Jelly Of Human Umbilical Cord Research Articles

Hipoxia modulates the secretion of growth factors of human umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cell (MSC) has great potential as therapies due its ability to regenerate tissue damage and promote tissue homeostasis. Preconditioning of MSC in low oxygen concentration has been shown to affect the therapeutic potential of these cells. This study aimed to compare the characteristic and secretion of trophic factors of MSCs cultured under hypoxia and normoxia. MSCs were isolated from Wharton's jelly of human umbilical cord (UC) tissue by explant method and characterized by flow cytometry. Following 24 h of CoCl2-induced hypoxic culture, the viability and metabolic activity of MSC were analyzed by trypan blue exclusion test and methyl thiazolyl tetrazolium (MTT) assay, respectively. The secretion of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) was assessed in conditioned medium using enzyme-linked immunosorbent assay (ELISA) method. Flow cytometry analysis showed >99% of the population of MSCs cells were positive for CD73 and CD90 and > 62% were positive for CD105. While the cell viability of MSC was not affected by hypoxic cultured condition, the metabolic activity rate of these cells was decreased under hypoxic conditioning. In line with reduced metabolic activity, hypoxic human UC-derived MSC produced less HGF than normoxic counterpart. Compared to normoxic MSC, hypoxic preconditioned MSC secreted higher level of VEGF in the conditioned medium (p < 0.05). Hypoxia decreased the metabolic activity of MSCs associated with the modulation of HGF and VEGF secretions. It is suggested that hypoxia may also affect the therapeutic capacity of MSC cells.

Evaluation of NLR Family CARD Domain Containing 3 and NLR Family CARD Domain Containing 5 Gene Expression in Interferon Gamma–Treated Mesenchymal Stem Cells from Wharton’s Jelly of Human Umbilical Cord

Background: Human umbilical cord Wharton’s jelly has provided a new source for mesenchymal stem cells (MSCs). The highly proliferative capacity with low immunogenicity and multi-differentiation potential of its stem cells make them applicable for transplantation purposes. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play various roles in antigen presentation of pathogens and damaged cells to suppress and/or modulate inflammation. Objectives: In this study, the expression levels of NLR family CARD domain containing 3 (NLRC3) and NLRC5 genes were analyzed and compared in both untreated and interferon gamma (IFN-γ)–treated Wharton’s jelly-derived MSCs (WJ-MSCs). Methods: MSCs were isolated from human umbilical cord Wharton’s jelly using standard tissue culture. The expression of NLRC5 and NLRC3 genes was analyzed in IFN-γ–treated WJ-MSCs (24 hours after treatment) and untreated WJ-MSCs (as a control) using quantitative real-time polymerase chain reaction (PCR). Results: It was found that IFN-γ treatment mimicking an inflammation scenario led to a statistically significant increase of NLRC3 and NLRC5 gene expression compared to untreated WJ-MSCs (P ≤ 0.05). Conclusions: It seems that higher expression of NLRC3 and NLRC5 genes in treated WJ-MSCs may make them a proper candidate to be used as a source for cell therapy in inflammatory conditions.

Human Wharton's jelly-derived mesenchymal stem cells alleviate concanavalin A-induced fulminant hepatitis by repressing NF-\u03baB signaling and glycolysis

BackgroundFulminant hepatitis is a severe life-threatening clinical condition with rapid progressive loss of liver function. It is characterized by massive activation and infiltration of immune cells into the liver and disturbance of inflammatory cytokine production. Mesenchymal stem cells (MSCs) showed potent immunomodulatory properties. Transplantation of MSCs is suggested as a promising therapeutic approach for a host of inflammatory conditions.MethodsIn the current study, a well-established concanavalin A (Con A)-induced fulminant hepatitis mouse model was used to investigate the effects of transplanting human umbilical cord Wharton's jelly-derived MSCs (hWJ-MSCs) on fulminant hepatitis.ResultsWe showed that hWJ-MSCs effectively alleviate fulminant hepatitis in mouse models, primarily through inhibiting T cell immunity. RNA sequencing of liver tissues and human T cells co-cultured with hWJ-MSCs showed that NF-κB signaling and glycolysis are two main pathways mediating the protective role of hWJ-MSCs on both Con A-induced hepatitis in vivo and T cell activation in vitro.ConclusionIn summary, our data confirmed the potent therapeutic role of MSCs-derived from Wharton's jelly of human umbilical cord on Con A-induced fulminant hepatitis, and uncovered new mechanisms that glycolysis metabolic shift mediates suppression of T cell immunity by hWJ-MSCs.

