Osteogenic Enrichment of Adipose Derived Stromal Cells

描述(申请人提供)：多行证据表明，人类脂肪来源的基质细胞(HASCs)有望在未来用于颅面部骨骼再生。与骨髓间充质细胞相比，人骨髓间充质干细胞可以通过简单的吸脂方法很容易地获得，并且很容易扩增。此外，人的ASCs经历了快速的成骨分化。在我们的实验室中，我们令人信服地观察到，无论是来自小鼠还是来自人类的ASCs，都有助于小鼠颅骨缺损的骨愈合。例如，小鼠顶骨(直径4毫米)的外科缺陷，即使在受伤后16周内不进行治疗也无法愈合。当HASC直接应用于骨传导支架时，仅在损伤后4周内即可观察到显著的骨愈合。也许最令人兴奋的是hASCs在人类患者身上的应用。在小型先导性研究中，使用hASCs，颅骨、上颌骨和下颌骨的缺陷要么已经愈合，要么愈合得更快。然而，目前对ASC特性和功能的了解仍然不足以进行更大规模的临床试验和应用，因为它们是高度异质性的细胞群体。虽然表面抗原的表达是这一理解的重要组成部分，但很可能需要更深入的分析才能充分表征hASCs。我们的目标是通过完成本提案中描述的具体目标来提供这一分析。我们期待这些研究的结果通过提供对hASCs内转录多样性的新理解来推动该领域的发展。我们预计，我们创新方法的发现将有助于在单个细胞水平上定义转录活性和细胞表面标记之间的关系。这一认识将使我们能够更具体地丰富对颅面部骨骼再生理想的细胞群体。 公共卫生相关性：在未成熟的动物和年幼的儿童中，头面部缺陷的成功再骨化更有活力。相反，骨骼成熟的动物和成年人类修复头骨缺陷的能力受到了损害。虽然在过去的一个世纪里已经开发了太多的治疗成人颅骨缺陷的策略，但目前可用的无数方法反映了每种治疗技术的不足。然而，结合发育生物学、器官发生学、干细胞生物学、生物工程学和材料科学的进展，出现了一种新的颅骨组织工程范式--再生医学。尽管越来越多的证据表明人类脂肪来源的基质细胞(HASCs)在再生应用中的用途，但人们对HASC群体中存在的异质性知之甚少。通过应用信息论方法基于相似的转录图谱来聚集单个细胞，可以系统地询问一组不同的细胞，如hASCs，以寻找功能不同的亚群，如骨祖细胞群体。这种丰富的人口将是用于颅面部骨骼再生医学的基于细胞的策略的理想选择。

DESCRIPTION (provided by applicant): Multiple lines of evidence suggest that human adipose-derived stromal cells (hASCs) hold promise for future use in craniofacial skeletal regeneration. Human ASCs are easily harvested by simple liposuction procedures and are readily expandable as compared to bone marrow mesenchymal cells. Moreover, human ASCs undergo rapid osteogenic differentiation. In our laboratory, we have observed convincingly that ASCs, whether derived from mouse or human origin, contribute to osseous healing of mouse calvarial defects. For example, a surgical defect in the mouse parietal bone (4mm in diameter) shows no healing when untreated even up to 16 weeks post injury. Upon direct hASC application with an osteoconductive scaffold, significant bony healing is observed in as little as 4 weeks post injury. Perhaps most exciting are those applications of hASCs to the human patient. In small pilot studies, defects of the cranium, maxilla, and mandible have been either healed or enabled to heal faster with the use of hASCs. The current understanding of ASC identity and function, however, remains insufficient to allow larger clinical trials and applications as they are a highly heterogeneous population of cells. While surface antigen expression is an important component to this understanding, it is likely that more in depth analysis will be required to fully characterize hASCs. We aim to provide this analysis through completion of the Specific Aims described in this proposal. We expect the results of these studies to move the field forward by providing a new understanding of the transcriptional diversity within hASCs. We anticipate that the findings from our innovative approach will help to define the relationship between transcriptional activity and cell surface markers on a single cell level. This understanding will allow us to more specifically enrich for a population of cells ideal for craniofacial skeletal regeneration. PUBLIC HEALTH RELEVANCE: Successful re-ossification of craniofacial defects is more robust in immature animals and young children. Conversely, skeletally mature animals and adult humans demonstrate an impaired ability to heal skull defects. While a plethora of strategies have been developed over the past century for treating adult calvarial defects, the myriad of methods currently available reflects the inadequacies of each therapeutic technique. By combining advances in developmental biology, organogenesis, stem cell biology, bioengineering, and material sciences, however, a new paradigm for calvarial bone tissue engineering has emerged - Regenerative Medicine. Despite accumulating evidence demonstrating the utility of human adipose derived stromal cells (hASCs) in regenerative applications, little is known about the heterogeneity that exists within the hASC population. By applying an information theoretic approach to cluster individual cells based on similar transcriptional profiles, it becomes possible it systematically interrogate a heterogeneous group of cells, such as hASCs, for functionally distinct subpopulations such as an osteoprogenitor population. This enriched population would be ideal to use for cell-based strategies for craniofacial skeletal regenerative medicine.