Name: isolation of rat adipose tissue mesenchymal stem cells for differentiation into insulin-producing cells
Uploaded: 2022-08-29T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells at JoVE.com

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。

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
<ol><li>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?...

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+stem+cell+mobilization-associated+molecules+on+multi-lineage+cells+from+adipose+tissue+and+bone+marrow.">Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.</a> Immunology Letters. 89 (2-3), 267-270 (2003).</li><li>Mosna, F., Sensebe, L., Krampera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow+and+adipose+tissue+mesenchymal+stem+cells:+A+user's+guide.">Human bone marrow and adipose tissue mesenchymal stem cells: A user's guide.</a> Stem Cells and Development. 19 (10), 1449-1470 (2010).</li><li>Camara, B. O. 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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Albumin, bovine serum Fraction V</td>
			<td>MP Biomedicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A3157</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody for Rat CD90, FITC</td>
			<td>Stem Cell Technologies</td>
			<td>60024FI</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A3912</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD105 Monoclonal Antibody, FITC</td>
			<td>Thermo Fisher Scientific, Invitrogen, USA</td>
			<td>MA1-19594</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 Polyclonal Antibody</td>
			<td>Thermo Fisher Scientific, Invitrogen, USA</td>
			<td>PA5-85917</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I, powder</td>
			<td>Gibco, Thermo Fisher, USA</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose anhydrous, extra pure</td>
			<td>Fisher Scientific, Germany</td>
			<td>G/0450/53</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific, Germany</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithizone staining</td>
			<td>Sigma-Aldrich, USA</td>
			<td>D5130</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - High Glucose 4.5 g/L</td>
			<td>Lonza, Switzerland</td>
			<td>12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Low Glucose 1 g/L</td>
			<td>Lonza, Switzerland</td>
			<td>12-707F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>Lonza, Switzerland</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse/RNAse free water</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute, Molecular biology grade</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>24103</td>
			<td></td>
		</tr>
		<tr>
			<td>Exendin-4</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>E7144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Thermo Fisher, Brazil</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, Molecular biology grade</td>
			<td>Fisher Scientific, USA</td>
			<td>BP2618500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (Anhydrous)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell Functional identification kit</td>
			<td>R&#38;D systems Inc., MN, USA</td>
			<td>SC006</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red Stain</td>
			<td>Sigma-Aldrich, USA</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Amphotericin</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 1X, without Ca/Mg</td>
			<td>Lonza, Switzerland</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Insulin ELISA Kit</td>
			<td>Cloud-Clone Corp., USA</td>
			<td>CEA682Ra</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic (Anhydrous)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic (Anhydrous)</td>
			<td>Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green Maxima</td>
			<td>Thermo Scientific, USA</td>
			<td>K0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, 0.2 micron</td>
			<td>Corning, USA</td>
			<td>431224</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Thermo Scientific, USA</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco Thermo Fisher, USA</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene-EDTA, 1X</td>
			<td>Lonza, Switzerland</td>
			<td>CC-5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Verso cDNA synthesis kit</td>
			<td>Thermo Scientific, USA</td>
			<td>AB-1453/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>M3148</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of rat adipose tissue mesenchymal stem cells for differentiation into insulin-producing cells

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。

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...

Watch this Scientific Journal Video about Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells at JoVE.com

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

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。

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

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

该方案的总体目标是分离和表征红色脂肪组织，干燥的间充质干细胞，并使用简单的方法将它们分化为产生胰岛素的细胞。间充质干细胞已经在遗传学领域找到了方法。它们可以从各种来源分离，例如骨髓，脂肪组织和围产期组织。他们接受优秀的文化特征。它们是多效的，可以潜在地治疗各种疾病。脂肪组织提供了间充质干细胞的丰富来源。这些脂肪MSCs可以很容易地从脂肪抽吸物中分离出来，与骨髓等其他来源相比，产量高。它们可以为自体和同种异体移植提供良好的来源。在本视频中，介绍了一种从大鼠附睾脂肪中分离和表征脂肪间充质干细胞的简单方案。此过程是一种简单高效的隔离方法。分离后，我们将使用简单，相对较短的方案忍受这些脂肪间充质干细胞分化为产生胰岛素的细胞。我们将从通过附睾脂肪的胶原消化来分离脂肪间充质干细胞开始，以获得更强的血管分数。然后间充质干细胞将以其可塑性粘附，以及CD标记物的表达和分化成多个中胚层谱系来表征。麻醉动物，然后使用宫颈脱位进行安乐死。用70%乙醇喷洒全身，首先切除腹部下部的皮肤和肌肉。然后用附睾脂肪暴露睾丸。轻轻切除附睾脂肪。注意不要用脂肪切开血管。在生物安全柜中，使用镊子和手术刀将脂肪组织切成小块。将脂肪组织转移到含有10毫升无菌PBS的离心管中。通过倾斜猎鹰管45秒开始清洗。然后将管子静置五分钟，分成两层。使用10毫升注射器取出含酶的PBS。重复此洗涤步骤四到五次，直到PBS变清。洗涤后，将脂肪组织重新悬浮在胶原蛋白溶液中，然后在振荡水浴中孵育，直到组织几乎均匀。之后，将组织胶原混合物涡旋15秒，然后离心5分钟。离心后，您将有三层。小心地丢弃基质血管部分的上清液。首先小心地丢弃顶部的油层，然后是回声层，而不干扰基质血管级分丸。在无菌BSA中重新悬浮基质血管部分并重新离心五分钟。整个离心，将完整的DMEM F-12培养基放入25厘米见方的烧瓶中。离心后，弃去上清液并将基质血管沉淀重新悬浮在完整的DMEM F-12培养基中。并转移到培养瓶中。放入37度5%二氧化碳培养箱中。第二天，取出未附着的细胞，并用新鲜培养基替换培养基。从隔离的第二天开始，成纤维细胞样细胞将开始出现。然后随着时间的流逝，它们的数量和大小会逐渐增加。通过传代，细胞将变得更加均匀。这些细胞表现出所有间充质干细胞表征标准。这些细胞使用含有烟酰胺β巯基乙醇和Exendin-4的简单方案分化为产生胰岛素的细胞。在整个分化步骤中，细胞开始失去它们的成纤维细胞形状，我认为簇状形态。当使用产生胰岛素的细胞显示阳性的深红色二硅醌染色时，再使用胰岛素产生细胞表达各种β细胞标志物，如Pdx-1，mafA和胰岛素。此外，当受到较高葡萄糖浓度的挑战时，诱导的胰岛素产生细胞在上清液中分泌更高水平的胰岛素。我们提供了一个详细的，逐步的方案，用于脂肪间充质干细胞的分离和表征。我们还在这里提供了一种相对简短，简单和有效的方案，用于将脂肪间充质干细胞分化为产生胰岛素的细胞。这为研究人员进入间充质干细胞领域并研究它们分化为产生胰岛素的细胞提供了门户。

Research

JoVE Journal

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

犬卵巢间充质干细胞的提取与分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...