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

检查骨骼干细胞多样性及其在椎间盘再生中的作用

基本信息

批准号：
10537672
负责人：
Malachia Hoover
金额：
$ 3.51万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-16 至 2024-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10537672
关键词：
3-Dimensional Acute Address Adipose tissue Affect Animals BMP2 gene Biological Assay Biomedical Engineering Biomimetics Bone Marrow Cartilage Cell surface Cells Chondrogenesis Connective Tissue Cues Data Degenerative polyarthritis Development Embryo Evaluation FGF2 gene Femur Fibrocartilages Flow Cytometry Foundations Gene Expression Profiling Genetic Variation Growth Factor Head Hematopoiesis Homeobox Genes Human Hyaline Cartilage In Vitro Injections Injury Intervertebral disc structure Knowledge Limb structure Marrow Medicine Methods Mus Musculoskeletal Diseases Natural regeneration Nature Operative Surgical Procedures Opioid Osteocytes Osteogenesis Output Pathway interactions Play Production Progressive Disease Property Recombinant Proteins Recombinants Regulation Reporting Repression Research Role SHH gene Signal Transduction Site Specific qualifier value Specimen Spinal Fusion Stem cell transplant Steroids Structure Surgical Models Tail Techniques Testing Tissues Vascular Endothelial Growth Factors Vertebral column age related base bone bone morphogenic protein cartilage regeneration chronic back pain clinical translation combat disc regeneration effective therapy experimental study genetic signature in vivo innovation intervertebral disk degeneration mesenchymal stromal cell minimally invasive morphogens mouse model novel osteogenic pre-clinical prevent progenitor prospective reduce symptoms repaired scaffold self-renewal skeletal skeletal stem cell skeletal tissue standard care stem cell based approach stem cell expansion stem cell fate stem cell therapy stem cells targeted treatment

项目摘要

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.

项目摘要/摘要。椎间盘 (IVD) 是一种高度特化的纤维软骨结构，其恶化速度更快与体内任何其他结缔组织相比，这种情况通常被称为 IVD 变性和 a。 IVD 修复的关键挑战是开发逆转纤维软骨的有效治疗方法由于其细胞内在的自我更新和分化的损伤特性，组织驻留物的利用干细胞有望作为一种基于干细胞的方法来对抗 IVD 退化。识别、分离和功能表征真正的骨骼干细胞 (SSC) 及其承诺的下游祖细胞，严格产生小鼠和人类的骨、软骨和骨髓基质32,33。 SSC 与间充质基质细胞不同，后者代表细胞类型的高度异质混合物57。此后，我们利用我们对 SSC 的了解来了解退行性骨骼状况，包括最近，我们使用我们的隔离方法来治疗骨关节炎、骨不连和年龄相关的骨质疏松症46,58,59。在小鼠和人类 IVD 中发现组织驻留 SSC 在该提案中，首要目标是确定决定 SSC 命运进入 IVD 组织的内在线索，并检查可能的外在线索在小鼠尾部 IVD 中使用新型微骨折手术模型指导 IVD 再生。数据表明，IVD SSC 的分化能力明显高于股骨 SSC 的软骨形成能力此外，我们发现小鼠 IVD SSC 专门表达 HOXA4 及其相关物质。骨骼命运的决定可以通过体外添加形态发生素 FGF2、SHH 和 WNT3A 来决定。发现小鼠尾部 IVD 的急性微骨折损伤不会放大驻留的 IVD SSC，并且将 IVD SSC 移植到微骨折 IVD 中无法生成纤维软骨，因此表明它们可能在此建议中，我们总体上需要额外因素的指导来进行体内软骨分化。假设 HOXA4 是 IVD 组织命运的内在调节因子，而 FGF2 是 IVD 组织命运的外在调节因子，调节 IVD SSC 中的这些通路可用作潜在的干细胞靶向治疗在目标 1 中，我们将通过调节 HOXA4 表达来解决这一假设。通过慢病毒转导在 IVD 和股骨（对照）SSC 中进行，然后在体外和体内进行分化测定来评估其软骨 IVD 输出在目标 2 中，我们将测试是否注射形态发生素。 FGF2、SHH和WNT3A可以改变微骨折常驻SSC再生受损的命运决定最终，这组基础和临床前实验将进一步定义体内 IVD 组织。提出了 SSC 多样性的概念，并为基于干细胞的疗法的临床转化奠定了基础预防和逆转 IVD 相关的肌肉骨骼疾病。