Defining the role of Wnt11 and Wnt5a in regulating hematopoietic and skeletal stem cell self-renewal potential during homeostasis and stress - (PLEASE KEEP IN WORD, DO NOT PDF) Significant progress has been made in identification of key components of the adult bone marrow (BM) mesenchymal stromal cell (MSC) niche that are critical for maintaining the function of rare, long-term self-renewing hematopoietic stem cells (LT-HSC). In particular, perivascular MSC expressing high levels of Cxcl12 and stem cell factor (Scf) and cell-surface markers including Lepr, Pdgfrα, and CD51, have been localized in very close proximity to LT-HSC and likely contribute to the maintenance of LT-HSC quiescence during steady-state hematopoiesis. We have shown that clonally-derived, Lepr+CD146+Pdgfrα+CD51+Scf+Cxcl12HI primary murine adult BM MSC lines with in vitro and in vivo multilineage developmental potential can significantly enhance the ability of LT-HSC to self-renew after 10 days of co-culturing 20 FACS-sorted LT-HSC with the MSC lines. Further, we show that LT-HSC self-renewal/survival could be further enhanced by increasing MSC expression of the evolutionarily-related noncanonical Wnt ligands, Wnt11 and Wnt5a, which potently suppressed canonical Wnt signaling. Conversely, ectopic expression of the canonical Wnt ligands, Wnt3a or Wnt10b, in MSC resulted in loss of transplantable LT-HSC after co-culture, rapid upregulation of an osteolineage gene expression profile in MSC, and upregulation of both endogenous Wnt11 and Wnt5a expression, suggesting activation of a negative feedback loop that would suppress further canonical Wnt signaling. In addition, we observed that loss of Wnt11 in Prx1+ MSC in vivo increases trabecular bone number in 3-week-old mice, which supports a function for Wnt11 in suppressing MSC differentiation specifically to the osteoblastic cell lineage. Based on these findings, we hypothesize that noncanonical Wnt signaling maintained by both Wnt11 and Wnt5a preserves self-renewal and multipotency of both LT-HSC and MSC within the BM niche. This hypothesis will be addressed in two Aims. Aim 1 will examine whether Wnt11 and Wnt5a coordinately control both LT-HSC and MSC self-renewal and multilineage developmental potential using LT-HSC/MSC co-cultures and primary MSC sublines where biallelic deletions of Wnt11 and Wnt5a can be induced with Cre recombinase. Aim 2 will assess the function of Wnt11 and Wnt5a in regulating LT-HSC and MSC homeostasis in vivo using Prx1-CreER or Wnt11-CreER mice to conditionally delete floxed alleles of Wnt11 and/or Wnt5a in adult BM MSC. Using Wnt11-CreERT;Rosa26-LSL-tdTomato;a-catulinGFP reporter mice, we will also determine the proximity of Wnt11+ MSC with respect to LT-HSC in BM and define the contribution of Wnt11+ MSC to the osteoblast, adipocyte and chondrocyte cell lineages in unperturbed conditions. Together, these studies will be important for understanding noncanonical Wnt signaling-dependent mechanisms that coordinate the life-long production of blood and bone-forming cells by LT-HSC and MSC, which are likely in direct cell-cell contact in vivo. This knowledge can then be applied to enhance the efficacy of stem cell transplantation, tissue repair, and targeted gene correction approaches that benefit patients in the clinic. SPECIFIC AIMS: Numerous studies have shed light on the cellular and molecular factors that control adult long-term self-renewing hematopoietic stem cell (LT-HSC) self-renewal and differentiation during steady-state and stress hematopoiesis. It is appreciated that a number of cell types including various mesenchymal stromal cell (MSC) subsets, sinusoidal endothelial cells (SEC), arterioles, neuronal cells, macrophage, and megakaryocytes all contribute to regulation of HSC homeostasis within the bone marrow (BM) niche 1-7. Using deep confocal imaging of optically cleared BM and highly specific LT-HSC markers like α-catulin, showed that the vast majority of quiescent and dividing LT-HSC are localized near SEC and MSC expressing leptin receptor (Lepr) and high levels of Cxcl128. Other imaging and cell ablation stud
