Embryonic Stem Cell Derived Hematopoietic Stem / Progenitor Cells In Experimental Study Of Adjuvant Chemotherapy

PART 1: PRIMARY BONE MARROW STROMAL CELLS ASSOCIATED WITH CYTOKINES INDUCE THE DIFFERENTIATION OF ES-D₃ TO HEMATOPOIETICCELLS IN VITROObjective: To study the efficiency of primary bone marrow stromal cellsassociated with cytokines, i.e. vascular endothelial growth factor (VEGF), stem cell factor (SCF) and thrombopoietin (TPO) in inducing the differentiation of ES-D3 cells to hematopoietic cells in vitro.Methods: BM samples of 6⁸ weeks old Kunming mice were used toprepare primary bone marrow stem cells (BMSC) feeder layer. Embryoid body (EB) were derived from ES-D3 cells, and then induced under four different system: control group(spontaneous differentiation), cytokines group (VEGF+SCF+TPO), BMSC group and BMSC+ VEGF+SCF+TPO group. The cells that differentiated for 7 days were used to carry through hematotopoietic colonies assay. The CD34,c-kiu, CD11b, Ter119 in cells that differentiated for 7 or 14 days were investigated by FACS and indirect immunofluorescant cell stain. Hematotopoietic transcription factors (Flk-1, GATA-2, SCL, β-H1 and β-major) were detected by RT-PCR.Results: Approximately 36.67%, 20.48%, 0.64% and 0.99% of the cells that differentiated for 7 days were shown to be CD34⁺ , c-kit⁺, CD11b⁺and Ter119⁺. Therefore, hematopoietic precursor cells are present within these cultures of 7-day-differentiated cells. About 0.64%, 20.49%, 18.04% and 16.02% of the cells that differentiated for 14 days were shown to be CD34⁺, c-kit⁺, CD11b⁺ and Ter119⁺. The cells that differentiated for 7 days generated high proliferation potential colonyforming cells (HPP-CFC) and burst-forming unit-erythroid (BFU-E). Immunofluorescence cell staining further affirmed that the cells that differentiated for 7 days hold CD34+and c-kit+ cells while the cells that differentiated for 14 days hold c-kit+, CDllb+ and Terll9+ cells. The vascular endothelial growth factor receptor Flk-1 was expressed both in undifferentiated and differentiated ES cells, which suggests that Flk-1 may have important roles in cells other than hematopoietic cells. GATA-2, SCL and P-Hl globin were expressed in the cells that differentiated for 7 days, consistent with the characteristic of primary hematopoietic colonies. In addition, the cells that differentiated for 14 days expressed mRNA for GATA-2, SCL, P-Hl and (3-major globin, which shows mix colonies in the cells. The BMSC+ VEGF+SCF+TPO group got the highest inducing rate according to the numbers of hematopoietic progenitors and colonies(P <0.05 or 0.01).Conclusion: Bone marrow stromal cells feeder layer associated with cytokines strongly promotes hematopoietic differentiation in vitro. Those induced hematopoietic progenitors could form hematotopoietic colonies and differentiated to mature cells. They also expressed genes associated with hematotopoietic transcription and differentiation.PART 2: HIGH DOSE CHEMOTHERAPY AND TRANSPLANTATION OF HEMATOPOIETIC PROGENITORS FROM MURINE ES-D3Objective: To study the capacity of female C57BL /6 mice to reconstitute the hematopoietic system following high dose chemotherapy by using embryonic stem cells grown on stromal cell feeder layer along with growth factor cocktail containing VEGF, TPO and SCF.Methods: Embryonic stem cells grew on stromal cell feeder layer along with growth factor cocktail containing VEGF, TPO and SCF for the purposes of differentiating to hematopoietic progenitors. Those induced differentiated cells were injected to female C57BL/6 mice that have been treated with high dose Topotecan chemotherapy. The first group served as a control group and received topotecan at a dose of 15mg/kg/day. The next sequential group of mice received topotecan at a dose of lOmg/kg/day, a third group received topotecan at a dose of 15mg/kg/day, and a fourth group received topotecan at a dose of 20mg/kg/day. After infusion, peripheral blood was examined twice a week for 8 weeks. On the 10th day after infusion, the spleen of one mouse from each group was examined pathohistologically. On the 40th day after infusion, the sex-determining region on the Y- chromsome (Sry gene) of cells in peripheral blood, bone marrow and spleen of the transplanted female mice, which was derived from male donors, was confirmed by PCR analysis.Results: We observed rapid white blood cell recovery, which suggested that differentiated cells infusion have a positive impact on hematopoiesis. The Sry gene in peripheral blood, bone marrow and spleen of transplanted female mice was confirmed by PCR analysis, which affirmed the existence of the chimera. WBC count show evident statistical difference among three experimental groups, the group that received topotecan of 15mg/kg recovered quickliest, treading on the heel of it was the group receiving 20mg/kg topotecan, the last is the group receiveing 1 Omg/kg topotecan (PO.01).Conclusion: ES-D3 cells grown under appropriate conditions are able to differentiate to hematopoietic progenitors capable of reconstituting hosts following myeloablative chemotherapy. The efficence of hematopoiesis is reletade to the dose of topotecan.PART 3: THE EFFECT OF HUMAN EMBRYONIC PRIMARY BONE MARROW STROMAL CELL FEEDER LAYER ON CULTURE OF HUMAN EMBRYONICSTEM CELLSObjective: To study the effect of human embryonic primary bone marrow stromal cell (BMSC) feeder layer on culture of human embryonic germ cells.Methods: Human fetus bone marrow prepared from thighbones wascultured to establish BMSC feeder layer; At the same time, fibroblast cells were separate from the same fetus, of culture medium were used as conditioned medium(CM).Some 13.5-day-old mouse embryos were used to establish primary mouse embryo fibroblast feeder layer (PMEF). Four groups, i.e. PMEF feeder layer, BMSC feeder layer, CM, BMSC feeder layer+CM, were designed. The growth characters of embryonic germ cells in different conditions were analyzed.Results: The results showed human embryonic primary BMSC feeder layer, under the collaboration of CM, play the same role with PMEF in proliferation and maintaining undifferentiation state of hEGC in vitro. Without the help of CM, the proliferate efficiency decreased. CM itself con't keep hEGC to proliferate. Conclusion: human embryonic primary bone marrow stromal cell feeder layer, under the collaboration of CM, can support hEGC expansion, without the infection of the heterogeneous protein, adapted to clinical applications.