Single cell RNA-Seq of mouse bone marrow-derived hematopoietic stem cells after 7-days in viro culture

Hematopoietic stem cells (HSCs) are cells able to self-renew and differentiate into all blood lineages. We previously have performed serum-free single-cell culture for HSC1 (CD201+CD150+CD48-CD41-CD34-LSK) and competitive repopulation with cultured cells. Single highly purified HSC1 were cultured with stem cell factor (SCF), SCF + G-CSF, or SCF + thrombopoietin (TPO) for 7 days. SCF directly acted on HSC1 and maintained their long-term multilineage reconstitution after serial transplantation. Compared with SCF alone, SCF+G-CSF neither enhanced the number of divisions of HSC1 nor enhanced their levels of reconstitution after serial transplantation. SCF+TPO significantly increased the number of divisions of HSC1. The levels of reconstitution of each lineage in SCF + TPO culture were significantly increased at early months after primary transplantation, however, it gradually decreased after secondary transplantation. Therefore, we performed single-cell RNA-Seq to examine the gene expression of cells cultured with different cytokine combinations. Overall design: scRNA-Seq were successfully performed on 43,48, and 48 HSC1 cells after cultured with SCF, SCF+G-CSF, and SCF+TPO, respecitvely