The Effect of Antioxidant Culture Conditions and Isolation Methods in Obtaining of Mesenchymal Stem Cells from The Wharton's Jelly of Human Umbilical Cord

The Effect of Antioxidant Culture Conditions and Isolation Methods in Obtaining of Mesenchymal Stem Cells from The Wharton's Jelly of Human Umbilical Cord

The Dynamics of Cell Properties during Long-Term Cultivation of Two Lines of Mesenchymal Stem Cells Derived from Wharton’s Jelly of Human Umbilical Cord

The cell properties of two MSC lines (MSCWJ-1 and MSCWJ-2) established from Wharton’s jelly of human umbilical cord were analyzed at the 6th, 13th, and 28th passages. The following were discovered. (1) The cell morphology during long-term cultivation was changed in both lines, with the cells being larger and having more debris in the MSCWJ-2 line than in the MSCWJ-1 line. (2) As shown by analysis of growth characteristics, long-term cultivation was accompanied with a significant increase of the average population-doubling time in both cell lines. This was correlated with morphological changes and a gradual increase in β-galactosidase activity, indicating the occurrence of replicative senescence. The interline changes concerned the level of proliferative activity, the index of proliferation, the time of the entry into the phase of replicative senescence (30 population doublings in the MSCWJ-2 line and 60 population doublings in the MSCWJ-1 line), and the percentage of senescent cells (80% in MSCWJ-2 line compared to 58% in the MSCWJ-1 line). (3) Numerical karyotypic analysis showed that both cell lines at early and late passages had normal karyotype 46, XX (MSCWJ-1 cells), and 46, XY (MSCWJ-2 cells). Structural karyotypic analysis showed a cytogenetic heterogeneity in both lines: low frequency of random clonal and nonclonal chromosomal rearrangements disappeared during long-term cultivation. Unlike in the case of the MSCWJ-1 line, the clonal chromosomal rearrangement of the short arm of chromosome 7 with a frequency of 39% was detected at the sixth passage in the MSCWJ-2 line but disappeared at the 13th passage. (4) Surface marker analysis of these cell lines at the 6th, 13th, and 28th passages revealed that these cells expressed surface antigens characteristic of human MSC (CD44, CD73, CD90, CD105, HLA-ABC), as well as vimentin, and did not express CD34 and HLA-DR. (5) Analysis of nondifferentiated hESC markers in both lines showed a decrease in the level of expression of the surface marker SSEA-4 and a lack of expression of the transcription factor SOX-2 at the 13th passage in the MSCWJ-2 and at the 28th passage in MSCWJ-1 cells. (6) Cell adhesion in both lines was lower in late passages compared to the cells at the sixth passage. The cells retained an osteogenic and adipogenic differentiation potential at late passages. A lack of chondrogenic differentiation in MSCWJ-2 cells, but not in MSCWJ-1 cells, was registered at late passages. The MSCWJ-2 cell line exhibited a frequently occurring clonal chromosomal rearrangement at an early passage, early replicative senescence with a large proportion of senescent cells, a significantly decreased index of proliferation during long-term cultivation, a decrease or disappearance of the expression of nondifferentiated ESCs of markers, and decreased differentiation potential. Given the metabolic cooperation between the cells, we can cautiously suppose that the clonal chromosomal rearrangement of the short arm of chromosome 7 detected at an early passage with high frequency is coupled with activation of cellular processes that proceed in the absence of this chromosomal aberration.

