Expansion and Directed Differentiation of Adult Side Population Stem Cells

成体侧群干细胞的扩增和定向分化

• 批准号: 7662277
• 负责人: DOUGLAS BURR COWAN
• 金额: $ 38.03万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-18 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7662277
• 关键词: Address; Adult; Binding; Biological Preservation; Bioreactors; Bone Marrow; Cardiac; Cell Fraction; Cell Lineage; Cell Therapy; Cells; DNA Minor Groove Binding; Development; Engraftment; Fluorescence-Activated Cell Sorting; Fluorescent Dyes; GTP-Binding Proteins; Goals; Hematopoietic System; Maintenance; Methods; Minor Groove; Mus; Myopathy; Population; Primary Cell Cultures; SP1 gene; Side; Skeletal Muscle; Source; Staining method; Stains; System; Techniques; Tissues; Xenobiotics; adult stem cell; base; design; effusion; fluorophore; human ABCG2 protein; human disease; primitive cell; reconstitution; regenerative; stem cell population

DESCRIPTION (provided by applicant): Identification of dependable methods that allow for the isolation and proliferation of adult stem cell populations has long been a significant obstacle in the development of cell-based therapies for human diseases. One particularly promising isolation technique is based on efflux of the DMA minor groove-binding fluorophore Hoechst 33342. Using fluorescence-activated cell sorting (FACS¿1/2), a sub-population of putative adult stem cells were originally identified in the bone marrow of mice. This rare cell fraction was termed the 'side population' or 'SP' due to a distinctive FACS¿1/2 profile that resulted from weak staining by Hoechst 33342. SP cells appeared dull compared to the majority of viable cells (called the main population or MP) due to fluorescent dye effusion through the ABCG2/Bcrp1 transporter. The expression and function of this transporter allows clearance of xenobiotic substances and is believed to be important in the maintenance of primitive cells as it is sharply down-regulated following lineage commitment. Though SP cells have been isolated from a large number of tissues in various mammalian species and appear to represent a multipotent adult stem cell population capable of differentiating toward myogenic, cardiogenic, and other cell lineages; there is currently no method to expand primary cultures of these cells. Therefore, our overall goal is to develop reproducible techniques to expand and differentiate adult bone marrow and skeletal muscle-derived side population (SP) stem cells to determine their regenerative and reparative potential for the treatment of cardiac and skeletal muscle disorders. To achieve this goal, we propose to address the following specific aims and hypotheses: 1) develop a reliable method for the preservation and expansion of side population cells (i.e. we will design a three-dimensional bioreactor system to propagate SP cells derived from two clinically-applicable adult tissue sources; namely, skeletal muscle and bone marrow), 2) determine the engraftment and reconstitution potential of the expanded SP cultures (i.e. SP expansion cultures will engraft to normal or damaged cardiac and skeletal muscle and reconstitute the entire hematopoietic system in lethally-irradiated mice), and 3) determine the conditions necessary to direct differentiation of expanded SP cells (i.e. we will devise approaches for the cardiogenic and myogenic specification of skeletal muscle and bone marrow-derived SP cells that have been expanded in culture.

描述（由申请人提供）：长期以来，鉴定允许分离和增殖成体干细胞群的可靠方法一直是开发基于细胞的人类疾病疗法的重大障碍。一种特别有前景的分离技术是基于 DMA 小沟结合荧光团 Hoechst 33342 的流出。使用荧光激活细胞分选 (FACS¿1/2)，最初在小鼠骨髓中鉴定出假定的成体干细胞亚群。这种罕见的细胞部分被称为“侧群”或“SP”，因为 Hoechst 33342 的弱染色导致了独特的 FACS¿1/2 图谱。由于荧光染料通过 ABCG2/Bcrp1 转运蛋白渗出，与大多数活细胞（称为主群或 MP）相比，SP 细胞显得暗淡。这种转运蛋白的表达和功能允许清除异生物质，并且被认为对于维持原始细胞很重要，因为它在谱系定型后急剧下调。虽然 SP 细胞已从各种哺乳动物物种的大量组织中分离出来，并且似乎代表了能够分化为肌源性、心源性和其他细胞谱系的多能成体干细胞群；目前还没有方法来扩大这些细胞的原代培养物。因此，我们的总体目标是开发可重复的技术来扩展和分化成人骨髓和骨骼肌来源的侧群（SP）干细胞，以确定它们治疗心脏和骨骼肌疾病的再生和修复潜力。为了实现这一目标，我们建议解决以下具体目标和假设：1）开发一种可靠的方法来保存和扩增侧群细胞（即我们将设计一个三维生物反应器系统来繁殖来自两种临床适用的成人组织来源的SP细胞；即骨骼肌和骨髓），2）确定扩增的SP培养物的植入和重建潜力 （即 SP 扩增培养物将移植到正常或受损的心脏和骨骼肌，并在致死照射的小鼠中重建整个造血系统），3）确定指导扩增的 SP 细胞分化所需的条件（即我们将设计用于在培养物中扩增的骨骼肌和骨髓来源的 SP 细胞的心源性和肌源性规范的方法。