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陷仍然是治疗复杂和不可愈合的骨科损伤的关键。这项建议侧重于使用人类胚胎干细胞(HESCs)和诱导多能干细胞(IPSCs)，为促进骨形成和骨折愈合的新疗法的开发提供了独特的优势。这一建议旨在验证一种假设，即来源于hESCs和ipSCs的细胞是具有成骨能力的中胚层前体细胞，并具有修复骨不连的能力。这些研究将采用两种相辅相成的方法。首先，我们将利用我们建立的具有启动子RUNX2(一种早期成骨细胞发育基因)的hESC系来驱动荧光蛋白mCitine(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。具体地说，包括地塞米松、抗坏血酸、重组人成纤维细胞生长因子-9、重组人骨形成蛋白-2、Wnt3a和雷帕霉素在内的成骨诱导剂将被用来刺激RUNX2-MCIT的表达。对MCIT细胞的鉴定将使我们能够鉴定来自IPSC的相似群体的成骨细胞，最终为使用针对患者的(自体)IPSC疗法铺平道路。第二个目标将利用hESC和IPSC来源的间充质干细胞/基质细胞(MSCs)和成骨细胞来确定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来源的细胞将比从人骨髓分离的MSCs(BM-MSCs)具有更强的成骨能力。具体地说，我们将推进我们的初步研究，证明hESC/IPSC来源的MSC比BM-MSCs具有更多的血管诱导潜力，从而促进体内愈合。在这里，将评估两种体内成骨模型：支架内细胞皮下植入和啮齿动物骨折修复模型，即在骨折不愈合处，将成骨细胞局部植入支架内。该项目结合了研究小组在干细胞生物学、整形外科手术、生物机械工程、骨生物学、组织学和成骨发育生物学方面的专业知识。这些研究的成功完成将推动人类多能干细胞的使用，以更好地确定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连修复。 公共卫生相关性：这些研究将对迅速增长的再生医学领域产生重要影响。更好地了解hESCs和ipscs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的细胞来源。此外，RUNX2启动子-报告系统可以发展为高通量筛选系统，在不使用外源细胞的情况下识别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.