ObjectiveTo study the characteristics of the human umbilical cord perivascular cells (HUCPVC) isolated from human first trimester umbilical cord perivascular layer tissues and the differentiation into islet-like cell clusters in vitro. MethodsThe HUCPVC derived from human first trimester umbilical cord which was donated by the volunteers were isolated and subcultured. The surface markers such as stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, OCT-4, TRA-1-60, and TRA-1-81 were detected by immunohistochemical method. The first trimester HUCPVC were induced to embryoid bodies (EB)-like cell aggregations and islet-like cell clusters in vitro through a simple stepwise culture protocol (5 steps). The expressions of specific markers[α-fetoprotein (AFP), Nestin, and smooth muscle actin (SMA)] were measured by immunohistochemical method; and the ability of glucose-stimulated insulin secretion was analyzed. ResultsThe first trimester HUCPVC were successfully isolated and could be passaged steadily more than 10 generations, which expressed SSEA-3, SSEA-4, OCT-4, TRA-1-61, and TRA-1-81. The first trimester HUCPVC were successfully induced into EB-like cell aggregations and islet-like cell clusters. The EB-like cell aggregations could express markers of three germ lineages:AFP, Nestin, and SMA. The islet-like cell clusters could release insulin significantly in response to elevated concentrations of glucose in vitro (t=7.444, P=0.002). The insulin contents were (23.2±5.3) mU/L and (7.0±0.5) mU/L in high and low glucose media, respectively. ConclusionThe first trimester HUCPVC has the ability to differentiate into islet-like cell clusters which can secret insulin in vitro.
Objective To investigate the method and conditions of isolation，proliferation of multipotent mesenchymal stem cells（MSCs）from human umbilical cord blood in vitro, and to induce osteogenic and adipogenic differentiation directly for identification. Methods Human umbilical cord blood was collected in asepsis condition, isolated by density gradient centrifugation,or sedimented red cell with methylcellulose, and then the same centrifugation was done, or obtained by negative immunodepletion of CD34+. These isolated mononuclear cells were used to carry on plastic adherent culture. To obtain single cellderived colonies, these cells were proliferated clonally in medium which consists of L-DMEM orMesencultTM medium and 10% fetal calf serum（FCS） respectively, then their differentiation potentiality to osteoblasts and lipoblasts was tested. Results The mononuclear cells isolated by sedimented and centrifugated way cultured in MesencultTM medium and 10%FCS were most available. These adhesive cells could become obviously short rodshape or shuttle-shape cells after 5-7 days.The colonies form well in 3rdpassage cells. The mononuclear cells obtained by onlycentrifugalized in density gradient were hard to form colony, isolated by immunomagnetic beads were hard to culture. The surface antigens of these colonies cells presented CD29, CD59, CD71 but not CD34,CD45 and HLADR etc. The colony cells differentiating into osteoblasts that produce mineralized matrices, stained by alizarin red, and differentiating into adipocytes that accumulate lipid vacuoles, stained by oil red. Conclusion MSCs can be isolated from human umbilical cord blood and proliferate it in vitro. The way that mononuclear cells are sedimented red cell by methylcellulose and cultured by MesencultTM medium and 10% FCS is the valid method of isolation. Proliferation colonies cells present matrix cell immunophenotypes, and candifferentiate into osteoblasts and adipocytes.
Objective To investigate the feasibility of differentiating human umbilical cord blood stem cells into hepatocytes. Methods Thirty-six BALB/c nude mice were randomly divided into experimental group and control group(18 in each of the group), and experimental group was again randomly divided into group A， B and C (six in each of the group). The mice in experimental group and control group were exposed to 350 cGy radiation produced by 60Ｃｏ. After 3 h, karyocytes at different concentrations in the fresh human umbilical cord blood were injected into the mice in experimental group A, B, C via their tail veins, and the equal volume of normal sodium (NS) was also injected into control group via tail veins. After one month, carbon tetrachloride (CCl4) was injected into experimental group A, B and control group via abdominal cavity, and the equal volume of normal sodium was injected into experimental group C. After two months, immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the expressions of human cytokeratin-18 (CK18), cytokeratin-19 (CK19) and albumin (ALB) in liver tissues of all mice. Results The expressions of CK18, CK19 and ALB in injured liver tissues were all positive, and the expressions of experimental group B were higher than those of experimental group A （P＜0.05）, but the expressions of CK18, CK19 and ALB in the liver tissues of control group and experimental group C, whose were not injured with CCl4, were all negative. Conclusion Human umbilical cord blood-derived stem cells can differentiate into hepatocytes and express ALB under special microenvironment after liver injured by CCl4 , and the expression level of ALB maybe directly related to the number of human umbilical cord blood stem cells.
