Human Cord Blood-derived Mesenchymal Stem Research Articles

Conditioned medium from human cord blood mesenchymal stem cells attenuates age-related immune dysfunctions.

Aging is accompanied with progressive deterioration of immune responses and tissue's function. Using 12-month-old mice as model, we showed that conditioned medium of human cord blood mesenchymal stem cells (CBMSC-CM) significantly reduced the population percentage of CD3-CD335+ NK and CD4+CD25+ regulatory T-cells in peripheral blood. The CBMSC-CM administration also increased naïve T-cells number and restored the ratio of naïve to memory T-cells in CD4+ T-cells population. These results indicated that CBMSC-CM improved the immune response efficiency of aged mice. Moreover, we also found CBMSC-CM treatment significantly reduced the number of senescenT-cells in kidney tissues. Finally, we demonstrated that CBMSC-CM remarkably attenuated hydrogen peroxide triggered T-cell response and ameliorated oxidative stress induced cellular senescence. All of these data suggest a prominent anti-aging effect of secretome of CBMSCs.

Cloning of human cord blood-mesenchymal stem cells for isolation of enriched cell population of higher proliferation and differentiation potential

Heterogeneity of Mesenchymal stem cells (MSCs) imposes limitations for their in vitro expansion and accounts for the lack of reproducibility in some clinical studies. So, this study was designed to isolate and enrich clones of multipotent and self-renewing MSCs from cord blood (CB). Enriched clones with higher proliferation and differentiation potential provide regenerative cells suitable for various clinical demands. MSCA and MSCB original (progenitor) cells were isolated from CB samples, and single cells were cloned by limiting dilution method, in mouse embryonic fibroblast conditioned media. Original MSCs and their single-cell derived clones were characterized by identifying their proliferation rate, immunophenotyping of surface antigens, expression of pluripotency and proliferation genes (Oct4, Sox2, Nanog, KLF4, c-Myc, and PDGFRA), and differentiation potential into multiple lineages (osteogenic, adipogenic, and chondrogenic). Some single-cell clones of MSCA showed a higher proliferation rate and greater differentiation potential than their original cells. However, original MSCB cells were of greater proliferation and differentiation potential than their derived single-cell clones, except for one clone which hadcomparable results. Cloning of MSCs was attainable when cultured in mouse embryonic fibroblast conditioned media. Single clones with higher proliferation and differentiation potential than their original progenitor cells were obtained by cloning of poorly functioning MSCs progenitor cells, enabling the selection of more therapeutically efficacious MSCs with better performance in clinical applications. Moreover, this study draws attention to the importance of CD105 as a possible MSCs biomarker associated with the multilineage commitment of MSCs.

Sonic Hedgehog, a Novel Endogenous Damage Signal, Activates Multiple Beneficial Functions of Human Endometrial Stem Cells.

Local endometrial stem cells play an important role in regulating endometrial thickness, which is an essential factor for successful embryo implantation and pregnancy outcomes. Importantly, defects in endometrial stem cell function can be responsible for thin endometrium and subsequent recurrent pregnancy losses. Therefore, many researchers have directed their efforts toward finding a novel stimulatory factor that can enhance the regenerative capacity of endometrial stem cells. Sonic hedgehog (SHH) is a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. In addition to this canonical function, we identified for the first time that SHH is actively secreted as a stem cell-activating factor in response to tissue injury and subsequently stimulates tissue regeneration by promoting various beneficial functions of endometrial stem cells. Our results also showed that SHH exerts stimulatory effects on endometrial stem cells via the FAK/ERK1/2 and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. More importantly, we also observed that endometrial stem cells stimulated with SHH showed markedly enhanced differentiation and migratory capacities and subsequent invivo therapeutic effects in an endometrial ablation animal model.

Resveratrol Prevents the Cellular Damages Induced by Monocrotophos via PI3K Signaling Pathway in Human Cord Blood Mesenchymal Stem Cells.

