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.

项目总结/摘要 在美国，每年有超过100万患者接受骨修复手术。自体髂骨植骨 仍然是骨缺损的首选治疗方法，但这种做法受到骨可用性和供体部位的限制 发病率或者，开发利用骨髓成骨潜力的疗法- 衍生的间充质干细胞（Bm-MSC）仍然是再生医学中的优先事项。然而，努力 由于对调控bm-MSC成骨活性的机制缺乏了解， in vivo.我们的总体目标是阐明调控骨化的机制， 使用自体骨髓间充质干细胞的骨再生策略。之前，我们证明了保存人类 骨髓间充质干细胞的成骨潜能依赖于与内皮细胞（EC）的持续接近。最近，我们 已经发现EC的类型显著影响体内bm-MSC的命运。具体来说，血管网络由 人骨小梁小动脉内皮细胞（tba-ECs）可自发诱导骨髓基质细胞向成骨细胞分化， MSC。相反，非骨EC不能。我们的初步数据表明，KITLG驱动器的表达 这种独特的骨诱导潜力。事实上，沉默tba-EC中的KITLG完全废除了骨生成 在体内植入后，而在非骨EC中过表达KITLG赋予了强大的骨诱导性， 特性.我们的数据还表明，KITLG在tba-EC中的表达受I型干扰素（IFN）的调节。 信号，一个以前未知的链接。我们的中心假设是组成性IFN-KITLG机制驱动 人tba-EC独特的骨诱导特性。我们还假设， 干细胞（iPSC）可以提供大量的替代tba-EC来源，消除了收获的需要。 自体骨为了验证这些假设，我们提出了三个具体目标。在Aim-1中，我们将剖析 人tba-EC通过KITLG表达介导骨生成的机制。我们将决定 KITLG亚型（可溶性与膜结合型）是不可或缺的，并分析了募集的c-Kit+的作用 造血祖细胞（c-Kit+ HPC）在骨生成中的作用。在Aim-2中，我们将确定 调节人tba-EC中KITLG表达的机制。我们将使用CRISPR/Cas9功能丧失 沉默I型IFN途径组分的方法，并阐明IFN信号传导之间的相互作用 介导子和KITLG基因的增强子-启动子区域。在目标3中，我们将采取战略， 人iPSC衍生的EC获得骨诱导功能，包括KITLG和IFN的瞬时活化 发信号。总之，这些研究将确定人类tba- 内皮细胞调节bm-MSCs的成骨分化，进而调节骨化。这些基本知识 将为促进骨修复和再生的策略奠定基础。