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. 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.
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 veteransPublic Health Relevance
