Proliferation And Differetiation Of Fetal Liver Derived Megakaryocytic Progenitor Cell And Its Regulation By Cell Cycle Control

Megakaryocytes (MKs) in fetal liver (FL) and bone marrow (BM) are small sized during ontogenesis and remain so until a few months after delivery . Our previous study demonstrated that although FL MK ploidy increased with increasing gestational age, it was not as high as adult MKs ploidy. It seems that endoreduplication and polyploidization may be retarded or delayed in the course of the development. In vitro cultures showed that fetal MK progenitor cells produced more MKs than adult MK progenitor cells did but that the cultured fetal MKs were smaller and had a lower ploidy thanadult MKs. This data suggested that the retardation of MK endoreduplication during ontogenesis might be related to intrinsic modification of MK progenitor cells. However, the mechanism of such retardation of endoreduplication remains unknown.Some investigations suggested that cyclins and CDK played an important role in the regulation of endomitosis of megakaryocyte. However, the mechanism of cyclins and CDK in the regulation of endomitosis of fetal megakaryocytes during ontogenesis and the relationship between cyclins/CDK and retardation of endomitosis of fetal mgakaryocyte is totally unknown. In one respect, this is because the proportion of megakaryocyte in fetal liver and bone marrow are low and it is very difficult to obtain enough megakaryocytes. On the other hand, it is also hard to obtain enough megakaryocytes in megakaryocyte culture system established before because the cells in these system did not only differentiate into megakaryocytic lineage, but also could differentiate into other hematopoietic lineage owing to multi-sort of cytokines were employed in these culture system. Therefore, number, DNA ploidy, and maturation stage of megakaryocyte was low in these system. In addition, some of the cytokines(eg: IL-3, GM-CSF) stimulated megakryocyte growth through the induced secretion of other cytokines by the adherent cells.This make the results obtained with multi-kinds of cytokines in culture seem ambiguous.Cloning and expression of TPO and the development of more sublte hematopoietic cells separation techniques made the investigation on fetal megakaryocytes possible.In this study, CD34+ cells were isolated from both human fetal liver and human adult bone marrow with magnetic beads. These cells were incubated in liquid suspension in the presence of thrombopoietin (TPO). The cell number, morphological characteristics, platelet-associated antigen phenotype, maturation stage and DNA ploidy of CD41+cells were examined from day 0 to day 12 in culture. The expression patterns of cyclin Bl, E, Dl, D2, and D3 were also analyzed. We found that (1) TPO stimulated the proliferation of fetal liver (FL) derived CD34+ cells from Ixl05/ml at the initial culture to 13.09?.06xl05/ml with a mean 73.14 fold increase of CD41+ cells after 12 days in culture. Adult BM derived CD34+ cells increased only to 1.31?.15xl05/ml with a mean 8.18 fold increase of CD41+ cells. (2) Although the membrane phenotype of both FL CD34+ derived MKs and BM CD34+ derived MKs analyzed with CD41a, CD42a, CD61 and CD34 were similar, all FL CD34+ derived MKs were in maturation stage I and II and in low ploidy (^4N) class. By comparison, BM CD34+ MKs possessed 15% MKs in maturation stage III and IV and with 23% MKs in high ploidy class (>4N). (3) Most of cultured FL derived CD34+ cells did not have a well-developed demarcation system (DM) and numerous a. -granules after 12 days incubation, von Willebrand factor (vWF) appears earlier on the cultured BM derived CD34+ cells than on FL derived CD34+ cells. (4) The expression of both cyclin E and cyclin Bl progressively increased in FL CD34+cells induced by TPO during 12 days in culture and high level of cyclin Bl was expressed on FL derived CD34+ cells of Gl phase after 12 days incubation. (5) The expression of cyclin Dl and D3 of FL CD34+cells induced by TPO during 12 days incubation initaially increased and then decrased. (6) Immunocytochemical analysis showed that cyclin D3was detected only in c...