体外分化胚胎干细胞为造血干细胞重建造血功能的研究

目的：探讨体外定向分化胚胎干细胞（ESCs）为造血干细胞（HSCs）对体内造血功能的重建作用。方法：将小鼠E14.1胚胎干细胞采用“三步诱导法”在体外分化发育为HSCs，造血克隆形成(CFU)实验观察其体外造血集落形成情况，免疫磁珠分选纯化HSCs移植给经亚致死剂量γ射线照射的雌性SCID小鼠，观察其植入及小鼠造血功能恢复情况。结果: 经过分阶段诱导，多种造血刺激因子联合应用能有效促进ESCs定向分化发育为HSCs,流式细胞仪检测HSCs特异性表面标志物CD34+/Sca-1+表达最高为（58.64±4.20）%，CFU培养能形成较多的红系、粒系/巨噬细胞系及混合细胞集落, Wright-Giemsa 染色显示为原始的造血细胞。此阶段的HSCs经分选纯化后移植给经γ射线照射后的小鼠，移植组小鼠+10 d造血功能开始恢复，观察40 d后除血小板恢复较慢外，白细胞、红细胞、血红蛋白等指标已接近正常，植入率为71.4%，存活率为43.0%，染色体检测证实已由受体鼠的XX转为供体鼠的XY。结论: 采用分阶段诱导的方法，可在体外定向诱导小鼠ESCs分化发育为HSCs，此来源的HSCs可以有效重建体内造血功能。

In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice

AIM: To investigate the potential of hematopoietic stem cells (HSCs) derived from mouse embryonic stem cells (ESCs) to reconstruct hematopoiesis in vivo. METHODS: Using a three-step method, a mice embryonic stem cell line, E14.1 was induced into hematopoietic stem cells. The cell markers with CD34+/ Sca-1+ were identified by flow cytometry analysis, then HSCs (1×109 cells/L) from third-step were injected into SCID mice for observing teratoma formation. To validate function of HSCs, colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted. RESULTS: The method of three-step differentiation, combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells (as high as 58.64%±4.20%) with more CFU-E, CFU-GM and CFU-GEMM populations. The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining. Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation, with 71.4% (5/7) successful engraftment rate. Three recipients showed that the cell population of the peripheral blood leukocytes, red blood cells and hemoglobin approached to normal index at 40 d after transplantation, but followed relative slow renew in platelet count. Survival rate in transplant group was 43%, compared to 100% mortality in control mice. Karyotyping assays confirmed the female mice with XY. CONCLUSION: The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs. HSCs derived from mouse ESCs can reconstruct hematopoiesis.