Human endothelial cell regulation of ossification

人内皮细胞对骨化的调节

• 批准号: 10680596
• 负责人: Juan M Melero-Martin
• 金额: $ 44.24万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-10 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680596
• 关键词: Affect; Autologous; Binding; Binding Sites; Biological Assay; Blood Vessels; Bone Marrow; Bone Regeneration; Bone Transplantation; CRISPR/Cas technology; ChIP-seq; Compressive Strength; DNA Binding; Data; Defect; Endothelial Cells; Enhancers; Exposure to; Foundations; Gene Expression Profile; Genomics; Goals; Harvest; Hematopoietic stem cells; Human; IFNAR1 gene; Immune system; Immunocompetent; Immunodeficient Mouse; Interferon Type I; Interferons; JAK1 gene; KITLG gene; Knock-in; Knowledge; Ligands; Link; Luciferases; Mediating; Mediator; Membrane; Messenger RNA; Molecular; Morbidity - disease rate; Mus; Nature; Notch Signaling Pathway; Osteoblasts; Osteogenesis; Pathway interactions; Patients; Physiologic Ossification; Procedures; Promoter Regions; Property; Protein Isoforms; Proto-Oncogene Protein c-kit; Regenerative Medicine; Regulation; Reporter; Role; STAT1 gene; STAT2 gene; Signal Pathway; Signal Transduction; Site; Source; TYK2; Testing; Therapeutic; Titrations; Transfection; Transgenic Mice; United States; Xenograft Model; arteriole; bone; bone marrow mesenchymal stem cell; bone repair; endothelial stem cell; human stem cells; implantation; in vivo; in vivo Model; induced pluripotent stem cell; loss of function; notch protein; osteogenic; overexpression; preservation; promoter; recruit; regeneration potential; single-cell RNA sequencing; stem cells; substantia spongiosa; therapy development

PROJECT SUMMARY/ABSTRACT Every year, >1 million patients undergo bone repair procedures in the United States. Autologous bone grafting remains the preferred treatment for bone defects, but this practice is limited by bone availability and donor site morbidity. Alternatively, the development of therapies that exploit the osteogenic potential of bone marrow- derived mesenchymal stem cells (bm-MSCs) continues to be a priority in regenerative medicine. However, efforts remain largely empirical due to a poor understanding of the mechanisms regulating bm-MSC osteogenic activity in vivo. Our overarching goal is to elucidate the mechanisms regulating ossification and develop therapeutic strategies for bone regeneration using autologous bm-MSCs. Previously, we showed that preserving human bm-MSCs' osteogenic potential depends on sustaining proximity to endothelial cells (ECs). More recently, we have found that the type of ECs drastically affects bm-MSC fate in vivo. Specifically, vascular networks lined by human trabecular bone arteriole ECs (tba-ECs) could spontaneously induce osteogenic differentiation of bm- MSCs. In contrast, non-bone ECs could not. Our Preliminary Data suggest that the expression of KITLG drives this unique osteoinductive potential. Indeed, silencing KITLG in tba-ECs completely abrogated osteogenesis upon implantation in vivo, whereas overexpressing KITLG in non-bone ECs conferred robust osteoinductive properties. Our data also suggest that KITLG expression in tba-ECs is regulated by type I interferon (IFN) signaling, a previously unknown link. Our central hypothesis is that a constitutive IFN-KITLG mechanism drives the distinct osteoinductive properties of human tba-ECs. We also postulate that educating induced pluripotent stem cells (iPSCs) could offer a plentiful source of surrogate tba-ECs, eliminating the need for harvesting autologous bone. To test these hypotheses, we propose three specific aims. In Aim-1, we will dissect the mechanism by which human tba-ECs mediates osteogenesis via KITLG expression. We will determine which KITLG isoform (soluble vs. membrane-bound) is indispensable and dissect the role of recruited c-Kit+ hematopoietic progenitor cells (c-Kit+ HPCs) in osteogenesis. In Aim-2, we will determine the molecular mechanism that regulates KITLG expression in human tba-ECs. We will use a CRISPR/Cas9 loss‐of‐function approach to silence components of the type I IFN pathway and unravel the interactions between IFN signaling mediators and the enhancer-promoter region of the KITLG gene. In Aim-3, we will pursue strategies to educate human iPSC-derived ECs to acquire osteoinductive function, including transient activation of KITLG and IFN signaling. In summary, these studies will define the cellular and molecular mechanisms by which human tba- ECs regulate the osteogenic differentiation of bm-MSCs and, in turn, ossification. This fundamental knowledge will form the foundation for strategies to promote bone repair and regeneration.

项目摘要/摘要 在美国，每年有100万名患者接受骨骼修复手术。自体骨移植 仍然是骨缺损的首选治疗方法，但这种做法受到骨可获得性和供骨部位的限制。 发病率。或者，开发利用骨髓成骨潜力的疗法-- 来源的间充质干细胞(BM-MSCs)仍然是再生医学的优先领域。然而，努力 由于对调控BM-MSC成骨活性的机制了解不深，大部分仍处于实验阶段 在活体内。我们的首要目标是阐明骨化的调节机制并开发治疗方法。 应用自体BM-MSCs进行骨再生的策略在此之前，我们展示了保存人类 BM-MSCs的成骨能力依赖于与内皮细胞(ECs)的亲和力。最近，我们 已发现内皮细胞的类型对BM-MSC在体内的命运有很大的影响。具体地说，血管网络由 人骨小动脉内皮细胞(TBA-ECs)可自发诱导BM-ECs向成骨方向分化。 MSCs。相比之下，非骨内皮细胞则不能。我们的初步数据表明，KITLG驱动基因的表达 这种独特的骨诱导潜能。事实上，沉默TBA-ECs中的KITLG完全取消了成骨 在体内植入后，在非骨内皮细胞中过表达KITLG可产生强大的成骨诱导作用 属性。我们的数据还表明，KITLG在TBA-ECs中的表达受I型干扰素(IFN)的调节 信令，一条以前未知的链路。我们的中心假设是，一个结构性的干扰素-KITLG机制驱动 人TBA-ECs独特的成骨特性。我们还假设，教育诱导的多能性 干细胞(IPSCs)可以提供大量替代TBA-ECs的来源，从而消除了采集的需要 自体骨。为了检验这些假设，我们提出了三个具体目标。在AIM-1中，我们将剖析 人TBA-ECs通过KITLG表达介导成骨的机制。我们将确定哪一个 KITLG亚型(可溶性与膜结合)是不可缺少的，并剖析了招募的c-Kit+的作用 造血祖细胞(c-Kit+HPC)与成骨在AIM-2中，我们将确定分子 调节人TBA-ECs中KITLG表达的机制。我们将使用CRISPR/CAS9功能丢失 沉默I型干扰素途径组分并解开干扰素信号之间相互作用的方法 介体和KITLG基因的增强子-启动子区域。在AIM-3中，我们将采取策略来教育 人IPSC来源的内皮细胞获得骨诱导功能，包括瞬时激活KITLG和干扰素 发信号。综上所述，这些研究将确定人类TBA- ECS调控BM-MSCs的成骨分化，进而促进成骨。这一基础知识 将构成促进骨骼修复和再生的策略的基础。