人AGM区基质细胞对脐血LTC-IC的支持作用

Supportive Effects of Human Aorta-Gonad-Mesonephros-Derived Stromal Cells on Umbilical Cord blood LTC-IC

为探讨人主动脉-性腺-中肾(AGM)区基质细胞对脐血长期培养启动细胞(LTC-IC)的造血支持作用,建立了人AGM区基质细胞与脐血CD34+细胞体外长期共培养体系。采用免疫磁珠方法分离人脐血CD34+细胞,接种于已制备好人AGM区基质细胞(hAGMS1-S5)饲养层的24孔板中共培养,同时设无饲养层组作为对照,分别于共培养5、6、7、8周时收获细胞行造血细胞集落培养,并采用极限稀释法(LDA)检测与人AGM区基质细胞共培养后的脐血CD34+细胞的LTC-IC含量。结果表明,无饲养层的对照组培养5周后不再产生造血细胞集落,而以hAGMS1-S5作为饲养层,共培养5周后造血细胞仍具有集落形成能力。hAGMS1-S5维持LTC-IC的作用组间比较有显著性差异(P<0.05),其中以hAGMS3与S4组支持作用最强。LDA检测结果显示,hAGMS3和S4维持LTC-IC的能力无显著性差异(P>0.05);与hAGMS3和S4共培养14天后脐血CD34+细胞中的LTC-IC扩增,分别是达到(176±46)%、(187±52)%,S3和S4两组间比较无显著性差异(P>0.05)。结论:人AGM区基质细胞S1-S5对脐血LTC-IC具有维持作用,特别是hAGMS3和S4两株细胞对脐血LTC-IC具有更好的维持及扩增作用。

The objective of this study was to explore the supportive effects of human aorta-gonad-mesonephros （AGM）-derived stromal cells on human umbilical cord blood long-term culture-initiating cells （LTC-IC）. A co-culture system was established with human AGM stromal cells and umbilical cord blood CD34^＋ cells. Different stromal cells derived from human AGM region （hAGM S1-S5 ） were plated on 24-well plates as feeder cells. CD34 ^＋ cells were positively selected from human umbilical cord blood through immunomagnetic bead selection method, seeded on the feeder cells, and co-cultured for 8 weeks. The hematopoietic cells were collected at 5,6,7 and 8 weeks for CFC analysis. Fre- quencies of LTC-IC in umbilical cord blood CD34 ^＋ cells after co-culture with AGM stromal cells were detected through limiting dilute analysis（LDA）. The results showed that there was no any hematopoietic CFC in the feeder cell-free culture system after 5 weeks of co-culture. However, in AGM feeder cells culture systems, there were still CFCs after 5 weeks of co-culture, which indicated that human AGM stromal cells could maintain LTC-IC in vitro. In groups of hAGM feeders, hAGMS3 and $4 had better supportive effects than other AGM groups （ P 〈 0.05 ）. The absolute number of LTC-IC in hAGM S3 and S4 culture systems got expansion up to （ 176 ±46）% and （ 187 ±52）% respectively without significant difference between hAGMS3 and S4 （P 〉0.05 ）. It is concluded that human AGM stromal cells S1- S5 support the maintenance of umbilical blood LTC-IC in vitro, while hAGMS3 and S4 cells have better effects on maintaining LTC-IC and expansion of LTC-IC.