从未受干扰和受伤的小鼠骨骼肌中分离和培养纤维脂肪祖细胞和肌肉干细胞的FACS-分离和培养
1Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS),National Institutes of Health (NIH), 2Flow Cytometry Section, National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS),National Institutes of Health (NIH)
Summary
纤维脂肪祖细胞（FAPs）和肌肉干细胞（MuSCs）的精确鉴定对于研究它们在生理和病理条件下的生物学功能至关重要。该协议为从成年小鼠肌肉中分离，纯化和培养FAP和MuSC提供了指南。
Abstract
纤维脂肪祖细胞 （FAP） 是一组骨骼肌驻留间充质基质细胞 （MSC），能够沿纤维化、成脂、成骨或成软骨谱系分化。FAP与肌肉干细胞（MuSC）一起在肌肉稳态，修复和再生中起着关键作用，同时积极维持和重塑细胞外基质（ECM）。在病理条件下，例如慢性损伤和肌肉萎缩症，FAPs经历异常激活并分化为产生胶原蛋白的成纤维细胞和脂肪细胞，导致纤维化和肌内脂肪浸润。因此，FAP在肌肉再生中起着双重作用，通过维持MuSC更新和促进组织修复或促进纤维化瘢痕形成和异位脂肪浸润，从而损害骨骼肌组织的完整性和功能。FAP和MuSCs的适当纯化是了解这些细胞在生理和病理条件下的生物学作用的先决条件。在这里，我们描述了一种使用荧光激活细胞分选（FACS）从成年小鼠的肢体肌肉中同时分离FAP和MuSC的标准化方法。该协议详细描述了来自整个肢体肌肉和受伤的胫骨前（TA）肌肉的单核细胞的机械和酶解离。随后使用半自动细胞分选仪分离FAP和MuSC，以获得纯细胞群。我们还描述了一种单独或在共培养条件下培养静态和活化FAP和MuSC的优化方法。
Introduction
骨骼肌是人体最大的组织，占成人体重的~40%，负责维持姿势、产生运动、调节基础能量代谢和体温1。骨骼肌是一种高度动态的组织，具有适应各种刺激的非凡能力，例如机械应力、代谢变化和日常环境因素。此外，骨骼肌响应急性损伤而再生，导致其形态和功能完全恢复2。骨骼肌可塑性主要依赖于一组常驻肌肉干细胞（MuSC），也称为卫星细胞，它们位于肌纤维质膜和基底层之间2，3。在正常情况下，MuSCs以静止状态存在于肌肉壁龛中，只有少数分裂来补偿细胞更新并补充干细胞库4。作为对损伤的反应，MuSCs进入细胞周期，增殖，并有助于新肌肉纤维的形成或在自我更新过程中返回生态位2，3。除MuSCs外，骨骼肌的稳态维持和再生依赖于称为纤维脂肪祖细胞（FAPs）的肌肉驻留细胞群的支持5，6，7。FAP是嵌入肌肉结缔组织中的间充质基质细胞，能够沿着纤维化，脂肪源性，成骨性或软骨细胞系分化5，8，9，10。FAP为MuSCs提供结构支持，因为它们是肌肉干细胞生态位中细胞外基质蛋白的来源。FAP还通过分泌细胞因子和生长因子来促进骨骼肌的长期维持，这些细胞因子和生长因子为肌生成和肌肉生长提供营养支持6，11。急性肌肉损伤后，FAPs迅速增殖以产生短暂的生态位，支持再生肌肉的结构完整性，并为维持MuSCs以旁分泌方式增殖和分化提供有利的环境5。随着再生的进行，FAP通过细胞凋亡从再生肌中清除，其数量逐渐恢复到基础水平12。然而，在有利于慢性肌肉损伤的情况下，FAPs会覆盖促凋亡信号传导并积聚在肌肉壁龛中，在那里它们分化成产生胶原蛋白的成纤维细胞和脂肪细胞，导致异位脂肪浸润和纤维化瘢痕形成12，13。
由于其多能性和再生能力，FAP和MuSCs已被确定为再生医学治疗骨骼肌疾病的前瞻性靶标。因此，为了研究它们的功能和治疗潜力，建立有效且可重复的FAP和MuSC分离和培养方案非常重要。
荧光激活细胞分选（FACS）可以根据形态特征（如大小和粒度）识别不同的细胞群，并允许基于使用针对细胞表面标志物的抗体进行细胞特异性分离。在成年小鼠中，MuSCs表达血管细胞粘附分子1（VCAM-1，也称为CD106）14，15和α7-整合素15，而FAP表达血小板衍生的生长因子受体α（PDGFRα）和干细胞抗原1（Sca1或Ly6A / E）5，6，9，12，16，17.在此描述的方案中，MuSCs被鉴定为CD31-/ CD45-/Sca1-/VCAM-1 + / α7-Integrin+，而FAP被鉴定为CD31-/CD45-/Sca1+/VCAM-1-/α7-Integrin-。或者，使用PDGFRαEGFP小鼠分离FAP作为CD31-/ CD45-/PDGFRα+/VCAM-1-/α7-整合素-事件18，19。此外，我们比较了PDGFRα-GFP+细胞的荧光信号与表面标记物Sca1鉴定的细胞之间的重叠。我们的分析表明，所有表达GFP的细胞对Sca1也呈阳性，这表明任何一种方法都可以用于FAP的鉴定和分离。最后，用特异性标记抗体染色证实了每个细胞群的纯度。
Protocol
