Name: isolation of adult human dermal fibroblasts from abdominal skin and generation of induced pluripotent stem cells using a non-integrating method
Uploaded: 2020-01-19T16:00:00
Description: Watch this Scientific Journal Video about Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method at JoVE.com

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人体组织标本是根据《赫尔辛基宣言》采集的，同时遵守费德里科二世大学医院的指导方针。参与这项研究的所有患者均提供书面同意。
1. 用品和文化媒体的准备
<ol><li>清洁和高压灭菌一把大手术剪刀，两套细钳子，两对微解剖剪刀，1 L无菌瓶，500 mL无菌瓶和250 mL无菌瓶。</li>
	<li>准备 100 mm 板、60 mm 板、35 mm 板、一次性手术刀、50 mL 无菌管、15 mL 无菌管和 100 mm 玻璃板。在...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choices+for+Induction+of+Pluripotency:+Recent+Developments+in+Human+Induced+Pluripotent+Stem+Cell+Reprogramming+Strategies.">Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent Stem Cell Reprogramming Strategies.</a> Stem Cell Reviews and Reports. 12, 54-72 (2016).</li><li>Revilla, 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=Current+advances+in+the+generation+of+human+iPS+cells:+implications+in+cell-based+regenerative+medicine.">Current advances in the generation of human iPS cells: implications in cell-based regenerative medicine.</a> Journal of Tissue Engineering and Regenerative Medicine. 10, 893-907 (2016).</li><li>Lee, Y. 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M., 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=Diversity+of+dermal+fibroblasts+as+major+determinant+of+variability+in+cell+reprogramming.">Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.</a> Journal of Cellular and Molecular Medicine. 23 (6), 4256-4268 (2019).</li><li>Shan, Z. Y., 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=Continuous+passages+accelerate+the+reprogramming+of+mouse+induced+pluripotent+stem+cells.">Continuous passages accelerate the reprogramming of mouse induced pluripotent stem cells.</a> Cellular Reprogramming. 16 (1), 77-83 (2014).</li><li>Schlaeger, T. M., 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=A+comparison+of+non-integrating+reprogramming+methods.">A comparison of non-integrating reprogramming methods.</a> Nature Biotechnology. 33 (1), 58-63 (2015).</li><li>Drews, K., Tavernier, G., Demeester, J., 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=The+cytotoxic+and+immunogenic+hurdles+associated+with+non-viral+mRNA-mediated+reprogramming+of+human+fibroblasts.">The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts.</a> Biomaterials. 33, 4059-4068 (2012).</li><li>Beissert, T., 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=Improvement+of+In+Vivo+Expression+of+Genes+Delivered+by+Self-Amplifying+RNA+Using+Vaccinia+Virus+Immune+Evasion+Proteins.">Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.</a> Human Gene Therapy. 28 (12), 1138-1146 (2017).</li><li>Mathieu, J., 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=Hypoxia+induces+re-entry+of+committed+cells+into+pluripotency.">Hypoxia induces re-entry of committed cells into pluripotency.</a> Stem Cells. 31 (9), 1737-1748 (2013).</li><li>Wang, Y., 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=Hypoxia+Enhances+Direct+Reprogramming+of+Mouse+Fibroblasts+to+Cardiomyocyte-Like+Cells.">Hypoxia Enhances Direct Reprogramming of Mouse Fibroblasts to Cardiomyocyte-Like Cells.</a> Cellular Reprogramming. 18 (1), 1-7 (2016).</li><li>Deng, Y., 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=Hypoxia+enhances+buffalo+adipose-derived+mesenchymal+stem+cells+proliferation,+stemness,+and+reprogramming+into+induced+pluripotent+stem+cells.">Hypoxia enhances buffalo adipose-derived mesenchymal stem cells proliferation, stemness, and reprogramming into induced pluripotent stem cells.</a> Journal of Cellular Physiology. 234 (10), 17254-17268 (2019).</li><li>Karagiannis, P., 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=Induced+Pluripotent+Stem+Cells+and+Their+Use+in+Human+Models+of+Disease+and+Development.">Induced Pluripotent Stem Cells and Their Use in Human Models of Disease and Development.</a> Physiological Reviews. 99 (1), 79-114 (2019).</li><li>Maherali, N., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3 (6), 595-605 (2008).</li><li>Asprer, J. 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iPSC正在迅速成为再生医学应用最有前途的细胞候选者，并作为疾病建模和药物测试的非常有用的工具3，8。此处介绍的协议描述了从具有皮肤冲孔活检大小的样本中生成人类 iPSC，其程序简单而高效，不需要任何特定设备或以前重新编程技术的经验。
优化成纤维细胞隔离和培养以提高成功机会至关重要，因为电镀密度和增殖率会影?...

