Roche Joseph A, Begam Morium, Galen Sujay S
Department of Health Care Sciences, Physical Therapy Program, College of Pharmacy and Health Sciences, Wayne State University;
Department of Health Care Sciences, Physical Therapy Program, College of Pharmacy and Health Sciences, Wayne State University.
J Vis Exp. 2017 Aug 24(126):55731. doi: 10.3791/55731.
Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.
骨骼肌由于驻留在组织中的、能生成肌纤维的（生肌）卫星细胞（SCs）而具有再生能力，这些卫星细胞在合适的条件下可以形成新的肌纤维。尽管卫星细胞可以从肌肉组织中获取并在体外培养，但所得的成肌细胞在移植到宿主肌肉中时，促进肌生成的效果并不理想。与成肌细胞移植相比，通过手术暴露宿主肌肉并将供体肌肉组织片段或带有卫星细胞的分离肌纤维移植到宿主肌肉上，能更好地促进肌生成。我们开发了一种新技术，称为微创肌肉植入（MIME）。MIME包括将手术针穿过宿主肌肉，将一块供体肌肉组织拉过针道，然后将供体组织留在宿主肌肉中，使其作为宿主肌肉卫星细胞的来源。在这里，我们详细描述了在所有细胞中均表达绿色荧光蛋白（GFP）的免疫缺陷小鼠模型中进行MIME的步骤。宿主小鼠的免疫缺陷降低了供体组织免疫排斥的风险，而GFP表达使宿主肌纤维（GFP +）和供体细胞衍生的肌纤维（GFP-）易于识别。我们的初步数据表明，MIME可用于将供体小鼠的趾长伸肌（EDL）肌肉植入宿主小鼠的胫前肌（TA）中。我们的数据还表明，在MIME后将肌毒素（氯化钡，BaCl2）注入宿主肌肉时，有证据表明宿主肌肉中有供体细胞衍生的肌生成，在单个宿主TA肌肉中，分别约有5％，26％，26％和43％的纤维没有宿主贡献、宿主贡献最小、宿主贡献中等和宿主贡献最大。