Isolation and Establishment of Mesenchymal Stem Cells from Wharton’s Jelly of Human Umbilical Cord

Mesenchymal stem cells (MSCs) are currently considered as 'medicinal signaling cells' and a promising resource in regard to cell-based regenerative therapy. Umbilical cord is a human term perinatal tissue which is easily attainable, and a promising source of stem cells with no associated ethical concerns. MSCs have been isolated from different regions of the umbilical cord and Wharton's jelly (WJ) is the gelatinous matrix that surrounds and provides protection to the umbilical cord blood vessels. Being more primitive, MSCs from human umbilical cord exhibit greater proliferative capacity and immunosuppressive ability as compared to adult stem cells which gives them a therapeutic advantage. To meet the requirements for cell therapy, it is important to generate MSCs at a clinical scale by following steps which are not time consuming or labor intensive. Here we present a simple, efficient protocol for isolation of MSCs from WJ of human umbilical cord by explant culture method which is reproducible and also, cost effective.

Human Umbilical Cord Mesenchymal Stem Cells Inhibit T Follicular Helper Cell Expansion through the Activation of iNOS in Lupus-Prone B6.MRL-Faslpr Mice

The aberrant generation or activation of T follicular helper (Tfh) cells contributes to the pathogenesis of systemic lupus erythematosus (SLE), yet little is known about how these cells are regulated. In this study, we demonstrated that the frequency of Tfh cells was increased in lupus-prone B6.MRL-Faslpr (B6.lpr) mice and positively correlated to plasma cell proportions and serum total IgG as well as anti-dsDNA antibody levels. Transplantation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cords (hUC-MSCs) ameliorated lupus symptoms in B6.lpr mice, along with decreased percentages of Tfh cells. In vitro studies showed that the differentiation and proliferation of Tfh cells were markedly suppressed by hUC-MSCs. The production of inducible nitric oxide synthase (iNOS) was dramatically upregulated in hUC-MSCs when cocultured with CD4+ T cells directly, while adding the specific inhibitor of iNOS into the coculture system significantly reversed the inhibitory effect of hUC-MSCs on Tfh cell generation. Interestingly, the efficacy of hUC-MSCs in inhibiting Tfh cells was impaired in the Transwell system, with the reduction of iNOS in both mRNA and protein levels. Taken together, our findings suggest that hUC-MSCs could effectively inhibit Tfh cell expansion through the activation of iNOS in lupus-prone B6.lpr mice, which is highly dependent on cell-to-cell contacts.

Evaluation of the potential of rhTGF- β3 encapsulated P(LLA-CL)/collagen nanofibers for tracheal cartilage regeneration using mesenchymal stems cells derived from Wharton's jelly of human umbilical cord

Tracheal injuries are one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, core-shell nanofibrous scaffold was fabricated to encapsulate bovine serum albumin & rhTGF-β3 (recombinant human transforming growth factor-β3) into the core of the nanofibers for tracheal cartilage regeneration. Characterization of the core-shell nanofibrous scaffold was carried out by scanning electron microscope (SEM), transmission electron microscope (TEM), laser scanning confocal microscopy (LSCM), and tensile mechanical test. The rhTGF-β3 released from the scaffolds in a sustained and stable manner for about 2months. The bioactivity of released rhTGF-β3 was evaluated by its effect on the synthesis of type II collagen (COL2) and glycosaminoglycans (GAGs) by chondrocytes. The results suggested that its bioactivity was retained during release process. The proliferation and morphology analyses of mesenchymal stems cells derived from Wharton's jelly of human umbilical cord (WMSCs) indicated the good biocompatibility of the fabricated nanofibrous scaffold. Meanwhile, the chondrogenic differentiation of WMSCs cultured on core-shell nanofibrous scaffold was evaluated by real-time qPCR and histological staining. The results suggested that the core-shell nanofibrous scaffold with rhTGF-β3 could promote the chondrogenic differentiation ability of WMSCs. Therefore, WMSCs could be a promising seed cells in the construction of tissue-engineered tracheal cartilage. Overall, the core-shell nanofibrous scaffold could be an effective delivery system for rhTGF-β3 and served as a promising tissue engineered scaffold for tracheal cartilage regeneration.

