摘要
目的:体外模拟胚胎早期AGM区造血微环境,诱导胚胎干细胞(ESCs)分化为造血干细胞(HSCs)。方法:将小鼠E14ESCs在含BMP-4及VEGF的半固体培养基中诱导为拟胚体(EB),分别于3、6、9、12、15d时收获EB,流式细胞术检测Flk-1+细胞含量。取Flk-1+细胞处于高峰期的EB细胞,在人AGM区基质细胞饲养层上进一步诱导分化,并设无饲养层对照,分别于3、6、9、12d时收获细胞计数、流式细胞术检测Sca-1+c-kit+细胞含量,并分析造血细胞集落形成能力。结果:诱导E14细胞形成EB过程中添加BMP4+VEGF的因子组Flk-1+细胞在第9d达峰值(27.53%±2.84%),与未添加因子组(8.77%±1.12%)比较差异显著(P<0.05)。将培养9d的EB细胞在hAGMS3、hAGMS4饲养层上进一步诱导分化,第6d时Sca-1+c-kit+细胞达峰值,分别为7.31%±1.21%、7.62%±1.52%,其绝对数分别扩增(2.57±0.48)倍、(2.35±0.36)倍,与无饲养层组比较显著差异(P<0.05)。该分化阶段的Sca-1+c-kit+细胞具有形成各系造血细胞集落的能力。结论:人胚早期AGM区基质细胞能促进小鼠ESCs定向分化为HSCs,为研究ESCs分化为HSCs的分子机制提供了实验模型。
AIM： To direct embryonic stem cells （ESCs） into hematopoietic stem cells （HSCs） in vitro by simulating the hematogenic microenvironment in human early embryonic aorta - gonad - mesonephero （AGM） region. METHODS ： Murine E14 embronic stem cell line was used for two - step differentiation. In the first step of primary differentiation, E14 ESCs were seeded into semisolid methylcellulose - based medium containing bone morphogenesis protein 4 （BMP4） and vascular endothelial growth factor （VEGF） for embryoid body （EB） formation. On days 3, 6, 9, 12 and 15, single EB cells were analyzed for Flk - 1^＋ cells amount through flow cytometry. In the second step, single cell from EB containing most Flk - 1^＋ cells was further co - cultured with human AGM stromal cells in non - contact system. On co - culture days of 3, 6, 9 and 12 days, cells were collected for cell count, flow cytometry for Sca - 1^＋ c - kit^＋ cells analysis, and colony forming cell assay. RESULTS： During the EB formation, BMP4 ＋ VEGF promoted Flk - 1^＋ cell genesis on day 9 at peak pencentage value of 27.53% ±2. 84%, which was statistically higher than that in control group as 8. 77 ±1.10 （P 〈 0. 05 ）. Collagenase - disassociated single cell from day 9 EB was co - cultured with human AGM stromal cells of hAGMS3 or hAGMS4 for further hematopeietic differentiation. On day 6 Sca - 1^＋ c - kit^＋ cells got to peak value as 7. 31%± 1.21% [ （2.57 ±0. 48） folds] and 7.62% ±1.52% [ （2. 35±0. 36） folds] in hAGMS3 and hAGMS4 feeder systems, respectively, both of which were greater than those values of no - stroma groups at the same culture duration （ P 〈 0. 05 ）. Colonogenic cell assay showed that these Sea - 1^＋ c - kit^＋ cells had ability of forming multiple lineage hematopeietic colonies. CONCLUSION： BMP4 in combination with VEGF promotes Flk - 1^＋ cell genesis during EB formation in vitro. Stromal cells from early human embryonic AGM region further enhance the directed differentiation of these primitive cells into HSCs. This two - step induction differentiation model can be used for molecular mechanism study of ESCs hematopeietic differentiation.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2007年第9期1747-1751,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30300377)
"十五"863计划资助项目(No.2003AA205008)
中国博士后科学基金资助项目(No.2003033432)
教育部博士点基金资助项目(No.20030558070)
关键词
胚胎干细胞
造血干细胞
细胞分化
Embryonic stem cells
Hematopeietic stem cells
Cell differentiation
