Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

Abstract

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

Hepatic leukemia factor-expressing paraxial mesoderm cells contribute to the developing brain vasculature

“…Moreover, Hlf expression does not mark EMPs within the yolk sac or endothelial cells of the yolk sac, AGM, or fetal liver ( Yokomizo et al, 2019 ). These observations were supported by the recent report by Tang et al (2021) . Compared with the unique Hlf expression in embryonic HSCs from E10.5 to E14.5, Hlf expression in non-hematopoietic cells remains elusive.…”

Multiple waves of fetal‐derived immune cells constitute adult immune system

“…Another recent study by Yokomizo et al 102 showed that FL HSCs do not contribute to FL hematopoiesis using lineage‐tracing mouse models. Hlf has recently been reported to be expressed in pre‐HSCs in the AGM region as well as in human HSCs 103–106 . They found that HlfCreERT2 specifically marked pre‐HSCs and not EMPs in the YS and that HlfCre ‐derived cells contributed to most hematopoietic progenitors in the FL, suggesting that Hlf + pre‐HSC populations contain greater percentages of HSC‐independent hematopoietic progenitors than HSCs.…”

“…Hlf has recently been reported to be expressed in pre-HSCs in the AGM region as well as in human HSCs. [103][104][105][106] They found that HlfCreERT2 specifically marked pre-HSCs and not EMPs in the YS and that HlfCre-derived cells contributed to most hematopoietic progenitors in the FL, suggesting that Hlf + pre-HSC populations contain greater percentages of HSC-independent hematopoietic progenitors than HSCs. Furthermore, Evi1 is highly expressed in the HSCs, and Evi1CreERT2 was able to specifically label HSCs in the FL.…”

Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

“…Bulk RNA-seq revealed that hPSC-derived day-10 hematopoietic progenitors and human embryonic DA CD144+ CD45+ HSCs 143 expressed comparable levels of HSC signature transcription factors, including HLF , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 HOPX , 160 NKX2.3 , 161 MEIS1 , 162 , 163 , 164 , 165 and HOXA genes ( HOXA5 , HOXA9 , and HOXA10 ) 62 , 65 , 74 ( Figures 6 B and S7 C). As a negative control, human DA CD144− CD45+ non-HSCs minimally expressed these HSC signature transcription factors ( Figures 6 B and S7 C).…”

“… 65 Hlf is required for HSC function 66 and constitutes an exquisitely specific HSC marker. 67 , 68 , 69 , 70 , 71 , 72 , 73 Likewise, HOXA5-10 are required for HSC function, 62 , 74 and forced expression of HOXA and other genes enhances the engraftment of hPSC-derived hematopoietic progenitors. 75 , 76 This underscores the importance of generating HLF + HOXA + hematopoietic progenitors from hPSCs and discovering the extracellular signals that ignite expression of these hallmark HSC markers.…”