Myogenic Potential of Extraocular Muscle Satellite Cells

描述（由申请人提供）：Duchenne肌营养不良症患者（DMD）的眼外肌（EOM）得以保留，并在体内大部分骨骼肌完全退化后继续发挥功能。DMD中这种保留的原因尚不清楚。与肢体骨骼肌不同，正常成人眼外肌保留了一群活化的卫星细胞，即成人骨骼肌中的再生细胞。卫星细胞在整个生命过程中，甚至在老化的眼外肌中积极融合成正常的肌纤维，导致肌纤维重塑的连续过程。眼外肌及其卫星细胞对损伤、去神经支配、疾病和衰老具有极强的弹性，在肢体肌肉正常萎缩时保持正常形态。此外，控制EOM发育的早期基因与控制体节发育的基因不同。我们将检验这一假设，即这种持续的重塑是眼外肌在DMD中得以保留的过程。我们将检查1）两种DMD小鼠模型（mdx和mdx/utrophin+/-（mdx/utrhet）小鼠）中肌源性前体细胞更新的速率，以及2）测试EOM中细胞分裂的抑制是否会阻止两种DMD小鼠模型中的细胞分裂。在DMD、衰老和损伤中保留眼外肌，以及骨骼肌中肌源性前体细胞群的差异表明，与成人肢体肌细胞相比，眼外肌中的肌源性前体细胞（mpcs）可能显著富集或表型不同。与肢体相比，成人眼外肌中的肌源性前体细胞多5-8倍。此外，来自EOM的mpcs比来自肢体的类似细胞更耐凋亡。我们将通过检查1）两种小鼠DMD模型（mdx和mdx/utrophin+/-杂合子（mdx/utrhet）小鼠）中的肌源性前体细胞更新速率，以及2）通过对EOM进行γ辐照抑制细胞分裂是否会阻止DMD模型中的保留，来检验持续重塑允许DMD中的EOM保留的假设。备用的眼外肌在DMD和他们的mpcs的差异表明，眼外肌mpcs可能是显着富集或表型不同的成年肢体mpcs相比。一是人口增加;这些细胞是CD 34+的，并且对于Sca 1、CD 31（一种内皮谱系标记）、CD 45（一种造血谱系标记）是阴性的，并且对于各种卫星细胞标记（EOMCD 34）是阴性的。这些EOMCD 34细胞存在于新生小鼠的眼外肌和四肢肌肉中，但仅在整个成年期的眼外肌中维持。这些细胞也存在于mdx和mdx/utrophin-/-小鼠的EOM中。我们的假设是，这个人口的EOMCD 34细胞丰富的成人EOM可能是，至少部分，负责保存的EOM在DMD。我们将在两个具体目标中检验这些假设。具体目标1：1）DMD小鼠模型中肌纤维重塑的速率是否增加？2)抑制成年眼外肌细胞分裂是否能阻止mdx/utrophin +/-杂合子小鼠眼外肌的保留？我们将从眼外肌和肢体肌肉中分离出单核细胞的特定亚群，并在具体目标2中提出：1）mpc是否对损伤更具抵抗力？2)眼外肌的mpcs比肢体肌肉的mpcs多能吗？3)眼外肌来源的肌源性前体细胞是否比肢体骨骼肌来源的细胞具有更强的增殖潜能？长期目标是从眼外肌中确定和分离出一种与肢体相比具有更大增殖和存活潜力的肌源性前体细胞类型。这些细胞将在成肌细胞转移模型中进行测试，以确定它们用于治疗DMD的潜力。这些细胞可能比其他细胞具有优势，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）患者的眼外肌（EOM）未受损伤，其原因尚不清楚。眼外肌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保留的原因。这可能是由于眼外肌中富含的肌源性前体细胞群具有更大的损伤、衰老和疾病存活能力。虽然DMD中的眼肌是备用的，但EOM中的再生速率与DMD的mdx小鼠模型的腿部肌肉中的再生速率相似。我们将试图证明，肌肉前体细胞分裂是负责保留的眼外肌使用辐射抑制细胞分裂。众所周知，眼外肌比肢体肌肉更容易在损伤后存活。这反过来表明，眼外肌中的肌肉祖细胞可能比肢体肌肉中的肌肉祖细胞更健壮和长寿。如果这些假设是真的，最终我们希望利用这一点，通过使用从EOM中鉴定出的肌源性前体细胞作为肌肉损伤和DMD小鼠模型成肌细胞治疗中的新的供体细胞来源。

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.