细胞重新编程是一种新技术，将身体的每一个体细胞转化为多能干细胞，称为iPSC1。将成人体细胞重新编程回到多能和未分化状态的可能性已经克服了与使用多能细胞（以前只能来自人类胚胎（胚胎干细胞或ESC）2、3、4）的多能细胞和伦理问题有关的不良可用性和伦理问题所施加的限制。2006年，高桥和山中信亚进行了一项开创性的研究，通过人工添加四个特定基因（Oct4、Sox2、Klf4、c-Myc）5，实现了成人体细胞从皮肤转化为多能细胞的首次转化。一年后，在汤姆森实验室进行的工作通过转导四个基因的不同组合（Oct4、Sox2、Nanog、Lin28）6，成功地将体细胞重新编程为iPSC。
iPSCs为不同领域的科学家和研究人员提供了许多机会，如再生医学和药理学，是研究和治疗不同疾病以及基因性反映患者特征的绝佳平台。iPSC的使用提供了几个优点，包括：由于细胞完全自体来源，免疫反应风险降低;创建细胞库的可能性，这是预测对新药及其副作用反应的重要工具，因为它们能够持续自我更新并产生不同的细胞类型;并有机会为药物管理7、8、9开发一种定制方法。
目前已知的各种技术，诱导重编程因素的表达，它们被包括两大类：非病毒和病毒载体的方法10，11，12，13。非病毒方法包括mRNA转染，miRNA感染/转染，PiggyBac，小圆载体和表皮质粒和外体体10，11，12，13。基于病毒的方法包括非整合病毒，如腺病毒、仙台病毒和蛋白质，以及整合病毒，如逆转录病毒和Lenti病毒10，11，12，13。
根据几项研究，这些方法在细胞重新编程的有效性方面没有显著差异，因此，选择合适的方法完全取决于所使用的细胞类型，以及随后的iPSCs应用获得14，15。上述所有方法都显示出缺点，例如，仙台病毒对所有细胞类型都有效，但需要大量通道才能获得iPSC;表皮体重新编程对血细胞是极好的，但需要修改成纤维细胞的标准培养条件;PiggyBac方法可能是一个有吸引力的替代方法，但人类细胞的研究仍然有限，弱10，11，12，13。外泌体是纳米囊泡，由细胞在生理上分泌到所有体液中。根据最近的研究，它们负责细胞间通信，可以在重要的生物过程中发挥作用，如细胞增殖、迁移和分化。外体体可以移植和转移mRNA和miRNA到受体细胞与完全自然的机制，因为他们共享相同的细胞膜16的组成。因此，外体是一种很有前途的新一代重新编程技术，但它们通过内容重新编程体细胞的潜力仍在调查中。基于病毒载体的方法使用经过修饰的病毒，以便将重新编程的基因传送到受体细胞。这项技术，尽管重新编程效率很高，但被认为是不安全的，因为细胞内的病毒整合可以导致感染、畸马细胞瘤和基因组不稳定17。
以下协议，以生成iPSC菌落结合山中和汤普森的重新编程鸡尾酒，并基于使用的方法，要求NM-RNA和免疫逃逸因子，并有可能在无异种条件下执行。使用这种方法的原理是在科学界传播一种协议，允许将成人成纤维细胞从腹部皮肤重新编程到iPSC18中快速、简单和高效的重新编程。
实际上，该方法的优点在于性能的简便性和获得 iPSC 所需的时间短。此外，该方法避免了细胞防御机制和病毒载体的使用，负责相关问题。
关于标准协议，进行了以下修改：（1） 在通道4处同步，在胰蛋白酶化前将0.1%的血清放入48小时;（2） 对培养细胞密度和试剂体积进行调整，以利用24孔多孔板代替6孔板;（3） 重新编程实验使用5%CO2培养箱，而不是具有大气（21%O2）或低氧（5%O2）条件的培养箱。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL serological pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>Sterile, polystyrene</td>
		</tr>
		<tr>
			<td>100 mm plates</td>
			<td>Falcon</td>
			<td>351029</td>
			<td>Treated, sterile cell culture dish</td>
		</tr>
		<tr>
			<td>15 mL sterile tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Centrifuge sterile tubes, polypropylene</td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Falcon</td>
			<td>353935</td>
			<td>Clear, flat bottom, treated multiwell cell culture plate, with lid, sterile</td>
		</tr>
		<tr>
			<td>25 mL serological pipet</td>
			<td>Falcon</td>
			<td>357525</td>
			<td>Sterile, polystyrene</td>
		</tr>
		<tr>
			<td>35 mm plates</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>Treated, sterile cell culture dish</td>
		</tr>
		<tr>
			<td>5 mL serological pipet</td>
			<td>Falcon</td>
			<td>357543</td>
			<td>Sterile, polystyrene</td>
		</tr>
		<tr>
			<td>50 mL sterile tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Centrifuge sterile tubes, polypropylene</td>
		</tr>
		<tr>
			<td>Advanced DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Gibco</td>
			<td>12491-015</td>
			<td>Store at 2-8 &#176;C; avoid exposure to light</td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Sigma- Aldrich</td>
			<td>D6429-500ml</td>
