Heparan Sulfate Proteoglycans in Human Hematopoiesis

描述(由申请人提供)： 背景：造血干细胞(HSC)移植可以治疗多种恶性肿瘤和非恶性血液病。脐带血(UCB)是一种很容易获得的HSC来源，但由于HSC数量不足，它在成人移植中的使用受到限制。更好地了解人HSC扩增所需的因素对于开发诱导HSC在体外长期存活和增殖的条件至关重要。虽然细胞因子和形态因子已经在培养中实现了短期扩增，但可移植的脐带血HSC在体外长期存活和扩增所需的因素和条件尚不清楚。在体内，HSC存在于高度专业化的“干细胞利基”中，可能由成骨细胞形成，在那里，细胞因子和基质/细胞表面大分子的特定组合定位，以调节干细胞池的大小，并控制受控的增殖和分化。在功绩补助的资助下，我们发现了一种这样的基质大分子，基质硫酸乙酰肝素(HS)，是在体外重现人类HSC生态位所需的关键成分，其中HS共同定位和调节细胞因子、趋化因子、形态发生因子(包括骨形态发生蛋白：BMPS)和基质成分在HSC上的活性。我们的小组还表明，HS支持体外、细胞因子介导的人脐血来源的再填充干细胞(NOD/SCID小鼠再填充细胞)的适度短期扩增，这些干细胞可以转移到二次受体。仅在细胞因子(不含HS)中培养时，HSC的再填充能力消失。最近，我们发现一些BMP，包括那些对HSC维持有抑制作用的BMP，是由UCB祖细胞本身产生的。与此一致的是，外源性BMP拮抗剂Gremlin1拮抗内源性自分泌/旁分泌BMP信号，进一步增强了原始造血祖细胞的长期维持和适度扩增。Gremlin1是一种细胞外肝素结合的BMP抑制物，在生长、分化、胚胎发育和各种疾病中发挥关键作用。它由成骨细胞和许多其他细胞产生，并调节几种已知的影响HSC命运的形态因子和途径(如WNTS、Sonic Hedgehog和Notch)。由于其他人和我们已经证明HS调节其他肝素结合的BMP拮抗剂的扩散、局部浓度和活性，我们推测这可能调节干细胞巢中的成骨细胞分泌的Gremlin1的作用。因此，Gremlin1和HS可能参与调节HSC的存活和增殖。假设：Gremlin1是干细胞利基的一个功能相关的成分，它在干细胞上的活性是通过与成骨细胞HS结合和相互作用来调节的。具体目标：(1)对HS和Gremlin1之间的相互作用进行结构和功能分析：确定结合动力学，确定每个分子上的结合结构域，并研究结合作用对Gremlin1功能的影响。(2)研究Gremlin1在干细胞生态位中的功能作用：利用在成骨细胞中过表达Gremlin1的突变小鼠和HS生物合成缺陷的小鼠，确定成骨细胞Gremlin1是否以及如何影响(A)内源性造血，(B)体外成骨细胞的干细胞支持功能，以及(C)移植后HSC的命运。(D)研究内源性成骨细胞HS如何影响Gremlin1诱导的HSC存活和增殖。意义，与退伍军人健康相关：了解Gremlin1与HS的相互作用以及他们对HSC的作用机制(S)将提高我们对干细胞生物学的理解，从而可能有助于开发支持干细胞在体外长期存活和增殖的条件。对Gremlin1上HS结合位点的结构和功能的研究也可能提高我们对疾病病理生理学的理解，并为未来多肽类药物/新药的开发提供指导，这些疾病在退伍军人中普遍存在，Gremlin1在这些疾病中发挥着病理生理作用，包括糖尿病肾病、玻璃体视网膜病变、骨量减少、肺部疾病和癌症。 公共卫生相关性： 对于成年退伍军人来说，造血干细胞(HSC)数量不足限制了更好的移植策略的使用，例如移植人脐血HSC，或清除癌细胞的自体HSC。我们的初步发现表明，多功能分子Gremlin1与硫酸肝素(Gremlin1似乎与之结合)有助于调节骨髓干细胞巢中的HSC。了解Gremlin1与硫酸肝素结合的结构和功能后果，以及Gremlin1在干细胞生态位中的作用，将有助于我们加深对HSC生物学的理解，并可能有助于为诱导移植的HSC扩增创造更好的条件。对Gremlin1上HS结合位点的结构和功能的研究也可能为将来开发治疗退伍军人中常见的其他疾病的新药提供参考，这些疾病中Gremlin1在其中发挥病理生理作用，如糖尿病肾病、玻璃体视网膜病变、骨量减少、肺部疾病和癌症。

DESCRIPTION (provided by applicant): Background: Hematopoietic stem cell (HSC) transplantation can cure several malignancies and non-malignant hematopoietic conditions. Umbilical cord blood (UCB) is an easily available source of HSC, but its use for transplanting adults is limited by insufficient HSC numbers. An improved understanding of factors required for human HSC expansion is essential for developing conditions that induce long-term in vitro survival and proliferation of HSC. Although short-term expansion in culture has been achieved by cytokines and morphogens, factors and conditions required for long-term in vitro survival and expansion of transplantable UCB HSC are unknown. In vivo, HSC reside in highly specialized "stem cell niches," likely formed by osteoblasts, wherein specific combinations of cytokines and matrix/cell surface macromolecules localize to regulate stem cell pool size and govern controlled proliferation and differentiation. With MERIT grant funding, we showed that one such matrix macromolecule, stromal heparan sulfate (HS), is a critical component required for recapitulation of the human HSC niche in vitro, wherein HS co-localize and modulate the activity of cytokines, chemokines, morphogens (including bone morphogenetic proteins: BMPs) and matrix components on HSC. Our group also showed that HS support ex vivo, cytokine-mediated modest short-term expansion of human UCB derived repopulating stem cells (NOD/SCID mouse repopulating cells) that can be transferred to secondary recipients. Repopulating HSC were lost when cultured in cytokines alone (without HS). More recently, we found that several BMPs, including those inhibitory to HSC maintenance, are produced by UCB progenitors themselves. Consistent