RUNX1 regulates transcription activity of WNT5A in mouse bone marrow derived mesenchymal stem cells / 中国实验血液学杂志

This study was purposed to investigate the effect of RUNX1 on transcription activity of WNT5A promoter in mouse bone marrow derived mesenchymal stem cells (MSC), and to explore the mechanism by which bone marrow environments regulate MSC. RT-PCR was used to detect the expression of RUNX1 in MSC isolated from mouse bone marrow and cultured in vitro; the chromatin immunoprecipitation (ChIP) was used to investigate the direct in vivo interaction between the RUNX1 and WNT5A promoter; retrovirus system was utilized to introduce the RUNX1 gene into MSC to detect the regulation of RUNX1 on the transcription activity of WNT5A promoter. The results showed that mouse bone marrow derived MSC was positive for Oil Red O, van Kossa and toluidine blue staining respectively and RUNX1 expressed in MSC. WNT5A promoter could be bound by RUNX1, and the expression level of WNT5A was enhanced with the increase of RUNX1. It is concluded that RUNX1 expresses in mouse bone marrow derived MSC, WNT5A is a direct target gene of RUNX1 and its transcriptional activity is regulated by RUNX1.

Effect of endothelium-specific deletion of PTEN on hemangioblast development in mouse embryo AGM region / 中国实验血液学杂志

This study was aimed to investigate whether endothelium-specific deletion of PTEN can affect hemangioblast development in the AGM region of mouse embryos. Based on Cre/loxP system, the Tie2CrePten(loxp/loxp) and Tie2CrePten(loxp/wt) mouse embryos were obtained. The genotype was identified by PCR. After treated with type I collagenase, the AGM region was dispersed into single-cell suspension, and then was cultured in blast colony-forming cell (BL-CFC) media. The number of BL-CFC was counted 4 or 5 days later. The hematopoietic capacity of BL-CFC was detected in methylcellulose culture system and the endothelial potential was assessed by tube-like structure formation on Matrigel. The results showed that the number of BL-CFC in AGM region of Tie2CrePten(loxp/loxp) mouse embryo decreased as compared with Tie2CrePten(loxp/wt) embryo. Whereas the hematopoietic capacity of mutant BL-CFC was enhanced, the endothelial potential, as evaluated by tube-like structure formation in vitro, was significantly reduced. It is concluded that the endothelial PTEN is capable of exerting regulatory functions on both the numbers and the dual potential of hemangioblast in mouse AGM region.

Separation of immortalized mesenchymal stem cell like stromal cells of mouse embryonic aorta-gonad-mesonephros region and their biological characteristics / 中国实验血液学杂志

To investigate the effects of microenvironment of aorta-gonad-mesonephros (AGM) on embryonic hematopoiesis, mesenchymal stem cell like stromal cells (MSC like stromal cells) derived from dorsal aorta (DA) in AGM region were separated and identified by their growth characteristics, related molecules expression and mesenchymal lineage potentials. Stromal cells from DA region in mouse embryos (E11.5) were isolated and cultured in vitro. After transfected by pSV3neo-SV40, the clones with G418 resistance were selected, and their growth characteristics were studied. The related molecules were analyzed by flow cytometry, and each clone was induced to differentiate into adipocytes, osteocytes, and chondrocytes. The results showed that most clones (20 clones) selected in the mouse DA region held the morphology of fibroblastoid cells. mDAF3 and mDAF18 could be grown in culture for more than 50 passages with G418 resistance, both have the potential to differentiate into adipocytes, osteocytes, and chondrocytes. At the logarithmic growth period, the cell population doubling time is about 24 hours. Surface markers, such as CD29, CD44, CD105 and Sca-1 were positively detected, while low levels of CD34, CD45, and CD31 were detected. It is concluded that immortalized mDAF3 and mDAF18 have the specific phenotype and differential potency of MSC, which suggests that MSC maybe exist in mouse embryonic DA region, where the MSC like stromal cells can be used as a cell model for further research on the modulation activity of DA microenvironment for embryonic hematopoiesis.

Relationship between hematopoietic development and endothelial cells (hemangioblast or hemogenic endothelium)--review / 中国实验血液学杂志

Developmental programs of blood and endothelium are closely correlated and remarkably conserved among species. To categorize the ontogeny relationship between hematopoietic and endothelial lineages, two putative models are presented here. In the yolk sac, hematopoietic and endothelial cells are more likely derived from a common precursor--the hemangioblast. By comparison, the hemogenic endothelium is proposed to characterize the generation of hematopoietic stem cells from mature endothelium in the P-Sp/AGM region. Furthermore, couples of molecules, including Scl, Flk-1 and Runx-1, are involved in formation and subsequent differentiation of the hemangioblast or hemogenic endothelium during embryonic hematopoiesis. In this article, the development of primitive and definitive hematopoiesis, two models associated with development of hematopoietic and vascular endothelial cells as well as molecules associated with development of hematopoietic and endotheliate cells were summaried.

