BMP6 Induction of Human Mesenchymal Stem Cell Osteoblast Differentiation

描述（由申请人提供）：我们的长期计划目标是在组织工程构建中利用成人骨髓源性间充质祖细胞（间充质干细胞- MSC）修复颅面和长骨缺损。MSC具有成为成骨细胞的强大潜力，并且当在动物模型中植入异位和原位位点时，人MSC（hMSC）可以形成骨。利用这些干细胞的治疗潜力的一个明显优势是，自体hMSC很容易从小骨髓抽吸物培养。在临床上，可以从患者收集骨髓，在体外培养hMSC，然后将其输送回患者体内。然而，为了最有效地使用hMSC治疗，我们必须能够完全控制它们的分化。这需要完全理解调节hMSC分化的生理相关因素，以及激活的下游信号传导和转录网络。我们的初步研究结果表明，骨形态发生-6（BMP 6）是一个重要的自分泌介质的hMSC分化。BMP 6是hMSC产生的唯一成骨BMP，并且用外源性BMP 6短期处理细胞促进成骨转录因子osterix的持续表达，导致成骨细胞分化。对于这个项目，我们提出了三个具体的目标：（1）我们将检查内源性BMP 6对成骨细胞分化的意义，（2）我们将研究BMP 6信号，指导成骨细胞分化，（3）我们将研究BMP 6诱导的osterix表达的重要性。这些目标将由一个研究小组在体外、体内和计算上实现，该研究小组包括一名兽医科学家和一名整形外科医生（都是生物化学家）以及一名计算生物学家。研究BMP 6功能和细胞信号传导的机制实验将在体外进行，并将利用原代hMSC和人胚胎干细胞（系WA 01和UC 06）。体内研究将使用临床前异位骨植入模型来检查hMSC的体内骨形成能力。数学建模将用于描述BMP 6诱导的成骨细胞分化的动力学，贝叶斯学习将用于发现BMP 6诱导的成骨细胞分化所特有的信号网络。这个为期五年的项目的完成不仅将证明BMP 6在治疗上控制hMSC成骨细胞分化的效用，而且还将揭示控制hMC成骨细胞分化的新型信号传导和转录网络。从长远来看，新靶点的确定将导致在组织工程构建体中指导hMSC分化以修复骨的增强的治疗选择的发展。

DESCRIPTION (provided by applicant): Our long-term programmatic goal is to utilize adult marrow-derived mesenchymal progenitors (mesenchymal stem cells - MSC) in tissue engineered constructs to repair craniofacial and long bone defects. MSC have robust potential to become osteoblasts and human MSC (hMSC) can form bone when implanted in heterotopic and orthotopic sites in animal models. A distinct advantage of exploiting the therapeutic potential of these stem cells is that autologous hMSC are easily cultivated from small marrow aspirates. Clinically, marrow could be collected from a patient, hMSC cultivated in vitro, and then delivered back to the patient. However, to most effectively use hMSC therapeutically, we must be able to fully control their differentiation. This requires a complete understanding of physiologically relevant factors that regulate hMSC differentiation, and in turn, downstream signaling and transcriptional networks that are activated. Our preliminary results show that bone morphogenetic-6 (BMP6) is an important autocrine mediator of hMSC differentiation. BMP6 is the only osteogenic BMP produced by hMSC and short-term treatment of cells with exogenous BMP6 promotes sustained expression of the osteogenic transcription factor osterix, leading to osteoblast differentiation. For this project we propose three specific aims: (1) we will examine the significance of endogenous BMP6 for osteoblast differentiation, (2) we will study BMP6 signaling that directs osteoblast differentiation, and (3) we will study the importance of BMP6-induced expression of osterix. These aims will be pursued in vitro, in vivo, and computationally by a team of investigators that includes a veterinary scientist and an orthopaedic surgeon - both trained as biochemists - and a computational biologist. Mechanistic experiments examining BMP6 function and cell signaling will be pursued in vitro and will utilize primary hMSC and human embryonic stem cells (lines WA01 and UC06). In vivo studies will use a preclinical heterotopic bone implantation model to examine the in vivo bone forming capacity of hMSC. Mathematical modeling will be used to describe kinetics of BMP6-induced osteoblast differentiation, and Bayesian learning will be used to discover signaling networks that are unique to BMP6-induced osteoblast differentiation. The completion of this five-year project will not only demonstrate the utility of BMP6 therapeutically to control hMSC osteoblast differentiation, but will also reveal novel signaling and transcriptional networks that govern hMC osteoblast differentiation. Long term, the identity of novel targets will lead to the development of enhanced therapeutic options for directing hMSC differentiation in tissue engineered constructs to repair bone.