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：
10723101
负责人：
Malachia Hoover
金额：
$ 3.62万
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/10723101
关键词：
3-Dimensional Acute Adipose tissue Affect Age-Related Osteoporosis Animals Biological Assay Biomedical Engineering Biomimetics Bone Marrow Bone Morphogenetic Proteins Cartilage Cell surface Cells Chondrogenesis Connective Tissue Cues Data Degenerative polyarthritis Deterioration 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 Joints 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 Rejuvenation Reporting Repression Research Role SHH gene Signal Transduction Site Specific qualifier value Specimen Spinal Fusion Steroids Structure Surgical Models Tail Techniques Testing Tissues Transplantation Vascular Endothelial Growth Factors Vertebral column barrier to testing bone cartilage regeneration cell type chronic back pain clinical translation combat disc regeneration effective therapy experimental study genetic signature in vivo innovation intervertebral disk degeneration lentivirally transduced 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）是一种高度专业化的纤维化结构，以更快的速度确定速度比体内任何其他连接的组织。这种情况通常称为IVD变性和 IVD修复的关键挑战是开发有效的治疗方法，从而逆转纤维球杆菌损害。由于它们的细胞自我更新和分化的固有特性，组织居民的利用干细胞有望成为基于干细胞的方法来对抗IVD变性。我们的实验室是第一个识别，孤立和功能表征真正的骨骼干细胞（SSC）及其承诺在小鼠和人类中严格引起骨骼，软骨和骨髓基质的下游祖细胞32,33。 SSC不同于间充质基质细胞，这些细胞代表了细胞类型的高度异质混合物57。从那以后，我们一直利用我们对SSC的知识来了解退行性骨骼状况骨关节炎，非工会和与年龄相关的骨质疏松症46,58,59。最近，我们使用隔离方法发现小鼠和人类IVD中的组织居民SSC。在此提案中，总体目标是确定将SSC命运决定到IVD组织的固有提示，并检查可能的外部线索在小鼠尾部IVD中使用新型的微裂缝手术模型引导IVD再生。我们的初步数据表明，IVD SSC比股骨SSC具有分化能力明显更大的软骨。体外和体内。此外，我们发现鼠标IVD SSC专门表达HOXA4及其骨骼命运的决定可以通过体外添加形态的FGF2，SHH和WNT3A来决定。我们也是发现小鼠尾部尾肠静脉IVD的急性微骨损伤不会扩大居民IVD SSC和将IVD SSC移植到微裂的IVD中，无法产生纤维球杆菌，因此表明它们可能可以需要指导体内软骨分化的其他因素。在这个建议中，我们的总体假设是Hoxa4是一种固有的调节剂，而FGF2是IVD组织命运的外部调节剂，并且在IVD SSC中调节这些途径可以用作潜在的干细胞靶向疗法用于对抗IVD变性。在AIM 1中，我们将通过调节HOXA4表达来解决这一假设通过慢病毒翻译在IVD和股骨（对照）SSC中，随后在体外和体内进行分化测定以评估其软骨IVD输出。在AIM 2中，我们将测试注射形态剂是否 FGF2，SHH和WNT3A可以改变微裂居民SSC的命运决定，以再生损坏 IVD组织在体内。最终，这组基本和临床前提出的实验将进一步定义概念SSC多样性并为基于干细胞的临床翻译奠定了基础防止和逆转IVD相关的肌肉骨骼疾病。