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

Abstract

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.

Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency

“…Importantly, OP9 cells secreted factors and signaling molecules to induce hematopoietic differentiation [ 30 , 31 ]. One study used single-cell q-PCR to track the dynamic gene expression of OP9-hESCs co-culture system at different stages and transiently over-expressed specific transcription factors (TFs) at different stages [ 32 ] and showed that over-expression of these TFs mainly promoted differentiation at the initial stage. Another recent study used 3D cell culture by co-culture of OP9 cells, and provided a minimal, scalable, biomimetic in vitro model of hematopoietic differentiation in the absence of exogenous cytokines [ 33 ].…”