1) for 3 weeks, hUC-MSCs possessed sweat gland cell-like morphology and expressed markers of sweat gland cells (CEA, CK14 and CK19) more efficiently than those of induction group 2. In reverse-transcription PCR and western blotting analysis, it was further confirmed that induced hUC-MSCs (group 1) also expressed a higher level of sweat gland developmental genes (EDA and EDAR) than group 2. These results together provided evidence that hUC-MSCs could possess a new emerging potential to differentiate into sweat gland cell-like cells with a higher efficacy under our new induction system. Thus, hUC-MSCs could be considered a new strategy for sweat glands restoration after skin injury as well as improvement of cutaneous regeneration.