Regulation of HSCs and HSC-Derived Osteoblasts in Osteogenesis Imperfecta

描述(申请人提供)：成骨不全(OI)是一种常染色体显性遗传性疾病，由编码I型胶原的两个基因(COL1A1或COL1A2)中的一个突变引起，是最常见的遗传性骨骼疾病。OI的特点是骨基质数量轻微到严重减少，导致反复骨折和骨畸形。在美国，OI的发病率估计为每20,000名活产儿中就有1名。目前还没有治愈OI的良方。治疗的目的是增加整体骨骼强度，防止骨折。这些药物的长期治疗有其自身的副作用；因此，有几种策略正在OI的实验中进行测试，以促进骨重建，其中之一是使用干细胞。我们已经证明，造血干细胞(HSC)移植可以改善OIM小鼠的骨缺损。我们还证明，在正常的骨转换和不稳定的骨折修复过程中，HSCs可以产生成骨细胞和骨细胞。我们在OIM小鼠身上的初步数据显示，移植了单个增强型绿色荧光蛋白(EGFP+)HSC的克隆群体后，移植小鼠的骨小梁和皮质参数有了显著改善。我们还表明，HSC移植可以在体内形成功能性成骨细胞，这些成骨细胞可以在体内沉积胶原并形成骨。这些研究和我们的数据表明，成骨细胞可以来自一种新的来源，即造血干细胞。因此，本研究旨在验证HSC可在OI中分化为成骨细胞的假说，了解促进其动员和成骨的分子因素在OI中可能具有治疗价值。目的1是通过移植小鼠的HSCs，在骨定向的I型胶原启动子的控制下，建立HSCs可以分化为成骨细胞的方法，从而可以对成骨细胞谱系的不同阶段的细胞进行特异性鉴定。利用造血细胞表达绿色荧光蛋白(GFP)和非造血细胞表达红色荧光蛋白(RFP)的小鼠，进行造血干细胞的谱系追踪。目标2的目的是确定影响HSC在OI中骨重建的因素，并确定HSC向成骨细胞分化的分子机制。影响HSCs动员及其向成骨细胞分化的因素将在体外和体内进行研究。这项研究的发现将具有重要意义，因为它们可以应用于促进和加速OI骨愈合的长期研究。这将为通过使用和调节HSCs治疗许多骨骼疾病和损伤开辟许多新的途径。

DESCRIPTION (provided by applicant): Osteogenesis imperfecta (OI), an autosomal dominant disorder caused by a mutation in one of the two genes that encode type I collagen (COL1AI or COL1A2), is the most common hereditary bone disease. OI is characterized by mild to severe reduction in the quantity of bone matrix that leads to repeated fractures and bone deformity. In the United States, the incidence of OI is estimated to be 1 per 20,000 live births. A present there is no cure for OI. Treatment is aimed at increasing overall bone strength to prevent fracture. Long-term treatment with the drugs has its own side effects; therefore several strategies are being tested experimentally in OI to enhance bone remodeling, one of them being the use of stem cells. We have shown that hematopoietic stem cell (HSC) transplantation can ameliorate the bone defects seen in oim mice. We also demonstrated that HSCs gives rise to osteoblasts and osteocytes during normal bone turnover as well as non- stabilized fracture repair. Our preliminary data from oim mice transplanted with a clonal population derived from a single enhanced green fluorescent protein (EGFP+) HSC demonstrates a significant improvement in the trabecular and the cortical parameters in engrafted mice. We also show that HSC transplantation leads to the formation of functional osteoblasts that deposited collagen and formed bone in vivo. These studies and our data suggest that osteoblasts can be derived from a novel source, i.e. HSCs. Thus, this proposal is to test the hypothesis that HSCs can give rise to osteoblasts in OI and understanding the molecular factors that promote its mobilization as well as osteogenesis could have therapeutic value in OI. Aim 1 is to establish that HSCs can give rise to osteoblasts employing transplantation studies with HSCs derived from mice having visual markers under the control of a bone directed collagen type I promoter, which allows for specific identification of cells at various stages of osteoblast lineage. Utilizing the mice expressing GFP in the hematopoietic cells and Red Fluorescent Protein (RFP) in the non-hematopoietic tissues we will do lineage tracing from the HSCs. The goals of Aim 2 are to characterize the factors modifying bone reconstitution by HSCs in OI and define the molecular mechanisms regulating HSC differentiation to osteoblasts. Effect of factors on mobilization of HSCs and their differentiation to osteoblasts will be studied both in vitro and in vivo. Findings from this study ill be significant in that they can be applied to long-term studies to enhance and accelerate bone healing in OI. This will open many new avenues of therapy for a number of bone diseases and injuries through the use and modulation of HSCs.