Expansion and Directed Differentiation of Adult Side Population Stem Cells

描述（由申请人提供）：长期以来，确定能够分离和增殖成体干细胞群的可靠方法一直是人类疾病细胞疗法发展的一个重大障碍。一种特别有前途的分离技术是基于DMA小凹槽结合荧光团Hoechst 33342的外排。利用荧光激活细胞分选（FACS¿1/2），最初在小鼠骨髓中鉴定出推定的成体干细胞亚群。这种罕见的细胞片段被称为“侧群”或“SP”，因为它具有独特的FACS¿1/2特征，是由Hoechst 33342染色造成的。由于通过ABCG2/Bcrp1转运体的荧光染料渗出，SP细胞与大多数活细胞（称为主细胞群或MP）相比显得暗淡。这种转运蛋白的表达和功能允许清除外源物质，并且被认为在原始细胞的维持中很重要，因为它在谱系承诺后急剧下调。虽然SP细胞已经从各种哺乳动物的大量组织中分离出来，并且似乎代表了一种多能的成体干细胞群体，能够向肌源性、心源性和其他细胞系分化；目前还没有办法扩大这些细胞的原代培养。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衍生侧群（SP）干细胞，以确定其再生和修复治疗心脏和骨骼肌疾病的潜力。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体细胞(即我们将设计一个三维生物反应器系统来繁殖来自两个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SP培养物的植入和重建潜力（即SP扩增培养物将植入正常或受损的心脏和骨骼肌，并重建致死辐照小鼠的整个造血系统）；3)确定指导扩增SP细胞分化所需的条件(即，我们将设计方法用于培养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。

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.