Clonal analysis and transcriptional profiling of ex vivo expanded mobilized peripheral blood hematopoietic stem and progenitor [scRNAseq]

Clonal analysis and transcriptional profiling of ex vivo expanded mobilized peripheral blood hematopoietic stem and progenitor We characterized mPB CD34+ cell population dynamics (n=2 donors) by single cell RNA sequencing (scRNAseq) during 8 days of expansion culture using commercial serum-free media (Miltenyi Bioscience) in the presence of the epigenetic modifier UM171 and early acting cytokines (SFT6: stem cell factor, 100ng/mL; flt3 ligand, 100ng/mL; thrombopoietin, 50ng/mL; interleukin 6, 50ng/mL).

Cultured cells were transduced with a lentiviral vector expressing a marker gene, to simulate a gene therapy context. CD34+ cells from donor 1 were analyzed before culture, on day 4 and on day 8 of expansion. For donor 2, in addition to the CD34+ bulk expansion culture, part of the CD34+ cells were FACS-sorted into subpopulations enriched in primitive HSPC (CD34+CD38-), committed granulocyte-monocyte progenitors (GMP) or megakaryocyte-erythrocyte progenitors (MEP), and subsequently expanded for 7 days.