Apelin: an endogenous peptide essential for cardiomyogenic differentiation of mesenchymal stem cells via activating extracellular signal-regulated kinase 1/2 and 5.

Growing evidence has shown that apelin/APJ system functions as a critical mediator of cardiac development as well as cardiovascular function. Here, we investigated the role of apelin in the cardiomyogenic differentiation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord in vitro. In this research, we used RNA interference methodology and gene transfection technique to regulate the expression of apelin in Wharton's jelly-derived mesenchymal stem cells and induced cells with a effective cardiac differentiation protocol including 5-azacytidine and bFGF. Four weeks after induction, induced cells assumed a stick-like morphology and myotube-like structures except apelin-silenced cells and the control group. The silencing expression of apelin in Wharton's jelly-derived mesenchymal stem cells decreased the expression of several critical cardiac progenitor transcription factors (Mesp1, Mef2c, NKX2.5) and cardiac phenotypes (cardiac α-actin, β-MHC, cTnT, and connexin-43). Meanwhile, endogenous compensation of apelin contributed to differentiating into cells with characteristics of cardiomyocytes in vitro. Further experiment showed that exogenous apelin peptide rescued the cardiomyogenic differentiation of apelin-silenced mesenchymal stem cells in the early stage (1-4 days) of induction. Remarkably, our experiment indicated that apelin up-regulated cardiac specific genes in Wharton's jelly-derived mesenchymal stem cells via activating extracellular signal-regulated kinase (ERK) 1/2 and 5.

Endothelial Progenitor Cells Derived From Wharton's Jelly of Human Umbilical Cord Attenuate Ischemic Acute Kidney Injury by Increasing Vascularization and Decreasing Apoptosis, Inflammation, and Fibrosis.

Ischemia-reperfusion (I/R) injury to the kidney, a major cause of acute renal failure in humans, is associated with a high mortality, and the development of a new therapeutic strategy is therefore highly desirable. In this study, we examined the therapeutic potential of implantation of endothelial progenitor cells (EPCs) isolated from Wharton's jelly of human umbilical cords in the treatment of renal I/R injury in mice. To visualize the localization of the transplanted EPCs, the cells were labeled with Q-tracker before injection into the renal capsule. Mice with renal I/R injury showed a significant increase in blood urea nitrogen and creatinine levels, and these effects were decreased by EPC transplantation. The kidney injury score in the mice with I/R injury was also significantly decreased by EPC transplantation. EPC transplantation increased the microvascular density, and some of the EPCs surrounded and were incorporated into microvessels. In addition, EPC transplantation inhibited the I/R-induced cell apoptosis of endothelial, glomerular, and renal tubular cells, as demonstrated by TUNEL staining, and significantly reduced reactive oxygen species production and the expression of the inflammatory chemokines macrophage inflammatory protein-2 and keratinocyte-derived cytokine, as shown by immunostaining and ELISA. Moreover, EPC transplantation reduced I/R-induced fibrosis, as demonstrated by immunostaining for S100A4, a fibroblast marker, and by Jones silver staining. To our knowledge, this is the first report that transplantation of EPCs from Wharton's jelly of human umbilical cords might provide a novel therapy for ischemic acute kidney injury by promoting angiogenesis and inhibiting apoptosis, inflammation, and fibrosis.

Functional recovery after acute intravenous administration of human umbilical cord mesenchymal stem cells in rats with cerebral ischemia-reperfusion injury.