ObjectiveTo investigate the differentiation potential of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes induced by rat fibrotic liver tissue extracts. MethodsLiver fibrosis was induced in the Sprague Dawley rats (weighting, 180-220 g) by repeated intraperitoneal injections of 3% thioacetamide-saline at a dose of 200 mg/kg twice a week for 4 weeks;fibrotic liver tissues were used to prepare liver homogenate supernatants. The HUCMSCs at passage 3 were cultured in DMEM/F12 with 10% fetal bovine serum (FBS) (control group) and in DMEM/F12 with 10%FBS and 50 g/L liver homogenate supernatants (experimental group) for 7 days. The morphological changes of the cells were recorded;the protein levels of cytokeratin 18 (CK18), alpha fetoprotein (AFP), and CYP3A4 were measured using Western blot. The glycogen storing ability of the cells was detected by periodic acid-schiff (PAS) staining. Furthermore, the synthesis of albumin (ALB) and blood urea nitrogen (BUN) was measured. ResultsIn experimental group, after 1 day of induction, the stem cells of fusiform shape began to lose sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with round and irregular shape at 7 days. Positive expressions of AFP, CK18, and CYP3A4 were observed in the experimental group, but negative expression in the control group. The concentrations of BUN and ALB were (0.43±0.07) mmol/L and (8.08±0.41) μg/mL in the control group and were (2.52±0.20) mmol/L and (41.48±4.11) μg/mL in the experimental group, showing significant differences (t=24.160, P=0.000;t=19.810, P=0.000). PAS staining results showed navy blue nucleus and lavender cytoplasm in the control group, but dark purple cell body and visible nucleus in the experimental group. ConclusionHUCMSCs could differentiate into hepatocyte-like cells induced by rat fibrotic liver tissue extracts, which have hepatocyte biomarkers (AFP, CK18, and CYP3A4) and hepatocyte-specific functions of glycogen storage, urea production and ALB secretion, so they could partially replace the function of hepatocytes, that may be one of the therapeutic mechanisms of stem cell transplantation.
ObjectiveTo explore the involvement of miR-126 and the role of mammalian target of rapamycin (mTOR)/hypoxia-induced factor 1 α (HIF-1 α) pathway in regulating human umbilical cord mesenchymal stem cells (hUCMSCs) exosomes (Exo) on vascular endothelial growth factor (VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells (HRECs). MethodsThe hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1% oxygen concentration. The cell model of high glucose and low oxygen was established. After modeling, divided HRECs into Exo group, phosphate buffer saline (PBS) group, PBS+anti-miR126 group, Exo+anti-miR126 group, PBS+anti-mTOR group, and PBS+anti-HIF-1 α group. High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100 μg/ml hUCMSC Exo. PBS+anti-mTOR group, PBS+anti-HIF-1 α group: 500 nmol/L mTOR inhibitor ADZ2014, 25 μmol/L HIF-1 α inhibitor YC-1 pretreatment for hREC 12 h, and then co-culture with PBS after High-glucose and hypoxia-induced. PBS+anti-miR126 group, Exo+anti-miR126 group: miR-126 LNA power inhibitor probe was transfected with high glucose, and co-cultured with PBS and hUCMSC Exo 6 h after transfection. Real-time polymerase chain reaction (qPCR) measured miRNA-126 expression levels in PBS, and Exo groups for 0, 8, 16 and 24 h. After 24 hof co-culture, the levels of mTOR and HIF-1 α in the cells of PBS and Exo groups were detected by immunofluorescence, Western blot and qPCR, respectively. Western blot, qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1 α groups. The expression of VE GF-A, mTOR, and HIF-1 α mRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR. Comparison between two groups was performed by t-test; one-way ANOVA was used for comparison between multiple groups. ResultsAt 0, 8, 16 and 24 h, the relative mRNA expression of miR-126 gradually increased in the Exo group (F=95.900, P＜0.05). Compared with the PBS group, The mTOR, HIF-1 α protein (t=3.466, 6.804), mRNA in HRECs in the Exo group, VEGF-A mRNA expression (t=8.642, 7.897, 6.099) were all downregulated, the difference was statistically significant (P＜0.05). The relative expression level of VEGF-Aprotein (t=3.337, 7.380) and mRNA (t=8.515, 10.400) was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1 α+PBS group, differences were statistically significant (P＜0.05). The relative expression of VEGF-A, mTOR, and HIF-1 α mRNA was significantly increased in the cells of the Exo+anti-miR126 group, the differences were all statistically significant (t=4.664, 6.136, 6.247; P＜0.05). ConclusionsmiR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1 α pathway.
