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

项目摘要 在确定关键的细胞、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)在稳态时调节HSC功能的生态位 造血术。即使有了更多的知识，人们对导致 调节体内HSC显著丧失后HSC数量的恢复。在这个提议中，我们利用克隆， 贴壁原代骨髓间充质基质/干细胞(MSC)，统一表达 成人MSC的特征，以确定调节HSC自我更新的关键因素。考虑到蜂窝和 BM利基的生理复杂性，无法评估其内在的自我更新促进作用 通过在体内删除给定的细胞类型来确定该细胞类型的潜能，因为这仅定义该细胞类型是否 通过直接或间接机制，维持肝星状细胞的存活、定位和/或功能 利基市场。通过使用高度明确的体外共培养系统，我们已经证明了克隆原代MSC具有 更高的内在能力，促进HSC细胞分裂，导致扩张(对称自我更新)和 这种能力与成骨细胞成熟的阶段呈负相关，因此成熟的成骨细胞 支持HSC自我更新的内在能力非常有限。这个系统很健壮，扩展了20倍 在共培养10天后出现功能性的LT-HSC，当WNT 根据金标准竞争性再繁殖细胞试验的测量，信号被阻断。尽管它有可能 有理由认为，任何体外系统都代表了体内HSC生态位的戏剧性过度简单化，即 然而，这些研究的结果将提供一个易于处理的模型来描述必要的组成部分 使用一种克隆的原代BM细胞类型调节HSC自我更新的Wnt信号可能是 与维持(或扩增)HSC的血管周围基质细胞最接近的功能类似物 在动态平衡和生理应激条件下。对LT-HSC调控因素的认识 扩增对加强临床应用至关重要，如基因治疗、骨髓移植和体细胞 遗传性血液疾病的基因矫正。这对于理解基本的分子机制也很重要。 由Wnt信号调控的对称性与非对称性干细胞分裂。总体假设 这一建议的一个特点是改变规范和非规范WNT信号功能之间的平衡，以 调节LT-HSC的自我更新或分化是否发生在原代Nestin+Lepr+BM MSC的背景下。 这一假设将通过：(1)确定规范和非规范WNT的贡献来解决 在原代骨髓来源的MSC中促进LT-HSC自我更新和分化的信号转导 克隆，(2)生化提纯和功能鉴定可溶性Wnt配体和其他因素 在LT-HSC/MSC共培养中被Wif1抑制，以及(3)确定WNT对LT-HSC自身 人Nestin+LEPR+MSC和CD34+细胞共培养的更新在小鼠和人之间是保守的。

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.