| Hematopoietic stem cells (HSCs) are highly potent cells that can regenerate hematopoiesis when transplanted into recipients. Bone marrow, umbilical cord blood and mobilized peripheral blood are the well-known sources for HSC transplantation, however, there are some concrete limitations in the clinical uses of the mentioned sources. For example, the low content of hematopoietic stem cells in umbilical cord blood cannot meet the demand for transplantation, and HLA barrier is also the main limitation in allogenous marrow transplantation. Embryonic stem cells (ES cell) have the capacity to proliferate unlimitedly and can be coaxed to differentiate into almost all hematopoietic lineages in vitro, thus providing a new potential source for HSC transplantation. However, until recently there have been few reports on the translatability of ES-derived hematopoietic cells into recipients, unless ES cells had been genetically modified. Previous data have demonstrated that mesenchymal stem cells (MSC) support in vitro hematopoiesis and promote the engraftment of HSC to bone morrow and spleen as well. Therefore, it might provide some useful information on transplantation of ES-derived hematopoietic cells to shed light on the regulatory contribution of MSC to ES differentiation.In this study, ES cells were initially co-cultured with the isolated MSC, and its differentiated cell components coupled with their functional characteristics were further analyzed to investigate the in vitro effects of MSC on ES differentiation into hematopoietic lineages. Firstly, MSC culture was developed by addition of the femora into culture to accelerate migration and proliferation of MSC, simulating the in vivo microenvironment in bone fracture repair. The biological properties of the adherent cells were evaluated by irnmunophenotyping and differentiation assays. The results showed that cells were negative for CD45and CD34, which are characteristic of hematopoietic cells and endothelia cells; and they were homogenously positive for CD29, CD44, CD 105 and Sca-1, which have been identified as specific surface markers on MSC. Furthermore, the gained cells could be differentiated into adipocytes and osteoblasts in the presence of inductive agents as previously described. The results above demonstrated that the cells isolated and proliferated were MSC. Further study with RT-PCR assays showed that MSC expressed SCF, TPO, IL-6, LIF and SDF-1 at mRNA level, indicating its regulation on hematopoiesis.Afterwards, CCE ES cells were induced into hematopoietic cells and the experiment was grouped into 3: A, ES cells with inductive agents only; B, plus MSC layer and C, plus NIH/3T3 layer. RT-PCR assays demonstrated that cells expressed Flk-1, GATA-1 and fl-Hl but not Oct4 after induction of G-CSF, SCF, TPO and EPO. The same result was observed when ES cells were induced and simultaneously co-cultured onto the layer of NIH/3T3 cells (group C). In group B, however, no expression of Flk-1, GATA-1 and J3-H1 except Oct4 was identified, indicating the suppressive effect of MSC on ES differentiation into hematopoietic cells. Further CFC assays showed that cells from group B maintained the capacity to form secondary EBs (2ndEBs) and no hematopoietic colonies appeared, whereas cells from the other two groups produced much more hematopoietic colonies and fewer 2ndEBs. The above results hinted that MSC might inhibit/postpone the initial differentiation of the ES cells into hematopoietic cells.To further clarify the complexity, 2ndEBs formed in the CFC assay system were re-plated in CFC culture. The EBs derived from group B produced much more colonies than those from the other two groups (p<0.05). When ES cells were co-cultured with MSC for 12 days, the number of colonies in group B increased to the utmost, reaching 668 + 58.59465 per 1×105EB cells. The CFCs contained in the suspended EBs and cells in the NIH/3T3 coculture group were at most 213.3 ± 35.5 and 278.7±35.3 respectively. In addition CFCs contained in the 2ndEBs from group A and C decreased dramat...
|
PDF Full Text Request