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 在确定肿瘤的关键细胞、分子和生理决定因素方面已经取得了重大进展， 成体骨髓（BM）造血干细胞（HSC）小生境，其在稳态期间调节HSC功能 造血即使有了更多的知识，对造成这种情况的因素仍然知之甚少。 在体内显著HSC损失后介导HSC数量的恢复。在这个提议中，我们利用克隆， 贴壁的原代BM间充质基质/干细胞（MSC），其均匀表达 标志，以确定调控HSC自我更新的关键因素。考虑到细胞和 由于BM生态位的生理复杂性，不可能评估固有的自我更新促进作用。 通过在体内删除该细胞类型来确定给定细胞类型的潜力，因为这仅定义该细胞类型是否是 通过直接或间接的机制，维持HSC的存活、定位和/或功能是必要的。 niche通过使用一个高度明确的体外共培养系统，我们已经证明克隆原代MSC具有一个高表达的细胞因子， 更高的内在能力，促进HSC细胞分裂，导致扩增（对称自我更新）， 这种能力与成骨细胞成熟的阶段呈负相关， 支持HSC自我更新的内在能力非常有限。这个系统是强大的，具有20倍的扩展 在共培养10天后出现功能性LT-HSC，当Wnt 信号传导被阻断，如通过金标准竞争性再增殖细胞测定所测量的。尽管它可以 正确地说，任何体外系统都代表了体内HSC生态位的戏剧性过度简化， 尽管如此，这些研究的结果仍将提供一个易于处理的模型，用于描述基本组成部分 使用克隆的原代BM细胞类型调节HSC自我更新的Wnt信号传导， 与维持（可能扩增）HSC的原型血管周围基质细胞功能最接近的类似物 在体内平衡和生理应激条件下。了解调节LT-HSC的因素 扩增对于提高基因治疗、骨髓移植和体细胞等临床应用至关重要 遗传性血液病的基因矫正它对于理解基本的分子机制也很重要 调节由Wnt信号传导调节的对称与不对称干细胞分裂。总体假设 改变经典和非经典Wnt信号传导功能之间的平衡， 调节LT-HSC自我更新或分化是否在原代Nestin+Lepr+ BM MSC的背景下发生。 这一假设将通过以下方式来解决：（1）确定典型和非典型Wnt的贡献 在原代骨髓来源的MSC的背景下促进LT-HSC自我更新和分化的信号传导 克隆，（2）生物化学纯化和功能表征可溶性Wnt配体和其他因子， 在LT-HSC/MSC共培养物中，WNT是否被Wif 1抑制，以及（3）确定WNT对LT-HSC自身的调节是否被WNT抑制。 人NESTIN+LEPR+ MSC和CD 34+细胞共培养物中的更新在小鼠和人之间是保守的。