Cai Yun, Zhang Xun-Chao, Huang Shao-Liang, Chen Hui-Qin, Wu Bei-Yan
Department of Pediatrics, SUN Yat-Sen University Third Affiliated Hospital, Guangdong Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2011 Oct;19(5):1189-94.
This study was purposed to directly induce murine embryonic stem cells (ESC) into hematopoietic stem cells (HSC) by simulating the spatial and temporal hematopoietic microenvironment changes in embryonic development, and to investigate the function of in vivo hematopoietic reconstitution of these HSC. E14 ESC were induced into embryoid body (EB) firstly. Then the cells from EB were further co-cultured with human aorta-gonad-mesonephros (AGM) region, fetal liver (FL) and bone marrow (BM) stromal cells in Transwell non-contact system in sequential orders. After 6 days of each co-cultured stage, the induced cells derived from EB were collected to analyze the Sca-1(+)c-Kit(+) cells by flow cytometry, check teratoma formation and transplant to BALB/C female mice exposed to lethal dose (60)Co γ-ray. The recipient mice were divided randomly into 5 groups: AGM, AGM + FL, AGM + FL + BM, irradiation control and normal control groups. The survival rates, hematopoietic reconstitution and engraftment of donor cells in the different groups were monitored. The results showed that Sca-1(+)c-Kit(+) cell level in EB cells co-cultured with human AGM region and FL stromal cells reached to peak value (21.96 ± 2.54)%. Teratomas could be found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region stromal cells, while there was no teratoma in the mice after subcutaneous injection of EB cells induced by human AGM region and FL stromal cells. The recipients in AGM group and irradiation control group all died. The survival rate was 77.8% in AGM+FL group, and 66.7% in AGM+FL+BM group. The peripheral blood cell count was near normal at day 21 after transplantation, and Sry gene copies from donor could be detected in recipient mice of these two groups. It is concluded that sequentially inductive system with feeder cells from human AGM region, fetal liver and bone marrow simulating embryonic defined hematopoiesis procedures can enhance the directed differentiation of ESC into HSC which can safely reconstitute hematopoiesis in vivo.
本研究旨在通过模拟胚胎发育过程中造血微环境的时空变化，直接将小鼠胚胎干细胞（ESC）诱导分化为造血干细胞（HSC），并研究这些HSC在体内的造血重建功能。首先将E14 ESC诱导形成胚状体（EB）。然后将EB来源的细胞依次与人主动脉-性腺-中肾（AGM）区、胎肝（FL）和骨髓（BM）基质细胞在Transwell非接触系统中共培养。在每个共培养阶段6天后，收集EB来源的诱导细胞，通过流式细胞术分析Sca-1(+)c-Kit(+)细胞，检测畸胎瘤形成，并移植到接受致死剂量（60）Co γ射线照射的BALB/C雌性小鼠体内。将受体小鼠随机分为5组：AGM组、AGM + FL组、AGM + FL + BM组、照射对照组和正常对照组。监测不同组中供体细胞的存活率、造血重建和植入情况。结果显示，与人AGM区和FL基质细胞共培养的EB细胞中Sca-1(+)c-Kit(+)细胞水平达到峰值（21.96 ± 2.54）%。皮下注射与人AGM区基质细胞共培养的EB细胞后，NOD-SCID小鼠体内可发现畸胎瘤，而皮下注射经人AGM区和FL基质细胞诱导的EB细胞后，小鼠体内未发现畸胎瘤。AGM组和照射对照组的受体均死亡。AGM+FL组的存活率为77.8%，AGM+FL+BM组的存活率为66.7%。移植后第21天外周血细胞计数接近正常，这两组受体小鼠体内均可检测到供体的Sry基因拷贝。结论是，用人AGM区、胎肝和骨髓的饲养细胞构建的顺序诱导系统模拟胚胎明确的造血过程，可增强ESC向HSC的定向分化，且这些HSC能在体内安全地重建造血功能
