Mammalian bone marrow (BM) regeneration after myeloablation requires orchestrated interplay between osteogenesis, angiogenesis and hematopoiesis. Specific subsets of endothelial cells (ECs) have been identified in mice, including sinusoidal ECs (Hooper et al., 2009) and H-endothelium (Kusumbe et al., 2014, 2016; Ramasamy et al., 2014), that drive and coordinate these processes. Their relevance to human biology, however, remains largely elusive.
Here, using a combinatory approach of cell purification and transcriptomics, we identify and characterize EC subsets associated with BM niche formation during ontogeny and regeneration in humans.
Massive parallel RNA sequencing of FACS-purified BM ECs (CD45-CD235-7AAD-CD31+CD9+) from AML patients in the regenerating phase after chemotherapy (n=3) revealed upregulated endoglin (CD105) expression, in comparison to ECs at diagnosis or from normal BM (n=7) (FPKM fold-change increase (FCI): 10.1 ± 4.9; GLM LRT Edge R; p=0.001). FACS analysis confirmed a strong enrichment of endoglin-expressing ECs in regenerative marrow (n=48) compared to normal BM (n=9) (10.81% ± 2.14 vs 0.48% ± 0.24 of CD31+CD9+ cells; p=0.043).
Similarly, in human fetal BM at 15-20 weeks after gestation (n=15), when angiogenesis, osteogenesis and hematopoiesis are tightly coupled, a striking predominance of endoglin-expressing ECs (62.8% ± 5.9 of CD31+CD9+ cells) was found, confirmed by in situ immunohistochemistry in comparison to adult core hip bone biopsies. Comprehensive transcriptional characterization by RNAseq of these endoglin-expressing 'human regeneration-associated EC' (hREC) from fetal bone (n=7) in comparison to endoglin- ECs revealed a remarkable phenotypic and molecular similarity to murine H-endothelium as well as activation of pathways and factors implicated in hematopoiesis, osteogenesis and angiogenesis. These included upregulated expression of CD31 (PECAM1) (FPKM FCI: 4.7 ± 1.0; p=0.04), mucin-like sialoglycoprotein endomucin (EMCN) (FPKM FCI: 435 ± 112; p <0.001), stromal cell-derived factor 1 (CXCL12) (FPKM FCI: 108 ± 34; p <0.001) and transcriptional activation of NOTCH (Normalized Enrichment score (NES): 1.51, False Discovery Rate-adjusted q-value (FDR): <0.10) and HIF1a (NES: 1.60, FDR <0.10) signaling pathways.
IL-33 was found to be highly and significantly overexpressed in both fetal and regenerative marrow ECs in comparison to steady-state normal BM ECs (FPKM FCI: 587 ± 128 and 174 ± 86, respectively, both p <0.001). Expression of IL-33 in endoglin-expressing ECs was confirmed at the protein level in both humans and mice. Recombinant human IL-33 (1) promoted the expansion of distinct subsets of hematopoietic precursor cells, specifically immunophenotypic multipotent progenitors (2.5-FCI ± 0.43; p<0.05) and multilymphoid progenitors (2.7-FCI ± 0.2; p<0.01), (2) increased metabolic activity in HUVECs (PrestoBlue Cell Viability assay; mean fluorescence intensity 1.40-FCI ± 0.02; p=0.03), and (3) promoted osteogenic differentiation of BM-derived mesenchymal cells (Alizarin red-staining and colorimetric assessment of calcium deposition (3.61-FCI ± 0.63; p<0.02)). Repressing IL-33 in HUVEC by shRNAs impaired their expansion in culture dramatically (FC decrease 6-7 fold; p=0.01). In vivo, enrichment of an endoglin-expressing subset of BM ECs was observed in mice exposed to a myeloablative dose of 5-fluorouracil (n=5) compared to steady-state mice (n=3) (FCI: 9.0 ± 1.7; p=0.01). Administration of recombinant murine IL-33 to C57BL/6 wild-type mice recapitulated the expansion of hematopoietic progenitors with myeloid and lymphoid potential (HPC1 subset) (FCI 2.6 ± 0.5; p=0.003), and resulted in a relative increase in CD31+CD105+ BM ECs (FCI 3.5 ± 0.3; p=0.002) as well as bone progenitor (Lin-Ter119-CD51+Sca-) cells (FCI 2.9 ± 0.7; p=0.06) within the niche.
Collectively, the data reveal the existence of an endothelial subset associated with BM regeneration and ontogeny in humans and identify IL-33 as a potential driver of marrow recovery. The molecular characterization of hREC is, thus, anticipated to instruct the discovery of novel determinants of EC-driven regeneration of the hematopoietic system after injury.
Raaijmakers: Novartis: Consultancy.
Asterisk with author names denotes non-ASH members.