Assessment of Human Multi-Potent Hematopoietic Stem/Progenitor Cell Potential Using a Single In Vitro Screening System

Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34+CD38−/low(CD45RA−CD90+) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.造血干细胞（hematopoietic stem cells）是贯穿生命全程、负责维持全身造血系统生成的核心细胞类群。其功能实现依赖于自我更新能力与多向分化潜能两大特性。因此，若要定量测定某一细胞群体中造血干细胞的数量，必须验证该群体同时具备这两项核心属性。尽管已有支持人源造血干细胞存活的异种移植模型（xenografts models）被报道，但此类体内实验系统仍存在明显局限，例如难以适配高通量筛选的研究需求。本研究构建了一套条件性四环素诱导系统（tetracycline inducible system），可精准调控小鼠基质MS5细胞（murine stromal MS5 cells）中人源NOTCH配体Delta-like 1的表达水平。我们将富集了祖细胞/干细胞（progenitor/stem cells）的造血未成熟细胞，与条件性表达Delta-like 1的MS5细胞进行共培养，并设置了适配多谱系分化的培养体系。实验结果表明：在共培养过程中诱导或抑制DL1的表达时，人源未成熟CD34+CD38−/low(CD45RA−CD90+)细胞可在单个培养孔乃至单细胞水平上，同步激活B细胞、T细胞、NK细胞、粒/单核细胞系及红系的分化潜能。此外，本研究还明确了低水平NOTCH激活（NOTCH activation）对人源B细胞及髓系/红系淋巴分化的干扰效应。该系统为精准定量同时兼具淋巴系与髓系分化潜能的人源造血未成熟细胞提供了一种全新的研究工具。