Jiabo Hu

Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro.

A method for isolating adult human bone marrow mesenchymal stem cells (MSCs) was established, and the ability of human MSCs to differentiate into cells with characteristics of cardiomyocytes in vitro was investigated. Selected MSC surface antigens were analyzed by flow cytometry. The MSCs at Passage 2 were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. Characteristics of the Putative myogenic cells were determined by Immunohistochemistry and transmission electron and confocal microscopies. The expression of myogenic specific genes was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, and DNA sequencing. The MSCs were spindle-shaped with irregular processes and were respectively Positive for CD13, CD29, CD44, CD71 and negative for CD3, CD14, CD15, CD33, CD34 CD38, CD45, and HLA-DR. The myogenic cells differentiated from MSCs were positive for beta-myosin heavy chain (beta-MHC), desmin, and alpha-cardiac actin. When the pyogenic cells were stimulated with low concentration of K+ (5.0 mM), an increase in intracellular calcium fluorescence was observed. Myofilament-like structures were observed in electron micrographs of the differentiated myogenic cells. The mRNAs of beta-MHC, desmin, alpha-cardiac actin, and cardiac troponin T were highly expressed in the myogenic cells. These results indicate that 5-azacytidine can induce human MSCs to differentiate in vitro into cells with characteristics commonly attributed to cardiomyocytes. Cardiomyocytes cultured from bone marrow sources are potentially valuable for repairing injured myocardium.

Human mesenchymal stem cells isolated from the umbilical cord

Mesenchymal stem cells (MSCs) are known as a population of multi‐potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. In this study human MSCs were successfully isolated from the umbilical cords. The research characteristics of these cells, e.g., morphologic appearance, surface antigens, growth curve, cytogenetic features, cell cycle, differentiation potential and gene expression were investigated. After 2 weeks of incubation, fibroblast‐like cells appeared to be dominant. During the second passage the cells presented a homogeneous population of spindle fibroblast‐like cells. After more than 4 months (approximately 26 passages), the cells continued to retain their characteristics. Flow cytometry analysis revealed that CD29, CD44, CD95, CD105 and HLA‐I were expressed on the cell surface, but there was no expression of hematopoietic lineage markers, such as CD34, CD38, CD71 and HLA‐DR. Chromosomal analysis showed the cells kept a normal karyotype. The cell cycle at the third passage showed the percentage of G0/G1, G2/M and S phase were 88.86%, 5.69% and 5.45%, respectively. The assays in vitro demonstrated the cells exhibited multi‐potential differentiation into osteogenic and adipogenic cells. Both BMI‐1 and nucleostemin genes, expressed in adult MSCs from bone marrow, were also expressed in umbilical cord MSCs. Here we show that umbilical cords may be a novel alternative source of human MSCs for experimental and clinical applications.

Lead acetate reduces the ability of human umbilical cord mesenchymal stem cells to support hematopoiesis in vitro

Plumbum (Pb) is a heavy metal toxin that causes many pathophysiological effects in various systems of the human body. It has previously been reported that excessive lead trioxide causes hematopoietic system toxicity. Mesenchymal stem cells (MSCs), as cells with self-renewal and multipotent differentiation potential, play a supportive role in hematopoietic function. Lead is well known to interfere with hemoglobin synthesis and affect erythrocyte morphology and survival. MSCs and the cytokines secreted by MSCs are the important components of the hematopoietic microenvironment. Thus, we hypothesized that lead may cause damage to MSCs, which may provide a new understanding of the mechanism of lead toxicity in the hematopoietic system. In the present study, cell count, MTT assay, apoptosis assay, osteogenic differentiation, cell histochemical staining, cell cycle analysis, colony forming assay and RT-PCR were used. The results showed that the proliferation of umbilical cord MSCs (UCMSCs) was affected if the concentrations of lead were higher than 10 µM. Following osteogenic differentiation, the rate of alkaline phosphatase and Von Kossa stain positivity in the experimental group was lower than that in the control group. In conclusion, these results demonstrate that lead suppresses the self-renewal and multipotent differentiation potential of UCMSCs, and induces an adverse effect on the expression of UCMSCs cytokines. MSCs also have a hematopoiesis-promoting function that is capable of supporting colony formation of bone marrow cells. Furthermore, we found that following treatment with lead acetate, the supportive function of UCMSCs on colony formation was inhibited. Taken together, lead acetate has a toxic effect on the self-renewal, multipotent differentiation potential and hematopoiesis-promoting function of UCMSCs.

Microvesicles released from human embryonic stem cell derived-mesenchymal stem cells inhibit proliferation of leukemia cells

Human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSCs) are able to inhibit proliferation of leukemia cells. Microvesicles released from human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSC‑MVs) might play an important part in antitumor activity. Microvesicles were isolated by ultracentrifugation and identified under a scanning electron microscopy and transmission electron microscope separately. After 48-h cocultured with hESC‑MSCs and hESC‑MSC‑MVs, the number of K562 and HL60 was counted and tumor cell viability was measured by CCK8 assay. The expression of proteins Bcl-2 and Bax were estimated by western blotting. Transmission electron microscope and western blot analysis were adopted to evaluate the autophagy level. Results showed that both hESC‑MSCs and hESC‑MSC‑MVs inhibited proliferation of leukemia cells in a concentration-dependent manner. hESC‑MSC‑MVs reduced the ratio of Bcl/Bax, enhanced the protein level of Beclin-1 and LC3-II conversion, thus upregulating autophagy and apoptosis. In conclusion, microvesicles released from human embryonic stem cell derived-mesenchymal stem cells inhibited tumor growth and stimulated autophagy and excessive autophagy might induce apoptosis.

Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells

An immortalized mouse fetal liver stromal cell line, named KM3, has demonstrated the potential to support the growth and maintenance of human embryonic stem cells (hESCs). In this study, the characteristics of KM3 cells were examined following cryopreservation at −70°C and in liquid nitrogen for 15, 30 and 60 days following treatment with 10 μg/ml mitomycin C. In addition, whether the KM3 cells were suitable for use as feeder cells to support the growth of hESCs was evaluated. The inhibition of mitosis without cell death was observed when the KM3 cells were treated with 10 μg/ml mitomycin C for 2 h. The morphology of the KM3 cells cryopreserved in liquid nitrogen for 60 days was not markedly changed, and the cell survival rate was 84.60±1.14%. By contrast, the survival rate of the KM3 cells was 66.40±2.88% following cryopreservation at −70°C for 60 days; the cells readily detached, were maintained for a shorter time, and had a reduced expression level of basic fibroblast growth factor. hESCs cultured on KM3 cells cryopreserved in liquid nitrogen for 60 days showed the typical bird’s nest structure, with clear boundaries and a differentiation rate of 16.33±2.08%. The differentiation rate of hESCs cultured on KM3 cells cryopreserved at −70°C for 60 days was 37.67±3.51%. These results indicate that the cryopreserved KM3 cells treated with mitomycin C may be directly used in the subculture of hESCs, and the effect is relatively good with −70°C short-term or liquid nitrogen cryopreservation.