Human Umbilical Mesenchymal Stem Research Articles

A Microfluidic Platform for Multi-Antigen Immunofluorescence Assays

Traditional cell-based assays such as cell immunoassay that utilizes plastic (chamber slides, dishes, microtiter plates), Magnetic bead, enzyme-linked immunsorbent assays (ELISA) [1], FACS cell sorting is labor intensive, time consuming, and requires a large numbers of cells or reagents. In this report, a microfluidic device integrated with cell culture, washing, fixation, and antigen-antibody reaction is presented for high-throughput immunoassay. Using this microfluidic device, each assay can be performed on a small number of cells and nanolitre or picolitre of reagents, this is particularly beneficial for rare or expensive cell types such as stem cells, or flow sorted cell populations. The capability of the microfluidic device was demonstrated for seeding human umbilical cord blood mesenchymal stem cells (UC-MSCs) in chambers and detecting the expression of surface markers (CD34, CD44, CD45, CD73, CD105, HLA-DR) by immunofluorescence assay.

The mixed human umbilical cord blood-derived mesenchymal stem cells show higher antitumor effect against C6 cells than the single in vitro

Objective: It is difficult for the treatment of neurologic tumors with current therapies, including glioma. Despite the advances in cancer therapeutics, the outcomes in these patients remain poor and, therefore, new modalities are required. Recent findings have demonstrated that human umbilical cord blood mesenchymal stem cells (UCB-MSCs) have the potential to inhibit glioma cell growth in vitro. Caspase-3 plays a critical role as an executioner of apoptosis and the level of caspase-3 expression determines the degree of apoptosis in cancer cell lines. CD133 (+) glioma displays a strong resistance to chemotherapy. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) are important cytokines in the generation of antitumor immunity.Methods: UCB-MSCs were harvested by density gradient separation. Green fluorescence protein stable C6 cells were established. Cytotoxicity was detected by visual survival cell assay, and caspase-3 activity was assessed using immunohistochemistry staining and western blot. Flow cytometry was used to test CD133 positive C6 cells. The concentrations of IL-2 and IFN-gamma proteins secreted from UCB-MSCs were then quantified using enzyme-linked immunosorbent assay. The cytotoxicities of IL-2 alone, IFN-gamma alone, and combination of IL-2 and IFN-gamma were compared against malignant glioma cells.Results: We noted a significant cytotoxicity of UCB-MSCs for malignant glioma cells. In addition, the toxicity of mixed UCB-MSCs was significantly higher than that of single UCB-MSCs for C6 glioma cells. Capase-3 levels in UCB-MSCs treatment at an effector/target (E/T) ratio of (5+5):1 were higher than those at an E/T ratio of 10:1. On the contrary, there was no change in the protein expression of capase-3 at an E/T ratio of 0:1. Immunohistochemistry staining and western blot revealed that the expression of capase-3 was higher in mixed UCB-MSCs treatment, when compared with single UCB-MSCs. We identified reductions of approximately 39 and 73% in the number of CD133 positive C6 cells treated with the single (10:1) and the mixed [(5+5):1] UCB-MSCs respectively as compared to the control group (0:1) in transwell inserts. However, the mixed UCB-MSCs secreted more immune response-related proteins (IL-2 and IFN-gamma) than the single UCB-MSCs. Combination of IL-2 and IFN-gamma might prove more cytotoxic on target cells than IL-2 alone and IFN-gamma alone.Discussion: The data collected herein confirm for the first time that the mixed UCB-MSCs were shown to have more cytotoxic effects than the single UCB-MSCs through increasing the expression of caspase-3 and decreasing the expression of CD133 in C6 glioma cells. In addition, the mixed UCB-MSCs secrete more immune response-related proteins (IL-2 and IFN-gamma) than the single UCB-MSCs. Combination of IL-2 and IFN-gamma was shown to have more cytotoxic effects than IL-2 alone and IFN-gamma alone. These results demonstrate that the mixed UCB-MSCs are a potential new therapeutic agent for glioma.