ObjectiveTo observe the growth characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) cultured on the alginate gel scaffolds and to explore the feasibility of hUCMSCs-alginate dressing for wound healing. MethodshUCMSCs were separated from human umbilical cords and cultured in vitro. After the 4th passage cells were co-cultured with alginate gel (experimental group), the cell growth characteristics were observed under the inverted phase contrast microscope. Vascular endothelial growth factor (VEGF) content was measured and the number of cells was counted at 0, 3, 6, and 9 days after culture; and the cell migration capacity was observed. The hUCMSCs were cultured without alginated gel as control. The model of full-thickness skin defects was established in 32 8-weekold Balb/c male mice and they were randomly divided into 4 groups (n=8): wounds were covered with hUCMSCsalginate gel compound (MSC-gel group), cell supernatants-alginate gel compound (CS-gel group), 10% FBS-alginate gel compound (FBS-gel group), and 0.01 mol/L PBS-alginate compound (PBS-gel group), respectively. Wound healing rates at 5, 10, and 15 days were observed and calculated; and the wound tissues were harvested for histological and immunohistochemical staining to assess new skin conditions at 15 days after operation. ResultshUCMSCs grew well with grape-like proliferation on the alginate gel, but no cell migration was observed at 7 days after cultivation. VEGF expression and cell number in experimental group were significantly less than those in control group at 3 days（P<0.05）; then they gradually increased, and VEGF expression and cell number were significantly more than those in control group at 9 days (P<0.05). The wound healing rates of MSC-gel and CS-gel groups were significantly higher than those of FBSgel and PBS-gel groups at 5, 10, and 15 days (P<0.05). The squamous epithelium, fibroblasts, sebaceous glands, capillaries and VEGF expression of the new skin in MSC-gel and CS-gel groups were significantly more than FBS-gel and PBS-gel groups (P<0.05). But there was no significance between MSC-gel and CS-gel groups (P>0.05). ConclusionhUCMSCs can continuously express VEGF in alginate gel, which is necessary for wound healing. The hUCMSCs-alginate compound is probably a good wound dressing.
Objective To observe the effects of cryopreservation and resuscitation on the biological characteristics of mesenchymal stem cells (MSCs) derived f rom human umbilical cord blood. Methods MSCs were isolated and cultured f rom human umbilical cord blood in vitro. The cells were passaged , and the third generation of MSCs were cryopreserved in－196 ℃ liquid nitrogen for 4 weeks with cryopreservation medium , which contained 10 % dimethyl sulfoxide (DMSO) and 90 % fetal calf serum ( FCS) . The morphology , proliferation and differentiation of MSCs were investigated and compared with those of MSCs before cryopreservation. Results There was no significant difference of morphology between pre-cryopreserved MSCs and the ones af ter resuscitation. It was observed that all MSCs were spindle-shaped and showed adherence growth characteristic before and af ter cryopreservation. The cell growth curves of MSCs were also similar before and af ter cryopreservation. Even though the curve of resuscitated MSCs descended a little as compared with that of pre-cryopreserved MSCs , there was no significant difference ( Pgt; 0. 05) . After 2-week adipocytic differentiation induction , fat drops could be found in the kytoplasm of MSCs and they were red when stained with oil-red O staining , which suggested that MSCs could be induced and differentiated into adipocytes. Af ter 4-week osteoblastic differentiation induction , MSCs could be induced and differentiated into osteoblasts , and calcium node showed black when stained with Von Kossa staining. There were no significant changes of the differentiating ability of MSCs into adipocyte and osteoblast before and after cryopreservation. Conclusion MSCs derived from human umbilical cord blood maintains their biological characteristics af ter cryopreservation and resuscitation.
