Single-cell qPCR facilitates the optimization of hematopoietic differentiation in hPSCs/OP9 coculture system

Human pluripotent stem cells (hPSCs)/OP9 coculture system is a widely used hematopoietic differentiation approach. The limited understanding of this process leads to its low efficiency. Thus, we used single-cell qPCR to reveal the gene expression profiles of individual CD34+ cells from different stages of differentiation. According to the dynamic gene expression of hematopoietic transcription factors, we overexpressed specific hematopoietic transcription factors (Gata2, Lmo2, Etv2, ERG, and SCL) at an early stage of hematopoietic differentiation. After overexpression, we generated more CD34+ cells with normal expression level of CD43 and CD31, which are used to define various hematopoietic progenitors. Furthermore, these CD34+ cells possessed normal differentiation potency in colony-forming unit assays and normal gene expression profiles. In this study, we demonstrated that single-cell qPCR can provide guidance for optimization of hematopoietic differentiation and transient overexpression of selected hematopoietic transcription factors can enhance hematopoietic differentiation.人多能干细胞（human pluripotent stem cells，hPSCs）与OP9细胞共培养体系是目前应用广泛的造血分化实验体系。由于对该分化过程的认知存在局限，该体系的造血分化效率普遍偏低。为此，我们采用单细胞qPCR（single-cell qPCR）技术，解析了不同分化阶段单个CD34阳性（CD34+）细胞的基因表达谱。基于造血转录因子的动态表达模式，我们在造血分化早期过表达了特定的造血转录因子（Gata2、Lmo2、Etv2、ERG及SCL）。过表达后，我们获得了更多CD34+细胞，且这些细胞的CD43与CD31表达水平正常——这两种分子是定义各类造血祖细胞的标志性标志物。此外，这些CD34+细胞在集落形成单位实验中展现出正常的分化潜能，且基因表达谱未出现异常。本研究证实，单细胞qPCR可为造血分化体系的优化提供指导，且瞬时过表达筛选得到的造血转录因子可有效提升造血分化效率。