人AGM区及胎肝基质细胞促进小鼠胚胎干细胞向造血干细胞分化和体内造血重建

Supportive effects of human AGM region and fetal liver stromal cells on differentiation of murine embryonic stem cells into hematopoietic stem cells and in vivo hematopoietic reconstitution

目的:探讨小鼠胚胎干细胞(ESCs)经人主动脉-性腺-中肾(AGM)区及胎肝(FL)基质细胞程序诱导后,向造血干细胞(HSCs)分化的效率及其造血功能。方法:将E14 ESCs诱导为拟胚体(EB),并在人AGM区及FL基质细胞饲养层上进一步诱导分化,培养6 d后收集细胞检测Sca-1+c-Kit+细胞含量、分析造血细胞集落形成能力及致瘤性。再将不同诱导阶段的EB来源细胞移植经致死量[60Co]γ射线辐照的BALB/c雌鼠,观察生存率、植入状况和造血重建。结果:(1)EB来源细胞经人AGM区及FL基质细胞程序诱导后Sca-1+c-Kit+细胞含量为(21.96±2.54)%,造血集落总数为(520±52)/105cells,明显优于诱导前及人AGM区基质细胞初步诱导者(P<0.05)。(2)NOD-SCID小鼠接种经人AGM区及FL基质细胞诱导的ESCs未见畸胎瘤。(3)BALB/c雌鼠移植经人AGM区及FL基质细胞诱导的EB来源细胞后生存率77.8%,14 d外周血细胞计数明显改善,存活受鼠均检测到供体来源sry基因,而移植人AGM区基质细胞诱导的EB细胞者15 d内全部死亡。结论:人AGM区及FL基质细胞能促进小鼠ESCs定向分化为HSCs,有效重建体内造血功能。

AIM：To investigate the differentiation of murine embryonic stem cells（ESCs） into hematopoietic stem cells（HSCs） by the supportive effects of human aorta-gonad-mesonephros（AGM） region and fetal liver （FL） stromal cells.METHODS： E14 ESCs were induced into embryoid body（EB） first.Then the cells from EB were further co-cultured with human AGM region and FL stromal cells in non-contact system.On day 6,the cells derived from EB were collected for Sca-1＋c-Kit＋ cell analysis by flow cytometry,colony forming unit（CFU） assay and teratoma formation checking.BALB/c female mice conditioned with lethal dose of γ-ray irradiation were transplanted with EB cells from different culture systems.The survival rates,engraftment of donor cells,reconstitution of hematopoietic were monitored.RESULTS： Sca-1＋c-Kit＋ cells in EB cells co-cultured with human AGM region and FL stromal cells had the value of（21.96±2.54）%,and the total CFU was as（520±52）/105 cells,which were statistically greater than those in EB cells only cultured with human AGM region stromal cells（P0.05）.No teratoma was found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region and FL stromal cells.In BALB/c female mice transplanted of EB cells co-cultured with human AGM region and FL stromal cells,the survival rate was 77.8%,and the peripheral blood cell count was obviously improved on day 14.PCR results showed the recipients all had sry gene copies from donor in bone marrow.The recipient mice transplanted with EB cells only cultured with human AGM region stromal cells all died within 15 days.CONCLUSION： Stromal cells from human AGM region and FL enhance the directed differentiation of ESCs into HSCs which can reconstruct hematopoiesis in vivo.