【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。更多还原

【Abstract】 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 hematopoietic 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 Sca-1+c-kit+ cells had ability of forming multiple lineage hematopoietic 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 hematopoietic differentiation.更多还原