[Differentiation of human umbilical cord derived mesenchymal stem cells into low immunogenic and functional hepatocyte-like cells in vitro].

OBJECTIVE: To investigate the biological function of hepatocyte-like cells derived from mesenchymal stem cells that isolated from human umbilical cord UC-MSCs in vitro, and to detect the changes in the immunogenicity of the differentiated hepatocyte-like cells (DHC). METHODS: Transdifferentiation of UC-MSCs into hepatic lineage in vitro was induced in modified two-step induction medium. The expressions of hepatic specific markers were detected by RT-PCR analysis and immunofluorescence staining at different time points after induction. The levels of albumin and urea in the supernatants of cultures were measured by enzyme-linked immunosorbent assay. Furthermore, the immunosuppressive property of DHC was detected by one-way mixed lymphocyte culture. RESULTS: The mRNA and proteins of alpha fetoprotein (AFP), albumin (ALB),and cytokeratin-19 (CK-19) were expressed in naive UC-MSCs at low levels. DHC highly expressed hepatic markers AFP, ALB, CK-19, and tryptophan 2, 3-dioxygenase 14 and 28 days after hepatic differentiation and were accompanied by an increased production of ALB and urea in supernatant in a time-dependent manner. DHC did not express human leukocyte antigen DR antigen and significantly decreased the lymphocyte proliferation. CONCLUSION: UC-MSCs are able to differentiate into functional hepatocyte-like cells in vitro, while the immunogenicity of DHC remains low.

STAT3 influences the characteristics of stem cells in cervical carcinoma.

The purpose of the study was to investigate the role of the signal transducer and activator of transcription 3 (STAT3), signal transduction protein in regulating the biological characteristics of stem cells in cervical carcinoma. Overexpressed plasmid of STAT3 was constructed and used to transfect SiHa into cervical carcinoma cells. STAT3-targeted specific siRNA was designed and produced. The effects of STAT3 upregulation (or inhibition) on the expression of NANOG, OCT4 and SOX2 markers of stem cells were measured, using western blot analysis and RT-qPCR. In addition, the tumor sphere experiment was also conducted to detect the formation of tumor spheres after the intervention of expression of STAT3 and the expression of STAT3, NANOG, OCT4 and SOX2 was detected in 35 cases of cervical carcinoma tissues and 31 cases of normal cervical tissues using immunohistochemistry. We determined whether the STAT3 overexpression plasmid was successfully constructed using enzyme digestion, PCR for bacterium solution, western blot analysis and RT-qPCR and found that the plasmid met the requirements of subsequent procedures. Compared with the empty plasmid group and STAT3 low expression group, the mRNA and protein expression of markers of stem cells, OCT4, SOX2 and NANOG were significantly elevated in the STAT3 overexpression group with statistically significant differences (P<0.05), the formation ratio of tumor spheres in the STAT3 overexpression group was also significantly higher than those in the other two groups (P<0.05). The positive expression of STAT3, OCT4, NANOG and SOX2 in the cervical squamous carcinoma group was also markedly higher than that in the chronic cervicitis group (P<0.05). This study led us to a conclusion that STAT3 can regulate the characteristics of stem cells in cervical carcinoma, and STAT3, NANOG, OCT4 and SOX2 are highly expressed in cervical squamous carcinoma, thus able to promote the progression of cervical carcinoma.

Comparison of hematopoietic supportive capacity between human fetal and adult bone marrow mesenchymal stem cells in vitro.

Hematopoietic stem cells (HSC) shift from fetal liver and spleen to bone marrow at neonatal stages and this movement may be due to inductive signals from different microenvironments. Mesenchymal stem cells (MSC) are the precursors of stromal cells in bone marrow microenvironments such as osteoblasts and endothelial cells. Some researchers speculated that fetal bone marrow before birth might be not perfectly suit HSC growth. However, it is still lack of direct evidence to prove this hypothesis. This study was aimed to compare the hematopoietic supportive capacity between human fetal and adult bone marrow MSC in vitro. Adult bone marrow MSC (ABM-MSC) were isolated from three healthy donors and fetal bone marrow MSC (FBM-MSC) were isolated from three fetuses between gestations of 19 to 20 weeks. After irradiation, MSC were co-cultured with CD34(+) cells isolated from umbilical cord blood in long-term culture-initiating cell (LTC-IC) assay. The colony number of colony forming cells (CFC) was counted and the phenotypic changes of co-cultured CD34(+) cells were analyzed by flow cytometry. Cytokine expressions in both kinds of MSC were detected by reverse transcription polymerase chain reaction (RT-PCR). The results showed that ABM-MSC had a stronger hematopoietic supportive capacity than FBM-MSC. Both of them enhanced the differentiation of CD34(+) cells into myeloid lineages. Cytokines were expressed differently in ABM-MSC and FBM-MSC. It is concluded that ABM-MSC possess more potential application in some treatments than FBM-MSC, especially in hematopoietic reconstitution.

More mesenchymal stem cells enriched from bone marrow aspirates by culturing bone marrow particles and mononuclear cells separately.

Bone marrow (BM) is the major source of mesenchymal stem cells (MSC). In most experiments, MSC were classically cultured from mononuclear cells (MNC) isolated by density gradient centrifugation method. However, several studies have demonstrated that this method was less efficient for MSC recovery. This study was aimed to investigate whether BM particles were the cause resulting in less efficiency of this method and how to isolate them. A total of 20 patients were enrolled in this study. MNC were cultured by standard adherence and BM particles were cultivated by primary explant culture. For BM from patients 1-10, MNC were first isolated and BM particles were then filtered out. The morphology and the fibroblastic colony number were compared between cultures of MNC and BM particles. For BM from patients 11-20, MNC isolation and BM particle filtration were processed in opposite order, then the immunophenotype and function between adherent cells expanded from MNC and BM particles were compared. In addition, for patients 11-20, the left BM aspirates were cultured too after BM particles and MNC were isolated separately. The results showed that adherent cells from BM particles were MSC. After BM particles were filtered out and cultured separately, MSC could be recovered completely from MNC isolated by density gradient centrifugation and no MSC were left in the residual BM aspirates. BM particles, which have been mostly discarded by the method of density gradient centrifugation, are another important source of MSC and they can be cultivated reliably by primary explant culture. It is concluded that more MSC are recovered from a single BM sample by culturing BM particles and MNC separately.

[Effect of fetal lung mesenchymal stem cells on differentiation of umbilical cord blood mononuclear cells into megakaryocytes].

In order to analysis the effect of fetal lung mesenchymal stem cell (FL-MSC) on differentiation of umbilical cord blood mononuclear cells (MNC) into megakaryocytes, the fresh umbilical cord blood MNC were isolated and divided into 2 groups in the culture added with TPO, IL-11 and heparin. In the first group MNC were cultured alone and in the second group MNC were cocultured with FL-MSC. The cells were collected at day 7, 10, 14 for cell counting and detection of CD41a and CD61 by flow cytometry. The morphology and ultrastructure of megakaryocytes were observed by immunohistochemistry method and transmission electron microscopy at day 14. The content of DNA was analyzed by flow cytometry at day 14 too. The results indicated that the of CD41a+ and CD61+ cells were obtained mostly in the second group at day 10 and were in 4.5 and 4.7 fold as much as the MNC cultured alone. The morphology and ultrastructure of megakaryocytes showed immature of nuclei in both of two groups. It is concluded that the FL-MSC could effectively enhance the production of CD41a+ and CD61+ cells, where the effect on nucleus development of the young megakaryocyte was not obviously shown.