[Analysis of the different effects of murine bone marrow endothelial cell conditioned medium on the growth of embryonic and adult hematopoietic stem/progenitor cells in vitro].

In the present study, the effects of murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of yolk sac and bone marrow hematopoietic stem/progenitor cells (HSPC) were investigated. Nonadherent cells of yolk sac and bone marrow were collected for semisolid culture assay of CFU-GM and HPP-CFC after being cultured in DMEM with 10% FBS, 10% mBMEC-CM and/or FL (5 ng/ml), TPO (2 ng/ml) for 24 hours. The number of CFU-GM and HPP-CFC was counted by day 7 and 14 respectively. Atlas cDNA Expression Array was used for analysis of cytokine receptor expression of yolk sac and bone marrow HSPC. The results showed that mBMEC-CM could support the expansion of CFU-GM and HPP-CFC in liquid culture system. The expansion effects of mBMEC-CM were enhanced by combination with FL and TPO. mBMEC-CM was more effective on expansion of bone marrow CFU-GM and HPP-CFC than that of yolk sac CFU-GM and HPP-CFC. The differential expression of cytokine receptors were detected between yolk sac and bone marrow HSPC. PDGF-Rbeta, PDGF-Ralpha and corticotropin releasing factor receptor (CRFR) were only expressed in yolk sac hematopoietic cells while IFN-gammaR, GM-CSFR, Dopamine D2R and follicle-stimulating hormone receptor were only expressed in bone marrow hematopoietic cells. In conclusion, mBMEC-CM could support the growth and proliferation of yolk sac and bone marrow HSPC, and this effect was further enhanced by addition of FL and TPO. mBMEC-CM was more effective on expansion of bone marrow HSPC than on expansion of yolk sac HSPC. The comparative study indicated that the different expressions of cytokine receptors existed between yolk sac and bone marrow hematopoietic cells, which might lead to the difference in expansion in vitro between embryonic and adult HSPC.

[Effects of murine bone marrow endothelial cell conditioned medium on the growth of yolk sac hematopoietic stem cells and progenitors].

OBJECTIVE: To investigate the effects of murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of yolk sac hematopoietic progenitors. METHODS: The serum-free mBMEC-CM was obtained from subcultures of murine endothelial cell line derived from bone marrow which was established in our laboratory. The murine yolk sacs were harvested on day 8.5 postcoitus (pc) and incubated with 0.1% collagenase in 10% fetal calf serum at 37 degrees C for 40 minutes. Yolk sac cells were incubated in tissue culture dishes at 37 degrees C for 1 hour. Nonadherent cells were collected for semisolid culture assay of granulocyte-macrophage colony forming unit (CFU-GM) and high proliferative potential-colony forming cell (HPP-CFC) after being cultured in DMEM with 10% mBMEC-CM and 10% FBS for 24 hours. The number of CFU-GM and HPP-CFC was counted at day 7 and day 14 respectively. RESULTS: The growth of CFU-GM and HPP-CFC was supported by mBMEC-CM with GM-CSF. mBMEC-CM could induce the proliferation and differentiation of yolk sac hematopoietic stem cells and progenitors in liquid culture system. The percentages of CFU-GM and HPP-CFC compared with the 0 hour control were (119.5 +/- 5.7)% and (130.8 +/- 9.8)% respectively after 24 hours liquid culture (P < 0.05). The expansion effects of mBMEC-CM on CFU-GM and HPP-CFC were enhanced by compounded with flt3 ligand (FL) and thrombopoietin (TPO). The percentages of CFU-GM and HPP-CFC compared with the 0 hour control were (132.0 +/- 6.2)% and (176.9 +/- 12.8)% respectively after 24 hours liquid culture (P < 0.01). CONCLUSION: Murine bone marrow endothelial cell conditioned medium could support the growth and proliferation of yolk sac hematopoitic stem cells and progenitors, and this promoting effect was further enhanced by addition of FL and TPO.

[Osteogenic differentiation of murine yolk sac mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the potential of yolk sac mesenchymal stem cells in osteogenic differentiation. METHODS: Murine yolk sacs were harvested on day 8.5 postcoitus, yolk sac cells were obtained after the yolk sacs were digested by 0.1% type I collagenase for 1 hour, the non-adherent cells were removed after being cultured for 1 hour. The adherent cells were cultured in DMEM containing of 5 ng/ml bFGF and 15% FBS, and passaged when they became subconfluent. The morphologic characteristics, and AKP, BMP-2, as well as type I, III collagen of the yolk sac adherent cells were observed and tested. The attached cells were treated with 10(-8) mol/L dexamethasone, 10 mmol/L beta-glycerophosphate, and 50 micrograms/ml vitamin C at passage 4. Alternations of morphological characteristic, AKP activity, collagen of type I, III and mineralization were detected. RESULTS: Pure mesenchymal stem cells which were of spindle shape, uniform in size, positive in type I, III collagen staining and weak positive in AKP activity could be induced to pleomorphism osteoblast-like cells in vitro. The cells were transformed from spindle shape to polygonal cells which were positive in type I collagen, negative in type III collagen, strong positive in BMP-2, and positive in Von Kossa's stain at week 8. The polygonal cells could form nodular structure and their AKP activity was increased. All these were coincidence with the characters of osteoblast. CONCLUSION: Yolk sac mesenchymal stem cell can be purified and induced to osteoblast in vitro.