大鼠骨骼肌中纤维-脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的鉴定、分离和表征
Summary
该协议概述了一种从大鼠骨骼肌中分离纤维脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的方法。在肌肉损伤模型中利用大鼠为分析提供了来自萎缩肌的组织可用性的增加，以及用于评估自由移动动物的肌肉力量和步态的更多经过验证的方法。
Abstract
纤维脂肪生成祖细胞（FAPs）是骨骼肌中的常驻间质细胞，与肌原祖细胞（MPs）一起在肌肉稳态，损伤和修复中起关键作用。目前用于FAPs鉴定和分离的方案使用流式细胞术/荧光激活细胞分选（FACS），迄今为止 在体内 评估其功能的研究仅在小鼠中进行。大鼠较大的固有尺寸允许对骨骼肌损伤模型中的FAPs进行更全面的分析，特别是在严重萎缩的肌肉中，或者当研究人员需要大量的组织质量进行多次下游测定时。大鼠还提供了更多不需要动物镇静或牺牲的肌肉功能测定选择，从而通过进行连续评估来最大限度地减少发病率和动物使用。针对小鼠优化的流式细胞术/ FACS方案具有物种特异性，特别是受市售抗体特性的限制。它们尚未针对将FAPs与大鼠或高度纤维化肌肉分离进行优化。基于表面标志物CD31，CD45，Sca-1和VCAM-1的差异表达，开发了一种流式细胞术/ FACS方案，用于从健康和去神经化大鼠骨骼肌中鉴定和分离FAPs和MP。由于大鼠特异性，流式细胞术验证的一抗受到严重限制，因此进行了靶向Sca-1的抗体的内部偶联。使用该协议，确认了成功的Sca-1偶联，并通过细胞培养和FACS分离的FAP和MP的免疫染色来验证FAP和MP的流式细胞术鉴定。最后，我们在延长（14周）大鼠去神经支配模型中报告了一种新的FAPs时间过程。这种方法为研究人员提供了在新型动物模型中研究FAPs的能力。
Introduction
纤维脂肪生成祖细胞（FAPs）是骨骼肌中常驻的多能祖细胞群，在肌肉稳态，修复和再生中起关键作用，相反，还介导对肌肉损伤的病理反应。顾名思义，FAPs最初被确定为具有分化成成纤维细胞和脂肪细胞1的潜力的祖细胞群体，并且据称是慢性损伤和疾病中骨骼肌纤维脂肪浸润的关键介质。进一步的研究表明，FAPs还具有成骨和软骨形成2，3，4的能力。因此，它们在文献中被更广泛地标记为间充质或基质祖3，5，6，7，8。在急性骨骼肌损伤中，FAPs通过短暂增殖间接帮助再生肌生成，为激活的肌肉卫星细胞及其下游肌原祖（MPs）对应物1，9，10提供有利的环境。在成功再生的同时，FAPs经历凋亡，使其数量恢复到基线水平1，9，10，11。相反，在慢性肌肉损伤中，FAPs会覆盖促凋亡信号，这导致它们持续存在9，10，11和异常的肌肉修复。
评估FAPs介导肌肉反应的细胞和分子机制的体内研究利用了迄今为止的小鼠动物模型1，7，9，10，11，12，13，14。虽然基因工程小鼠是用于这些分析的强大工具，但动物的小尺寸限制了在长期局部损伤模型中研究的组织可用性，其中肌肉萎缩可能是深刻的，例如创伤性去神经支配。此外，肌肉力量和身体功能的测量需要需要终止小鼠的离体或原位测量，或需要手术和/或全身麻醉的体内方法，以评估肌肉收缩性能15，16，17，18，19，20.在大鼠中，除了对更复杂的运动行为（例如步态分析，例如坐骨神经痛功能指数，CatWalk分析）的分析之外，还存在经过良好验证和全球利用的肌肉功能分析，并在清醒和自发移动的动物中进行21，22，23，24.这还优化了动物实验中最低发病率的原则，以及使用的研究动物的数量。因此，大鼠为FAPs研究者提供了更大的损伤肌肉体积的灵活性，用于蛋白质和细胞分析，并且能够对警觉动物中的肌肉复合物静态和动态功能活动和行为进行连续评估。
FAPs主要分别使用流式细胞术和荧光激活细胞分选（FACS）从全肌肉样品中鉴定和分离。这些是基于激光的测定，能够根据特征特征（例如大小，粒度以及细胞表面或细胞内标记物的特定组合）鉴定多个特定细胞群25。这在骨骼肌等器官系统的研究中非常有利，因为稳态和再生是由大量细胞类型协调的复杂多因素过程。一项开创性的研究确定了FAPs以及MP，在小鼠骨骼肌中使用流式细胞术方法1。他们证明FAPs本质上是间充质的，因为它们缺乏来自内皮（CD31），造血（CD45）或肌源性（整合素-α7 [ITGA7]）起源的细胞特异性的表面抗原，但表达间充质干细胞标志物Sca-1（干细胞抗原1）1并在培养物中分化成纤维化和脂肪生成细胞。其他研究表明，基于替代干细胞标志物，血小板衍生的生长因子受体α（PDGFRα）2，7，8的表达，成功分离肌肉间充质祖细胞，进一步的分析显示这些可能与FAPs3相同。FAPs现在通常在流式细胞术中使用Sca-1或PDGFRα作为阳性选择标记物1，9，10，11，12，13，14，26，27，28，29，30，31.PDGFRα的使用对人体组织是首选的，然而，作为小鼠Sca-1的直接人类同系物尚未被鉴定32。此外，其他细胞表面蛋白已被报道为MP的标志物（例如，VCAM-1），为ITGA7提供了一种潜在的替代方案，作为FAPs分离期间肌源谱系细胞的指标33。
