Isolation of novel mesenchymal stem cell subpopulations

新型间充质干细胞亚群的分离

• 批准号: 8038578
• 负责人: BUDDHADEB DAWN
• 金额: $ 18.5万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-15 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038578
• 关键词: Adherence; Adult; Antigens; Arts; Bone Marrow; Cardiac; Cause of Death; Cell Count; Cell Culture Techniques; Cells; Cellular biology; Cessation of life; Clinical Trials; Confocal Microscopy; Country; Culture Media; Development; Developmental Biology; Diffuse; Discipline; ENG gene; Enzyme-Linked Immunosorbent Assay; Exhibits; Fibroblast Growth Factor 2; Flow Cytometry; Foundations; Future; Gene Expression; Gene Proteins; Genetic; Goals; Growth Factor; Heart failure; In Vitro; Infarction; Insulin-Like Growth Factor I; Investigation; LIF gene; Laboratories; Left Ventricular Function; Length; Maintenance; Mediating; Medical; Mesenchymal Stem Cells; Modeling; Molecular Biology; Molecular Profiling; Morbidity - disease rate; Morphology; Myocardial Infarction; Myocardial Ischemia; Myocardium; Nature; PTPRC gene; Patients; Perfusion; Phenotype; Plastics; Population; Property; Protein Chemistry; Proto-Oncogene Protein c-kit; Protocols documentation; Public Health; Quality of life; Reverse Transcriptase Polymerase Chain Reaction; Role; Serial Passage; Simulate; Staging; Stromal Cell-Derived Factor 1; Surface Antigens; Techniques; Testing; Therapeutic; Time; Vascular Endothelial Growth Factors; angiogenesis; base; combinatorial; cytokine; heart cell; immunocytochemistry; improved; in vivo; insight; interdisciplinary approach; myogenesis; novel; osteogenic; outcome forecast; paracrine; pluripotency; prevent; primitive cell; protein expression; public health relevance; relating to nervous system; repaired; stem; stem cell biology; stem cell therapy; stemness

DESCRIPTION (provided by applicant): Although therapy with adult bone marrow (BM)-derived unfractionated mesenchymal stem cells (MSCs) improves cardiac function, remodeling, and perfusion after MI, the extents of MSC-induced angiogenesis, myogenesis, and antiapoptotic and `paracrine effects' have been inconsistent in studies from different laboratories. These apparent discrepancies might have stemmed from the heterogeneous nature of unfractionated `MSCs', and the use of dissimilar expansion protocols in different laboratories. Our fundamental hypothesis is that a primitive and homogenous MSC subpopulation isolated on the basis of surface antigen expression will exhibit greater endothelial and cardiomyogenic differentiation potential, and secrete greater amounts of cardioprotective growth factors/cytokines; and that optimization of medium is critical for the expansion of MSC subpopulation specifically intended for cardiac repair without significant changes in antigenic phenotype, gene expression, differentiation potential, and secretory profile. Accordingly, Aim 1 will thoroughly examine whether compared with unfractionated MSCs, an antigenically-defined MSC subpopulation exhibits a more primitive phenotype, expresses endothelial/cardiomyogenic lineage markers, and undergoes greater endothelial/cardiomyogenic differentiation. Expression of markers of pluripotency and lineage commitment will be compared using real-time RT-PCR in unfractionated MSCs and MSC subpopulations isolated by flow cytometry based on the expression of CD45, c-kit, Flk-1, Sca-1, CD90, and CD105. Endothelial and cardiomyogenic differentiation will be rigorously quantitated (morphology, immunocytochemistry, confocal microscopy). To predict in vivo efficacy, differentiation (and secretory profile in Aim 2) will be assessed in culture conditions that simulate the in vivo milieu. Aim 2 will examine whether an antigenically-defined MSC subpopulation secretes greater amounts of cardioprotective growth factors/cytokines, and whether gene expression profiles correlate with secretory profiles. Aim 3 will conclusively establish the optimal medium composition for the expansion of select MSC subpopulation(s) with no or minimal alterations in surface antigen expression, gene expression, differentiation potential, and secretory abilities. Four primary media (DMEM, IMDM, 1-MEM, and Mesencult) and five factors (LIF, IGF-1, FGF-2, PDGF-B, and Wnt3a) will be examined. Antigen and gene expression, differentiation, and secreted factors will be examined during serial passages. In summary, this exploratory/developmental proposal aims to identify a novel MSC subpopulation most suitable for cardiac repair, and to establish the optimal medium that will expand these cells without significant changes in surface antigen expression/gene expression/differentiation potential/secretory attributes for future cardiac repair in vivo. The results of the proposed investigations will not only provide novel insights into MSC biology, but may also identify a cellular population ideally-suited for cardiac repair in patients with ischemic heart disease and post-MI heart failure. PUBLIC HEALTH RELEVANCE: Ischemic heart disease is the single most prevalent cause of death and morbidity in all Western countries. Despite significant advances in medical treatments, the prognosis for patients with large heart attacks is poor with eventual progression to end-stage heart failure and death. Although recent studies indicate that therapy with adult primitive cells can repair dead heart muscle, the results have been variable, and the mechanisms remain unclear. Thus, the identification of an ideal cell for heart repair, a major goal of this exploratory proposal would have enormous therapeutic implications for patients with ischemic heart disease and heart failure. The optimization of culture medium for expansion of this cell population will help generate a large number of cells for potential use in vivo. The results of the proposed studies will therefore be highly relevant for improving public health, and improving the length and quality of life of millions of patients with ischemic heart disease and heart failure.