Objective To investigate the effect of blood microenvironment of rats with hepatic fibrosis on differentiation of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes and its mechanisms. Methods Eighteen male adult Sprague Dawley rats [weighing, (200±20) g] were used, liver fibrosis was induced in 12 rats by repeated intraperitoneal injections of thioacetamide. The serum was separated after successful model preparation, and the serum of 6 normal rats was collected. ELISA assay was used to detect the concentrations of epidermal growth factor (EGF), hepatocyte growth factor (HGF), oncostatin M (OSM), and basic fibroblastic growth factor (bFGF). Passage 3 HUCMSCs were divided into 3 groups: cells were cultured for 7 days in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from rats with hepatic fibrosis (group A), in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from normal rats (group B), and in DMEM/F12 containing 10% fetal bovine serum (group C). The morphological changes of the cells were observed. The expressions of α-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunofluorescence. The protein levels of albumin (ALB), tryptophan 2, 3-dioxygenase (TPH2), and CYP3A4 and MAPK/ERK signal pathway protein (P-ERK) were detected using Western blot. The content of blood urea nitrogen (BUN) was measured by diacetyl m onoxime method. Results HE staining showed that the liver tissue of rats was in accordance with the change of fibrosis, indicating successful model preparation. In serum of normal rats and rats with hepatic fibrosis, the concentrations of EGF were (21.42±0.32) pg/mL and (17.57±0.31) pg/mL respectively, showing significant difference (t=14.989, P=0.000); the concentrations of OSM were (129.96±0.65) pg/mL and (98.44±1.32) pg/mL respectively, showing significant difference (t=37.172, P=0.000); the concentrations of HGF were below the detection limit and (1.03±0.12) ng/ mL respectively; and the concentrations of bFGF were lower than the detection limit in both groups. No morphological changes of cells were observed in both groups at 7 days, and there was no significant difference between groups. At 7 days after culture, the cells in group A could express human hepatocyte biomarkers of AFP, CK18 and hepatocyte-specific-function proteins of ALB, TPH2, and CYP3 A4 while cells in groups B and C did not. Western blot showed that cells in each group could express P-ERK protein. The relative level of P-ERK protein in group A was significantly higher than that in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The BUN concentration of group A [(0.74±0.07) mmol/ L] was significantly higher than that of groups B [(0.40±0.04) mmol/ L] and C [(0.38±0.04) mmol/L] (P < 0.05), but no significant difference was shown between groups B and C (P > 0.05). Conclusion Under the condition of hepatic fibrosis, the level of HGF will increase while EGF and OSM will decrease. The formed blood microenvironment will activate MAPK/ERK signal pathway in HUCMSCs, induce them differentiate into hepatocytes.