虽然流式细胞术/FACS是研究FAPs在骨骼肌1、9、10、11、13、29中的作用和致病潜力的强大方法，但它在技术上受到其所需试剂的特异性和优化的限制。由于流式细胞术鉴定和分离FAPs已经在小鼠动物模型1、9、10、11、29中得到开发和进行，这给希望在其他模式生物中研究FAPs的研究人员带来了挑战。许多因素 – 例如要处理的最佳组织尺寸，以及试剂和/或抗体特异性和可用性 – 因所用物种而异。
除了在新型动物模型中研究FAPs的技术障碍外，它们在很大程度上是在急性有毒环境中研究的 – 通常通过肌内化学注射或心脏毒素。对FAPs长期动力学的评估主要限于评估杜氏肌营养不良症，使用mdx小鼠模型9，10，11和组合肌肉损伤模型，如巨大的肩袖撕裂，其中同时在肩部肌肉组织上进行肌腱横断和去神经支配26，27，28.FAPs对慢性创伤性去神经支配的唯一侮辱的反应，在重工业，农业和出生创伤（臂丛神经损伤）34，35，36，37中常见的工作场所事故中发生，具有显着的发病率，尚未得到很好的表征，通常仅限于短期时间范围11，38。
我们描述了一种从大鼠的健康以及严重萎缩和纤维化骨骼肌中识别和分离FAPs和MP的方法。首先，使用组织消化和流式细胞术染色方案鉴定CD31-/CD45-/Sca-1+/VCAM-1-FAPs和CD31-/CD45-/Sca-1-/VCAM-1+MPs，并通过对FACS分离的细胞进行培养和免疫细胞化学染色，随后对我们的发现进行验证。使用这种方法，我们还在大鼠的长期孤立去神经支配损伤模型中报告了一种新的FAPs时程。
Protocol
执行该协议的调查人员必须获得当地动物伦理委员会/护理委员会的许可。所有动物工作均由圣迈克尔医院团结健康多伦多动物护理委员会（ACC #918）批准，并根据加拿大动物护理委员会（CCAC）制定的指导方针进行。流式细胞术方案的示意图如图 1所示。如果下游应用是FACS和随后的细胞培养，则应使用适当的无菌技术完成所有步骤。 1. 肌肉收割 ?…
Representative Results
使用包括 Sca-1 和 VCAM-1 在内的新型抗体组合，通过流式细胞术鉴定 FAP 和MP用于识别大鼠肌肉中FAPs的门控策略基于小鼠29的流式细胞术方案，其门控CD31（内皮）和CD45（造血）阳性细胞（称为谱系[Lin]），并检查来自谱系阴性（Lin-）群体的FAPs标志物Sca-1和MP标记ITGA7的荧光谱。在缺乏市售的荧光团偶联和流式细胞术验证的大鼠Sca…
Discussion
对于希望研究由于生物学或技术原因在小鼠中不可行的损伤模型的研究人员来说，针对大鼠肌肉的优化，经过验证的FAPs分离方案至关重要。例如，小鼠不是研究慢性局部或神经退行性损伤（如长期去神经支配）的最佳动物模型。从生物学上讲，小鼠的短寿命和快速衰老使得由于衰老混杂因素的去神经支配而难以准确描绘肌肉特征。从技术角度来看，由于严重萎缩导致的肌肉质量急剧减少不足以进…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢渥太华大学的流式细胞术核心设施和基南生物医学科学研究中心（KRC），多伦多圣迈克尔斯医院Unity Health在优化本手稿中介绍的流式细胞术/ FACS方案方面的专业知识和指导。这项工作由医学设计新想法2018年基金（MbDNI-2018-01）资助给JB。
Materials
| 5 mL Polypropylene Round-Bottom Tube | Falcon | 352063 | |
| 5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap | Falcon | 352235 | |
| 10 cm cell culture dishes | Sarstedt | 83.3902 | |
| 12-well cell culture plate | ThermoFisher | 130185 | |
| 12 mm glass coverslips, No.2 | VWR | 89015-724 | |
| 10 mL Syringe | Beckton Dickenson | 302995 | |
| 15 mL centrifuge tubes | FroggaBio | 91014 | |
| 20 gauge needle | Beckton Dickenson | 305176 | |
| 25mL Serological pipette | Sarstedt | 86.1685.001 | |
| 40µm cell strainer | Fisher Scientific | 22363547 | |
| 50mL centrifuge tubes | FroggaBio | TB50 | |
| AbC Total Antibody Compensation Beads | ThermoFisher | A10497 | |
| Ammonium Chloride, Reagent Grade | Bioshop | AMC303.500 | |
| APC Conjugation Kit, 50-100µg | Biotium | 92307 | |
| Aquatex Aqueous Mounting Medium | Merck | 108562 | |
| Biolaminin 411 LN | Biolamina | LN411 | |
| Bovine Serum Albumin (BSA) | Bioshop | ALB001 | |