580. CXCR1 Mediated Migratory Capacity of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells toward Gliomas

580. CXCR1 Mediated Migratory Capacity of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells toward Gliomas

Abstract 3322: Downregulation of XIAP by cord blood stem cells is associated with inhibition of heat shock protein 90 in glioblastoma

Abstract Heat shock protein 90 (HSP90) is highly expressed under stressful conditions in eukaryotic cells showing chaperone activity including the suppression of protein aggregation and signaling pathways. HSP90 is upregulated in various human tumors, including glioblastoma, and targeting its function might provide new therapeutic opportunities. Previously, we have shown that human umbilical cord blood-derived mesenchymal stem cells (hUCBSC) downregulate XIAP in glioblastoma cells in vitro and in vivo. In the present study, we report that downregulation of XIAP by hUCBSC is associated with inhibition of HSP90. Under in vivo conditions, hUCBSC induce apoptosis by downregulation of XIAP in which XIAP migrates from the cytosol to the nucleus in mice brain xenografts. Immunoprecipitation experiments proved that SMAC is associated with Cytochrome C and XIAP in the mitochondria, indicating mitochondrial apoptosis. Simultaneously, cytosolic HSP90 is downregulated and co-localized with TNFR2 in hUCBSC-treated tumors, proving that TRAIL-mediated apoptosis is taking place in hUCBSC-treated tumor xenografts. Also, expression of mitochondrial HSP90 (TRAP-1) is downregulated in glioma cells. The downregulation of XIAP by hUCBSC is shown to be under the transcriptional control of SP1 under both in vitro and in vivo conditions. These results have been confirmed by using shRNA for XIAP. Taken together, these results prove that hUCBSC are efficient in the inhibition of HSP90, and hUCBSC have the potential to be therapeutic tools for the treatment of glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3322. doi:10.1158/1538-7445.AM2011-3322

Experimental study on the tropism of umbilical cord blood mesenchymal stem cells for gastric carcinoma xenografts

Objective To investigate the possibility of human umbilical cord blood mesenchymal stem cells' migrating to the gastric carcinoma xenografts. Methods Gastric cancer cells SGC-7901 were injected subcutaneously into one side of the inguinal groove in the hairless mice to establish the animal model.Gastric cancer-bearing mice were divided randomly into two groups and five in each group (n =5). Then mesenchymal stem cells and the fibroblast HFL-Ⅰ labeled with fluorescent dye SP-DiI were injected at the opposite side. Ten days later, the mice were put to death, and the gastric carcinoma xenografts, liver, spleen,lung and the tissue of injected point were obtained. Cryosections from frozen tissues were processed for fluorescent microscopy and the distribution of MSC and fibroblasts in different organs were observed. Adjacent sections were stained with HE. Results Human umbilical cord blood mesenchymal stem cells were mainly distributed in gastric carcinoma xenografts (0.0150±0.0079), but were found only a little in liver (0.0010±0.0005), spleen (0.0015±0.0012), lung (0.0014±0.0008) and injected point (0.0043±0.0039). Fibroblasts were mainly observed in the tissue of injected point, but none in other tissues (P ＜0.01). Conclusion Human umbilical cord blood mesenchymal stem cells have the tendency of migrating to tumor sites, suggesting that it can be hopefully used in the diagnosis and treatment of gastric cancer. Key words: Mesenchymal stem cells; Gastric carcinoma; Animal model; Tropism

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Upregulate Myelin Basic Protein in Shiverer Mice

Human umbilical cord blood is a rich source of pluripotent mesenchymal stem cells and possesses significant advantages over other stem cell sources such as the embryo and bone marrow. In the present study, we aimed to investigate the potential of human umbilical cord blood-derived pluripotent mesenchymal stem cells (hUCB) to myelinate the axons of shiverer mice brains. We also investigated the effect of hUCB treatment on regulation of myelin basic protein in vitro in PC-12 cells, which are normally not myelinated. The results of our study clearly demonstrated that hUCB survive and migrate in vivo and has the potential to myelinate shiverer mice brains. The expression level of myelin basic protein, a major component of the myelin sheath, has been significantly increased in vivo and in vitro as revealed by Western blot, reverse transcription (RT)-polymerase chain reaction, immunohistochemistry, immunocytochemistry, and fluorescent in situ hybridization results. Further, transmission electron microscopic images of hUCB-treated shiverer mice brains showed several layers of myelin around the axons compared with a thin and fragmented layer of myelin in untreated animals. Moreover, the frequency of shivering was diminished 1 month after hUCB treatment in shiverer mice. Our results strongly indicated that hUCB transplantation could be an effective means of treating demyelinating or hypomyelinating disorders.

