Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators

Abstract

SUMMARY Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on differential single-cell gene expression analysis of mesenchymal osteolineage cells close to and further removed from hematopoietic stem/progenitor cells to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. Amongst the genes which were preferentially expressed in proximal cells, we functionally examined three secreted or cell surface molecules not previously connected to HSPC biology: the secreted RNase Angiogenin, the cytokine IL18 and the adhesion molecule Embigin and discovered that all of these factors are HSPC quiescence regulators. Our proximity-based differential single cell approach therefore reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance understanding of microenvironmental regulation of stem cell function.

Secreted protein Del-1 regulates myelopoiesis in the hematopoietic stem cell niche

“…and perivascular stromal cells, like CAR cells, (9,(14)(15)(16), and by cells that mediate the reconstitution of hematopoiesis after transplantation, like OSL cells (3,17,18). Del-1 promotes steady-state myelopoiesis.…”

“…Analysis of BM revealed that this difference was due to impaired reconstitution of myelo- of hematopoietic progenitors, particularly LT-HSCs, toward the myeloid lineage. After transplantation, hematopoietic progenitor cells localize in proximity to blood vessels at the endosteal area of the BM (3,17,18,39), which could be attributed to irradiation-induced disruption of sinusoids, though not of endosteal arterioles (3). The expression of Del-1 in the peri-arteriolar region and by OSL cells, which also contribute to the restoration of hematopoiesis after transplantation, prompted us to further investigate in more detail how Del-1 supports the regeneration of myelopoiesis.…”

“…Recent advances in BM imaging reveal a perivascular distribution of HSCs in their niche, which comprises several cell populations. Endothelial (4)(5)(6)(7)(8)(9)(10)(11) and perivascular stromal cells (9,(12)(13)(14), including CXCL12-abundant reticular cells (CAR cells) (15,16), are considered the main cell populations constituting the homeostatic HSC niche, whereas osteolineage cells and osteoblasts have a significant role in the post-transplant HSC niche (3,17,18). Niche cells regulate HSC homeostasis through paracrine growth factor and chemokine signals as well as via adhesive interactions (3,5,9,19,20).…”

“…In particular, Del-1 is expressed by those niche cells that have a major role in the maintenance of HSCs, i.e., arteriolar endothelial cells and perivascular CAR cells (3,6,7,9,15). In addition, Del-1 is expressed by cells of the osteoblastic lineage that crucially mediate the engraftment of HSCs in the post-transplantation niche (3,17,18). This spatial distribution of Del-1 raised the possibility that it might be involved in the regulation of hematopoiesis.…”

Regulation of normal and leukemic stem cells through cytokine signaling and the microenvironment

“…These include CXCL-12, angiopoietin-1, stem cell factor (SCF), jagged-1, and thrombopoietin [56]. Recent work has taken this a step further using single cell analysis to define populations in close proximity to HSCs [57]. Cells within short distances of HSPCs had upregulation of cell surface proteins Vcam1, Adam9, and Amot as well as immune response genes Map3K14, CXCL12, and IL-18.…”