Single cell transcriptome analysis reveals markers of naïve and lineage-primed hematopoietic progenitors derived from human pluripotent stem cells

Abstract

14During embryogenesis the hematopoietic system develops through distinct waves that 15 generate progenitors with increasing lineage potential, ultimately producing 16 haematopoietic stem cells (HSCs). In vitro differentiation of human pluripotent stem cells 17 (hPSCs) follows the early steps of haematopoietic development but the production of 18HSCs has proven more challenging. To study the dynamics and heterogeneity of 19 hematopoietic progenitor cells generated in vitro from hPSCs, we performed RNA 20 sequencing of over 10000 CD235a -CD43 + single cells. We identified the transcriptome of 21 naïve progenitors and those primed toward erythroid, megakaryocyte and leukocyte 22 lineages, and revealed their markers by clustering, trajectory analyses and functional 23 assays. CD44 marks naïve clonogenic progenitors that express the transcription factor, 24 LMO4 and can be expanded upon BMP4 stimulation. Naïve progenitors give rise to primed 25 CD326 + erythroid, ICAM2 + CD9 + megakaryocyte, and monocyte, neutrophil and eosinophil 26 progenitors. We have generated an online dataset of human hematopoietic progenitors 27and their transcriptional remodelling upon lineage priming. 28 29Taken together these data indicate that CD235a -CD43 + cells, from differentiating hPSCs, 126 contains definitive hematopoietic progenitors and excludes cells derived from the first, 127 primitive wave. We anticipated that CD235a -CD43 + compartment would also comprise the 128 early stages of lineage priming, capturing the hierarchy of early human developing 129 progenitors. 130 131 132

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development

“…Nearby, and closely related to cycling hematopoietic precursors are naive hematopoietic progenitors (cluster 9) expressing RUNX1C-GFP (Figure 6A) but not CD34 and cell-cycle-associated genes. Naive hematopoietic progenitors express elevated levels of CPA3, GATA2, HDC, SRGN, KIT, CD43, CD44, and LMO4 (Figure S4D), as reported by Fidanza et al (2019).…”

“…Hematopoietic cell clusters were identified from differentially expressed lineage-specific markers reported in the literature (violin plots shown in Figure S4). We have adopted Fidanza's terminology to name human PSCderived hematopoietic clusters (Fidanza et al, 2019(Fidanza et al, , 2020. For example, cluster 18 was identified as granulocyte-primed hematopoietic progenitors because of elevated levels of myeloperoxidase and proteinase 3 (Figure S4E).…”

Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy

“…The resulting HE also expressed SOX17 ( Figures S1B – S1D ), a transcription factor expressed in AGM vascular structures and required for HSPC generation ( Clarke et al, 2013 ; Kim et al, 2007 ; Ng et al, 2016 ). Transforming growth factor beta (TGFβ) inhibitor SB431542 (SB) significantly promoted EHT for generation of CD45+CD43+ HSPCs ( Chang et al, 2022 ) that co-expressed definitive markers CD44 ( Fidanza et al, 2019 ; Oatley et al, 2020 ) and RUNX1c ( Ng et al, 2016 ) ( Figures S1E – S1H ). The resulting HSPCs also maintained a high viability after freeze thaw ( Figures S1I and S1J ).…”

Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy

“…The employment of TGFβ inhibitor SB431542 (SB) (Bertrand et al, 2010; Boisset et al, 2010; Kissa and Herbomel, 2010) promoted EHT process for the generation of CD45+CD43+ HSPCs ( Fig. S1E-F ) that co-expressed definitive hematopoiesis markers CD44 (Fidanza et al, 2019; Oatley et al, 2020) ( Fig. S1G ) and RUNX1c (Ng et al, 2016) ( Fig.…”