| Calcium Chloride | Bioshop | CCL444.500 | |
| Collagenase Type II | Gibco | 17101015 | |
| CountBright Plus Absolute Counting Beads | ThermoFisher | C36995 | |
| Dexamethasone | Millipore Sigma | D4902 | |
| Dispase | Gibco | 17105041 | |
| Dulbecco’s Modified Eagle Medium (DMEM) (1X) | Gibco | 11995-065 | (+)4.5 g/L D-Glucose (+)L-Glutamine (+)110 mg/L Sodium Pyruvate |
| EDTA | FisherScientific | S311 | |
| FACSClean Solution | Beckton Dickenson | 340345 | |
| FACSDiva Software | Beckton Dickenson | — | |
| FACSRinse Solution | Beckton Dickenson | 340346 | |
| Fetal Bovine Serum | Sigma | F1051 | |
| Flow Cytometry Sheath Fluid | Beckton Dickenson | 342003 | |
| FlowJo Software | Beckton Dickenson | — | |
| Fluorescent Mounting Medium | Dako | S302380-2 | |
| Goat anti-mouse Alexa Fluor 555 secondary antibody | Invitrogen | A21424 | |
| Goat anti-rabbit Alexa Fluor 488 secondary antibody | Invitrogen | A11008 | |
| Goat anti-rabbit Alexa Fluor 555 secondary antibody | Invitrogen | A21429 | |
| Goat Serum | Gibco | 16210-064 | |
| Ham's F10 Media | ThermoFisher | 11550043 | (+) Phenol Red (+) L-Glutamine (-) HEPES |
| Hank’s Balanced Salt Solution (HBSS) (1X) | Multicell | 311-513-CL | |
| Heat Inactivated Horse Serum | Gibco | 26050-088 | |
| Hemocytometer | Reichert | N/A | |
| HEPES, minimum 99.5% titration | Sigma | H3375 | |
| Horse Serum | ThermoFisher | 16050130 | |
| Human Transforming Growth Factor β1 (hTGF-β1) | Cell Signaling | 8915LF | |
| Humulin R | Lilly | HI0210 | |
| IBMX | Millipore Sigma | I5879 | Also known as 3-Isobutyl-1-methylxanthine |
| Isopropanol | Sigma | I9516 | Also known as 2-propanol |
| Lewis Rat, Female | Charles River Kingston | 004 (Strain Code) | 200-250 grams used |
| LSRFortessa X-20 Benchtop Cytometer | Beckton Dickenson | — | |
| Microcentrifuge | Eppendorf | EP-5417R | |
| MoFlo XDP Cell Sorter | Beckman Coulter | — | |
| Mouse Anti-CD31::FITC Antibody | Abcam | ab33858 | Clone TLD-3A12 |
| Mouse Anti-CD45::FITC Antibody | Biolegend | 202205 | Clone OX-1 |
| Mouse Anti-CD106::PE Antibody | Biolegend | 200403 | Also known as VCAM-1 |
| Mouse Anti-MHC Antibody | Developmental Studies Hybridoma Bank (DSHB) | N/A | Also known as MF20 |
| Mouse Anti-Pax7 Antibody | Developmental Studies Hybridoma Bank (DSHB) | N/A | |
