实验背景: 造血干细胞(hematopoietic stem cells, HSCs)是目前研究最为深入的一类干细胞，它具有自我更新及多向分化两大重要特点，能够在保持干细胞池相对稳定的同时增殖分化为复杂造血系统中的各系血细胞。然而，HSCs在骨髓及脐带血中比例极低，基础研究及临床移植中用到的HSCs又多为异质性的细胞群体，即HSCs及定向分化的造血祖细胞混合体，虽然脐带血的应用大大降低了临床用造血干细胞对组织相容性抗原（HLA）的要求，但脐带血仍无法满足成人移植所需的造血干细胞量。基于此，临床移植面临HSCs数目不足及移植用HSCs无统一的质量标准等问题亟待解决。小分子化合物在HSCs调控领域以其浓度可精确调控、结构多样方便优化、易于处理等特点显示出极大优势，已有研究证实，SR1及UM71两种小分子化合物单独使用及组合使用均可显著扩增HSPCs(hematopoietic stem/progenitor cells)，其扩增倍数可达到100倍以上。近年兴起的单细胞技术，可有效解决细胞异质性问题，细胞表面标记研究结合先进的流式细胞分选平台，使得纯化获取单个hHSC成为可能。已有研究表明，造血干细胞的表面标记除了经典的CD34、CD38外，CD90、CD45RA以及HSCs特异性标记CD49f，的表面标记组合可将HSCs与造血祖细胞充分区分开来，从而获得单纯的HSCs。体外造血功能实验CFC(colony forming cells)及CAFC(cobblestone area forming cells)可用于体外验证HSCs功能，其中CFC侧重于观测多能祖细胞的分化能力，CAFC可反映培养系统中具有长期造血重建能力的HSCs。

实验目的： 本研究主要通过利用对HSCs有明显扩增作用的小分子化合物SR1及UM171，构建多参数单细胞的流式分选及培养体系，通过对分选得到的单个hHSC体外培养后流式检测及形态分析，以及体外造血功能实验，验证本单个hHSC体外培养体系的稳定性和可靠性，以便用于后续调控HSCs功能的小分子化合物筛选。

实验方法： 首先，我们通过CD34⁺细胞体外培养确定SR1及UM171的使用浓度，再通过实验摸索得到单细胞培养最适宜的培养基体系，利用BD INFLUX 流式分选仪将单个DAPI^-CD34⁺CD38^-CD90⁺CD45RA^-CD49f⁺细胞分选到96孔板中进行14d体外培养，并完成培养后流式分析及细胞形态检测，分析单个hHSC培养体系中小分子化合物的作用。利用经典的体外造血功能实验作一对比，以此验证体系的稳定性。

结果： CD34⁺细胞体外培养确定了化合物使用浓度为SR1(1μmol/L)，UM171（35nmol/L）,SR1+UM171(750nmol/L+35nmol/L)。单个hHSC体外培养14天流式结果显示，化合物处理组较对照组相比，有效孔数明显增加，在各孔细胞总数无明显差异的情况下，化合物处理组CD34⁺细胞及CD34⁺CD49f⁺细胞绝对数及比例显著增高（P<0.05），几乎所有有效孔细胞均向髓系、淋系两大方向分化，未出现偏系分化，细胞染色结果与流式检测结果一致。CFC结果显示，处理组克隆总数及各种克隆数目较对照组显著增加，且克隆形态及各种克隆比例无差异。CAFC泊松分布结果表明，处理组HSCs频率较对照组明显上升(P<0.05) 。

结论: 此构建的单个hHSC体外培养体系可保证SR1及UM171处理条件下，HSC保持自我更新同时仍具有多向分化潜能，缩短了体外扩增及验证性实验周期，初步解决了HSCs研究面临的异质性问题，为之后筛选靶向HSC的小分子化合物奠定了基础。

关键词：单细胞；造血干细胞；小分子；化合物筛选

Background: Hematopoietic stem cells (HSCs) are the stem cells which have been deeply investigated. HSCs can maintain stability of stem cell pool while differentiate into various cell populations due to their self-renewal and hierarchical differentiate potential. However, most studies and clinical trials are actually conducted with a mixture of differentiated cell subpopulations that include stem cells and more lineage-committed progenitors, owing to the high heterogeneity in blood cell populations and the rarity of HSCs. The identification of small molecule compounds that target HSCs is attractive in stem cell research due to the unique properties and structural diversity of these compounds. Among these small chemicals, SR1 and UM171 offered the most promise for HSPC expansion, inducing even up to over 100-fold increase in HSPC number. Different combinations of cell surface markers enable the isolation of pure HSCs, flow cytometry now can provide an advanced multiparameter technique for separating and analyzing single cell. Researches showed that surface marker of HSCs including not only the classical marker CD34 and CD38, but also CD90、CD45RA and specific marker of HSCs CD49f, this combination of surface marker could separate HSCs with HPCs. CFC and CAFC assays can be conducted to verify function of HSC, CFC could show differentiate ability of multipotent progenitors, furthermore, CAFC assay could reflect HSC that holds ex vivo long-term hematopoietic capacity.

Objective: In this study, we used small molecule compounds SR1 and UM171, which have been reported to have the ability of proliferating HSPC. We set up a platform for multiparameter sing-cell sorting. Furthermore, we employed ex vivo short-term and long-term functional assays to verify our screening platform, with the aim of applying this method for further screening of small molecule compounds.

Methods: We used bulk culture to determine concentration of SR1 and UM171. We constructed the best applied culture system for ex vivo single-cell culture, then sorted single DAPI^-CD34⁺CD38^-CD90⁺CD45RA^-CD49f⁺ cell into wells, detected cell counts and phenotypes both by FCM and cell morphology assay after cultured for 2 weeks. Conducted CFC and CAFC assays to verify the stability of this system.

Results: Determined compound concentration through CD34⁺ cells bulk culture, SR1(1μmol/L)，UM171（35nmol/L）,SR1+UM171(750nmol/L+35nmol/L). After 14-day ex vivo single hHSC culture, flow cytometry resulted that treatment with SR1 and UM171 showed an obvious increase when compared with the control group, both for wells formed colonies and absolute number of CD34⁺ and CD34⁺CD49f⁺ cells(P<0.05). Cellilar morphology assays supported the flow cytometry results, almost all wells showed a balance differentiation to both lymphoid and myeloid cell lineages. CFC assays represented that treatment with small-molecule compounds significantly improved the colony-forming capacity of cultured cells. All kinds of colonies were found and there was no difference in the distribution or morphology of colonies between treatment and control group. CAFC assay showed compound-treated group enhanced the frequency of HSCs compared with control group (P<0.05).

Conclusion: Treatment of sorted cells with SR1 and UM171 in this ex vivo single hHSC assay showed that HSC could retain its self-renewal ability, at the same time, differentiated into different hematopoietic subpopulations. This approach could shorten the time-consuming and costly assay，primarily resolved heterogeneity of HSCs. Our studies thus provided a rapid and efficient single-HSC sorting and culture strategy for further applied to chemical screening.

Key words: single cell; hematopoietic stem cell; small molecule compound;chemical screening