Cell therapy is a potential approach for treatment of strokes. Mesenchymal stem cells (MSCs) are a potential cell source for clinical use because they are safe and easy to obtain. A peptide solution can promote neural regeneration. Previously, such a solution was stereotactically injected into the brain of rats with cerebral infarction, resulting in improvement in the animal's neurological function and reduction in the infarction volume, but the injury was relatively severe. The current study established a rat model of cerebral ischemia-reperfusion (I/R) injury. MSCs isolated from Wharton's jelly of human umbilical cords (HUMSCs) were injected intravenously immediately after cerebral I/R injury(3 × 10(6) cells per rat). Twenty-four h and 14 d after surgery, animal behavior was evaluated using the Rogers test and infarct lesion volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining. Fourteen d after surgery, brain tissues were collected at 14 d to study migration/implantation of HUMSCs, cellular proliferation, neural regeneration and astrocyte activation. Compared to cerebral I/R injury alone, HUMSC treatment improved function at 14 d after surgery, with no reduction in infarct volume or migration or implantation of cells into the damaged brain areas. Nevertheless, 14 d after surgery, HUMSC administration increased cellular proliferation and the level of neurofilament 200 level and decreased the level of glial fibrillary acidic protein. After cerebral I/R injury, acute intravenous administration of HUMSCs could promote recovery by activating endogenous neural regeneration and inhibiting astrocyte activation, without migration and implantation directly into lesions.

Differentiation of human umbilical cord-derived mesenchymal stem cells into hepatocytes in vitro.

The stem cell based therapy is a potential alternative to liver transplantation. The aim of the study is to investigate the hepatocytic differentiation ability of human umbilical cord derived mesenchymal stem cells (HUC-MSCs) in vitro. The HUC-MSCs were isolated from Wharton's jelly of human umbilical cord. The cells were identified by assessing the stem cell markers. The HUC-MSCs were characterized by multipotency of differentiation. We modified the hepatogenic differentiation protocol and examine the function of differentiated cell by Periodic acid-Schiff (PAS) staining and Low-Density Lipoprotein (LDL) uptake. The protein expressions of Total protein (TP), Albumin (ALB), Globulin (GLB), Urea (BUN) and α-fetoprotein (AFP) were also detected. The cells of the hepatogenic differentiation group showed the function of hepatocyte. Protein expressions of TP, ALB, GLB BUN and AFP improve that the HUC-MSCs are able to different into the functional hepatogenic-like cell. The above founding indicated that the HUC-MSCs are able to different into the functional hepatogenic-like cell by present method.

Temporal studies into attachment, VE-cadherin perturbation, and paracellular migration of human umbilical mesenchymal stem cells across umbilical vein endothelial monolayers.

Mesenchymal stem cells from Wharton's jelly of human umbilical cords (WJ-MSC) are a valuable alternate source of stem cells. Their role in situ and whether they can interact and cross intact endothelial monolayers requires elucidation. The aim of this study was to investigate the dynamic interactions between WJ-MSC and human umbilical vein endothelial cells (HUVEC), including attachment, transit times, extravasation pathway, and the effects of WJ-MSC on junctional vascular endothelial (VE)-cadherin. HUVEC were grown to near confluence in endothelial media and to full confluence in mixed media before the addition of PKH26-labelled WJ-MSC. Time lapse fluorescence microscopy showed stem cells undergoing membrane blebbing followed by amoeboid movement on HUVEC monolayers before rounding up and changing shape toward the spindle-shaped morphology during/after transmigration to subendothelial positions. Cells demonstrated a time lag of 60 min before paracellular extravasation, confirmed by confocal microscopy. Forty-six percent of attached cells crossed in the first 2 h. By 16 h, a majority of cells had transmigrated with >96% of cells crossing by 22 h. There were concomitant changes in endothelial junctional VE-cadherin with statistically significant increases in discontinuous staining at 2 h, return to control values at 16 h, even as from 22 h onward HUVEC displayed increased percentage of junctions with continuous staining and upregulation of protein. Our data suggests that WJ-MSC crosses the endothelial barrier through the paracellular pathway and can influence junctional organization of HUVEC with discreet perturbation of VE-cadherin preceding transmigration followed by upregulation once the adluminal side is reached. The latter may reflect a perivascular support function of WJ-MSC in the umbilical cord.

The effect of gestational diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells.