| Neutral Buffered Formalin, 10 % | Sigma | HT501128 | |
| Oil Red O | Millipore Sigma | O0625 | |
| PE-Cy7 Conjugation Kit | Abcam | ab102903 | |
| Penicillin-Streptomycin | Sigma | P4333 | |
| Phosphate Buffered Saline, pH 7.4 (1X) | Gibco | 10010-023 | (-)Calcium Chloride (-)Magnesium Chloride |
| Potassium Bicarbonate, Reagent Grade | Bioshop | PBC401.250 | |
| Rabbit Anti-Fibroblast Specific Protein 1 (FSP-1) Antibody | Invitrogen | MA5-32347 | FSP-1 also known as S100A4 |
| Rabbit Anti-Integrin-a7 Antibody | Abcam | ab203254 | |
| Rabbit Anti-Laminin Antibody | Sigma | L9393 | |
| Rabbit Anti-Perilipin-1 Antibody | Abcam | ab3526 | |
| Rabbit Anti-Sca-1 Antibody | Millipore Sigma | AB4336 | |
| Rabbit Recombinant Anti-Collagen Type I Antibody | Abcam | ab260043 | Also known as Col1a1 |
| Rabbit Recombinant Anti-PDGFR Alpha Antibody | Abcam | ab203491 | |
| Recombinant Human FGF-basic | Gibco | PHG0266 | |
| Sodium Azide | Sigma | S2002 | |
| Triton-X-100 | Fisher Scientific | BP151 | |
| Troglitazone | Millipore Sigma | T2573 | |
| Tween-20 | Bioshop | TWN510 |
Riferimenti
- Joe, A. W. B., et al. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nature Cell Biology. 12, 153-163 (2010).
- Wosczyna, M. N., Biswas, A. A., Cogswell, C. A., Goldhamer, D. J. Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification. Journal of Bone and Mineral Research. 27 (5), 1004-1017 (2012).
- Uezumi, A., Ikemoto-Uezumi, M., Tsuchida, K. Roles of nonmyogenic mesenchymal progenitors in pathogenesis and regeneration of skeletal muscle. Frontiers in Physiology. 5, 1-11 (2014).
- Biswas, A. A., Goldhamer, D. J. FACS fractionation and differentiation of skeletal-muscle resident multipotent Tie2+ progenitors. Methods in Molecular Biology. 1460, 255-267 (2016).
- Biferali, B., Proietti, D., Mozzetta, C., Madaro, L. Fibro-adipogenic progenitors cross-talk in skeletal muscle: The social network. Frontiers in Physiology. 10, 1-10 (2019).
- Wosczyna, M. N., Rando, T. A. A muscle stem cell support group: Coordinated cellular responses in muscle regeneration. Developmental Cell. 46 (2), 135-143 (2018).
- Uezumi, A., Fukada, S. I., Yamamoto, N., Takeda, S., Tsuchida, K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nature Cell Biology. 12 (2), 143-152 (2010).
