Development of an adipose-derived stem cell isolation matrix

描述(申请人提供)：间充质干细胞(MSCs)是一种成体干细胞群体，通常从骨髓抽吸物中分离出来，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。一般来说，为了达到这些应用所需的细胞数量，细胞通常在培养中扩增几代；然而，这些细胞的体外扩增已被证明诱导基因表达的变化，并影响这些细胞的分化能力。这表明，使用骨髓间充质干细胞作为治疗将是最有效的新鲜分离的细胞数量很多，但需要不切实际的数量的骨髓才是可行的。脂肪来源的干细胞(ASCs)已被认为是一种替代骨髓来源的MSCs的临床适应症。据估计，与类似质量的骨髓中的MSCs数量相比，每克脂肪组织中这些细胞的丰度要高出500倍。此外，与骨髓相比，脂肪组织更丰富、更容易获得，患者发病率更低。因此，我们假设ASC浓缩系统的开发将允许护理点分离大量自体细胞，从而消除在植入前进行体外扩增的需要。在这项提案中，我们将使用我们的核心平台技术-噬菌体展示生物扫描-来识别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。在平行的研究计划中，我们已经确定了能够从骨髓抽提物制剂中分离出MSCs的多肽，并且与其他类型的细胞的交叉反应最小，从而验证了这一提议的方法。我们将首先使用噬菌体展示鉴定与ASC结合的多肽，然后将这些多肽偶联到树脂上。这种多肽包裹的基质将用于优化培养细胞的ASC结合和洗脱条件，并从脂吸物的粗细胞部分中浓缩ASCs。最后，我们将表征ASC表面标记及其分化为三个谱系的能力，以确保暴露于我们的多肽不会影响这些细胞的干细胞性质。根据我们对MSC分离的结果，我们设想，由固体支持基质与我们的ASC结合多肽组成的产品将允许分离自体ASCs--一种比骨髓更容易获得和丰富的细胞群--用于点护理治疗。 与公共卫生相关：成人干细胞开始出现在新的医疗产品中。通常，这些细胞是来自骨髓的间充质干细胞。然而，它们在骨髓中的数量很少，需要在体外分离和扩大细胞，然后才能将它们整合到市场上的产品中。在这里，我们寻求开发一种新型的干细胞结合试剂来从脂肪组织中提取干细胞。脂肪来源的干细胞比骨髓来源的干细胞多得多。这一增加可能会使脂肪来源干细胞的护理点使用成为可能，使外科医生能够在外科手术中从自体来源获取和部署细胞。我们目前设想这种基于多肽的试剂在用于组织工程应用之前，将用于无培养的ASC分离方法。

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.