			<td>Store at 2-8 &#176;C; avoid exposure to light</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma- Aldrich</td>
			<td>F9665-500ml</td>
			<td>Store at -20 &#176;C. The serum should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Sigma- Aldrich</td>
			<td>H1387-1L</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td>Store at -20 &#176;C. It should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>INVITROGEN</td>
			<td>13778-030</td>
			<td>Synthetic siRNA Transfection Reagent; store at 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>CORNING</td>
			<td>354234</td>
			<td>Basement Membrane Matrix, store at -20 &#176;C. Avoid multiple freeze-thaws.</td>
		</tr>
		<tr>
			<td>Neubauer Chamber</td>
			<td>VWR</td>
			<td>631-1116</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>NutriStem XF Culture Medium</td>
			<td>Biological Industries</td>
			<td>05-100-1A-500ml</td>
			<td>Xeno-free, serum-free, low growth factor human ESC/iPSC culture medium. Store at -20 &#176;C. Upon thawing, the medium may be stored at 2-8 &#176;C for 14 days. Media should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles.</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-062-100ml</td>
			<td>Reduced-Serum Medium; store at 2-8 &#176;C; avoid exposure to light</td>
		</tr>
		<tr>
			<td>Penicillin and Streptomycin</td>
			<td>Sigma- Aldrich</td>
			<td>P4333-100ml</td>
			<td>Store at -20 &#176;C. The solution should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma- Aldrich</td>
			<td>P9333</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma- Aldrich</td>
			<td>P5665</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>RNase-free 0.5 mL tubes</td>
			<td>Eppendorf</td>
			<td>H0030124537</td>
			<td>RNase-free sterile, microfuge tubes, polypropylene</td>
		</tr>
		<tr>
			<td>RNase-free 1.5 mL tubes</td>
			<td>Eppendorf</td>
			<td>H0030120086</td>
			<td>RNase-free sterile, microfuge tubes, polypropylene</td>
		</tr>
		<tr>
			<td>RNaseZAP</td>
			<td>INVITROGEN</td>
			<td>AM9780</td>
			<td>Cleaning agent for removing RNase</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma- Aldrich</td>
			<td>S5761</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma- Aldrich</td>
			<td>S7653</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma- Aldrich</td>
			<td>94046</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>StemRNA 3rd Gen Reprogramming Kit</td>
			<td>Reprocell</td>
			<td>00-0076</td>
			<td>Third Generation NM-RNAs-based Reprogramming Kit for Cellular Reprogramming of Fibroblasts, Blood, and Urine. Store at or below -70 &#176;C.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma- Aldrich</td>
			<td>T4049-100ml</td>
			<td>Store at -20 &#176;C. It should be aliquoted into smaller working volumes to avoid repeated freeze/thaw cycles</td>
		</tr>
	</tbody>
</table>