with this, antagonism of endogenous autocrine/paracrine BMP signaling by exogenously provided BMP antagonist Gremlin1 further augmented long-term maintenance and modest expansion of primitive hematopoietic progenitors. Gremlin1 is an extracellular heparin-binding BMP inhibitor that plays a critical role in growth, differentiation and embryonic development and in various diseases. It is produced by osteoblasts and many other cells, and modulates several morphogens and pathways (e.g. Wnts, Sonic hedgehog and Notch) known to influence HSC fate. Because others and we have shown that HS regulates the diffusion, local concentration and activity of other heparin-binding BMP antagonists, we speculate that may modulate the effects of Gremlin1 secreted by osteoblasts in the stem cell niche. Gremlin1 and HS may thus contribute to the regulation of HSC survival and proliferation. Hypothesis: Gremlin1 is a functionally relevant component of the stem cell niche, where its activity on stem cells is modulated by binding to and interacting with osteoblast HS. Specific Aims: (1) Perform a structural and functional analysis of interactions between HS and Gremlin1: determine binding kinetics, identify binding domains on each molecule, and examine the effect of binding interactions on Gremlin1 function. (2) Examine the functional role of Gremlin1 in the stem cell niche: Using mutant mice that over-express Gremlin1 in osteoblasts, and mice deficient in HS biosynthesis, determine if and how osteoblast Gremlin1 influences (a) endogenous hematopoiesis, (b) the stem-cell supportive function of osteoblasts in vitro, and (c) HSC fate following transplantation. (d) Examine how endogenous osteoblast HS influences Gremlin1-induced HSC survival and proliferation. Significance, relevance to veterans< health: Understanding the interactions of Gremlin1 with HS, and the mechanism(s) of their action on HSC will improve our understanding of stem cell biology and may thus help develop conditions that support long-term stem cell survival and proliferation in vitro. The structure-function studies on HS-binding sites in Gremlin1 may also improve our understanding of disease pathophysiology, and inform future development of peptide-mimetics/novel drugs for the treatment of several disorders prevalent in veterans in which Gremlin1 plays a pathophysiological role, including diabetic nephropathy, vitreoretinopathy, osteopenia, lung disease and cancer. PUBLIC HEALTH RELEVANCE: Insufficient hematopoietic stem cell (HSC) numbers limit the use of preferable transplantation strategies such as transplantation of human umbilical cord blood HSC, or autologous HSC purged of cancer cells, for adult Veterans. Our preliminary findings indicate that the multi-functional molecule Gremlin1, together with heparan sulfate (to which Gremlin1 appears to bind), contributes to regulation of HSC in the bone marrow stem cell niche. Understanding the structural aspects and functional consequences of binding interactions of Gremlin1 with heparan sulfate, and the role of Gremlin1 in the stem cell niche, will enhance our understanding of HSC biology, and may help develop better conditions for inducing HSC expansion for transplantation. The structure- function studies on HS-binding sites in Gremlin1 may also inform future development of novel drugs for other disorders prevalent in Veterans in which Gremlin1 plays a pathophysiological role, such as diabetic nephropathy, vitreo-retinopathy, osteopenia, lung disease and cancer.