The role of resveratrol (RV) as a neuroprotectant is well recognized, and cellular molecules involved in imparting the physiological effect have been well illustrated. However, some ambiguity still prevails as the specific receptor, and downstream signaling molecules are not yet clearly stated. So, we investigated the signaling pathway(s) involved in its cellular protection in the human umbilical cord blood mesenchymal stem cell (hUCB-MSC) derived neuronal cells. The mesenchymal stem cells were exposed to various concentrations (10, 100, 1000μM) of monocrotophos (MCP), a known developmental neurotoxic organophosphate pesticide, for a period of 24h. The MAPK signaling pathways (JNK, p38, and ERK) known to be associated with MCP-induced damages were also taken into consideration to identify the potential connection. The biological safe dose of RV (10μM) shows a significant restoration in the MCP-induced alterations. Under the specific growth conditions, RV exposure was found to promote neuronal differentiation in the hUCB-MSCs. The exposure of cells to a specific pharmacological inhibitor (LY294002) of PI3K confirms the significant involvement of PI3K-mediated pathway in the ameliorative responses of RV against MCP exposure. Our data identifies the substantial role of RV in the restoration of MCP-induced cellular damages, thus proving to have a therapeutic potential against organophosphate pesticide-induced neurodegeneration.

PKA-GSK3β and β-Catenin Signaling Play a Critical Role in Trans-Resveratrol Mediated Neuronal Differentiation in Human Cord Blood Stem Cells

The role of resveratrol (RV), a natural polyphenol, is well documented, although its role on neurogenesis is still controversial and poorly understood. Therefore, to decipher the cellular insights of RV on neurogenesis, we investigated the potential effects of the compound on the survival, proliferation, and neuronal differentiation of human cord blood-derived mesenchymal stem cells (hCBMSCs). For neuronal differentiation, purified and characterized hCBMSCs were exposed to biological safe doses of RV (10μM) alone and in combination with nerve growth factor (NGF-50ng). The cells exposed only to NGF (50ng/mL) served as positive control for neuronal differentiation. The genes showing significant involvement in the process of neuronal differentiation were further funneled down at transcriptional and translational level. It was observed that RV promotes PKA-mediated neuronal differentiation in hCBMSCs by inducing canonical pathway. The studies with pharmacological inhibitors also confirmed that PKA significantly induces β-catenin expression via GSK3β induction and stimulates CREB phosphorylation and pERK1/2 induction. Besides that, the studies also revealed that RV additionally possesses the binding sites for molecules other than PKA and GSK3β, with which it interacts. The present study therefore highlights the positive impact of RV over the survival, proliferation, and neuronal differentiation in hCBMSCs via PKA-mediated induction of GSK3β, β catenin, CREB, and ERK1/2.

Neurotrophic factor mediated neuronal differentiation of human cord blood mesenchymal stem cells and their applicability to assess the developmental neurotoxicity

Plasticity and developmental capacity of stem cells have now been established as a promising tool to restore the degenerative disorders. The linearity differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, chondrogenic, osteogenic and even in neuronal subtypes has been demonstrated. The number of xenobiotics such as dexamethasone, insulin, isobutyl 1-methyle xanthine and retinoic acid has been reported for the potential to differentiate hMSCs into neuronal subtypes. But, the applicability of indigenous neurotrophic factor-nerve growth factor (NGF) has not been explored for the purpose. Thus, the present investigations were carried out to study the NGF induced neuronal differentiation of hMSCs. Following the isolation, purification and characterization of hMSCs were allowed to differentiate into neuronal subtypes under the influence of NGF (50 ng/mL). At various concentrations of NGF, the neuronal makers were analysed at both mRNA and protein levels. Cells, exposed with NGF were showing the significant and gradual increase in the neuronal markers in differentiating cells. The magnitude of expression of markers was maximum at day 4 of differentiation. NGF at 50 ng/mL concentration was found to induce neuronal differentiation of hMSCs into neuronal subtypes.

Gene Profiles in a Smoke-Induced COPD Mouse Lung Model Following Treatment with Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The Hbb and Hba, genes with oxygen transport and antioxidant functions, were increased on days 1 and 14. A serine protease inhibitor was also increased at a similar time point after injection of hCB-MSCs. Gene Ontology analysis indicated that the levels of genes related to immune responses, metabolic processes, and blood vessel development were altered, indicating host responses after hCB-MSC injection. These gene expression changes suggest that MSCs induce a regeneration mechanism against COPD induced by cigarette smoke. These analyses provide basic data for understanding the regeneration mechanisms promoted by hCB-MSCs in cigarette smoke-induced COPD.

