Osteoprogenitor Subpopulations within Adipose-derived Stroma

描述（由申请人提供）：脂肪源性基质细胞（ASC）具有成骨分化的能力。这些出生后细胞可用于设计基于细胞的再生疗法，用于使人衰弱的遗传性疾病、退行性疾病和创伤性或手术后组织缺陷的骨骼修复。虽然我们在研究ASCs骨分化的细胞生物学基础方面取得了实质性进展，但我们受到异质细胞群的限制，并且需要鉴定具有最大骨潜能的特定富集的骨祖细胞亚群。目前的研究利用异质细胞，并有几个缺点。由于存在多种细胞类型，亚群基因表达变化可能减弱，甚至完全掩盖。确定是否有一组祖细胞能够骨分化将是非常有益的，无论是在提高我们的知识出生后多能细胞和优化临床治疗策略。初步数据表明，它确实是可能的，以丰富异质性ASC的骨祖细胞使用荧光激活细胞分选（FACS）。第一个特定目标侧重于对成年小鼠腹股沟脂肪垫来源的细胞原代培养物进行细胞分级分离实验。根据先前鉴定为成体干细胞标志物的单个候选标志物（即标志物+与标志物-）的表达，对细胞进行细分。将在体外测定这些细胞与未分级的ASC相比增殖和向下分化成骨谱系的潜力。在第二个特定目标中，将在第一个特定目标中鉴定的具有最大骨潜能的ASC亚群置于体内小鼠颅骨缺损模型中，以确定它们是否增强骨骼再生。总之，这些目标将使用流式细胞术与体外和体内成骨细胞分化试验相结合，以前瞻性地描绘骨祖细胞的细胞表面标志物分布，这些骨祖细胞存在于收获的ASC的异质混合物中。皮肤病对多种医学学科构成了严峻的挑战。与此同时，它也给我们的医疗基础设施带来了巨大的社会经济负担，每年超过10亿美元。目前的临床骨骼再生方法包括使用自体移植物、同种异体材料和假体产品，每种方法都有其固有的缺点。利用细胞积木祖细胞的再生潜力的能力预示着一个令人兴奋的和新的范式管理骨骼疾病。

DESCRIPTION (provided by applicant): Adipose-derived stromal cells (ASCs) have the capacity to undergo osteoblastic differentiation. These postnatal cells may be used to design cell-based regenerative therapies for skeletal repair in debilitating genetic disorders, degenerative diseases, and traumatic or post-surgical tissue deficits. While we have made substantial progress in studying the cell biology underlying osteodifferentiation of ASCs, we are limited by working with a heterogeneous cell population, and specific enriched, subpopulations of osteoprogenitor cells with the greatest osteopotential need to be identified. Current investigations utilize heterogeneous cells and have several disadvantages. Subpopulation gene expression changes may be attenuated, or even completely masked, due to the presence of multiple cell types. Determining whether there is a single set of progenitor cells capable of osteodifferentiation would be extremely beneficial, both in terms of advancing our knowledge of postnatal multipotent cells and for optimizing clinical therapeutic strategies. Preliminary data suggest that it is indeed possible to enrich heterogeneous ASCs for osteoprogenitors using fluorescent-activated cell sorting (FACS). The first Specific Aim focuses on performing cell fractionation experiments on primary cultures of cells derived from inguinal fat pads of adult mice. Cells will be subdivided according to expression of individual candidate markers (i.e. marker+ vs. marker-) previously identified as markers of adult stem cells. These cells will be assayed in vitro for their potential to proliferate and differentiate down the osteogenic lineage compared to unfractionated ASCs. In the second Specific Aim, the ASC subpopulations identified in the first Specific Aim with greatest osteopotential will be placed in an in vivo mouse calvarial defect model to determine if they enhance skeletal regeneration. Together, these Aims will use flow-cytometry in concert with both in vitro and in vivo osteoblastic differentiation assays to prospectively delineate the cell-surface marker profile of osteoprogenitors residing within the heterogeneous mix of harvested ASCs. Skeletal disease poses a serious challenge to a variety of medical disciplines. At the same time, it also poses a significant socioeconomic burden on our healthcare infrastructure exceeding $1 billion annually. Current clinical approaches to regeneration of the skeleton include use of autogenous grafts, allogenic materials and prosthetic products, each with its own inherent disadvantages. The ability to harness the regenerative potential of cellular building block progenitor cells portends an exciting and novel paradigm for management of skeletal disease.