- Uezumi, A., et al. Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle. Journal of Cell Science. 124 (21), 3654-3664 (2011).
- Lemos, D. R., et al. Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors. Nature Medicine. 21 (7), 786-794 (2015).
- Malecova, B., et al. Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy. Nature Communications. 9 (1), (2018).
- Madaro, L., et al. Denervation-activated STAT3-IL-6 signalling in fibro-adipogenic progenitors promotes myofibres atrophy and fibrosis. Nature Cell Biology. 20 (8), 917-927 (2018).
- Heredia, J. E., et al. Type 2 innate signals stimulate fibro/adipogenic progenitors to facilitate muscle regeneration. Cell. 153 (2), 376-388 (2013).
- Fiore, D., et al. Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration. Stem Cell Research. 17 (1), 161-169 (2016).
- Kang, X., et al. Interleukin-15 facilitates muscle regeneration through modulation of fibro/adipogenic progenitors. Cell Communication and Signaling. 16 (1), 1-11 (2018).
- Lovering, R. M., Roche, J. A., Goodall, M. H., Clark, B. B., Mcmillan, A. An in vivo rodent model of contraction-induced injury and non-invasive monitoring of recovery. Journal of Visualized Experiments. (51), e2782 (2011).
- Iyer, S. R., Valencia, A. P., Hernández-Ochoa, E. O., Lovering, R. M. In vivo assessment of muscle contractility in animal studies. Methods in Molecular Biology. 1460, 293-307 (2016).
- Mintz, E. L., Passipieri, J. A., Lovell, D. Y., Christ, G. J. Applications of in vivo functional testing of the rat tibialis anterior for evaluating tissue engineered skeletal muscle repair. Journal of Visualized Experiments. (116), e54487 (2016).
- Hakim, C. H., Wasala, N. B., Duan, D. Evaluation of muscle function of the extensor digitorum longus muscle ex vivo and tibialis anterior muscle in situ in mice. Journal of Visualized Experiments. (72), e50183 (2013).
- Moorwood, C., Liu, M., Tian, Z., Barton, E. R. Isometric and eccentric force generation assessment of skeletal muscles isolated from murine models of muscular dystrophies. Journal of Visualized Experiments. (71), e50036 (2013).
- Gerlinger-Romero, F., et al. Non-invasive assessment of dorsiflexor muscle function in mice. Journal of Visualized Experiments. (143), e58696 (2019).
- Iohom, G., et al. Long-term evaluation of motor function following intraneural injection of ropivacaine using walking track analysis in rats. British Journal of Anaesthesia. 94 (4), 524-529 (2005).
- Brown, C. J., et al. Self-evaluation of walking-track measurement using a sciatic function index. Microsurgery. 10 (3), 226-235 (1989).
- Bozkurt, A., et al. CatWalk gait analysis in assessment of functional recovery after sciatic nerve injury. Journal of Neuroscience Methods. 173 (1), 91-98 (2008).
- Deumens, R., Jaken, R. J. P., Marcus, M. A. E., Joosten, E. A. J. The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection. Journal of Neuroscience Methods. 164 (1), 120-130 (2007).
- McKinnon, K. M. Flow cytometry: An overview. Current Protocols in Immunology. 2018, 1-11 (2018).
- Jensen, A. R., et al. Neer Award 2018: Platelet-derived growth factor receptor α co-expression typifies a subset of platelet-derived growth factor receptor β-positive progenitor cells that contribute to fatty degeneration and fibrosis of the murine rotator cuff. Journal of Shoulder and Elbow Surgery. 27 (7), 1149-1161 (2018).
- Mosich, G. M., et al. Non-fibro-adipogenic pericytes from human embryonic stem cells attenuate degeneration of the chronically injured mouse muscle. JCI Insight. 4 (24), (2019).
- Lee, D., et al. HMGB2 is a novel adipogenic factor that regulates ectopic fat infiltration in skeletal muscles. Scientific Reports. 8 (1), 1-12 (2018).
- Low, M., Eisner, C., Rossi, F. Fibro/Adipogenic Progenitors (FAPs): Isolation by FACS and Culture. Muscle Stem Cells: Methods and Protocols. , 179-189 (2017).
- Giuliani, G., et al. SCA-1 micro-heterogeneity in the fate decision of dystrophic fibro/adipogenic progenitors. Cell Death and Disease. 12 (1), 1-24 (2021).