Apple ethanol extract promotes proliferation of human adult stem cells, which involves the regenerative potential of stem cells

Tissue regeneration using adult stem cells (ASCs) has significant potential as a novel treatment for many degenerative diseases. Previous studies have established that age negatively affects the proliferation status and differentiation potential of ASCs, suggesting a possible limitation in their potential therapeutic use. Therefore, we hypothesized that apple extract might exert beneficial effects on ASCs. The specific objectives were to investigate the proliferative effect of apple ethanol extract on human adipose tissue-derived mesenchymal stem cells (ADSCs) and human cord blood-derived mesenchymal stem cells (CB-MSCs), and identify the possible molecular mechanisms. Apple extract promoted proliferation of ADSCs and CB-MSCs as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Click-iT 5-ethynyl-2′-deoxyuridine flow cytometry assays. In addition, phosphorylation of p44/42 MAPK (ERK), mammalian target of rapamycin (mTOR), p70 S6 kinase (p70S6K), S6 ribosomal protein (S6RP), eukaryotic initiation factor (eIF) 4B and eIF4E was induced stepwise in ADSCs. Furthermore, apple extract significantly induced the production of vascular endothelial growth factor and interleukin-6 in both ADSCs and CB-MSCs. Similarly, apple extract-induced phosphorylation of the mTOR/p70S6K/S6RP/eIF4B/eIF4E pathway was blocked by pretreatment with PD98059, a specific ERK inhibitor. These results indicate that apple extract-induced proliferation of ADSCs under serum-free conditions is mediated by ERK-dependent cytokine production. Moreover, the beneficial effect of apple extract on proliferation of ASCs may overcome the limitation in therapeutic use of stem cells in tissue regeneration and maintenance of stem cell homeostasis.

Effects of hypoxia on proliferation of human cord blood-derived mesenchymal stem cells.

The purpose of our study was to examine the influence of hypoxia on proliferation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). The mononuclear cells were separated by density gradient centrifugation from human umbilical cord blood and then, respectively, cultured under hypoxia (5% O2) or normoxia (20% O2). Their cell morphology, cell surface markers, β-galactosidase staining, cell growth curve, DNA cycle, and the expression of hypoxia-inducible factor-1α (HIF-1α) were evaluated. We found that hypoxia, in part via HIF-1α, improved the proliferation efficiency, and prevented senescence of hUCB-MSCs without altering their morphology and surface markers. These results demonstrated that hypoxia provides a favorable culture condition to promote hUCB-MSCs proliferation in vitro, which is a better way to obtain sufficient numbers of hUCB-MSCs for research and certainly clinical application.

Diet composition transiently modulates proliferative and potency features of human cord blood-derived mesenchymal stem cells

Mesenchymal stem cells (MSC) emerged in the last few years as a promise in regenerative medicine and have been actively tested in several clinical trials worldwide. However, the lack of common standards and a precise definition of MSC preparations remain a major obstacle in research and application. In this study, we compared the effects during culture of two different MSC commercial media (aMEM and SPE-IV) on the proliferative capacities, phenotypic and molecular features in human cord blood derived-MSC lines. Moreover, as miRNA are markers of stem cell multipotency and regulators of somatic cell reprogramming, we performed a miRNome analysis in both conditions. As a result, we observed that SPE-IV promoted a faster growth and modulated stemness and proliferation associated genes such as PDGFRB, p16 and p21. Notably, in aMEM miR-335 and miR-302b, both proposed as putative stemness markers, were upregulated together with miRNAs reported to decrease adipo- and osteogenesis confirming the observed reduced differentiation potential after growth in this condition. Intriguingly, phenotypic divergences were entirely due to culturing conditions and, most importantly, completely transitory since, after medium switch, the cells were able to revert their signatures. Thus, it emerges as crucial keeping constant the experimental settings, starting from culturing conditions, to avoid misleading characterization of stemness and/or potency markers when the eventual goal is unequivocal definition of such parameters for future clinical choice.

