Molecular signatures of heterogeneous stem cell populations are resolved by linking single cell functional assays to single cell gene expression

The discovery of significant heterogeneity in the self-renewal durability of adult haematopoietic stem cells (HSCs) has challenged our understanding of the molecules involved in population maintenance throughout life. Gene expression studies in bulk populations are difficult to interpret since multiple HSC subtypes are present and HSC purity is typically less than 50% of the input cell population. Numerous groups have therefore turned to studying gene expression profiles of single HSCs, but again these studies are limited by the purity of the input fraction and an inability to directly ascribe a molecular program to a durable self-renewing HSC. Here we combine single cell functional assays with flow cytometric index sorting and single cell gene expression assays to gain the first insight into the gene expression program of HSCs that possess durable self-renewal. This approach can be used in other stem cell systems and sets the stage for linking key molecules with defined cellular functions. Overall design: single-cell RNA-Seq of haematopoietic stem cells

成年造血干细胞（adult haematopoietic stem cells, HSCs）自我更新耐久性存在显著异质性，这一发现对我们关于贯穿生物体全生命周期的群体维持相关分子的认知提出了挑战。由于样本中存在多种造血干细胞亚型，且输入细胞群体中造血干细胞的纯度通常不足50%，针对群体细胞的基因表达研究结果往往难以解读。因此，诸多研究团队转而开展单个造血干细胞的基因表达谱分析，但此类研究仍受限于输入组分的纯度，且无法直接将特定分子程序与具备持久自我更新能力的造血干细胞建立对应关系。本研究将单细胞功能测定、流式细胞术指数分选与单细胞基因表达测定相结合，首次揭示了具备持久自我更新能力的造血干细胞的基因表达程序。该方法可推广应用于其他干细胞研究系统，为关联关键分子与明确的细胞功能奠定了研究基础。实验整体设计：造血干细胞的单细胞RNA测序（single-cell RNA-Seq）