Reduction of blast-colony forming cell development and bi-potential commitment by neutralizing TGF-beta1 in vitro / 中国实验血液学杂志

To study the regulation of TGF-beta(1) on the development of hemangioblast, embryonic stem cell-derived blast forming cells (BL-CFC) were used as the model of hemangioblast in vitro. TGF-beta(1) or anti-TGF-beta(1) neutralization antibody was added in the medium of embryoid body (EB) generation for observating influence of TGF-B(1) addition in different culture stages on number of BL-CFC and differentiation of BL-CFC to endothelial and hematopoietic cells. The results showed that antagonizing TGF-beta(1) in the course of EB growth could significantly reduce the number of BL-CFC (P<0.01), and the frequency of Flk-1(+) cells was also decreased consistently. Furthermore, the BL-CFC derived from EB pretreated with TGF-beta(1) demonstrated remarkably elevated hematopoietic and endothelial potential, whereas such bi-potential was impaired in the group with neutralizing antibody. It is concluded that TGF-beta(1), a conventional negative regulator in hematopoiesis and angiogenesis exert positive effects on the development and differentiation capacities of BL-CFC in vitro.

Suppression of ras mediated signaling attenuates hematopoietic differentiation of embryonic stem cells of mice in vitro / 中国实验血液学杂志

To investigate the possible involvement of Ras signaling in the hematopoietic differentiation of embryonic stem cells (ES cells), ES cells were transfected with RasN17, the dominant-negative mutant of Ras. Western blot was used to test the effect of RasN17 expression on Erk1/2 and Akt phosphorylation, semi-quantitative RT-PCR was used to detect expression of gene related to hematopoiesis in differentiation of ES cells. The results showed that the expression of RasN17 in the ES cells remarkably downregulated the phosphorylation of Erk1/2 and Akt simultaneously. Moreover, the expression of several markers related with hematopoiesis including Runx1, SCL and beta-major globin, were significantly suppressed in the EB expressing RasN17, whereas the transcription of Flk1, a gene required earlier than SCL in development of hematopoietic and endothelial lineages, was not influenced. It is concluded that the activation of Ras is pivotal for in vitro hematopoietic differentiation of ES cells.

Effect of bone marrow mesenchymal stem cells on hematopoietic differentiation of murine embryonic stem cells / 中国实验血液学杂志

Mesenchymal stem cells (MSCs), precursors of diverse stromal cells, can support hematopoiesis in vitro and can promote the implantation of hematopoietic stem cells in vivo when co-transplanted with CD34(+) cells. The aim of this study was to investigate the potential effect of MSCs on the hematopoietic development of embryonic stem cells (ES cells) and the feasibility of a novel system in which ES cells will be co-cultured with MSCs. The murine bone marrow MSCs were isolated and cultured and then their phenotype and differentiation function were identified with FCM and histochemical technique. The CCE cells, murine ES cell line, were co-cultured with the isolated MSCs and the hematopoietic differentiation of CCE cells was observed with hematopoietic clonogenic assay and RT-PCR. The results showed that the morphology of MSCs became gradually homogeneous with the passage culture of cells. After passage 4, the marker of Sca-1, CD29, CD44 and CD105 were highly expressed, however, CD34 and CD45, the specific marker of hematopoietic and endothelial cells, could hardly be identified. The isolated MSCs differentiated into adipocytes and osteoblasts in specific induction culture system. After maintaining culture on mouse embryonic fibroblasts, CCE cells were plated in suspended culture system with only differentiation inductive agents and co-culture system in which MSCs were added. Compared with CCE cell suspended culture, the cells differentiated into embryoid body were obviously enhanced and there were no colony-forming cells in the co-culture system of ES cells and MSCs. In addition, transcription factor Oct-4 in co-cultured CCE cells was expressed and hematopoietic markers, Flk-1, GATA-1 and beta-H1, were negative. The ability of embryoid bodies derived from the co-culture system to produce hematopoietic colonies was markedly higher than that from the suspended culture system. It is concluded that MSCs inhibit the initial differentiation of ESC and enhance hematopoietic differentiation ability of the co-cultured ES cells.

Generation of high proliferative potential hematopoietic progenitor cells from embryonic stem cell-derived BL-CFC / 中国实验血液学杂志

The blast colony-forming cells (BL-CFC), which are detected within embryoid bodies derived from embryonic stem cells (ES cells) differentiated for 2.5-3.5 days, have dual-potential of differentiation to hematopoietic and endothelial cells. In this investigation the culture method of BL-CFC was established and colony forming assay, immunofluorescent technique as well as nested RT-PCR was employed to identify the differentiation capacity of adherent and nonadherent cells derived from individual blast colony. The results showed that the adherent cells could intake DiI-Ac-LDL and expressed the endothelium-specific surface markers including CD31, UEA-I and VE-cadherin. In addition, nonadherent cells were capable of developing primitive or/and definitive hematopoiesis potential. High proliferative potential colony-forming cells (HPP-CFC) bearing self-renewal capacity was found in 20% of BL-CFC. It is concluded that BL-CFC derived from embryonic stem cells can generate high proliferative potential hematopoietic progenitor cells. However, the whether BL-CFC can reconstitute the adult bone marrow hematopoiesis in long-term remains to be further determined.