Biocompatibility of electrospun graphene oxide–poly(ε-caprolactone) fibrous scaffolds with human cord blood mesenchymal stem cells derived skeletal myoblast

Biocompatibility of electrospun graphene oxide (GO)–poly (ε-caprolactone)-(PCL) composite fibrous scaffolds has been investigated using skeletal muscle cells obtained from human umbilical cord blood (UCB) derived mesenchymal stem cells (CB-hMSCs). Such CB-hMSCs derived human skeletal muscle cells (CB-hSkMCs) adhered, proliferated and formed myotubes when cultured on GO–PCL fibrous scaffold suitable for skeletal muscle tissue engineering applications.

Experimental Study on the Effect of Intravenous Stem Cell Therapy on Intestinal Ischemia Reperfusion Induced Myocardial Injury

The myocyte death that follows intestinal ischemia reperfusion (I/R) injury is a major factor contributing to high mortality and morbidity in ischemic heart disease. The purpose of stem cell (SC) therapy for myocardial infarction is to improve clinical outcomes. The present study aimed at investigating the possible therapeutic effect of intravenous human cord blood mesenchymal stem cells (HCBMSCs) on intestinal ischemia reperfusion induced cardiac muscle injury in albino rat. Thirty male albino rats were divided equally into control (Sham-operated) group, I/R group where rats were exposed to superior mesenteric artery ligation for 1 hour followed by 1 hour reperfusion. In SC therapy group, the rats were injected with HCBMSCs into the tail vein. The rats were sacrificed four weeks following therapy. Cardiac muscle sections were exposed to histological, histochemical, immunohistochemical and morphometric studies. In I/R group, multiple fibers exhibited deeply acidophilic sarcoplasm with lost striations and multiple fibroblasts appeared among the muscle fibers. In SC therapy group, few fibers appeared with deeply acidophilic sarcoplasm and lost striations. Mean area of muscle fibers with deeply acidophilic sarcoplasm and mean area% of fibroblasts were significantly decreased compared to I/R group. Prussion blue and CD105 positive cells were found in SC therapy group among the muscle fibers, inside and near blood vessels. Intestinal I/R induced cardiac muscle degenerative changes. These changes were ameliorated following HCBMSC therapy. A reciprocal relation was recorded between the extent of regeneration and the existence of undifferentiated mesenchymal stem cells.

3D study of capillary network derived from human cord blood mesenchymal stem cells and differentiated into endothelial cell with VEGFR2 protein expression

Background and Aim: New blood forming vessels are produced by differentiation of mesodermal precursor cells to angioblasts that b ecome endothelial cells (ECs) which in turn give rise to primitive capillary netw ork. Human cord blood (HCB) contains large subsets of mononuclear cells (MNCs) that can be differentiated into endothelial-like cells in vitro . Methods: Human mononuclear progenitor cells were purified f rom fresh umbilical cord blood by the expression of CD34 and FLK-1 antigens expressed in both angioblasts and hematopoetic stem cells. The H CB derived mesenchymal stem cells (MSCs) can differentiate into adipocyte, osteocyte, chondrocyte and ECs. Results: In this study, the differentiation of human cord b lood mesenchymal stem cells (hCBMSCs) into endothelial-like cells was ind uced in presence of vascular endothelial growth factor (VEGF) and insulin-like g rowth factor (IGF-1). The differentiated ECs were then examined for their abi lity to express VEGF receptor- 2 (VEGFR2) and von Willebrand factor (vWF). These cells were adopted to grow, proliferate and develop into a capillary network in a semisolid gel matrix in vitro . Conclusion: The capillary network formation in each well of 24 -well plate was found to be 80% in presence of VEGF (40 ng/ml) and IGF-1 (20 ng/ml) of culture media, suggesting that the capillary network format ion is associated with endothelial-like cells derived from hCBMSCs by expression of their markers .

