Ex vivo expansion of skeletal muscle satellite cells

骨骼肌卫星细胞的离体扩增

基本信息

批准号：
10570269
负责人：
Dawn D Cornelison
金额：
$ 17.19万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
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：
2022-02-10 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10570269
关键词：
Acceleration Acute Adult Aging Antibodies Basal lamina Base Sequence Basic Science Binding Biological Cell Compartmentation Cell Cycle Cell Nucleus Cell Therapy Cell Transplantation Cell membrane Cell surface Cells Characteristics Chemicals Chromatin Communities DNA Data Dedications Degenerative Disorder Disease Duchenne muscular dystrophy Dystrophin Engraftment Ephrin-A5 Ephrins Epigenetic Process Extracellular Domain Failure Family Frequencies Funding Funding Mechanisms Gene Expression Gene Expression Profiling Genes Goals Growth Half-Life Homeostasis Human In Vitro Individual Injury Label Life Mediating Methods Mitogens Molecular Mus Muscle Muscle Fibers Muscle function Muscle satellite cell MyoD Protein Myoblasts Natural regeneration Neuromuscular Diseases Pathology Patient Isolation Patients Phenotype Population Positioning Attribute Process Proliferating Property Proteins RNA Refractory Reproducibility Research Personnel Resistance Rest Serum Signaling Molecule Skeletal Muscle Satellite Cells Source Specificity Speed Structure Techniques Technology Testing Therapeutic Therapeutic Uses Tissues Translational Research Trauma Uncertainty adult stem cell aptamer cost cost effective cost effectiveness density extracellular genetic manipulation immunogenicity in vivo in vivo engraftment inhibitor injured muscle form muscle regeneration novel postnatal precursor cell prevent progenitor proliferation potential receptor repaired response to injury satellite cell self-renewal skeletal muscle growth small molecule stem cell population stem cells success timeline tool transcription factor translational applications translational potential volumetric muscle loss

项目摘要

SUMMARY Because all nuclei in differentiated, functional myofibers have permanently exited the cell cycle, the satellite cell population acts as a source of new nuclei when tissue growth, repair, or regeneration is required. Satellite cells during homeostasis rest in a quiescent, nonproliferative state sandwiched between the cell membrane and the basal lamina of a differentiated myofiber. In this state they express the satellite stem cell transcription factor Pax7 but little to no transcription factors of the MyoD family. In response to injury or disease, satellite cells are activated to re-enter the cell cycle, upregulate MyoD and other myogenic transcription factors, and expand as myoblasts to generate new myogenic cells, then commit to terminal differentiation and fuse into new or existing myofibers. Through mechanisms that are not yet well understood a fraction of satellite cell progeny will re-enter the quiescent state and repopulate the stem cell pool. This process is rapid and highly effective, and in most cases is sufficient to maintain muscle mass and function throughout life. However, in the case of muscle degenerative diseases such as Duchenne's muscular dystrophy or massive trauma leading to volumetric muscle loss, endogenous satellite cells are insufficient or unable to repair the muscle leading to long-term pathology. A longstanding goal in the field has been isolating patient- or donor-derived satellite cells and expanding them ex vivo (potentially in concert with manipulations such as repair of the dystrophin gene) then engrafting them therapeutically, however this approach has not yet met with good success. One major hurdle is the difficulty of maintaining satellite cells and their progeny in a proliferative progenitor state in vitro: they tend to commit to terminal differentiation even under high-mitogen conditions, and even those cells that remain proliferative largely lose their stem cell character (e.g., ability to self-renew as satellite cells in vivo). We have exciting new data showing that in the absence of the cell surface signaling molecule ephrin-A5, myoblasts will not only not commit to terminal differentiation but they will, when grown at high densities, instead exit the cell cycle and express high levels of Pax7, thus resembling quiescent satellite cells in at least two key respects. When repassaged at low density, these cells will re-enter the cell cycle and expand again without committing to terminal differentiation. To leverage this result into a potential translational application, we propose to attempt to transiently and reversibly inhibit (rather than delete) ephrin-A5, to allow myoblast expansion in culture without loss to differentiation and ideally to enhance stem cell character on engraftment. We have chosen to develop RNA aptamers as ephrin-A5 inhibitors in order to take advantage of aptamers' high specificity of binding, absence of immunogenicity, potential for additional chemical functionalization, and speed and cost-effectiveness to generate. If successful, this technique has the potential to significantly advance cell-based therapies for DMD and volumetric muscle loss and to accelerate basic science by allowing researchers to generate of large numbers of progenitor cells over extended culture periods.

概括因为分化的，功能性肌纤维中的所有核已永久退出了细胞周期，所以卫星当需要组织生长，修复或再生时，细胞种群充当新核的来源。卫星体内平衡期间的细胞休息在细胞膜之间的静止，非增殖状态和分化的肌纤维的基底层。在这种状态下，他们表达卫星干细胞转录因子PAX7，但几乎没有Myod家族的转录因子。为了应对伤害或疾病，卫星细胞被激活以重新进入细胞周期，上调MYOD和其他肌源性转录因子，以及作为成肌细胞扩展以产生新的肌源细胞，然后进行终端分化并融合到新的或现有的肌纤维。通过尚未充分了解一小部分卫星细胞后代的机制将重新输入静止状态并重新填充干细胞池。这个过程是快速而高效的，在大多数情况下，足以维持肌肉质量和一生的功能。但是，在肌肉退化性疾病，例如Duchenne的肌肉营养不良或巨大的创伤体积肌肉损失，内源性卫星细胞不足或无法修复导致的肌肉长期病理。该领域的长期目标是隔离患者或供体衍生的卫星细胞并将它们扩展到vivo（可能与诸如肌营养不良基因修复之类的操作协同合作）然后对它们进行治疗，但是这种方法尚未取得良好的成功。一个主要障碍是在体外保持卫星细胞及其后代的困难：体外：他们即使在高落下条件下，甚至是那些细胞，甚至保持增殖很大程度上失去了其干细胞特征（例如，在体内自我更新为卫星细胞的能力）。我们有令人兴奋的新数据，表明在没有细胞表面信号传导分子以ephrin-a5的情况下，成肌细胞不仅不会承诺终端差异化，而且在高密度生长时，它们会退出细胞周期并表达高水平的PAX7，因此在至少两个键中类似于静态卫星细胞尊重。当以低密度重新加倍时，这些单元将重新输入细胞周期，并再次扩展而无需致力于终端分化。为了将这一结果用于潜在的翻译应用，我们建议尝试瞬时和可逆地抑制（而不是删除）ephrin-a5，以允许肌细胞培养物的扩展而不会损失分化，理想情况下可以增强植入的干细胞特征。我们选择将RNA适体作为ephrin-A5抑制剂开发，以利用适体的优势。结合的高特异性，缺乏免疫原性，潜在的额外化学功能化和产生的速度和成本效益。如果成功，该技术有可能显着基于细胞的DMD和体积肌肉损失的预先基于细胞的疗法，并通过允许研究人员在延长的培养期间生成大量祖细胞。