Methodologic considerations on how to identify human hematopoietic stem cells

Recently, human CD34⁺ hematopoietic stem cells (HSCs) have been purified to a frequency of approximately one in three cells, a population denoted as CD34⁺CD38^-CD45RA^-CD90^+/- endothelial protein C receptor (EPCR)⁺ HSCs. This work aimed to evaluate the methodology for CD34⁺ HSC isolation, exploring differences in antibody clones, conjugates, source of cells, and additional cell surface antigens (integrin-α6, CLEC9A, and GPRC5C) to enhance the purity of these EPCR⁺ HSCs. We are emphasizing here the importance of experimental planning and antibody panel selection concerning the isolation of these human HSCs from multiple sources and providing important notes on the pitfalls of the reagents used for such purposes. Our results should enable a better reproducibility of results between laboratory tests as well as further pursuits of work toward improving the enrichment of human HSCs.