The effects of hedgehog on RNA binding protein Msi1 during the osteogenic differentiation of human cord blood-derived mesenchymal stem cells

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are useful tools for regenerative medicine due to their capacity for self-renewal and multi-lineage differentiation. The appropriate clinical application of MSCs for regenerative medicine requires an integrated understanding of multiple signaling pathways that regulate cell proliferation, stemness and differentiation. However, the potential molecular mechanisms mediating these functions are not completely understood.The effects of hedgehog (Hh) signaling on the osteogenic differentiation of MSCs are still controversial, and the underlying mechanisms are unclear. In the present study, we evaluated the direct effects of Hh signaling on the osteogenic differentiation of hUCB-MSCs and investigated potential downstream regulatory mechanisms responsible for Hh signaling. We observed that Hh signaling acts as a negative regulator of osteogenic differentiation through the suppression of RNA-binding Msi1, which in turn suppresses the expression of Wnt1 and the miR-148 family, especially miR-148b. Moreover, Hh and Msi1 are considered to be potential stemness markers of hUCB-MSCs due to their differentiation-dependent expression profiles. This study provides new insights into mechanisms regulating MSC differentiation and may have implications for a variety of therapeutic applications in the clinic.

A Phase I Study of Human Cord Blood-Derived Mesenchymal Stem Cell Therapy in Patients with Peripheral Arterial Occlusive Disease

Half of patients with critical limb ischemia (CLI) are ineligible for revascularization at diagnosis. The aim of this study was to assess the safety and feasibility of intramuscular human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) therapy in patients with CLI due to atherosclerosis obliterans (ASO) or thromboangiitis obliterans (TAO). A total of eight patients (all male, median age 52 years, range 31∼77) with CLI were enrolled in this phase I trial. All patients were considered ineligible for further revascularization to improve CLI. We injected 1×10(7) hUCB-MSCs per single dose intramuscularly into the affected limb. The primary end points of safety were occurrence of adverse events (procedure-related complication, allergic reaction to hUCB-MSCs, graft-versus-host disease, cardiovascular and cerebrovascular events) and improvement of symptoms/clinical parameters (healing of foot ulcer, ankle-brachial index, and pain-free walking distance). Angiogenesis was measured with conventional angiography and scored by an independent reviewer. There were four adverse events in three patients. One patient, developed whole body urticaria after injection on treatment day, which disappeared after one day of antihistamine treatment. The other adverse events included diarrhea, oral ulceration, and elevation of serum creatinine level; all conditions improved without treatment. Abnormal results of laboratory parameters were not detected in any patients. Three of four ulcerations (75%) healed completely. Angiographic scores increased in three of eight patients. This phase I study demonstrates that intramuscular hUCB-MSC injection is a safe and well tolerated treatment for patients with end-stage CLI due to ASO and TAO.

Generation of Glucose-Responsive, Insulin-Producing Cells from Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells

We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge. These data suggest that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.

Effect of stem cell therapy on amiodarone-induced liver injury in albino rats

Introduction Sustained liver injury causes the development of fibrosis. For patients with end-stage fibrosis (cirrhosis) with subsequent portal hypertension, liver failure, and hepatocellular cancer, liver transplantation is the only effective method of treatment. However, it is associated with several complications and side effects. Aim of the work The present study aimed to determine the possible effect of human cord blood mesenchymal stem cell therapy on liver injury using amiodarone as a model of induced liver damage in albino rats. Materials and methods Twenty-three adult male albino rats were divided into three groups: group I (control) included six rats that were given 0.5ml Tween 80 orally for 2 weeks, group II included 10 rats that were given 5.4mg of amiodarone orally for 2 weeks, and group III included seven rats that were injected with stem cells in the tail vein following confirmation of liver damage and held for 4 weeks before sacrifice. Liver specimens were processed. Sections were subjected to the following stains: H&E, Masson's trichrome, and Prussian blue. Immunohistochemical studies were carried out for CD44 and human APF. Digital image analysis was used to determine the area % of collagen fibers and the optical density of α-fetoprotein-positive cells. The results were compared statistically. Results In group II, congested dilated blood sinusoids were observed. Some hepatocytes showed dark nuclei. Some hepatocytes appeared with dark nuclei and a strong acidophilic cytoplasm; others were ballooned. Mallory bodies were observed. Some portal areas showed intense mononuclear cellular infiltration. Extensive collagen fibers existed around some central veins and portal tracts and increased area % of collagen fibers was observed. Most histological findings were improved in group III. In addition, multiple-positive α-fetoprotein immunostained cells were detected and proved morphometrically by their increased mean optical density in comparison with group II. Conclusion It can be concluded that cord blood mesenchymal stem cell therapy induces amelioration in morphological changes associated with amiodarone-induced liver injury, provided therapy is initiated early in the development of the injury.

