Defining the role of Wnt11 and Wnt5a in regulating hematopoietic and skeletal stem cell self-renewal potential during homeostasis and stress

定义 Wnt11 和 Wnt5a 在稳态和应激过程中调节造血和骨骼干细胞自我更新潜力的作用

基本信息

批准号：
10731650
负责人：
CHRISTOPHER KLUG
金额：
$ 29.7万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2026-05-31
项目状态：
未结题

项目摘要

(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 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. Modified

（请保持言语，不要PDF）在识别成年骨髓（BM）间充质基质细胞（MSC）的关键成分方面取得了重大进展，这对于维持稀有的长期自我更新造血干细胞（LT-HSC）的功能至关重要。特别是，表达高水平的CXCL12和干细胞因子（SCF）以及包括LEPR，PDGFRα和CD51在内的细胞表面标记的MSC已被定位在与LT-HSC的非常接近的位置，并可能有助于维持在稳态的静态血压内LT-HSC戒烟。 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.此外，我们表明，通过增加与进化相关的非环wnt配体，Wnt11和Wnt5a的MSC表达，可以进一步增强LT-HSC自我更新/生存率，从而有可能抑制规范的Wnt信号传导。相反，MSC中规范Wnt配体Wnt3a或Wnt10b的依托表达导致共培养后可移植的LT-HSC丧失，在MSC中快速上调骨素基因表达谱在MSC中的基因表达谱，并在Entogenos Wnt1111和Wnt5a表达中的上调，提出了poctient of Feedback of Feedback loop的上调。此外，我们观察到，体内PRX1+ MSC中WNT11的损失会增加3周大的小鼠的小梁骨数，这支持WNT11的功能，以抑制MSC分化为特异性分化为成骨细胞谱系。基于这些发现，我们假设Wnt11和Wnt5a维持的非规范WNT信号传导可保留LT-HSC和MSC在BM利基市场中的自我更新和多稳定性。该假设将以两个目的解决。 AIM 1将检查WNT11和WNT5A是否使用LT-HSC/MSC共培养和主要的MSC Sublines来控制LT-HSC和MSC自我更新和多琳的发育潜力，其中Wnt11和Wnt5a的BioLELIC缺失可以用CRE重组酶诱导。 AIM 2将评估Wnt11和Wnt5a在使用PRX1-CREER或WNT11-CREER小鼠体内调节LT-HSC和MSC稳态中的功能，以有条件地删除成人BM MSC中Wnt11和/或Wnt5a的等位基因。使用wnt11-creert; rosa26-lsl-tdtomato; a-catulingFP报告基因小鼠，我们还将确定Wnt11+ MSC与BM中LT-HSC的接近性，并定义Wnt11+ MSC对成骨细胞，脂肪细胞和脑核细胞层的贡献，并在未列表中均为未列出。总之，这些研究对于理解非规范Wnt信号依赖性机制至关重要，该机制可以通过LT-HSC和MSC来协调血液和骨形成细胞的生产，这些机制可能在体内直接细胞 - 细胞接触中。然后，可以应用这些知识来提高干细胞移植，组织修复和靶向基因校正方法的效率，从而使诊所患者受益。具体目的：大量研究阐明了控制成年长期自我更新造血干细胞（LT-HSC）自我更新和分化过程中的细胞和分子因素。值得注意的是，包括各种间充质基质细胞（MSC）亚群，正弦内皮内皮细胞（SEC），小动脉，神经元细胞，巨噬细胞和巨核细胞在内的许多细胞类型都有助于调节骨骨髓内HSC稳态（BM）Niche Niche 1-7。使用光学清除的BM和高度特异性LT-HSC标记（如α-catulin）的深共聚焦成像，表明绝大多数静态和分裂的LT-HSC位于SEC附近，MSC表达瘦素受体（LEPR）和高水平的CXCL128。