进行的所有动物实验均按照国家关节炎，肌肉骨骼和皮肤病研究所（NIAMS）动物护理和使用委员会（ACUC）批准的机构指南进行。执行此协议的调查人员必须遵守当地的动物伦理准则。 注意：该协议详细描述了如何从成年雄性和雌性小鼠（3-6个月）的后肢和受伤的胫骨前（TA）肌肉中分离FAP和MuSC，并提供共培养FAP和MuSC的指南。实验程序的概述如图 1所示?…
Representative Results
该协议允许从野生型成年小鼠（3-6个月）的未受伤后肢中分离约100万个FAP和多达350，000个MuSC，对应于FAP的产量为8%，MuSC的产量为3%的总事件。当从受伤后 7 天受损的 TA 中分选细胞时，将两到三块 TA 肌肉汇集以获得多达 300，000 个 FAP 和 120，000 个 MuSC，分别对应于 11% 和 4% 的产量。FAP和MuSC的分选后纯度值通常高于95%。 用于分离FAP和MuSC的门控策略如图 2所…
Discussion
建立高效且可重复的方案来鉴定和分离纯成体干细胞群是了解其功能的第一步，也是最关键的一步。分离的FAP和MuSC可用于在移植实验中进行多组学分析，作为肌肉疾病的潜在治疗方法，或者可以进行基因改造，用于干细胞治疗中的疾病建模。
此处描述的方案为从成年小鼠后肢肌肉获得的FAP和MuSC的鉴定，分离和培养提供了标准化指南。使用基于FACS的技术分离FAP和MuSC的纯群体…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们要感谢Tom Cheung（香港科技大学）关于MuSC隔离的建议。这项工作由NIAMS-IRP通过NIH拨款AR041126和AR041164资助。
Materials
| 5 mL Polypropylene Round-Bottom Tube | Falcon | 352063 | |
| 5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap | Falcon | 352235 | |
| 20 G BD Needle 1 in. single use, sterile | BD Biosciences | 305175 | |
| anti-Alpha 7 Integrin PE (clone:R2F2) (RatIgG2b) | The University of British Columbia | 53-0010-01 | |
| APC anti-mouse CD31 Antibody | BioLegend | 102510 | |
| APC anti-mouse CD45 Antibody | BioLegend | 103112 | |
| BD FACSMelody Cell Sorter | BD Biosciences | ||
| BD Luer-Lok tip control syringe, 10-mL | BD Biosciences | 309604 | |
| Biotin anti-mouse CD106 Antibody | BioLegend | 105703 | |
| C57BL/6J mouse (Female and Male) | The Jackson Laboratory | 000664 | |
| B6.129S4-Pdgfratm11(EGFP)Sor/J mouse | The Jackson Laboratory | 007669 | |
| Corning BioCoat Collagen I 6-well Clear Flat Bottom TC-treated Multiwell Plate | Corning | 356400 | |
| Corning BioCoat Collagen I 12-well Clear Flat Bottom TC-treated Multiwell Plate | Corning | 356500 | |
| Corning BioCoat Collagen I 24-well Clear Flat Bottom TC-treated Multiwell Plate | Corning | 356408 | |
| DAPI Solution (1 mg/mL) | ThermoFisher Scientific | 62248 | |
| Disposable Aspirating Pipets, Polystyrene, Sterile | VWR | 414004-265 | |
| Donkey anti-Goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | ThermoFisher Scientific | A-11055 | |
| Falcon 40 µm Cell Strainer, Blue, Sterile | Corning | 352340 | |
| Falcon 60 mm TC-treated Cell Culture Dish, Sterile | Corning | 353002 | |
| Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 15-mL | VWR | 352196 | |
| Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 50-mL | Corning | 352070 | |
| Falcon Round-Bottom Tubes, Polypropylene, Corning | VWR | 60819-728 | |
| Falcon Round-Bottom Tubes, Polystyrene, with 35um Cell Strainer Cap Corning | VWR | 21008-948 | |
| Fibroblast Growth Factor, Basic, Human, Recombinant (rhFGF, Basic) | Promega | G5071 | |
| FlowJo 10.8.1 | |||
| Gibco Collagenase, Type II, powder | ThermoFisher Scientific | 17101015 | |
| Gibco Dispase, powder | ThermoFisher Scientific | 17105041 | |
| Gibco DMEM, high glucose, HEPES | ThermoFisher Scientific | 12430054 | |
| Gibco Fetal Bovine Serum, certified, United States | ThermoFisher Scientific | 16000044 | |
| Gibco Ham's F-10 Nutrient Mix | ThermoFisher Scientific | 11550043 | |
| Gibco Horse Serum, New Zealand origin | ThermoFisher Scientific | 16050122 | |
| Gibco PBS, pH 7.4 | ThermoFisher Scientific | 10010023 | |
| Gibco PBS (10x), pH 7.4 | ThermoFisher Scientific | 70011044 | |
| Gibco Penicillin-Streptomycin-Glutamine (100x) | ThermoFisher Scientific | 10378016 | |
| Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 555 | ThermoFisher Scientific | A-21127 | |
| Hardened Fine Scissors | Fine Science Tools Inc | 14090-09 | |
| Invitrogen 7-AAD (7-Aminoactinomycin D) | ThermoFisher Scientific | A1310 | |
| Mouse PDGF R alpha Antibody | R&D Systems | AF1062 | |
| Normal Donkey Serum | Fisher Scientific | NC9624464 | |
| Normal Goat Serum | ThermoFisher Scientific | 31872 | |
| Pacific Blue anti-mouse Ly-6A/E (Sca 1) Antibody | BioLegend | 108120 | |
| Paraformaldehyde, 16% | Fisher Scientific | NCC0528893 | |
| Pax7 mono-clonal mouse antibody (IgG1) (supernatant) | Developmental Study Hybridoma Bank | N/A | |
| PE/Cyanine7 Streptavidin | BioLegend | 405206 | |
| Student Vannas Spring Scissors | Fine Science Tools Inc | 91500-09 | |
| Student Dumont #5 Forceps | Fine Science Tools Inc | 91150-20 | |
| Triton X-100 | Sigma-Aldrich | T8787 |
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Citar este artículo
Riparini, G., Simone, J. M., Sartorelli, V. FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle. J. Vis. Exp. (184), e63983, doi:10.3791/63983 (2022).