Hematopoietic stem cells (HSCs) can self-renew and can differentiate to a variety of specialized blood cells). The proliferation and homing of HSCs are strictly regulated both in the system level and local level. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, potent species-specific stimulator of granulocyte-macrophage, eosinophil, megakaryocyte and erythroid progenitors. In clinical purpose GM-CSF has been used as hematopoietic growth factor. It can promote the mobilization of HSC from bone marrow to peripheral blood. The number of HSCs mobilized into blood can be modulated by the kinds of cytolines. However, the information for the cytokine which promote mobilization is limited. Basic fibroblast growth factor (bFGF or FGF-2) induces the change of niche and affects the maintenance and differentiation of HSCs. FGF-2 positively regulates hematopoiesis, by acting on stroma cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. In this study, we investigated the effect of FGF-2 on HSCs mobilization and proliferation compare to GM-CSF. GM-CSF and FGF-2 were injected for 2 or 5 days into peritoneum of CD-1 mice (6～8 wks old) and sampling the bone marrow and peripheral blood. The bone marrow cells and peripheral blood were analyzed using FACS. In GM-CSF group, the number of HSCs was significantly increased by 2 days of injection but was significantly decreased in 5 days of injection. On the other hand the number of HSCs was significantly increased by the administration of FGF2 both in 2 days and 5 days. GM-CSF and FGF-2 are all increased the number of HSC both in bone marrow and peripheral blood. From these results, it is revealed that chronic administration of GM-CSF does not cause of the increase the number of HSCs. On the other hand, FGF2 can stimulate the proliferation of HSC without inhibition by the treatment period. It is suggested that GM-CSF and FGF2 may use different mechanisms to stimulate the HSCs proliferation. IP: 220.149.***.

Coculture of HSC with bone marrow-derived mesenchymal stem cells (BM-MSCs) is one of used methods to increase cell numbers before transplant to the patients. However, because of difficulties to purify HSCs after coculture with BM-MSCs, it needs to develop a method to overcome the problem. In the present study, we have examined whether a culture insert placed over a feeder layer might support the expansion of HSCs within the insert. cells isolated from the umbilical cord blood by using midiMACS were divided into three groups. A group of 1 cells were grown on a culture insert without feeder layer (Direct). The same number of HSCs was directly cocultured with BM-MSCs (Contact). The third group was placed onto an insert below which BM-MSCs were grown (Insert). To distinguish feeder cells from HSCs, BM-MSCs was pre-labeled fluorescently with PKH26 and 1 cells were seeded in the culture dishes. After culture for 13 days, the expansion factor (x) of HSCs that were grown without feeder layer (Direct) was In contrast, the number of HSCs directly cocultured with feeder layer was 59.6 0.5 and that of HSCs cultured onto an insert was The percentage of BM-MSCs cells remained being fluorescent was after culture. Immune-phenotypically large proportion of cultured cells were founded to be differentiated into myeloid/monocyte progenitor cells. The ability of BM-MSCs, fetal lung, cartilage and brain tissue cells to support ex vivo expansion of HSCs was also examined using the insert. After 11 days of coculture with each of these cells, the expansion factor of HSCs was 15.0, 39.0, 32.0 and 24.0, respectively. Based upon these observations, it is concluded that the coculture method using insert is very effective to support ex vivo expansion of HSCs and to eliminate the contamination of other cells used to coculture wth HSCs.