Stomal cells derived from Wharton's jelly of human umbilical cord (WJMSCs) are considered as the potential therapeutic agents for regeneration and are getting famous for stem cell banking. Our study aims to evaluate the effects of gestational diabetes on proliferation capacity and viability of WJMSCs. Mesenchymal stromal cells were isolated from Wharton's jelly of human umbilical cords from normal and gestational diabetic (DWJMSCs) mothers. Growth patterns of both types of cells were analyzed through MTT assay and population doubling time. Cell survival, cell death and glucose utilization were estimated through trypan blue exclusion assay, LDH assay and glucose detection assay respectively. Angiogenic ability was evaluated by immunocytochemistry and ELISA for VEGF A. Anti-cancerous potential was analyzed on HeLa cells. DWJMSCs exhibited low proliferative rate, increased population doubling time, reduced cell viability and increased cell death. Interestingly, DWJMSCs were found to have a reduced glucose utilization and anti-cancerous ability while enhanced angiogenic ability. Gestational diabetes induces adverse effects on growth, angiogenic and anti-cancerous potential of WJMSCs.

Abstract 1977: Therapeutic miRNAs targeted selectively to tumors by mesenchymal stem cell derived microparticles

Abstract Introduction: MiRNAs have been implicated in the development of some if not all cancer types and have been identified as attractive targets for therapy. However, systemic delivery of bare miRNAs faces its own set of limitations because of degradation by RNases and filtration and excretion by the kidneys. In this work we propose miRNAs as therapeutic agents. We have developed a novel system for miRNA delivery applicable for both local and systemic administration with the use of mesenchymal stem cell (MSC) microparticles (MPs). Methodology: MSCs were isolated from the Wharton's jelly of human umbilical cords. The MSC cultures were subjected to serum deprivation, leading to the formation of MPs (secreted membrane vehicles &amp;lt;1μm) which were harvested and characterized by SEM, PCR, FACS and Fluorescence Microscopy. Breast (MDA-MB-231 &amp; MCF-7), colon (RKO &amp; HT-29) and ovarian adenocarcinoma (SKOV3) cell lines were then exposed to MPs. The response to treatment was evaluated by cell morphology, proliferation, migration, gene expression and apoptosis. Furthermore, the therapeutic potential of MPs was tested in vivo in xenograft tumor mouse models. Innovative imaging modalities such as in vivo flow cytometry and whole body fluorescence-bioluminescence were employed to dynamically investigate the biodistribution and homing kinetics of MPs in mice. Results: In vitro experiments confirmed that MSC-derived MPs can be internalized by the various cancer cell lines and induce a biological effect as evidenced by membrane damage, cell shrinkage and blebbing in the recipient cell. Significantly, there was evidence that MPs induce apoptosis, inhibit cell proliferation and tumor growth attenuation in a dose/time-dependent manner. The pro-apoptotic and anti-migration effects of MPs in cancer cells were almost completely abrogated by RNase treatment before administration to cultures. In vivo studies demonstrated that we were able to monitor and quantify fluorescently labelled MPs in circulation and to detect and image the biodistribution and incorporation in cells and organs in healthy and tumor-bearing mice. Conclusion: MSC-derived MPs containing miRNAs possess tumor inhibitory properties both in vitro and in vivo. Administration of MPs after RNase treatment induces the loss of anti-cancer properties suggesting a horizontal transfer of small RNAs from MPs to cancer cells. MPs formulated to contain specific miRNAs, could affect the action of genes associated with carcinogenesis, neovascularization, metastasis and other cancer characteristics, thus leading to therapeutic benefit. Citation Format: Marianna Prokopi, Agamemnon Epenetos, Andreas Anayiotos, Costas Pitsillides, Konstantinos Kapnisis, Christina Kousparou. Therapeutic miRNAs targeted selectively to tumors by mesenchymal stem cell derived microparticles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1977. doi:10.1158/1538-7445.AM2014-1977

A Nanoscaffold Impregnated With Human Wharton's Jelly Stem Cells or Its Secretions Improves Healing of Wounds

