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Principal Investigator: CHRISTOPHER KLUG
Organization: UNIVERSITY OF ALABAMA AT BIRMINGHAM
Fiscal Year: 2024
Award: $284,416
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
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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 studies have further highlighted the close approximation of LT-HSC with perivascular MSC that express a Nestin-GFP transgene 9 and other cell-surface markers and secreted factors including CD51, Pdgfrα, CD146, and stem cell factor (Scf)10-17. Deletion of Scf from either perivascular MSC or endothelial cells using Lepr-Cre or Tie2-Cre depleted HSC numbers, which was not observed when Scf was deleted from hematopoietic cells, osteoblasts, or Nestin-Cre+ cells 18. Since membrane-bound Scf is essential for maintaining LT-HSC in vivo 19,20, LT-HSC are likely in direct contact with Scf-expressing perivascular MSC during steady-state hematopoiesis in adult mice.
Using clonally-derived, Lepr+CD146+Pdgfrα+CD51+Scf+Cxcl12HI primary murine adult BM MSC lines with in vitro and in vivo multilineage developmental potential, we showed that Wnt signaling modulated the ability of LT-HSC to self-renew in long-term co-cultures of 20 FACS-sorted LT-HSC and the clonal MSC lines 21. These MSC lines express high levels of Wnt ligands that activate noncanonical Wnt signaling, particularly Wnt11 and Wnt5a, which potently suppress the canonical (α-catenin-dependent) pathway. This was consistent with the lack of expression of the canonical Wnt target gene, Axin2, in both LT-HSC and MSC in the co-cultures 21. Wnt11 and Wnt5a are also highly expressed in the most primitive, self-renewing adult BM skeletal stem cell subset (mSSC) that gives rise to bone and cartilage in vivo 22, in Nestin-GFP+ stromal cells 23, in Osterix-expressing osteolineage cells 24, and in adult BM stromal progenitor cells by single-cell RNAseq 25. In preliminary co-culture studies, we show that activation of canonical Wnt signaling in the MSC lines through ectopic expression of either Wnt3a or Wnt10b stimulates loss of LT-HSC self-renewal and/or survival and potently activates an osteolineage gene expression program in MSC. Conversely, overexpression of Wnt11 or Wnt5a in MSC enhanced LT-HSC long-term repopulating activity and blocked activation of an osteolineage gene signature in MSC. Further, deletion of Wnt11 in MSC using Prx1-Cre increased deposition of trabecular bone in newborn mice, which suggests an important role for Wnt11 in suppressing osteolineage differentiation of MSC induced by canonical Wnt signaling. Based on these findings, we hypothesize that noncanonical Wnt signaling in MSC maintained by Wnt11 and Wnt5a is essential to preserve the self-renewal and multilineage differentiation potential of both LT-HSC and MSC in the BM niche in order to sustain life-long blood cell and bone production. This hypothesis will be addressed in the following Specific Aims:
Aim 1: Functionally define 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 with Cre-inducible, biallelic floxed alleles of both Wnt11 and Wnt5a. Transplantation studies will address whether the absence of either Wnt11 or Wnt5a, or both Wnt11/Wnt5a, in MSC results in loss of LT-HSC reconstitution potential after LT-HSC/MSC co-culture in vitro. The ability of MSC to self-renewal in the absence of Wnt11/Wnt5a will be assessed by CFU-F assays, while biased and irreversible activation of MSC commitment to the osteoblast lineage due to the inability to suppress canonical Wnt signaling in MSC will be assessed using osteogenic, adipogenic and chondrogenic growth conditions in vitro and in vivo.
Aim 2: Define the unique and redundant functions of Wnt11 and Wnt5a in sustaining adult BM LT-HSC and MSC self-renewal and multilineage developmental potential in vivo during homeostasis. Here, we will use Prx1-Cre, Prx1-CreERT and Wnt11-CreERT mice to delete Wnt11, Wnt5a, or both Wnt11 and Wnt5a, to determine whether they are both essential for maintaining LT-HSC and MSC self-renewal in vivo. We will use Wnt11-CreERT;Rosa26-LSL-tdTomato;a-catulinGFP mice to determine localization of Wnt11-expressing MSC with respect to LT-HSC in adult BM, and for lineage tracing to determine the contribution of tdTomato+ MSC to the osteoblast, adipocyte and chondrocyte cell lineages in an unperturbed setting in vivo.
Defining factors that preserve life-long maintenance of both blood-forming LT-HSC and bone-forming MSC has been a fundamental challenge since loss of factors controlling an essential function like self-renewal of either stem cell population would likely result in embryonic lethality. This study will utilize inducible, floxed alleles of the noncanonical Wnt ligands, Wnt11 and Wnt5a, to elucidate their role in coordinate regulation of both adult LT-HSC and MSC self-renewal. Developing a deeper understanding of factors regulating crosstalk between LT-HSC and MSC, which are likely in direct cell-cell contact in adult BM, will significantly enhance our ability to manipulate LT-HSC and MSC in vitro, with the goal of improving the efficacy of stem cell transplantation, tissue repair, and targeted gene correction approaches that can then be applied in the clinic.
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