Effect of Stem Cell Therapy on Amiodarone Induced Fibrosing Interstitial Lung Disease in Albino Rat

The fibrosing forms of interstitial lung disease (ILD) are associated with significant morbidity and mortality. ILD may be idiopathic, secondary to occupational, infection, complicate rheumatic diseases or drug induced. Efficacy of antifibrotic agents is as far as, limited and uncertain. No effective treatment was confirmed for pulmonary fibrosis except lung transplantation. The present study aimed at investigating the possible effect of human cord blood mesenchymal stem cell (MSC) therapy on fibrosing ILD. This was accomplished by using amiodarone as a model of induced lung damage in albino rat. Seventeen adult male albino rats were divided into 3 groups. Rats of amiodarone group were given 30 mg/kg of amiodarone orally 6 days/ week for 6 weeks. Rats of stem cell therapy group were injected with stem cells in the tail vein following confirmation of lung damage and left for 4 weeks before sacrifice. Obstructed bronchioles, thickened interalveolar septa and thickened wall of pulmonary vessels were found and proved morphometrically. Reduced type I pneumocytes and increased area% of collagen fibers were recorded. All findings regressed on stem cell therapy. Cord blood MSC therapy proved definite amelioration of fibrosing interstitial lung disease provided therapy starts early in the development of the pathogenesis.

In vivo hepatic differentiation potential of human cord blood-derived mesenchymal stem cells

Although recent studies have demonstrated the in vitro hepatic differentiation potential of mesenchymal stem cells (MSCs), the evidence supporting the in vivo engraftment of MSCs, hepatic differentiation and improvement of hepatic function is still lacking. We investigated in vivo hepatic differentiation potential and therapeutic effect of cord blood derived-MSCs (CBMSCs) transplantation in a cirrhotic rat model. CBMSCs (2 x 10(6)) were infused in Wistar rats with thioacetamide-induced chronic liver injury. Biochemical markers, liver fibrosis and engraftment of CBMSCs were assessed. Infused CBMSCs were detected in the perivascular or fibrous region of the liver and did not acquire mature hepatic phenotypes. There was no difference in biochemical markers and in the area of liver fibrosis between the experimental and placebo groups. After infusion of CBMSCs in our experimental cirrhotic rat model we did not observe an improvement of liver function and liver fibrosis. Inversely, CBMSCs could have a pro-fibrogenic potential suggesting that a cautious approach is required in future research.

Effects of Human Cord Blood Mesenchymal Stem Cells on Cutaneous Wound Healing in Lepr db Mice

In the present study, we used the diabetic mouse as a model of delayed wound healing to investigate the effects of human cord blood mesenchymal stem cells (CB-MSC) on wound healing. A delayed wound healing model was used by db/db mice. Study models were divided by an injection of human CB-MSC with phosphate buffered solution (PBS) by a different method. One was a locally topical injection, the other was a systemic injection via the end tail vein. Both models were treated with 2.0 × 10(6) CB-MSC after an 8-mm full thickness defect was made by a skin punch biopsy on the back. We evaluated the wound size, transforming growth factor (TGF)-β, and vascular endothelial growth factor histologic evaluation, and vessel counts. Engraft of CB-MSC was detected by an antihuman antibody. Wound healing was accelerated in the experimental group in the topical injection model with statistical significance on the 6th, 9th, and 12th day (P < 0.05). In the systemic injection model, wound healing was completed from the 9th day, but there was no statistical significance. TGF-β increased in the first week and decreased in the third week in the experimental groups of both models. But there were opposite results in the control groups of both models. The statistical differences were found in first and third week in topical injection and in the third week in systemic injection (P < 0.05). Vascular endothelial growth factor increased in all groups and in all models as the wound healing. But statistical significance did not show between all experimental and control groups. Anti-human antibody immunochemical staining was positive in the wound. We concluded that CB-MSC had a positive effect on wound healing. Statistically significant results were noted in the topical injection model. We also reported good effects on the systemic injection model, although we did not find any statistical significance. CB-MSC may influence wound healing by TGF-β.