Myogenic Potential of Extraocular Muscle Satellite Cells

描述(申请人提供)：眼外肌(EOM)在Duchenne肌营养不良症(DMD)患者中幸免于难，并在体内大多数骨骼肌完全退化后继续发挥功能。DMD中这种备用的原因尚不清楚。与四肢骨骼肌不同，正常成人EOM保留了一群激活的卫星细胞，卫星细胞是成人骨骼肌中的再生细胞。在EOM中，卫星细胞在整个生命过程中主动融合为正常的肌纤维，即使在老化的EOM中也是如此，导致肌纤维重塑的持续过程。EOM及其卫星细胞对损伤、失神经、疾病和衰老具有极强的弹性，在肢体肌肉正常萎缩的情况下保持正常的形态。此外，早期控制EOM发育的基因与控制体节发育的基因是不同的。我们将验证这样一种假设，即这种持续的重塑是DMD中EOM得以幸免的过程。我们将检测1)两种DMD小鼠模型--MDX和MDX/utroin+/-(MDX/UTRAHET)小鼠的成肌前体细胞周转率，以及2)抑制EOM中的细胞分裂是否会阻止它们在两种DMD小鼠模型中的存在。在DMD、衰老和损伤中EOM的存在以及骨骼肌成肌前体细胞群体的差异表明，EOM中的成肌前体细胞(MPC)可能与成人肢体肌肉细胞相比显著丰富或表型不同。成人EOM的肌源性前体细胞数量是肢体的5~8倍。此外，来自EOM的MPC比来自肢体的类似细胞更耐凋亡。我们将通过检查1)两种DMD模型(MDX和MDX/utroin+/-杂合子(MDX/UTRAHET)小鼠)中肌源性前体细胞的周转率，以及2)伽玛照射抑制EOM的细胞分裂是否阻止DMD模型中的EOM保留，来检验持续重塑允许DMD模型中的EOM保留的假设。DMD中EOM的保留及其MPC的差异表明，与成人肢体MPC相比，EOM MPC可能显著丰富或表型不同。增加了一个群体；这些细胞CD34+，内皮细胞系标志Sca1，CD31阴性，造血系标志CD45阴性，各种卫星细胞标志(EOMCD34)阴性。这些EOMCD34细胞存在于新生小鼠的EOM和四肢肌肉中，但只在EOM中保持整个成年期。这些细胞也存在于MDX和MDX/utroin-/-小鼠的EOM中。我们的假设是，这种在成人EOM中丰富的EOMCD34细胞群可能至少在一定程度上是DMD中EOM减少的原因。我们将在两个具体目标上检验这些假设。具体目的1问：1)DMD小鼠模型的肌纤维重建率增加了吗？2)抑制成年EOM的细胞分裂是否阻止了MDX/utroin+/-杂合子小鼠EOM的减少？我们将从EOM和四肢肌肉中分离出特定的单核细胞亚群，并有针对性地提出以下问题：1)MPC对损伤的抵抗力更强吗？2)EOM的MPC是否比四肢肌肉的MPC更具多能性？3)EOM的肌源性前体细胞是否比肢体骨骼肌的MPC具有更大的增殖潜力？长期的目标是从EOM中定义和分离出一种肌源性前体细胞类型，与肢体相比具有更大的增殖和生存潜力。这些细胞将在成肌细胞转移模型中进行测试，以确定它们在治疗DMD方面的潜力。这些细胞可能比其他细胞更有优势，因为自体移植是可能的。公共卫生相关性：Duchenne肌营养不良症(DMD)患者眼外肌(EOM)被保留，这种保留的原因尚不清楚。EOM在整个生命过程中不断重塑的独特能力表明，这种能力可能是造成这种备用的原因。这可能是由于EOM内丰富的肌源性前体细胞群，具有更强的损伤、衰老和疾病生存能力。虽然眼部肌肉在DMD中幸免于难，但EOM中的再生速度与DMD的MDX小鼠模型的腿部肌肉中的再生速度相似。我们将试图通过使用辐射抑制细胞分裂来证明肌肉前体细胞分裂是导致EOM减少的原因。众所周知，EOM比四肢肌肉更能耐受损伤。这反过来表明，EOM中的肌肉前体细胞可能比肢体肌肉中的肌肉前体细胞更健壮和长寿。如果这些假设是真的，我们最终希望通过使用从EOM中识别出的成肌前体细胞作为新的供体细胞来源来利用这一点，在肌肉损伤和DMD模型的小鼠的成肌细胞治疗中。

DESCRIPTION (provided by applicant): The extraocular muscles (EOM) are spared in Duchenne muscular dystrophy patients (DMD) and continue to function after most skeletal muscles in the body have completely degenerated. The reason for this sparing in DMD is unknown. Unlike limb skeletal muscle, normal adult EOM retain a population of activated satellite cells, the regenerative cell in adult skeletal muscle. Satellite cells actively fuse into normal myofibers in the EOM throughout life, even in aging EOM, resulting in a continuous process of myofiber remodeling. The EOM and their satellite cells are extremely resilient to injury, denervation, disease, and aging, retaining normal morphology when limb muscle would normally atrophy. In addition, the early genes controlling EOM development are distinct from those that control somite development. We will test the hypothesis that this continuous remodeling is the process by which EOM are spared in DMD. We will examine 1) the rate of myogenic precursor cell turnover in two mouse models of DMD, the mdx and mdx/utrophin+/- (mdx/utrhet) mice, and 2) test whether inhibition of cell division in the EOM prevent their sparing in the two mouse models for DMD. The sparing of the EOM in DMD, aging, and injury and differences in the myogenic precursor cell populations in skeletal muscle suggest myogenic precursor cells (mpcs) within EOM may be significantly enriched or phenotypically distinct compared with adult limb muscle cells. There are 5-8 fold more myogenic precursor cells in adult EOM compared to limb. Additionally, mpcs from