Development of an adipose-derived stem cell isolation matrix

描述（由申请人提供）：间充质干细胞（MSC）是一种成体干细胞群，通常从骨髓抽吸物中分离，能够自我更新、在培养中扩增并分化为脂肪细胞、软骨细胞和成骨细胞。这些细胞正在探索用于组织工程应用，如脊柱融合，骨缺损修复和软骨再生。通常，为了获得这些应用所需的细胞数量，通常将细胞在培养物中扩增多次传代;然而，这些细胞的离体扩增已显示诱导基因表达的变化并影响这些细胞分化的能力。这表明使用MSC作为治疗剂在大量新鲜分离的细胞的情况下将是最有效的，但需要不切实际的骨髓量才可行。脂肪来源的干细胞（ASC）已被提出作为骨髓来源的MSC在几个临床适应症的替代。这些细胞被估计为500倍更丰富的每克脂肪组织相比，在一个类似的质量的骨髓间充质干细胞的数量。此外，脂肪组织比骨髓更丰富，更容易获得，患者发病率更低。因此，我们假设ASC富集系统的开发将允许大量自体细胞的即时分离，从而消除在植入前进行离体扩增的需要。在这项提案中，我们将采用我们的核心平台技术-噬菌体展示生物淘选-以确定ASC结合肽，并测试其从脂肪组织中分离这些成体干细胞的能力。在平行的研究项目中，我们已经鉴定出能够从骨髓抽吸物制备物中分离MSC的肽，其与其他细胞类型的交叉反应性最小，从而验证了该提议的方法。我们将首先使用噬菌体展示鉴定ASC结合肽，然后将肽偶联到树脂上。这种肽包被的基质将用于使用培养的细胞优化ASC结合和洗脱条件，并从脂肪抽吸物的粗细胞级分中富集ASC。最后，我们将表征ASC表面标志物及其分化成三种谱系的能力，以确保暴露于我们的肽不会影响这些细胞的干细胞样质量。基于我们的MSC分离结果，我们设想由与我们的ASC结合肽偶联的固体支持基质组成的产品将允许分离自体ASC-一种比骨髓更容易获得和丰富的细胞群-用于即时治疗用途。 公共卫生相关性：成人干细胞开始出现在新的医疗产品中。通常，这些细胞是来源于骨髓的间充质干细胞。然而，它们在骨髓中的低数量要求在将其掺入市售产品之前离体分离和扩增细胞。在这里，我们寻求开发一种新的干细胞结合试剂，从脂肪组织中收获干细胞。脂肪来源的干细胞比骨髓来源的干细胞多得多。这种增加可能使脂肪来源的干细胞的即时使用成为可能，允许外科医生在外科手术中从自体来源收获和部署细胞。我们目前设想这种基于肽的试剂在用于组织工程应用之前用于ASC分离的无培养方法。

DESCRIPTION (provided by applicant): Mesenchymal stem cells (MSCs) are an adult stem cell population, typically isolated from bone marrow aspirates, and are capable of self-renewal, expansion in culture, and differentiation into adipocytes, chondrocytes, and osteoblasts. These cells are being explored for use in tissue engineering applications, such as spinal fusions, bone defect repairs, and cartilage regeneration. In general, to attain cell numbers required for these applications, the cells are typically expanded in culture over a number of passages; however, the ex vivo expansion of these cells has been shown to induce changes in gene expression and affect the ability of these cells to differentiate. This suggests that the use of MSCs as a therapeutic would be most efficacious with high numbers of freshly isolated cells, but would require impractical amounts of bone marrow to be feasible. Adipose derived stem-cells (ASCs) have been proposed as an alternative to bone marrow-derived MSCs in several clinical indications. These cells are estimated to be 500-fold more abundant per gram of adipose tissue compared to the number of MSCs in a similar mass of bone marrow. Furthermore, adipose tissue is more abundant and more easily obtained with less patient morbidity than bone marrow. Therefore, we hypothesize that the development of an ASC enrichment system would allow for point-of-care isolation of large numbers of autologous cells, thereby eliminating the need for ex vivo expansion prior to implantation. In this proposal, we will employ our core platform technology-phage display biopanning-to identify an ASC-binding peptide and test its ability to isolate these adult stem cells from adipose tissue. In parallel research programs, we have identified peptides capable of isolating MSCs from bone marrow aspirate preparations with minimal cross-reactivity with other cell types, thereby validating the approach of this proposal. We will first identify ASC-binding peptides using phage display, then couple the peptides to a resin. This peptide-coated matrix will be used for the optimization of ASC binding and elution conditions using cultured cells and to enrich ASCs from the crude cellular fraction of lipoaspirates. Finally, we will characterize the ASC surface markers and their ability to differentiate into three lineages to ensure that exposure to our peptide did not affect the stem-like qualities of these cells. Based on our results with MSC isolation, we envision that a product consisting of a solid support matrix coupled to our ASC-binding peptide would allow for the isolation of autologous ASCs-a cell population that is more easily accessible and abundant than that of bone marrow-for point-of-care therapeutic use. PUBLIC HEALTH RELEVANCE: Adult stem cells are beginning to appear in new medical products. Typically, these cells are mesenchymal stem cells derived from bone marrow. However, their low numbers in bone marrow require the cells to be isolated and expanded ex vivo prior to their incorporation into a marketed product. Here, we seek to develop a novel stem cell-binding reagent to harvest stem cells from adipose tissue. Adipose-derived stem cells are far more numerous than those derived from bone marrow. This increase could potentially enable point-of-care use of adipose-derived stem cells, allowing a surgeon to harvest and deploy cells from autologous sources in a surgical procedure. We currently envision this peptide-based reagent to be used in a culture-free method of ASC isolation prior to their use in tissue engineering applications.