A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.1189-1189
• Main Authors: Walasek, Marta A., Chau, Crystal, Barborini, Christian, Richardson, Matthew, Szilvassy, Stephen J., Louis, Sharon A., Thomas, Terry E., Eaves, Allen C., Wognum, Albertus W.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-128830

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was o