EOM are more resistant to apoptosis than similar cells from limb. We will test the hypothesis that continuous remodeling allows for EOM sparing in DMD by examining 1) the rate of myogenic precursor cell turnover in two mouse DMD models, the mdx and mdx/utrophin+/- heterozygote (mdx/utrhet) mice, and 2) whether inhibition of cell division by gamma irradiation of the EOM prevents sparing in the DMD models. Sparing of the EOM in DMD and differences in their mpcs suggest that the EOM mpcs may be significantly enriched or phenotypically distinct compared with adult limb mpcs. One population is increased; these cells are CD34+ and negative for Sca1, CD31, an endothelial lineage marker, CD45, an hematopoietic lineage marker, and negative for various satellite cell markers (EOMCD34). These EOMCD34 cells are present in the EOM and limb muscles of neonatal mice, but only maintained throughout adulthood in EOM. These cells are also present in the EOM of mdx and mdx/utrophin-/- mice. Our hypothesis is that this population of EOMCD34 cells enriched in adult EOM may be, at least in part, responsible for the sparing of EOM in DMD. We will test these hypotheses in two Specific Aims. Specific aim 1 asks: 1) is the rate of myofiber remodeling increased in the DMD mice models? 2) Does inhibition of cell division in adult EOM prevent sparing in the EOM of mdx/utrophin +/- heterozygote mice? We will isolate specific subpopulations of mononucleated cells from EOM and limb muscles and in specific aim 2 ask: 1) Are the mpcs more resistant to injury?2) Are the mpcs from EOM more multipotent than those from limb muscle? 3) Do the myogenic precursor cells from EOM have greater proliferative potential than those derived from limb skeletal muscle? The long term goal is to define and isolate a myogenic precursor cell type from EOM that has greater proliferative and survival potential compared with limb. These cells would be tested in a myoblast transfer model to determine their potential for use in the treatment of DMD. These may offer advantages over other cells, as autologous transplants would be possible. PUBLIC HEALTH RELEVANCE: The extraocular muscles (EOM) are spared in Duchenne muscular dystrophy (DMD), and the cause of this sparing is unknown. The unique ability of EOM to continuously remodel throughout life suggests that this ability may be responsible for this sparing. This may be due to an enriched myogenic precursor cell population within the EOM that has a greater ability to survive injury, aging and disease. While the eye muscles are spared in DMD, the rate of regeneration in the EOM is similar to that in the leg muscles of the mdx mouse model of DMD. We will attempt to demonstrate that muscle precursor cell division is responsible for sparing of the EOM by using irradiation to inhibit cell division. It is well known that the EOM survive injury better than limb muscle. This in turn suggests that muscle progenitors in EOM may be more robust and long-lived than those from limb muscles. If these hypotheses are true, ultimately we hope to exploit this by using identified myogenic precursor cells from EOM as a new source of donor cells in myoblast therapy in mice models of muscle injury and DMD.