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最为广泛的造血分化诱导体系之一。由于当前对该分化过程的机制认知不足，导致其造血分化效率偏低。为此，本研究采用单细胞qPCR技术，解析了不同分化阶段单个CD34阳性细胞的基因表达谱。基于造血转录因子的动态表达特征，我们在造血分化早期阶段，对特定的造血转录因子（Gata2、Lmo2、Etv2、ERG及SCL）实施了过表达操作。过表达处理后，我们获得了更多CD34阳性细胞，且其CD43与CD31的表达水平符合正常标准——这两种标志物是定义各类造血祖细胞的关键指标。此外，经集落形成单位实验检测，这些CD34阳性细胞展现出正常的分化潜能，其基因表达谱亦无异常。本研究证实，单细胞qPCR技术可为造血分化体系的优化提供科学指导，而对筛选得到的造血转录因子进行瞬时过表达，能够有效提升造血分化效率。