Ex vivo expansion of skeletal muscle satellite cells

骨骼肌卫星细胞的离体扩增

• 批准号: 10390539
• 负责人: Dawn D Cornelison
• 金额: $ 20.63万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-10 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10390539
• 关键词: 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; 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 Isolators; 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; Testing; Therapeutic; Therapeutic Uses; TimeLine; Tissues; Translational Research; Trauma; Uncertainty; adult stem cell; aptamer; base; cost; cost effective; cost effectiveness; density; extracellular; genetic manipulation; immunogenicity; in vivo; inhibitor; injured; muscle form; muscle regeneration; novel; postnatal; precursor cell; prevent; progenitor; receptor; repaired; response to injury; satellite cell; self-renewal; skeletal muscle growth; small molecule; stem cell population; stem cells; success; tool; transcription factor; translational applications; translational potential; volumetric muscle loss

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 由于分化的功能性肌纤维中的所有细胞核已永久退出细胞周期，卫星 当需要组织生长、修复或再生时，细胞群充当新细胞核的来源。卫星 体内平衡期间的细胞处于夹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，它们表达卫星干细胞转录 因子 Pax7，但很少甚至没有 MyoD 家族的转录因子。为了应对伤害或疾病，卫星 细胞被激活重新进入细胞周期，上调 MyoD 和其他生肌转录因子，并且 成肌细胞扩张以产生新的肌原细胞，然后进行终末分化并融合成新的肌原细胞 或现有的肌纤维。通过尚未充分了解的机制，卫星细胞后代的一小部分 将重新进入静止状态并重新填充干细胞池。这个过程快速且高效， 在大多数情况下足以维持终生的肌肉质量和功能。然而，在以下情况下 肌肉退行性疾病，例如杜氏肌营养不良症或导致严重创伤的 体积肌肉损失，内源性卫星细胞不足或无法修复肌肉导致 长期病理。该领域的一个长期目标是分离患者或供体来源的卫星细胞 并在体外扩增它们（可能与肌营养不良蛋白基因修复等操作相配合） 然后进行治疗性移植，但这种方法尚未取得良好成功。一个专业 障碍是在体外维持卫星细胞及其后代处于增殖祖细胞状态的困难： 即使在高有丝分裂原条件下，它们也倾向于进行终末分化，甚至那些细胞 保持增殖能力很大程度上失去了干细胞特性（例如，在体内作为卫星细胞自我更新的能力）。我们 令人兴奋的新数据表明，在缺乏细胞表面信号分子 ephrin-A5 的情况下， 成肌细胞不仅不会进行终末分化，而且当它们以高密度生长时，它们会发生终末分化。 退出细胞周期并表达高水平的 Pax7，因此至少在两个关键方面类似于静止卫星细胞 尊重。当以低密度重新传代时，这些细胞将重新进入细胞周期并再次扩增，而无需 致力于终端差异化。为了利用这一结果进入潜在的转化应用，我们 建议尝试暂时和可逆地抑制（而不是删除）ephrin-A5，以允许成肌细胞 在培养物中扩增而不损失分化，并理想地增强移植时的干细胞特性。 我们选择开发RNA适体作为ephrin-A5抑制剂，以充分利用适体的优势 结合的高特异性、无免疫原性、额外化学功能化的潜力，以及 生成速度和成本效益。如果成功的话，这项技术有可能显着 推进针对 DMD 和体积肌肉损失的细胞疗法，并通过允许 研究人员在延长的培养期内产生大量的祖细胞。