Wound healing is a major problem in diabetic patients and current methods of treatment have met with limited success. Since skin cell renewal is under the control of mesenchymal stem cells (MSCs) treatment of wounds has been attempted with the application of exogenous bone marrow MSCs (hBMMSCs). However, hBMMSCs have the limitations of painful harvest, low cell numbers and short-lived stemness properties unlike MSCs from the Wharton's jelly of human umbilical cords (hWJSCs). Since nanoscaffolds provide three dimensional architectural patterns that mimic in vivo stem cell niches and aloe vera has antibacterial properties we evaluated the use of an aloe vera-polycaprolactone (AV/PCL) nanoscaffold impregnated with green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs + AV/PCL) or its conditioned medium (hWJSC-CM + AV/PCL) for healing of excisional and diabetic wounds. In skin fibroblast scratch-wound assays exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL, fibroblasts migrated significantly faster from edges of scratches into vacant areas together with increased secretion of collagen I and III, elastin, fibronectin, superoxide dismutase, and metalloproteinase-1 (MMP-1) compared to controls. After one application of GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL excisional and diabetic wounds in mice showed rapid wound closure, reepithelialization, and increased numbers of sebaceous glands and hair follicles compared to controls. The same wounds exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL also showed positive keratinocyte markers (cytokeratin, involucrin, filaggrin) and increased expression of ICAM-1, TIMP-1, and VEGF-A compared to controls. AV/PCL nanoscaffolds in combination with hWJSCs appear to have synergistic benefits for wound healing.

Cytokine and growth factor expressions profile of mesenchymal stem cells from Wharton's jelly of human umbilical cord

Objective To characterize Wharton's jelly-derived mesenchymal stem cells from human umbilical cord (WMSCs) by observing their cytokine and growth factor expressions.Methods We isolated WMSCs from the umbilical cords from healthy full-term newborns.We verified the stemness by colony-forming unit-fibroblast (CFU-F) assay,detected the cell surface marker expression by flow cytometry,and observed the cytokine and growth factor expressions by protein chip assay.Results Human WMSCs were found to synthesize substantial levels of 78 of the 79 factors on the chip,with the only exception of IL-13.WMSCs were shown to produce significantly high levels [mean optical density (OD) ＞ 0.800] of the pro-inflammatory cytokines IL-lα,IL-1β,IL-6 and IL-8,as well as the chemokines growth related oncogene (GRO) and GRO-α.The expressed growth factors verified the osteogenesis and chondrogenesis differentiation potentials of WMSCs.In addition,the expression profile included 6 immune suppressive-related factors and tumor growth suppressors,which may reduce the chance of immune rejection and tumorigenesis of the allograft cells.Expressions of metalloproteases inhibitor,TIMP-1 and TIMP-2,could be important for the generation of functional tissue as they may prevent the breakdown of newly synthesized extracellular cell matrix (ECM) and protect the perivascular niche.Expressions of nerve growth factors (BDNF,NT-3,NT-4 and GDNF) indicate WMSCs have a potential to be induced and differentiated into nerve cells.Conclusions WMSCs are easy to obtain.The cytokines they express can reduce the chance of immune rejection and tumorigenesis and protect the implanted cells from metalloproteases digestion.So WMSCs may be a promising cellular candidate for use in regenerative medicine and fabrication of engineering tissue or organ. Key words: Umbilical cord; Mesenchymal stem cells; Cytokine; Cytokine antibody micro-array

Characteristics of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord and for fabrication of non-scaffold tissue-engineered cartilage

Once cartilage is damaged, it has limited potential for self-repair. Autologous chondrocyte implantation is an effective treatment, but patients may suffer during cartilage harvesting and the donor-site morbidity may accelerate joint degeneration. Using autologous mesenchymal stem cells (MSCs) derived chondrocytes is another selection, while it also causes some injuring. The umbilical cord, an ecto-embryo tissue may be an ideal source of cells, because of its accessibility, abundant resources, painless procedures for harvesting, and lack of ethical issues. We isolated MSCs from Wharton's jelly of human umbilical cord (WMSCs), which expressed CD44, CD105 and CD271 but not CD34 and CD45 with flow cytometry analysis. RT-PCR showed not only positive expression of CD90, c-kit, Sca1, SH2 and SH3 but also positive expression of the chondrocyte markers Sox-9 and Col-2A1. WMSCs cultured in high-density in the presence of transforming growth factor β1 and dexamethasone showed cartilage extracellular matrix-secretion and integrated into a thin piece of cell-based membrane. The cell-based thin membrane cultured in rotary cell culture system formed a round, opaque, glistening non-scaffold cartilage-like tissue, larger and condenser than what was formed with conventional pellet culture. Glycosaminoglycan and type II collagen content significantly increased after 3-week culture. The human WMSCs express characteristics of pre-chondrocytes, low immunogenicity and are easy to be obtained with higher purity because there have no hematopoietic cells in Wharton's jelly, so it may be a new seed cells more suitable for constructing tissue-engineered cartilage.

Chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells in type I collagen-hydrogel for cartilage engineering

IntroductionA potent mesenchymal stem cell (MSC) population was recently isolated from the Wharton's jelly of human umbilical cord (UC). The aim of the current experiments was to determine the potential of human UC-derived MSC (UC-MSC) in cartilage healing. Materials and methodsChondrogenic differentiation of collagen hydrogel-embedded cells was induced in standard chondrocyte conditioning medium and further detected by real-time PCR, histochemistry and immunohistochemistry analyses. Cell viability and apoptosis of the MSCs in the collagen I hydrogels were monitored using apoptosis detection kit. ResultsCells isolated from UC were positive for MSC biomarkers and negative for haematopoietic lineage and endothelial biomarkers and possess the capacity to differentiate along osteogenic lineage. UC-MSCs embedded in collagen hydrogel can undergo chondrogenesis characterised by significantly increased expressions of collagen II, aggrecan, COMP (cartilage oligomeric matrix protein) and sox9 after exposed cells-embedded hydrogels to chondrogenic factors. The most of cells remained viable throughout the hydrogels after 3weeks of cultivation in chondrogenic differentiation medium. ConclusionsCollagen hydrogel can provide an appropriate 3-D environment for the chondrogenesis of UC-MSCs. UC-MSCs embedded in biocompatible scaffold may have great potential for cartilage engineering.

Lovastatin protects chondrocytes derived from Wharton’s jelly of human cord against hydrogen-peroxide-induced in vitro injury

Our aim was to improve the survival and reduce the apoptosis of chondrocytes derived from mesenchymal stem cells from Wharton's jelly of human umbilical cord (WJMSCs) by Lovastatin supplementation under hydrogen-peroxide-induced injury conditions to simulate the osteoarthritic micro-environment. Chondrocytes were differentiated in vitro from WJMSCs. The cultured WJMSCs expressed CD90 (84.07%), CD105 (80.84%), OCT4 (26.90%), CD45 (0.42%) and CD34 (0.48%) as determined by flow cytometry. Increased aggregation of proteoglycans observed by Safranin-O staining accompanied by increased expression of COL2A1, ACAN, SOX9 and BGN shown by immunocytochemistry and reverse transcription with the polymerase chain reaction (PCR) confirmed the chondrogenic differentiation of the WJMSCs. The in vitro differentiated chondrocytes were subjected to oxidative stress by exposure to 200 μM hydrogen peroxide, either in the presence or absence of Lovastatin (2 μM) for 5 h. Lovastatin treatment resulted in decreased apoptosis, senescence and LDH release and in increased viability and proliferation of WJMSC-derived chondrocytes. Real time PCR analysis showed markedly up-regulated expression of prosurvival, proliferation and chondrogenic genes (BCL2L1, BCL2, AKT, PCNA, COL2A1, ACAN, SOX9 and BGN) and significantly down-regulated expression of pro-apoptotic genes (BAX, FADD) in the Lovastatin-treated group in comparison with injured cells. The reduced expression of VEGF and p53 as determined by enzyme-linked immunosorbent assay and PCR suggests the suitability of the use of Lovastatin in adjunct to WJMSC-derived chondrocytes for the treatment of osteoarthritis. We conclude that Lovastatin protects WJMSC-derived chondrocytes from hydrogen-peroxide-induced in vitro injury.