其他成像和细胞消融研究进一步突出了LT-HSC与血管周围MSC的紧密近似，该MSC表达了Nestin-GFP变换9和其他细胞表面标记和分泌的因子，包括CD51，PDGFRα，CD146和干细胞因子（SCF）10-17。使用LEPR-CRE或TIE2-CRE耗尽从血管周围MSC或内皮细胞中删除SCF的HSC数量，当SCF从造血细胞中删除时未观察到，这在造血细胞，骨细胞，骨细胞或Nestin-Cre+细胞中的scf+ scf 18。与SCF保持LT-HSC INS SCF的可能性至关重要。成年小鼠稳态造血的血管周期MSC。使用克隆衍生的LEPR+CD146+PDGFRα+CD51+SCF+CXCL12HI原发性鼠类成人BM MSC线具有体外和体内多收益的发育潜力，我们表明Wnt信号通过LT-HSC在长期共同培养中自我更新的能力进行了21 faceSsscormscclon的能力，并构图了20 faceSssss sefs ints facssss sefs clon-sclon clon-lt-hscons and lt-hsc and lt-hsc and lt-hsc clon-hsc and clon-hsc and clon clon-hsc and clon-hsc and clon-hsc and clon-hsc。高水平的Wnt配体激活了非规范Wnt信号传导Wnt11和Wnt5a，它们可能会抑制规范（α-catenin依赖性）途径。 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 ecopic expression of either Wnt3a or Wnt10b stimulates loss of LT-HSC self-renewal and/or survival and potentially activates an osteolineage gene expression program in MSC.相反，MSC中Wnt11或Wnt5a的过表达增强了LT-HSC的长期重现活性，并阻止了MSC中骨硅烷基因的激活。此外，使用PRX1-CRE在MSC中的缺失增加了小梁骨在新生小鼠中的沉积，这表明Wnt11在抑制典型Wnt信号引起的MSC的骨磷酸分化中的重要作用。基于这些发现，我们假设Wnt11和Wnt5a维持的MSC中的非规范WNT信号传导对于保留BM NICHE中LT-HSC和MSC的自我更新和多层分化潜力至关重要，以维持生命的血细胞和骨产生。该假设将在以下具体目的中解决： AIM 1：在功能上定义WNT11和WNT5A是否使用LT-HSC/MSC共培养以及具有CRE诱导的，双重型，双质的Flox flox flox Elleles cre-wnt11和Wnt5a的lt-HSC/MSC共培养和主要的MSC Subline，是否可以协同控制LT-HSC和MSC自我更新和多琳的发育潜力。移植研究将解决MSC中的Wnt11或Wnt11/Wnt5a的缺乏，或者在体外LT-HSC/MSC共培养后是否会导致LT-HSC重建潜力的损失。在缺乏WNT11/WNT5A的情况下，MSC自我更新的能力将通过CFU-F测定法进行评估，而MSC对成骨细胞谱系的偏见和不可逆转的激活，由于无法在MSC中抑制MSC，因此使用骨化性，依赖性，依赖性，依赖性，依赖性和浓度，由于无法抑制MSC的构成Wnt信号。 AIM 2：在体内稳态期间，定义WNT11和WNT5A的独特和冗余功能，以及MSC的自我更新和MSC自我更新和多级发育潜力。在这里，我们将使用PRX1-CRE，PRX1-CREERT和WNT11-CREERT小鼠删除Wnt11，Wnt5a或Wnt11和Wnt5a，以确定它们是否对于维持LT-HSC和MSC自我更新至关重要。 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. 定义维护终身维持血液形成的LT-HSC和骨形成MSC的因素一直是一个基本挑战，因为失去控制基本功能的因素丧失，例如任何一种干细胞种群的自我更新可能会导致胚胎致死性。这项研究将利用非规范Wnt配体Wnt11和Wnt5a的诱导型，flox的等位基因，以阐明它们在坐标调节成人LT-HSC和MSC自我恢复中的作用。对LT-HSC和MSC之间进行串扰的因素有更深入的了解可能会在成年BM中直接接触，这将显着增强我们在体外操纵LT-HSC和MSC的能力，目的是提高干细胞移植，组织修复以及靶向基因矫正接近的效率。修改的