137P In vitro differentiation of human umbilical vein mesenchymal stem cells into hepatocyte-like cells

Human umbilical vein, as a readily available source of stem cells, is one of the good alternative source for mesenchymal stem cells. Human umbilical cord vein mesenchymal stem cells (UVMSCs) have been recently isolated and demonstrated the ability to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this study, we have investigated whether human UVMSC are also able to differentiate into hepatocyte-like cells. Hepatic differentiation was done with a 2-step protocol and with the use of hepatocyte growth factor and oncostatin M. The cells were cultured with hepatocyte growth factor (HGF) and oncostatin M (OSM). During 4 weeks of induction most of the cells displayed a cuboidal morphology. Immunological analysis indicated that UVMSCs-derived hepatocyte-like cells expressed liver-specific protein markers such as albumin (ALB) and cytokeratin-18 (CK-18). The hepatocyte-like cells also displayed several characteristics of hepatocytes, including expression of transthyretin, glucose-6-phosphatase (G6P), cytokeratin-8,18 (CK-8,18), alpha-fetoprotein (AFP), hepatocyte nuclear factor-3β (AFP) and ALB. The result of indocyanine green (ICG) cell uptake as a test substance to evaluate hepatocyte-like cells function was positive for differentiated cells. Glycogen storage was examined by periodic acid–Schiff (PAS) staining and accumulation of intracellular glycogen were detected in hepatocyte-like cells. Based on these observations it can be concluded that UVMSCs encompass suitable hepatogenic potential which may provide a source of stem cells for cell therapy in liver diseases.

The experimental study on differentiation of human umbilical cord blood mesenchymal stem cells into neural cells induced by lithium chloride

Objective To study the possibility of inducing human umbilical cord blood mesenchymal stem cells(MSCs) to differentiate into neuron-like cells by lithium chloride(LiCl) in vitro.Methods Human umbilical cord blood was collected from mature neonates.All samples were obtained sterilely with 20 U/ml heparin.The cord mononuclear cells were isolated with lymphocyte separation medium(density 1.077 g/ml), then purified by wall sticking screening and expanded with slight sugar DIEM containing 15% FBS.The third passage of the expanded MSCs were pre-inducted with DIEM containing 15% FBS and 20 ng/ml bFGF for 24 hours, then induced with DIEM without serum but 3 mol/L LiCl for 6 days in group A.The MSCs were induced with DIEM containing 3 mol/L Licl for 7 days in group B.The MSCs were normally cultured with DIEM containing 15% FBS in group C.The morphological changes of the cells were observed under phase contrast microscope.The neuron specific markers containing neuron specific enolase(NSE), microtubule associated protein2(MAP2) and glial fibrillary acid protein(GFAP) were evaluated by indirect immunocyto-chemistry staining.Results After inducted for 3 days, morphological changes were observed obviously in group A and B.6 days later, the differentiated cells showed typical neuronal morphology.The expression of NSE and MAP2 were positive for the majority cells in group A and B, and that of group A[(73.6 ± 7.8)%, 75.5 ± 8.5)% respectively]were obviously higher than group B[(31.0 ± 4.3)%,(33.5 ± 5.0)% respectively], few expressed GFAP in both groups.Conclusion The combination of LiCl and growth factor may induce the human umbilical cord blood MSCs into neuron-like cells in vitro. Key words: Human umbilical cord blood; Mesenchymal stem cells; Lithium chloride; Neuion; Differentiation

Abstract 3323: Induction of apoptosis in glioma cells by downregulation of X-linked inhibitor of apoptosis protein (XIAP)

Abstract XIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the family of apoptosis inhibitors. XIAP blocks apoptosis by inhibiting both the initiator (caspase 9) and effector (caspase 3) caspases, and therefore, prevents cell death induced by various triggers. One mechanism through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of XIAP. When XIAP is overexpressed, cancer cells are rendered resistant to apoptosis, both intrinsically and in response to chemotherapy and radiotherapy. It is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth, and survival of malignant cells. In order to downregulate XIAP in glioma cells, we used human umbilical cord blood mesenchymal stem cells (hUCB) and siRNA of XIAP (siXIAP). We observed the effect of both hUCB and siXIAP on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In either case, proliferation of the glioma cells was significantly inhibited. Both treatments increased cytotoxicity of glioma cells and led to cell death. However, cytotoxicity is more significant in siXIAP treatments compared to hUCB cocultures. Both hUCB and siXIAP induced TNF-alpha-mediated apoptosis in glioma cells, which was confirmed by TUNEL assay, FACS analyses and immunoblotting. Apoptosis is characterized by loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic protein BAD. Cell death of glioma cells is marked by downregulation of Akt and phospho-Akt molecules. Taken together, our results indicate the therapeutic potential of XIAP and hUCB in treating malignant gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3323.

In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs' in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.

