Examining Skeletal Stem Cell Diversity and its Role in Intervertebral Disc Regeneration

项目概要/摘要。 椎间盘（IVD）是一种高度特化的纤维软骨结构， 比身体里的其他结缔组织都要多这种情况通常被称为IVD变性， IVD修复的关键挑战是开发有效的治疗方法， 损害由于它们的细胞自我更新和分化的内在特性， 干细胞有望成为对抗IVD变性的基于干细胞的方法。我们的实验室是第一个 鉴定、分离和功能表征真正骨骼干细胞（SSCs）及其定向的 在小鼠和人类中严格产生骨、软骨和骨髓基质的下游祖细胞32，33。 SSC不同于代表细胞类型的高度异质混合物的间充质基质细胞57。 从那以后，我们利用我们对SSC的了解来了解退行性骨骼疾病，包括 骨关节炎、骨不连和年龄相关性骨关节炎46，58，59。最近，我们使用隔离方法， 发现小鼠和人IVD中的组织驻留SSC。本提案的总体目标是 确定决定SSC进入IVD组织的内在线索，并检查可能 在小鼠尾侧IVD中使用新型微骨折手术模型引导IVD再生。我们的初步 数据表明，在分化能力方面，IVD SSC明显比股骨SSC更具有软骨形成能力， 无论是在体外还是在体内。此外，我们发现小鼠IVD SSC仅表达HOXA 4， 骨骼命运的决定可以通过在体外添加形态发生剂FGF 2、SHH和WNT 3A来决定。我们也 发现小鼠尾部IVD的急性微骨折损伤不会扩大驻留的IVD SSC， 将IVD SSC移植到微骨折的IVD中不能产生纤维软骨，因此表明它们可能 需要额外的因子指导体内软骨分化。在这份提案中，我们的总体 假设HOXA 4是IVD组织命运内在调节剂，而FGF 2是IVD组织命运的外在调节剂， 并且在IVD SSC中调节这些途径可以用作潜在的干细胞靶向治疗 用于对抗IVD退化。在目标1中，我们将通过调节HOXA 4表达来解决这一假设。 在IVD和股骨（对照）SSC中通过慢病毒转导，随后在体外和体内进行 分化测定以评估它们的软骨生成IVD输出。在目标2中，我们将测试形态发生剂的注射是否 FGF 2、SHH和WNT 3A可以改变微骨折的驻留SSC的命运决定，以再生受损的SSC。 体内IVD组织。最终，这组基础和临床前拟议实验将进一步定义 概念SSC多样性，并为基于干细胞的治疗的临床转化奠定基础， 预防和逆转IVD相关的肌肉骨骼疾病。

Project Summary/Abstract. The intervertebral disc (IVD) is a highly specialized, fibrocartilaginous structure that deteriorates at a rate faster than any other connective tissue in the body. This condition is commonly referred to as IVD degeneration and a critical challenge for IVD repair is the development of effective treatments that reverses the fibrocartilage damage. Due to their cell intrinsic properties of self-renewal and differentiation, the utilization of tissue resident stem cells holds promise as a stem cell-based approach to combat IVD degeneration. Our lab was the first to identify, isolate and functionally characterize bona fide skeletal stem cells (SSCs) and their committed downstream progenitors that give rise strictly to bone, cartilage, and marrow stroma in mice and humans32,33. SSCs are distinct from mesenchymal stromal cells which represent highly heterogenous mixtures of cell types57. We’ve since have leveraged our knowledge of SSCs to understand degenerative skeletal conditions including osteoarthritis, non-unions and age-related osteoporosis46,58,59. More recently, we used our isolation methods to discover tissue resident SSCs within mouse and human IVDs. In this proposal, the overarching objective is to identify the intrinsic cues that dictate SSC fate into IVD tissue as well as examine the extrinsic cues that may guide IVD regeneration using a novel microfracture surgical model in the mouse caudal IVD. Our preliminary data suggests that IVD SSCs are distinctly more chondrogenic than femur SSCs in their differentiation capacity both in vitro and in vivo. Additionally, we found that mouse IVD SSCs exclusively express HOXA4 and their skeletal fate decisions can be dictated by the addition of morphogens FGF2, SHH and WNT3A in vitro. We also found that acute microfracture injury of caudal IVDs in the mouse tail does not amplify resident IVD SSCs and transplantation of IVD SSCs into microfractured IVD fail to generate fibrocartilage, thus suggesting they may require the guidance of additional factors for cartilage differentiation in vivo. In this proposal, our overall hypothesis is that HOXA4 is an intrinsic regulator, while FGF2 is an extrinsic regulator of IVD tissue fate, and that modulating these pathways in IVD SSCs can be used as a potential stem cell-targeting therapy for combating IVD degeneration. In Aim 1, we will address this hypothesis by modulating HOXA4 expression in IVD and femur (control) SSCs via lentiviral transduction and subsequently perform in vitro and in vivo differentiation assays to assess their cartilagenic IVD output. In Aim 2, we will test if the injection of morphogens FGF2, SHH and WNT3A can change the fate decision of microfractured resident SSCs to regenerate damaged IVD tissue in vivo. Ultimately, this set of basic and pre-clinical proposed experiments will further define the concept the SSC diversity and set the foundation for the clinical translation of stem cell-based therapies for preventing and reversing IVD-related musculoskeletal diseases.