Osteogenic Repair from Human Pluripotent Stem Cells

描述（由申请人提供）：发展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可愈合的骨科损伤的关键。这一建议的重点是利用人类胚胎干细胞（hESCs）和诱导多能干细胞（iPSCs）为开发新的治疗方法提供了特定的优势，以改善骨形成和骨折愈合。本研究旨在验证hESCs和iPSCs衍生的细胞作为具有成骨潜能的中胚层祖细胞，具有修复骨不愈合骨折的能力的假设。这些研究将采用两种互补的方法。首先，我们将利用我们生成的表达报告细胞系，该细胞系具有启动子RUNX2（早期成骨细胞发育基因）来驱动荧光蛋白mCitrine （mCit）的表达。鉴定和分离RUNX2/mCit+这些细胞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，将使用地塞米松、抗坏血酸、rhFGF-9、rhBMP-2、Wnt3a和雷帕霉素等骨诱导性药物刺激RUNX2-mCit表达。mCit+细胞的鉴定将有助于鉴定来自iPSC的类似成骨细胞群体，最终为利用患者特异性（自体）iPSC为基础的治疗铺平道路。第二个目标将利用hESC和ipsc衍生的间充质干细胞/基质细胞（MSCs）和成骨细胞来确定最佳表型细胞群和成骨生长和修复的条件。我们假设，与从人骨髓分离的间充质干细胞（BM-MSCs）相比，hESC和ipsc衍生的细胞具有更高的成骨潜能。具体来说，我们将推进我们的初步研究，证明hESC/ ipsc衍生的MSC比BM-MSCs具有更多的血管诱导潜力，从而促进体内愈合。本研究将评估两种体内成骨模型：皮下植入支架内的细胞和在不愈合骨折部位局部植入支架内的成骨细胞的啮齿动物骨折修复模型。总之，该项目结合了干细胞生物学、骨科外科、生物力学工程、骨生物学、组织学和成骨发育生物学等研究小组的专业知识。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骼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愈合骨科骨折修复。

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.