描述（由申请人提供）：尽管对成年骨髓（BM）衍生的未分离间充质干细胞（MSCS）的治疗改善了MI后的心脏功能，重塑和灌注，但MSC诱导的血管生成，肌生成，肌生成，肌生成，肌生成，抗肌动症和抗激素和旁皮细胞激素作用已在具有不同的实验室中。这些明显的差异可能源于未分离的“ MSC”的异质性质，以及在不同实验室中使用不同扩展方案的使用。我们的基本假设是，根据表面抗原表达分离的原始和同质的MSC亚群将表现出更大的内皮和心肌分化潜力，并分泌更多的心脏保护生长因子/细胞因子。培养基的优化对于专门用于心脏修复的MSC亚群的扩展至关重要，而抗原表型，基因表达，分化潜力和分泌谱的显着变化。因此，AIM 1将彻底检查与未分流的MSC相比，抗原定义的MSC亚群是否表现出更原始的表型，表达内皮/心脏基体生成谱系标记，并经历更大的内皮/心脏和心脏造成的分化。将使用基于流式细胞术分离的无分流MSC和MSC亚群在基于CD45，C-KIT，FLK-1，SCA-1，SCA-1，CD90和CD105中的表达中比较多能性和谱系承诺标记的表达。将严格定量内皮和心肌分化（形态，免疫细胞化学，共聚焦显微镜）。为了预测体内功效，将在模拟体内环境的培养条件下评估分化（AIM 2中的分泌概况）。 AIM 2将检查抗原定义的MSC亚群是否分泌更多的心脏保护生长因子/细胞因子，以及基因表达谱是否与分泌谱相关。 AIM 3将最终建立最佳的培养基组成，以扩展精选的MSC亚群，而表面抗原表达，基因表达，分化潜力和分泌能力无需最小或最小的改变。将检查四个主要培养基（DMEM，IMDM，1-MEM和Mesencult）和五个因素（LIF，IGF-1，FGF-2，PDGF-B和WNT3A）。在串行通道中将检查抗原和基因表达，分化和分泌因子。总而言之，该探索性/发育建议旨在确定最适合心脏修复的新型MSC亚群，并建立最佳的培养基，该培养基将扩展这些细胞而不会在体内抗原表达/基因表达/分化潜在/分泌属性/分泌属性上显着变化，以用于体内未来心脏修复。拟议的调查结果不仅将提供有关MSC生物学的新见解，而且还可以鉴定出缺血性心脏病和MI后心力衰竭患者心脏修复非常适合心脏修复的细胞种群。公共卫生相关性：缺血性心脏病是所有西方国家最普遍的死亡和发病率。尽管药物治疗取得了重大进展，但心脏病发作大的患者的预后却很差，最终进展到终阶段心力衰竭和死亡。尽管最近的研究表明，成人原始细胞的治疗可以修复死心肌，但结果是可变的，并且机制尚不清楚。因此，识别理想的心脏修复细胞，该探索性建议的主要目标对缺血性心脏病和心力衰竭患者具有巨大的治疗意义。扩展该细胞种群的培养基的优化将有助于产生大量细胞在体内潜在使用。因此，拟议研究的结果将与改善公共卫生以及改善数百万缺血性心脏病和心力衰竭患者的寿命长度和质量相关。