Osteoprogenitor Subpopulations within Adipose-derived Stroma

描述(申请人提供)：脂肪来源的基质细胞(ASCs)具有向成骨细胞分化的能力。这些出生后的细胞可以用来设计基于细胞的再生疗法，用于治疗衰弱的遗传性疾病、退行性疾病以及创伤或手术后的组织缺失的骨骼修复。虽然我们已经在研究ASCs成骨分化的细胞生物学方面取得了实质性进展，但我们受到异质细胞群体的限制，需要确定具有最大成骨潜力的特定丰富的成骨细胞亚群。目前的研究利用了异质细胞，并有几个缺点。由于多种细胞类型的存在，亚群基因表达的变化可能会减弱，甚至完全被掩盖。确定是否存在一组能够成骨分化的祖细胞将是非常有益的，无论是在促进我们对出生后多潜能细胞的了解方面，还是在优化临床治疗策略方面。初步数据表明，使用荧光激活细胞分选(FACS)确实有可能为骨祖细胞丰富异种ASCs。第一个具体目标是在成年小鼠腹股沟脂肪垫细胞的原代培养上进行细胞分离实验。细胞将根据先前被识别为成体干细胞标记的单个候选标记(即标记与标记-)的表达进行细分。与未分离的ASCs相比，这些细胞将在体外检测其增殖和向成骨谱系分化的潜力。在第二个特定目标中，第一个特定目标中确定的具有最大骨势的ASC亚群将被放置在活体小鼠颅骨缺损模型中，以确定它们是否促进骨骼再生。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分析相结合，前瞻性地描绘居住在采集的ASCs的异种混合物中的成骨前体细胞的细胞表面标记谱。骨骼疾病对各种医学学科构成了严重的挑战。与此同时，它也给我们的医疗保健基础设施带来了巨大的社会经济负担，每年超过10亿美元。目前临床上再生骨骼的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。利用细胞构建块祖细胞的再生潜力的能力预示着一种令人兴奋的新的骨骼疾病管理范例。

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.