Transwell培养体系中人AGM区基质细胞支持脐血CD34^+细胞造血

Supportive Effects of Stromal Cells of Human Embryonic Aorta-gonad-mesonephros (AGM) on Umbilical Cord Blood CD34^+ Stem/Progenitor Cells in Transwell Culture System

【目的】探讨Transwell系统中人胚主动脉-性腺-中肾(AGM)区基质细胞对脐带血造血干/祖细胞的造血能力长期维持及扩增的作用。【方法】采用免疫磁珠方法分离人脐带血CD34+细胞,接种于底层铺有人AGM区基质细胞的Transwell培养板的Inserts中,非接触共培养7-35 d,每星期取样检测细胞总数,用半同体培养基分析CFU-C及HPP-CFU集落形成数,流式细胞术检测CD34+、CD34+CD38-细胞百分率。【结果】在Transwell中非接触共培养条件下,人AGM区基质细胞培养体系较胚胎躯干基质细胞和无饲养层培养体系对有核细胞总数、CFC和CD34+细胞均具有明显的扩增作用,共培养14 d的CD34+、CD34+CD38-造血干/祖细胞均获得峰值扩增(2.62±0.8和2.15±1.04,P<0.05),而MNC总数和CFC均在21 d获得最大扩增(32.5±4.3和4.2±0.6倍,P<0.05).克隆分析发现CFU-Mix、CFU-GM、BFU-E在共培养4-5星期后均仍然可见。原始祖细胞HPP-CFC在3星期也得到2.23倍的扩增,较空白及hFT对照组均有显著性差异(P<0.05),并在共培养35 d 后仍可见HPP-CFU集落形成。【结论】人AGM区基质细胞hAGM-S3/hAGM-S4均具有造血支持作用,在非接触共培养条件下中可长期维持脐血中造血干/祖细胞的多系造血能力和高增殖潜能达21～35 d,对脐血CD34+/

[Objective]To explore the supportive effects of stromal cells of human aorta-gonad-mesonephros region on umbilical cord blood CD34+ cells in transwell non-contact culture system. [Methods] CD34+ cells were positively selected from human umbilical cord blood through immunomagnetic beads selection method and seeded into inserts of 24-transwell plate, and non-contact co-cultured for 1~5 weeks. Different stromal cells from human AGM region or fetal trunk were cultured on the bottom of each well as feeder cells. Then at every week, total nucleated cells number were count, frequencies of CD34+ and CD34+CD38- cells detected by flow cytometry, and CFU-C and HPP-CFU determined by semi-solid medium clonal culture. [Results] Of different tissue-derived stromal cells in the transwell non-contact culture system, human AGM cells supported proliferation of nucleated cells, CD34+ cells and CFC for 2~3 weeks more than groups of fetal trunk fibroblasts and controls without feeder cells. CD34+ and CD34+CD38- hematopoietic stem/progenitor cells were expanded 2.62 and 2.15 tolds respectively after 2 weeks co-culture (P<0.05), while mononucleated cells number, colony forming cells were 32.5 and 4.20 times expanded after 21 days of co-culture with AGM cells in vitro (P<0.05). In colony analysis, CFU-Mix, CFU-GM, and BFU-E could be detected under phase contrast microscope after 4~5 weeks of co-culture. More primitive progenitor cells HPP-CFC could be expanded 2.23 folds at 3 weeks of co-culture with AGM stromal cells, which showed AGM cells were better than hFT cells in HPP-CFC expansion (P<0.05). HPP-CFU could still be detected even after 5 weeks of cord blood cells co-culture with AGM stromal cells. [Conclusions] In non-contact culture system, human AGM stromal cell lines hAGM-S3 and hAGM-S4 both significantly supported the expansion of human umbilical cord blood CD34+ cells, and could maintain the multi-lineage differentiation and high proliferation potential abilities of those hematopoielic stem/progenitor cells up to 3~5 weeks in vitro.