- Wosczyna, M. N., et al. Mesenchymal stromal cells are required for regeneration and homeostatic maintenance of skeletal muscle. Cell Reports. 27 (7), 2029-2035 (2019).
- Upadhyay, G. Emerging role of lymphocyte antigen-6 family of genes in cancer and immune cells. Frontiers in Immunology. 10, 819 (2019).
- Boscolo Sesillo, F., Wong, M., Cortez, A., Alperin, M. Isolation of muscle stem cells from rat skeletal muscles. Stem Cell Research. 43, 101684 (2020).
- Ciaramitaro, P., et al. Traumatic peripheral nerve injuries: Epidemiological findings, neuropathic pain and quality of life in 158 patients. Journal of the Peripheral Nervous System. 15 (2), 120-127 (2010).
- Noble, J., Munro, C. A., Prasad, V. S. S. V., Midha, R. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. Journal of Trauma and Acute Care Surgery. 45 (1), (1998).
- Malik, S. Traumatic peripheral neuropraxias in neonates: A case series. Journal of Clinical and Diagnostic Research. 8 (10), 10-12 (2014).
- Smith, B. W., Daunter, A. K., Yang, L. J. S., Wilson, T. J. An update on the management of neonatal brachial plexus palsy-replacing old paradigms a review. JAMA Pediatrics. 172 (6), 585-591 (2018).
- Rebolledo, D. L., et al. Denervation-induced skeletal muscle fibrosis is mediated by CTGF/CCN2 independently of TGF-β. Matrix Biology. 82, 20-37 (2019).
- Walls, P. L. L., McRae, O., Natarajan, V., Johnson, C., Antoniou, C., Bird, J. C. Quantifying the potential for bursting bubbles to damage suspended cells. Scientific Reports. 7 (1), 1-9 (2017).
- Yuen, D. A., et al. Culture-modified bone marrow cells attenuate cardiac and renal injury in a chronic kidney disease rat model via a novel antifibrotic mechanism. PLOS One. 5 (3), 9543 (2010).
- Fukada, S. I. The roles of muscle stem cells in muscle injury, atrophy and hypertrophy. Journal of Biochemistry. 163 (5), 353-358 (2018).
- Itabe, H., Yamaguchi, T., Nimura, S., Sasabe, N. Perilipins: A diversity of intracellular lipid droplet proteins. Lipids in Health and Disease. 16 (1), 1-11 (2017).
- Chapman, M. A., Mukund, K., Subramaniam, S., Brenner, D., Lieber, R. L. Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis. American Journal of Physiology – Cell Physiology. 312 (2), 131-143 (2016).
- Hillege, M., Galli Caro, R., Offringa, C., de Wit, G., Jaspers, R., Hoogaars, W. TGF-β regulates Collagen Type I expression in myoblasts and myotubes via transient Ctgf and Fgf-2 Expression. Cells. 9 (2), 375 (2020).
- Kafadar, K. A., Yi, L., Ahmad, Y., So, L., Rossi, F., Pavlath, G. K. Sca-1 expression is required for efficient remodeling of the extracellular matrix during skeletal muscle regeneration. Biologia dello sviluppo. 326 (1), 47-59 (2009).
- Batt, J. A. E., Bain, J. R. Tibial nerve transection – a standardized model for denervation-induced skeletal muscle atrophy in mice. Journal of Visualized Experiments. (81), e50657 (2013).
- Carlson, B. M. The biology of long-term denervated skeletal muscle. European Journal of Translational Myology. 24 (1), (2014).
- Kennedy, E., et al. Embryonic rat vascular smooth muscle cells revisited – A model for neonatal, neointimal SMC or differentiated vascular stem cells. Vascular Cell. 6 (1), 1-13 (2014).
- Pannérec, A., Formicola, L., Besson, V., Marazzi, G., Sassoon, D. A. Defining skeletal muscle resident progenitors and their cell fate potentials. Development (Cambridge). 140 (14), 2879-2891 (2013).
Citazione di questo articolo
Te, L. J. I., Doherty, C., Correa, J., Batt, J. Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat. J. Vis. Exp. (172), e61750, doi:10.3791/61750 (2021).