Myogenic Potential of Extraocular Muscle Satellite Cells

描述（由申请人提供）：杜氏肌营养不良患者（DMD）的眼外肌（EOM）得以保留，并在身体大部分骨骼肌完全退化后继续发挥功能。DMD中这种节省的原因尚不清楚。与肢体骨骼肌不同，正常成人EOM保留了激活的卫星细胞群，这是成人骨骼肌中的再生细胞。在EOM的整个生命过程中，即使在衰老的EOM中，卫星细胞也会主动融合到正常的肌纤维中，从而导致肌纤维重构的持续过程。EOM及其卫星细胞对损伤、失神经支配、疾病和衰老具有极强的弹性，在肢体肌肉通常萎缩时仍能保持正常形态。此外，控制EOM发育的早期基因与控制somite发育的早期基因不同。我们将验证这一假设，即这种持续的重塑是DMD中EOM被保留的过程。我们将检测1)mdx和mdx/utrophin+/- (mdx/ utrheet)小鼠两种DMD小鼠模型的肌原性前体细胞更新率，以及2)在EOM中抑制细胞分裂是否会阻止它们在两种DMD小鼠模型中保留。DMD、衰老和损伤中EOM的保留以及骨骼肌中肌原性前体细胞群的差异表明，与成人肢体肌肉细胞相比，EOM中的肌原性前体细胞（mpcs）可能显着丰富或表型不同。成人EOM中肌原性前体细胞比肢体多5-8倍。此外，来自EOM的mpcs比来自肢体的类似细胞更能抵抗凋亡。我们将检验连续重塑允许DMD中EOM保留的假设，通过检验1)两种小鼠DMD模型，mdx和mdx/utrophin+/-杂合子（mdx/ utrheet）小鼠的肌原性前体细胞周转率，以及2)γ射线照射EOM抑制细胞分裂是否会阻止DMD模型中的EOM保留。DMD中EOM的保留及其mpcs的差异表明，与成人肢体mpcs相比，EOM mpcs可能显着丰富或表型不同。一是人口增加；这些细胞CD34阳性，Sca1、CD31（内皮谱系标记）、CD45（造血谱系标记）阴性，各种卫星细胞标记（EOMCD34）阴性。这些EOMCD34细胞存在于新生小鼠的EOM和肢体肌肉中，但仅在EOM中维持整个成年期。这些细胞也存在于mdx和mdx/utrophin-/-小鼠的EOM中。我们的假设是，在成人EOM中富集的EOMCD34细胞群可能至少在一定程度上负责DMD中EOM的保留。我们将在两个具体目标中检验这些假设。具体目标1问：1)DMD小鼠模型中肌纤维重塑率是否增加？2)抑制成年EOM细胞分裂是否会阻止mdx/utrophin +/-杂合子小鼠EOM的保留？我们将从EOM和肢体肌肉中分离出特定的单核细胞亚群，并在特定的目标2中问：1)mpcs是否更能抵抗损伤？2) EOM的mpcs是否比肢体肌肉的mpcs更具多能性？3)外源性肌源性前体细胞是否比来源于肢体骨骼肌的前体细胞具有更大的增殖潜能？长期目标是从EOM中定义和分离出一种与肢体相比具有更大增殖和存活潜力的肌原性前体细胞。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：杜氏肌营养不良症（DMD）患者眼外肌（EOM）不受影响，其原因尚不清楚。EOM在整个生命过程中不断重塑的独特能力表明，这种能力可能是这种保留的原因。这可能是由于EOM内丰富的肌原性前体细胞群具有更强的抗损伤、抗衰老和抗疾病能力。虽然DMD小鼠的眼部肌肉不受影响，但EOM的再生速度与mdx DMD小鼠模型的腿部肌肉的再生速度相似。我们将尝试证明，肌肉前体细胞分裂是负责通过辐照来抑制细胞分裂的EOM节约。众所周知，EOM比肢体肌肉更能在损伤后存活。这反过来表明，EOM中的肌肉祖细胞可能比肢体肌肉中的肌肉祖细胞更健壮，寿命更长。如果这些假设是正确的，最终我们希望利用从EOM中鉴定出的肌原性前体细胞作为成肌细胞治疗小鼠肌肉损伤和DMD模型的新供体细胞来源。

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.