Gene expression at single cell level in human umbilical cord blood cells enriched for hematopoietic stem cells and cultured in conditions that maintain HSCs ex vivo.

The capacity for self-renewal of human hematopoietic stem cells (HSCs) is lost upon typical culture conditions, which often include high levels of multiple hematopoietic cytokines, serum, and/or support cells. We found that culture of HSCs in CR medium (basal culture medium with inhibitors of GSK-3 (CHIR99021) and mTORC1 (Rapamycin) and no serum or cytokines) maintains self-renewal in human HSCs for at least 7 days without loss of repopulating activity (Huang et al PMID 23142822). Here we performed single cell RNA-seq to determine transcriptomic changes associated with HSC maintenance in CR. We compared a population of freshly isolated human hematopoietic cells enriched for HSCs (CD34+CD38-CD45RA-CD90+) to cells cultured for 2 days in basal medium (vehicle control), CR, and CR and low dose cytokines (CRCY). The data show that, compared to Day 0 uncultured cells, the HSC pool is maintained when CR is included (CR or CRCY medium) but not in the vehicle control. The most highly enriched transcript in the HSC population was the chaperone/UPR sensor GRP78/HSPA5. This population was also highly enriched for immediate early response genes and components of the AP-1 complex (FOS and JUN family transcription factors) which mediate the immediate early response to stress. Human umbilical cord blood CD34+ cells (StemCell Technologies, mixed donor, cat# 70008, lot# 1901310270) were sorted for CD34+CD38-CD45RA-CD90+ cells, which enriches for hematopoietic stem cells (HSCs). Sorted cells were cultured with vehicle (DMSO), 3 µM CHIR99021 + 5 nM Rapamycin (CR), or CR with low dose cytokines (CRCY) for 2 days and were then collected for single cell RNA-seq using the 10XGenomics platform. Freshly thawed CD34+ cells from the same pool of donors (Day 0) were sorted and subjected to single cell analysis in parallel (Day 0 control).