Osteogenic Repair from Human Pluripotent Stem Cells

描述（由申请人提供）：开发新的治疗方法来修复骨折和其他骨缺损仍然是治疗复杂和不愈合骨科损伤的关键。该提案的重点是使用人类胚胎干细胞（hESC）和诱导多能干细胞（iPSC）为开发新疗法以改善骨形成和骨折愈合提供特定优势。该提案旨在验证以下假设：源自hESC和iPSC的细胞作为具有成骨潜能的中胚层祖细胞，并具有修复骨不连骨科骨折的能力。这些研究将采取两种相辅相成的办法。首先，我们将利用我们已经产生的具有启动子RUNX 2（早期成骨细胞发育基因）的表达报告基因hESC系来驱动荧光蛋白mCitrine（mCit）的表达。RUNX 2/mCit+这些细胞的鉴定和分离将使我们能够确定介导成骨细胞发育的关键信号通路。具体而言，包括地塞米松、抗坏血酸、rhFGF-9、rhBMP-2、Wnt 3a和雷帕霉素的骨诱导将用于刺激RUNX 2-mCit表达。mCit+细胞的鉴定将有助于鉴定来源于iPSC的成骨细胞的类似群体，最终为利用基于患者特异性（自体）iPSC的疗法铺平道路。第二个目标将利用hESC和iPSC衍生的间充质干细胞/基质细胞（MSC）和成骨细胞来定义成骨生长和修复的最佳表型细胞群和条件。我们假设hESC和iPSC衍生的细胞与从人骨髓分离的MSC（BM-MSC）相比具有增加的成骨潜力。具体来说，我们将推进我们的初步研究，证明hESC/iPSC衍生的MSC具有比BM-MSC更大的血管诱导潜力，从而增强体内愈合。在此，将评价两种体内成骨模型：在支架内皮下植入细胞和在骨不连骨折部位的支架内局部植入成骨细胞的啮齿动物骨折修复模型。总之，该项目结合了研究小组的专业知识，精通干细胞生物学，整形外科，生物力学工程，骨生物学，组织学和成骨发育生物学。这些研究的成功完成将促进人类多能干细胞的使用，以更好地定义介导人类骨发育的细胞和遗传机制，并将这些研究转化为基于干细胞的骨不连矫形骨折修复。 公共卫生相关性：这些研究将对快速发展的再生医学领域产生重要影响。更好地了解hESC和iPSC的成骨发育将转化为治疗复杂和不可愈合的骨科损伤的新细胞来源。此外，RUNX 2启动子-报告基因系统可以发展为高通量筛选系统，以在不使用外源细胞的情况下鉴定促进成骨的新化合物。

DESCRIPTION (provided by applicant): The development of novel therapeutic approaches to repair fractures and other bony defects remains a critical necessity to treat complex and non-healing orthopedic injuries. This proposal focuses on use of human embryonic stem cells (hESCs) and induced-Pluripotent Stem Cells (iPSCs) offer specific advantages for development of new therapies to improve bone formation and fracture healing. This proposal is designed to test the hypothesis that cells derived from hESCs and iPSCs serve as mesoderm progenitor cells with osteogenic potential and have the ability to repair non-union orthopedic fractures. These studies will pursue two complementary approaches. First, we will utilize an expression-reporter hESC line that we have generated that has the promoter RUNX2 (an early osteoblast developmental gene) to drive expression of the fluorescent protein, mCitrine (mCit). Identification and isolation of RUNX2/mCit+ these cells will allow us to define key signaling pathways that mediate the development of osteogenic cells. Specifically, osteoinductive including dexamethasone, ascorbic acid, rhFGF-9, rhBMP-2, Wnt3a and rapamycin will be used to stimulate RUNX2-mCit expression. The identification of mCit+ cells will us to identify similar populations of osteogeneic cells derived from iPSCs, eventually paving the way for the utilization of patient-specific (autologous) iPSC- based therapies. The second aim will utilize hESC and iPSC-derived mesenchymal stem/stromal cells (MSCs) and osteogenic cells to define the optimal phenotypic cell population and conditions for osteogenic growth and repair. We hypothesize that hESC- and iPSC-derived cells will have increased osteogenic potential compared to MSCs isolated from human bone marrow (BM-MSCs). Specifically, we will advance our preliminary studies that demonstrate that hESC/iPSC-derived MSC have more vascular inductive potential than BM-MSCs leading enhanced healing in vivo. Here, two in vivo osteogenic models will be evaluated: subcutaneous implantation of cells within scaffolds and a rodent fracture repair model with osteogenic cells locally implanted within scaffolds at the non-union fracture site. Together, this project combines expertise of research groups with proficiency in stem cell biology, orthopedic surgery, biomechanical engineering, bone biology, histology and osteogenic developmental biology. Successful completion of these studies will advance the use of human pluripotent stem cells to better define cellular and genetic mechanisms that mediate human bone development and translate these studies to stem cell-based repair of non-union orthopedic fractures. PUBLIC HEALTH RELEVANCE: These studies will have important impact in the rapidly growing area of regenerative medicine. Better understanding of osteogenic development from hESCs and iPSCs will translate to novel source of cells to treat complex and non-healing orthopedic injuries. Additionally, the RUNX2 promoter-reporter system can advance to a high-throughput screening system to identify new compounds to promote osteogeneis without use of exogenous cells.