Osteogenic Enrichment of Adipose Derived Stromal Cells

描述（由申请人提供）：多种证据表明，人类脂肪来源的基质细胞（hASCs）在颅面骨骼再生中具有未来的应用前景。与骨髓间充质细胞相比，人类ASCs很容易通过简单的吸脂程序获得，并且易于扩展。此外，人类ASCs经历快速成骨分化。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人类的ASCs，都有助于小鼠颅骨缺损的骨愈合。例如，在小鼠顶骨（直径4mm）的手术缺陷中，即使在受伤后16周，未经治疗也没有愈合。在骨导电性支架直接应用hASC后，在损伤后4周内观察到明显的骨愈合。也许最令人兴奋的是hasc在人类病人身上的应用。在小规模的初步研究中，头盖骨、上颌骨和下颌骨的缺陷已经愈合，或者使用hASCs能够更快地愈合。然而，目前对ASC身份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，因为它们是一个高度异质性的细胞群。虽然表面抗原表达是这种理解的重要组成部分，但可能需要更深入的分析来充分表征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种分析。我们期望这些研究的结果通过提供对hASCs转录多样性的新理解来推动该领域的发展。我们预计，我们的创新方法的发现将有助于在单细胞水平上定义转录活性和细胞表面标记物之间的关系。这种理解将使我们能够更有针对性地丰富颅面骨骼再生的理想细胞群。

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.