分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞
Summary
脂肪组织来源的间充质干细胞（Ad-MSCs）可能是分化为产生胰岛素的细胞（IPC）的MSCs的潜在来源。在该协议中，我们提供了用于分离和表征大鼠附睾Ad-MSCs的详细步骤，然后是用于从同一只大鼠Ad-MSC生成IPC的简单，简短的协议。
Abstract
间充质干细胞（MSCs） – 特别是那些从脂肪组织（Ad-MSCs）中分离出来的干细胞 – 作为一种可再生的，丰富的干细胞来源而受到特别关注，不会引起任何伦理问题。然而，目前隔离Ad-MSC的方法并不标准化，并且采用需要特殊设备的复杂协议。我们使用一种简单，可重复的方法从Sprague-Dawley大鼠的附睾脂肪中分离出Ad-MSCs。分离的Ad-MSCs通常在分离后3天内出现，因为贴壁细胞显示成纤维细胞形态。这些细胞在分离后1周内达到80%的汇合度。之后，在第3-5代（P3-5），通过对特征性MSC分化簇（CD）表面标志物（如CD90，CD73和CD105）进行免疫表型分析，以及诱导这些细胞在成骨，脂肪和软骨成因谱系中的分化，对分离的Ad-MSCs进行全面表征。反过来，这意味着分离细胞的多能性。此外，我们通过结合高葡萄糖Dulbecco的改良鹰培养基（HG-DMEM），β-巯基乙醇，烟酰胺和exendin-4，通过简单，相对较短的方案诱导分离的Ad-MSCs向胰岛素产生细胞（IPC）谱系的分化。首先，通过测量特异性β细胞标志物（如MafA，NKX6.1，Pdx-1和Ins1）的表达水平以及生成的IPC的二噻酮染色，对IPCs进行遗传评估。其次，评估也通过葡萄糖刺激胰岛素分泌（GSIS）测定在功能上进行。总之，Ad-MSCs可以很容易地分离出来，表现出所有MSC表征标准，并且它们确实可以在实验室中为糖尿病研究提供丰富的可再生IPC来源。
Introduction
间充质干细胞（MSCs），也称为间充质基质细胞，是再生医学中使用最广泛的细胞类型之一1，2。它们被归类为成体干细胞，其特征在于多系分化潜力和自我更新能力3。间充质干细胞可以从各种来源分离和获得，包括脂肪组织，骨髓，外周血，脐带组织和血液，毛囊和牙齿4，5。
从脂肪组织中分离干细胞被认为既有吸引力又有希望，因为它们易于获取， 体外快速扩增和高产量6。脂肪组织来源的间充质干细胞（Ad-MSCs）可以从不同的物种中分离出来，例如人类，牛，小鼠，大鼠以及最近的山羊7。已经证明，Ad-MSCs现在是组织工程和基因/细胞治疗的潜在候选者，甚至可用于开发用于长期修复软组织损伤或缺陷的自体替代品7，8。
国际细胞和基因治疗学会（ISCT）定义了MSC必须展示的三个最低标准，以进行完整的表征9。首先，它们必须是塑料粘附的。其次，间充质干细胞表面标志物（如 CD73、CD90 和 CD105）应表达，且缺乏造血标志物 CD45、CD34、CD14 或 CD11b、CD79α 或 CD19 以及 HLA-DR 的表达。最后，间充质细胞应表现出分化成三种间充质谱系的能力：脂肪细胞、骨细胞和软骨细胞。有趣的是，MSCs也可以分化成其他谱系，如神经元细胞，心肌细胞，肝细胞和上皮细胞10，11。
事实上，间充质干细胞具有独特的性质，使它们能够作为潜在治疗剂应用于不同疾病的再生疗法中。间充质干细胞可以分泌可溶性因子以诱导免疫调节环境，从而提供治疗益处12.此外，间充质干细胞可以向损伤部位和肿瘤微环境迁移以提供靶向治疗;然而，其机制尚未完全阐明13。此外，MSCs具有分泌外泌体的能力，外泌体是纳米级的细胞外囊泡，携带着大量非编码RNA，蛋白质和可溶性因子，最近成为MSCs在各种疾病中治疗潜力的新机制14。
更重要的是，间充质干细胞通过基因修饰15，16或通过在体外培养基中利用各种外在诱导因子来分化成产生胰岛素的细胞（IPC）的潜力引起了显着的关注17。IPC诱导期差异很大，因为它取决于所使用的诱导方案和利用的外在因素。分化过程可持续数天至数月，需要外源性诱导因子的组合，这些因子必须在不同阶段添加和/或提取。许多用于内分泌胰腺分化的因子是生物活性化合物，已被证明可以促进胰岛素分泌β细胞的增殖或分化/新生和/或增加IPCs的胰岛素含量18，19，20，21。值得注意的是，据报道，间充质干细胞还通过几种机制（包括其分泌组）以及广泛的免疫调节作用对糖尿病及其并发症具有治疗作用22，23，24。
在该协议中，我们提出了一个详细的逐步协议，用于从大鼠附睾脂肪中分离和表征Ad-MSCs，然后是一个简单的，相对较短的协议，用于从Ad-MSC生成IPC。
Protocol
所有实验均根据批准的指南进行，所有程序均由埃及开罗英国大学药学院伦理委员会批准。Lopez和Spencer采用了Ad-MSC隔离协议，修改了15。 1. 从大鼠附睾脂肪垫中分离Ad-MSCs 使用体重不超过1个月（每次隔离两次）的体重为250-300g的雄性Sprague-Dawley大鼠。麻醉动物，然后通过宫颈脱位对它们实施安乐死。用70%乙醇喷洒安乐死动物。切除腹部下?…
Representative Results
Ad-MSC 的隔离和表征如图 2所示，从脂肪组织中分离的细胞从分离的第二天开始显示出圆形和成纤维细胞样细胞的异质群体（图2A）。分离后4天，成纤维细胞的数量和大小开始增加，并通过第1代作为同质群体生长（图2B，C）。这些细胞继续作为塑性贴壁的成纤维细胞生长，如图3所示，满足MSC特征的第一…
Discussion
在该协议中，我们设法提出了从大鼠附睾脂肪中分离Ad-MSCs的详细方案，并将这些Ad-MSC分化为IPC。事实上，大鼠附睾脂肪是用于获得Ad-MSCs的易于获得的脂肪组织来源，并且不需要任何特殊设备，无论是用于收集还是用于处理15，26，27。分离的Ad-MSCs表现出优异的培养扩增，并表现出所有被定义为MSC的标准。所使用的协议先前进行…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢Rawda Samir Mohamed博士，硕士，兽医专家，埃及英国大学（BUE）药学院兽医专家，帮助解剖大鼠。
我们还要感谢埃及英国大学（BUE）大众传播学院为制作和编辑本手稿的视频所做的努力。
我们要感谢埃及英国大学（BUE）英语助理讲师Fatma Masoud小姐，MSc对手稿的修订和英语校对。
这项工作由埃及开罗英国大学药学院药物研究与发展中心（CDRD）部分资助。
Materials
| Albumin, bovine serum Fraction V | MP Biomedicals | ||
| Alcian Blue 8GX | Sigma-Aldrich, USA | A3157 | |
| Alizarin Red S | Sigma-Aldrich, USA | A5533 | |