Experiment research of the function of human umbilical cord blood mesenchymal stem cells in the regeneration of rat's sciatic nerve

Objective To evaluate the efectiveness of using mesenchymal stem cells(MSCs)derived from HUCB(human umbilical cord blood)to a tissue engineered bioartificial nerve on bridging a 1 0 mm sciatic nerve gap.Methods The cord blood mononuclear cells were isolated by lymphocyte separation medium,purified and expanded with MesencultTM medium and acidic environment to produce adherent layer(MSCs).Thirty SD female rats were randomly divided into three groups.Group A:Human amnion tubes were seeded by HUCBMSCs together with fibrin sealant.Group B:Human amnion tubes were seeded only with fibrin sealant.group C:autografts.9 weeks later,a series of examinations were performed which included morphological,sciatic nerve function index,weight of gastrocnemius,histological staining and immunostaining of S100.Results The HUCB-derived mononuclear cells,when seeded in specific medium,gave rise to adherent cells (MSCs).At 9 weeks after the operations,all the examinations of group A was better than group B(P＜0.05).Conclusion HUCBMSCs can be isolated,purified, cultivated and expanded MesencultTM medium.HUCBMSCs can promote the nerve to regenerate in reparing the sciatic nerve gap. Key words: Umbilical cord blood; Mesenchymal stem cells; Sciatic nerve; Nerve regeneration; Tissue engineering

In vitro differentiation of human umbilical cord blood mesenchymal stem cells into neural cells

Objective To isolated and culture vitro human umbilical cord blood mesenehymai stem cells (MSCs) in vitro and observe the growth characteristics and the nerve cell differentiation induced conditions of umbilical cord blood mesenchymal stem cells in vitro.Methods Umbilical cord blood MSCs were induced into nerve cells,and the morphological changes of MSCs were observed in vitro.At the 5th day when umbilical cord blood MSCs were induced,neuronal cell-specific marker was assayed by immunofluorescence staining; and the control group cultured with low sugar DMEM medium were compared.Results Umbilical cord blood MSCs in the induced group grew and spreaded,Racial dendrite had formed and connected with each other.NSE immunoreactive positive ceils were found in this group,the characteristics of neurons were shown.But in the control group MSCs did not express this neuron morphological character; and the result of NSE immunofluorescence was negative.Conclusion The umbilical cord blood MSCs may be induced to differentiate into neural cells by chemical methods directed in vitro,which can be used in nerve repairment and regeneration and other fields. Key words: Umbilical mesenehymal stem cell; Neural ceil; Differentiation

Study on the expansion of umbilical cord blood mononuclear cells supported by human umbilical cord blood mesenchymal stem cells

Objective To explore the effects on umbilical cord blood mesenchymal stem cells (UCB-MSCs) combined with exogenous cytokines in supporting the expansion of umbilical cord blood mononuclear cells(UCB MNCs). Methods Mesenchymal stem cells (MSCs) were isolated from umbilical cord blood, and then were detected the cellular surface antigens. The UCB-MNCs were euhured in a serum-free system for 18 days. On day 0, 7, 10, 14 and 18, total mononuclear cells、CD34 cells、CD133 cells、cellular cycle and isolated and cultured from umbilical cord blood were negative for CD34 and CD133, but positive for CD29, The expansion combined with UCB-MSCs and exogenous cytokines had better effect than another groups. The above parameters reached the peak values on day 10. The culture system could maintain at least 18 efficiently support the expansion of UCB-MNCs. Key words: Umbilical cord blood; Cytokines; Mesenehymal stem cells; Cell culture

Growth of Human Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells on the Terpolyester Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate)

As a new member of the polyhydroxyalkanoate (PHA) family, the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) was evaluated for its biocompatibility for human umbilical cord Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). More WJ-MSC adhesion and proliferation were observed on PHBVHHx film compared with films made of poly(L-lactic acid) (PLA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Higher DNA synthesis by WJ-MSCs was detected on PHBVHHx film than on PLA, PHBV and PHBHHx films. PHBVHHx film had a rougher surface and more adsorption of extracellular matrix (ECM) proteins including collagen I, fibronectin and vitronectin, compared with PLA, PHBV and PHBHHx films. PHBVHHx film was also more hydrophobic than PLA and PHBV. These results demonstrated that PHBVHHx could be a promising biomaterial in medical implant applications for supporting the growth of cells, including WJ-MSCs.

Human umbilical cord blood mesenchymal stem cells differentiate into functional hepatocypte-like cells in vitro

Human umbilical cord blood mesenchymal stem cells differentiate into functional hepatocypte-like cells in vitro