Integration of single-cell transcriptomes and biological function reveals distinct behavioral patterns in bone marrow endothelium

Abstract

Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue’s homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs’ ECs to decode mechanistic information and explore therapeutics.

An epigenetically distinct HSC subset supports thymic reconstitution

“…Additionally, we observed functional differences within Kit lo HSCs in the context of aging, specifically a competitive disadvantage in Old Kit lo HSCs, potentially attributable to their differences in Zbtb1 expression. Consistent with prior research, our study suggests Zbtb1 modulates Notch-dependent 56 and cellular survival programs 24,69 . However, further mechanistic studies are needed to elucidate the precise transcriptional mechanisms underlying this regulation.…”

“…Consistent with prior research, our study suggests Zbtb1 modulates Notch-dependent 60 and cellular survival programs. 29, 74 However, further mechanistic studies are needed to elucidate the precise transcriptional mechanisms underlying this regulation. While the definitive role of Zbtb1 in HSCs is not established, it may not have a direct role at the HSC stage; instead, its expression could be indicative of epigenetic states that facilitate or permit Zbtb1 expression and function at later stages of development, in lymphocyte progenitors.…”

“…Recent reports have shown remarkable functional heterogeneity of HSC [21][22][23][24][25][26] subsets, highlighting discordance between current HSC phenotypic definitions to their molecular identities [11][12][13] . To identify HSCs with an enriched multilineage program and gain insights into their transcriptional and epigenetic framework, we performed multiome single-cell RNA and ATAC sequencing on hematopoietic stem cells (LT-HSCs: Lineage -CD34 -CD48 -CD150 + Sca-1 + c-Kit + ) from 2-mo (young) or 24-mo (old) female mice.…”

Endomucin knockout leads to delayed retinal vascular development and reduced ocular pathological neovascularization

“…The importance of EMCN extends to in vivo models as well, where its knockout has been demonstrated to disrupt vascularization in the developing mouse retina 26 . In the context of tip cell formation, which is essential for initiating new blood vessel sprouts, EMCN may influence the selection and behavior of these specialized endothelial cells as indicated by previous study that noted that Emcn+ bone marrow ECs expressed markers associated with endothelial tip cells and crucial for sprouting during vascular morphogenesis, such as Apln, Cxcr4, Kdr, Dll4, Angpt2, Sox17 45 . This is consistent with what we observed during the retinal vasculature development.…”

Recapitulation of human endochondral ossification by SOX9+ sclerotomal progenitors.

“…Immunostaining of bone marrow markers further revealed that vascular architecture and bone marrow stroma were formed in the grafts (Figure 3I), with COL I and EMCN to mark trabecular bone intertwined by sinusoidal vessels 60 , LEPR to mark recruited host stromal cells 61 , and CXCL12 and SCF to mark cytokines secreted by the stromal cells 61,62 .…”