ObjectiveTo investigate the safety and efficacy of human umbilical cord mesenchymal stem cells (MSCs) by intra-articular injection for degenerative knee osteoarthritis. MethodsBetween January 2015 and January 2016, 36 patients with moderate or severe degenerative knee osteoarthritis were randomly devided into 2 groups (n=18). Intra-articular injection of 2.5-3.0 mL human umbilical cord MSCs suspension containing (2-3)×107 cells was performed once a month for 2 times as a course of treatment in the cell treatment group; sodium hyaluronate by intra-articular injection was used once a week for 5 times as a course of treatment in the control group. There was no significant difference in gender, age, body mass index, side, stage of osteoarthritis, course of disease, and preoperative Lysholm score of the knee joint, the Western Ontario and McMaster Universities osteoarthritis index (WOMAC), and SF-36 scale score between 2 groups (P > 0.05). The clinical efficacy was evaluated by SF-36 scale score, Lysholm score, and WOMAC score. ResultsAll patients of 2 groups received a course of treatment. The patients were followed up for 6 months. After injection, the incidences of pain and swelling in the cell treatment group were significantly higher than those in the control group (χ2=16.200, P=0.000; χ2=11.688, P=0.000), but no significant difference was found in the incidence of effusion (χ2=2.118, P=0.146). In the cell treatment group, Lysholm score at 1-6 months after treatment, WOMAC score and SF-36 scale score at 2-6 months after treatment were significantly better when compared with scores before treatment (P < 0.05), and no recurrence of knee pain was observed during follow-up. In the control group, there was no significant difference in Lysholm score and SF-36 scale score between pre-and post-treatment (P > 0.05); there were significant differences in WOMAC score between pre-treatment and at 1, 2, 3 months after treatment (P < 0.05); at 3 months after treatment, 11 patients had joint pain symptoms again. No significant difference was found in the knee joint function score and SF-36 scale score at 1 and 2 months after treatment between 2 groups (P > 0.05), but the scores of the cell treatment group were significantly better than those of the control group at 3 and 6 months (P < 0.05). ConclusionIt can significantly improve the joint function and quality of life to use intra-articular injection of human umbilical cord MSCs for treating degenerative knee osteoarthritis. It takes effect after 1 month and the treatment effect can be sustained for 6 months.
ObjectiveTo evaluate the effect of using Schwann-like cells derived from human umbilical cord blood mesenchymal stem cells (hUCBMSCs) as the seed cells to repair large sciatic nerve defect in rats so as to provide the experimental evidence for clinical application of hUCBMSCs. MethodsFourty-five male Sprague Dawley (SD) rats in SPF grade, weighing 200-250 g, were selected. The hUCBMSCs were harvested and cultured from umbilical cord blood using lymphocyte separating and high molecular weight hydroxyethyl starch, and then was identified. The hUCBMSCs of 3rd generation were induced to Schwann-like cells, and then was identified by chemical derivatization combined with cytokine. The acellular nerve basal membrane conduit was prepared as scaffold material by the sciatic nerve of SD rats through repeated freezing, thawing, and washing. The tissue engineered nerve was prepared after 7 days of culturing Schwann-like cells (1×107 cells/mL) on the acellular nerve basal membrane conduit using the multi-point injection. The 15 mm sciatic nerve defect model was established in 30 male SD rats, which were randomly divided into 3 groups (10 rats each group). Defect was repaired with tissue engineered nerve in group A, with acellular nerve basal membrane conduit in group B, and with autologous sciatic nerve in group C. The nerve repair was evaluated through general observation, sciatic function index (SFI), nerve electrophysiology, weight of gastrocnemius muscle, and Masson staining after operation. ResultsThe hUCBMSCs showed higher expression of surface markers of mesenchymal stem cells, and Schwann-like cells showed positive expression of glia cell specific markers such as S100b, glial fibrillary acidic protein, and P75. At 8 weeks after operation, the acellular nerve basal membrane conduit had no necrosis and liquefaction, with mild adhesion, soft texture, and good continuity at nerve anastomosis site in group A; group B had similar appearance to group A; adhesion of group C was milder than that of groups A and B, with smooth anastomotic stoma and no enlargement, and the color was similar to that of normal nerve. SFI were gradually decreased, group C was significantly greater than groups A and B, group A was significantly greater than group B (P<0.05). The compound action potential could be detected in anastomotic site of 3 groups, group C was significantly greater than groups A and B, and group A was significantly greater than group B in amplitude and conduction velocity (P<0.05). Atrophy was observed in the gastrocnemius of 3 groups; wet weight's recovery rate of the gastrocnemius of group C was significantly greater than that of groups A and B, and group A was significantly greater than group B (P<0.05). Masson staining showed that large nerve fibers regeneration was found in group A, which had dense and neat arrangement with similar fiber diameter. The density and diameter of medullated fibers, thickness of myelinated axon, and axon diameter of group C were significantly greater than those of groups A and B, and group A was significantly greater than group B (P<0.05). ConclusionTissue engineered nerves from hUCBMSCs-derived Schwann-like cells can effectively repair large defects of the sciatic nerve. hUCBMSCs-derived Schwann-like cells can be used as a source of seed cells in nerve tissue engineering.