| Ammonium hydroxide | Fisher Scientific, Germany | ||
| Antibody for Rat CD90, FITC | Stem Cell Technologies | 60024FI | |
| Bovine serum albumin | Sigma Aldrich | A3912 | |
| Calcium Chloride | Fisher Scientific, Germany | ||
| CD105 Monoclonal Antibody, FITC | Thermo Fisher Scientific, Invitrogen, USA | MA1-19594 | |
| CD34 Polyclonal Antibody | Thermo Fisher Scientific, Invitrogen, USA | PA5-85917 | |
| Chloroform | Fisher Scientific, USA | ||
| Collagenase type I, powder | Gibco, Thermo Fisher, USA | 17018029 | |
| D-Glucose anhydrous, extra pure | Fisher Scientific, Germany | G/0450/53 | |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific, Germany | BP231-100 | |
| Dithizone staining | Sigma-Aldrich, USA | D5130 | |
| DMEM – High Glucose 4.5 g/L | Lonza, Switzerland | 12-604F | |
| DMEM – Low Glucose 1 g/L | Lonza, Switzerland | 12-707F | |
| DMEM/F12 medium | Lonza, Switzerland | BE12-719F | |
| DNAse/RNAse free water | Gibco Thermo Fisher, USA | 10977-035 | |
| Ethanol absolute, Molecular biology grade | Sigma-Aldrich, Germany | 24103 | |
| Exendin-4 | Sigma-Aldrich, Germany | E7144 | |
| Fetal Bovine Serum (FBS) | Gibco Thermo Fisher, Brazil | 10270-106 | |
| Formaldehyde 37% | Fisher Scientific | ||
| Hydrochloric acid (HCl) | Fisher Scientific, Germany | ||
| Isopropanol, Molecular biology grade | Fisher Scientific, USA | BP2618500 | |
| L-Glutamine | Gibco Thermo Fisher, USA | 25030-024 | |
| Magnesium Chloride (Anhydrous) | Fisher Scientific, Germany | ||
| Mesenchymal Stem Cell Functional identification kit | R&D systems Inc., MN, USA | SC006 | |
| Nicotinamide | Sigma-Aldrich, Germany | N0636 | |
| Oil Red Stain | Sigma-Aldrich, USA | O0625 | |
| Penicillin-Streptomycin-Amphotericin | Gibco Thermo Fisher, USA | 15240062 | |
| Phosphate buffered saline, 1X, without Ca/Mg | Lonza, Switzerland | BE17-516F | |
| Potassium Chloride | Fisher Scientific, Germany | ||
| Rat Insulin ELISA Kit | Cloud-Clone Corp., USA | CEA682Ra | |
| Sodium Bicarbonate | Fisher Scientific, Germany | ||
| Sodium Chloride | Fisher Scientific, Germany | ||
| Sodium Phosphate Dibasic (Anhydrous) | Fisher Scientific, Germany | ||
| Sodium Phosphate Monobasic (Anhydrous) | Fisher Scientific, Germany | ||
| SYBR Green Maxima | Thermo Scientific, USA | K0221 | |
| Syringe filter, 0.2 micron | Corning, USA | 431224 | |
| TRIzol | Thermo Scientific, USA | 15596026 | |
| Trypan blue | Gibco Thermo Fisher, USA | 15250061 | |
| Trypsin-Versene-EDTA, 1X | Lonza, Switzerland | CC-5012 | |
| Verso cDNA synthesis kit | Thermo Scientific, USA | AB-1453/A | |
| β-mercaptoethanol | Sigma-Aldrich, Germany | M3148 |
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Citar este artigo
Kassem, D. H., Habib, S. A., Badr, O. I., Kamal, M. M. Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells. J. Vis. Exp. (186), e63348, doi:10.3791/63348 (2022).