人胚AGM区造血干／祖细胞的分离及其在含AGM区基质细胞体系中的培养

本研究分离人胚主动脉-性腺-中肾（AGM）区造血干／祖细胞（HSPC）并在含人AGM区基质细胞的培养体系中进行培养，以了解人AGM区基质细胞对HSPC的作用。以免疫组织化学方法检测人胎龄28—45天胚胎AGM区细胞CD34、Flk-1、VEGF的表达；分离AGM来源的HSPC，通过直接接种和经Transwell非直接接触两种方法接种于含人AGM区基质细胞系（hAGMS3和hAGMS4）饲养层的培养体系，观察HSPC生长情况和卵石区形成细胞（cobblestone area—forming cells，CAFC）并计数卵石区（cobblastone area，CA）；用间接免疫荧光方法检测培养体系中悬浮细胞和CAFC的cD34、Flk-1表达；收获细胞进行半固体造血集落培养以检测两种培养方法对AGM—HSPC的集落形成能力影响。结果表明：人AGM区造血细胞表达cD34和Flk-1。两种接种方法均显示有CFC形成，直接接触培养能形成CD34和Flk-1双阳性的CAFC，集落总数明显高于非接触培养（hAGMS3体系：1647-1-194VS389-1-31．P〈0．05；hAGMs4体系：1586±75VS432±35，P〈0．05）。结论：①人胚AGM区含有CD34^＋Flk-1^＋的HSC。②首次建立含人胚AGM基质细胞的AGM—HSPC培养体系，直接接触培养和非直接接触培养均能促进AGM—HSC的生长，AGM基质细胞与HSC直接接触发挥重要作用。

Isolation and Culture of Human Embryonic AGM Derived HSPCs in Hematopoietic Culture Systems Created by AGM Stromal Cells

This study was purposed to isolate human embryonic AGM derived HSPCs and investigate the effect of AGM stromal cells on AGM-derived HSPCs. Immunohistochemical sections of human AGM tissue were investigated for CD34, Flk-1 and VEGF expression. Human AGM-derived single cells were isolated and seeded onto pre-treated feeder of human AGM stromal cells (hAGMS3 and hAGMS4) by direct contact and non-contact co-culture in Transwell culture system. Growth characteristics of HSPCs with cobblestone area-forming cells (CAFCs) were observed and number of cobblestone area (CA) was counted. Indirect immunofluorescent assay was used to detect CD34 and Flk-1 expression on the surface of suspended cells as well as CAFCs in contact co-culture system. The cells after culture for 2 weeks were collected from both contact and non-contact co-culture systems for CFU assay. The result showed that hematopoietic cells in AGM tissue expressed CD34 and Flk-1. Both of the hematopoietic culture systems could produce CFCs. Nevertheless, direct contact co-culture produced CD34(+)Flk-1(+) CAFC and more CFUs than those from indirect non-contact culture (hAGMS3 system: 1647 +/- 194 vs 389 +/- 31, p < 0.05; hAGMS4 system: 1586 +/- 75 vs 432 +/- 35, p < 0.05). It is concluded that there were CD34(+)Flk-1(+) HSCs in human embryonic AGM region. The hematopoietic co-culture systems composed of AGM-derived HSPCs and AGM stromal cells are successfully established, both direct contact and Transwell non-contact co-culture can expand AGM-derived definitive HSPCs. Cell-cell contact between AGM-derived HSPCs and AGM stromal cells are of most importance to maintain and expand AGM-HSPCs.