Defining pathways promoting HSC self-renewal by mesenchymal stem/stromal cells

定义间充质干细胞/基质细胞促进 HSC 自我更新的途径

• 批准号: 9126155
• 负责人: CHRISTOPHER KLUG
• 金额: $ 11.76万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9126155
• 关键词: Address; Adult; Agonist; Automobile Driving; Back; Biological Assay; Blood Cells; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; CD34 gene; Cell Count; Cell Death; Cell Line; Cell Survival; Cell division; Cell physiology; Cells; Chemicals; Coculture Techniques; Complex; Endothelial Cells; Epigenetic Process; Equilibrium; Extracellular Matrix; Generations; Genetic; Goals; Gold; Growth Factor; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Homeostasis; Human; In Vitro; Inherited; Interleukin-11; Knowledge; LEPR gene; Life; Ligands; Light; MCAM gene; Maintenance; Measures; Mediating; Mesenchymal; Mesenchymal Stem Cells; Modeling; Molecular; Mus; Neurons; Osteoblasts; Oxygen measurement, partial pressure, arterial; Pathway interactions; Physiological; Process; Production; Recovery; Regulation; Role; Signal Pathway; Signal Transduction; Site; Somatic Cell; Staging; Stem cells; Stress; Stromal Cells; System; Transplantation; adult stem cell; analog; beta catenin; cell type; clinical application; cytokine; gene correction; gene therapy; in vivo; inhibitor/antagonist; knock-down; knowledge translation; macrophage; man; meetings; nestin protein; novel; overexpression; research study; self-renewal; shear stress; stem; stem cell division; stem cell niche; stem cell population; success; transcription factor

Project Summary Significant progress has been made in defining the key cellular, molecular and physiologic determinants of the adult bone marrow (BM) hematopoietic stem cell (HSC) niche that regulate HSC function during steady-state hematopoiesis. Even with this increased knowledge, little remains known about the factors responsible for mediating recovery of HSC numbers following significant HSC loss in vivo. In this proposal, we utilize clonal, adherent primary BM mesenchymal stromal/stem cells (MSC) that uniformly express molecules that are hallmarks of adult MSC, to define critical factors regulating HSC self-renewal. Given the cellular and physiologic complexity of the BM niche, it is not possible to evaluate the inherent self-renewal-promoting potential of a given cell type by deleting that cell type in vivo as this only defines whether that cell type is necessary, through direct or indirect mechanisms, to maintain HSC survival, localization, and/or function within the niche. By using a highly defined in vitro co-culture system, we have shown that clonal primary MSC have a much higher intrinsic ability to promote HSC cell divisions leading to expansion (symmetric self-renewal) and that this ability is inversely correlated with the stage of osteoblast maturation such that mature osteoblasts have very limited intrinsic ability to support HSC self-renewal. This system is robust, with a 20-fold expansion of functional LT-HSC occurring after 10 days of co-culture and up to ~100-fold expansion occurring when Wnt signaling is blocked as measured by the gold-standard competitive repopulating cell assay. Although it could rightly be argued that any in vitro system represents a dramatic oversimplification of the in vivo HSC niche, the results from these studies will nevertheless provide a tractable model for delineating the essential components of Wnt signaling that are regulating HSC self-renewal using a clonal, primary BM cell type that is likely the closest functional analog to the prototypic perivascular stromal cell that maintains (and perhaps expands) HSC during homeostasis and under physiologic stress conditions. Understanding factors regulating LT-HSC expansion is vital for enhancing clinical applications like gene therapy, BM transplantation, and somatic cell gene correction of inherited blood disorders. It is also important for understanding basic molecular mechanisms regulating symmetric versus asymmetric stem cell divisions regulated by Wnt signaling. The overall hypothesis of this proposal is that altering the balance between canonical and non-canonical Wnt signaling functions to regulate whether LT-HSC self-renewal or differentiation occurs in the context of primary Nestin+Lepr+ BM MSC. This hypothesis will be addressed by: (1) determining the contributions of canonical and non-canonical Wnt signaling to promotion of LT-HSC self-renewal and differentiation in the context of primary BM-derived MSC clones, (2) biochemically purifying and functionally characterizing soluble Wnt ligands and other factors being inhibited by Wif1 in LT-HSC/MSC co-cultures, and (3) determining whether WNT regulation of LT-HSC self- renewal in human NESTIN+LEPR+ MSC and CD34+ cell co-cultures is conserved between mouse and man.

项目摘要 在定义关键细胞，分子和生理决定因素方面取得了重大进展 成人骨髓（BM）造血干细胞（HSC）构成调节HSC功能期间HSC功能 造血。即使知识越来越多，对负责的因素几乎没有了解 在体内明显的HSC损失后，介导HSC数的恢复。在此提案中，我们利用克隆人 粘附的原代BM间充质基质/干细胞（MSC）均匀表达分子 成人MSC的标志，以定义调节HSC自我更新的关键因素。给定细胞和 BM利基市场的生理复杂性，无法评估固有的自我更新促进 通过在体内删除该细胞类型，给定单元类型的潜力，因为这仅定义了该单元类型是否为 通过直接或间接机制，必须维持HSC生存，定位和/或功能 利基。通过使用高度定义的体外共培养系统，我们表明克隆主要MSC具有 促进HSC细胞分裂导致扩展的固有能力更高（对称的自我更新）和 这种能力与成骨细胞成熟的阶段成反比，使成熟的成骨细胞 支持HSC自我更新的内在能力非常有限。该系统很健壮，扩展20倍 在共培养10天后发生的功能性LT-HSC，当Wnt时发生高达〜100倍的扩张 信号被阻断，如金标准竞争性重新流动细胞测定法所测量。虽然可以 正确地说，任何体外系统都代表了体内HSC壁ni的戏剧性过度简化， 然而，这些研究的结果将提供一个可划定基本组件的可拖动模型 使用克隆的主要BM细胞类型来调节HSC自我更新的Wnt信号传导 与原型周围基质细胞的最接近功能类似物，该细胞维持（甚至可以扩展）HSC 在体内稳态和生理压力条件下。了解调节LT-HSC的因素 扩展对于增强基因治疗，BM移植和体细胞等临床应用至关重要 遗传性血液疾病的基因校正。这对于理解基本分子机制也很重要 调节由Wnt信号调节的对称与非对称干细胞分裂。总体假设 该建议的是，改变规范和非典型的Wnt信号函数之间的平衡 调节LT-HSC自我更新还是分化发生在主巢+ LEPR+ BM MSC的背景下。 该假设将通过：（1）确定规范和非规范Wnt的贡献 在主要BM衍生的MSC的背景下，促进LT-HSC自我更新和分化的信号传导 克隆，（2）生化纯化和功能表征可溶性Wnt配体的表征，其他因素是 WIF1在LT-HSC/MSC共培养中抑制 人巢+ LEPR+ MSC的更新和CD34+细胞共培养在小鼠和人之间保守。