isolation of adult human dermal fibroblasts from abdominal skin and generation of induced pluripotent stem cells using a non-integrating method

诱导多能干细胞（iPSCs）可被视为迄今为止一个有前途的多能细胞来源，用于管理目前无法治疗的疾病、重建和再生受伤组织以及开发新药。尽管与使用 iPSC 相关的所有优势，例如拒绝风险低、道德问题减少，以及有可能从年轻和老年患者那里获得这些差异，而他们的重新编程潜力没有任何差异，但需要克服的问题仍然很多。事实上，使用病毒和整合病毒进行的细胞重新编程会导致感染，而引入所需基因会导致受体细胞的基因组不稳定，从而损害其在临床上的使用。特别是，有许多关于使用c-Myc基因的关注，从几项研究中，它因其突变诱导活性而广为人知。纤维细胞已成为细胞重新编程的合适细胞群，因为它们易于分离和培养，并且通过微创性皮肤冲活检进行收获。此处描述的协议提供了整个过程的详细分步描述，从样品加工到获取细胞培养、试剂和用品的选择、清洁和制备，以及通过商业方式对细胞重新编程基于非改性RNA（NM-RNA）的重新编程套件。所选择的重新编程试剂盒允许对iPSC进行有效的人体皮成纤维细胞重新编程，并且在第一次转染后24小时就可以看到小菌落，即使对标准数据表进行修改也是如此。该协议中使用的重新编程过程具有安全重新编程的优点，无需病毒载体方法引起的感染风险，减少了细胞防御机制，并允许生成无异种的 iPSC，所有为进一步临床应用而必须提供的关键功能。

该协议的目的是使用基于NM-RNA的非集成重编程方法重新编程从腹部皮肤分离的皮肤成纤维细胞，以诱导特定因子的表达。为了实现这一目标，从接受腹部粘动手术的患者的皮肤标本中分离出人类皮肤成纤维细胞，通过结合NM-RNA和微RNA技术的商业即用重新编程试剂盒，产生了Oct4、Sox2、Klf4、cMyc、Nanog、Lin28重新编程因子和E3、K3、B18免疫逃逸因子。协议的时间表如图1所示。
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Watch this Scientific Journal Video about Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method at JoVE.com

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

细胞重新编程需要引入关键基因，这些基因调节和维持多能细胞状态。所述方案使诱导多能干细胞（iPSCs）菌落的形成来自人类皮肤成纤维细胞，无需病毒/整合方法，但使用非修饰RNA（NM-RNA）结合免疫逃逸因子减少细胞防御机制。

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

以下协议通过快速、高效地重新编程从皮肤冲孔活检中分离出的人类皮肤纤维细胞，提供自体人类诱导多能干细胞的说明。这些程序可以由在细胞重新编程方面经验很少或没有经验的研究人员进行，并且通过适当的调整，允许无异种诱导多能干细胞的产物。自体IPS细胞可用于再生医学应用和建模疾病，如遗传性疾病或癌症，以调查潜在的分子机制和开发更具体的疗法。工作负载很高。在仔细规划实验之前研究协议非常有帮助，包括应准备试剂和材料的步骤。该程序的视觉演示有助于经验很少或没有经验的研究执行细胞分离和重新编程，避免与可能的微生物和RNA污染相关的常见错误。在无菌罩下，用HPSS溶液从腹部清洗新鲜获得的人体皮肤样本，用HPSS溶液清洗。用细钳和剪刀去除头发和脂肪，用手术刀解剖，获得两毫米一毫米大小的碎片，避免疤痕，脏或烧伤的组织区域。在每个35毫米的盘中放置四个小碎片，盖上无菌盖玻璃，并添加1.5毫米的 I-DMEM。在37摄氏度和5%的二氧化碳下孵育板约15天，或直到细胞达到85%汇合。每三天更换一次培养培养，每天使用倒置相对比显微镜检查细胞的生长。现在，用细钳子抬起盖杯，将它们倒置在 100 毫米的盘中，用 1X 无菌 PBS 清洗。取出样品的碎片并丢弃它们。用 1X 无菌 PBS 清洗板。取出 1X PBS，在放置在 100 毫米盘中的盖眼镜中加入 3 毫米的 Trypsin-EDTA，并在 35 毫米的盘子中加入 1 毫米的 Trypsin-EDTA。在37摄氏度下孵育5分钟，吸收5%的二氧化碳。然后，通过向 100 毫米盘添加 5 毫米 TSS 和在每个 35 毫米的培养皿中添加 2 毫米 TSS 来阻断三辛化。将悬浮液收集到15毫米无菌管中，在4摄氏度下以400倍G离心，5分钟。吸制上能物，在12毫米的 I-DMEM 中重新暂停颗粒。将悬浮液分成三毫米等分，然后将它们转移到一个60毫米的板中。在37摄氏度下孵育，二氧化碳含量为5%。每天更换媒体。保持细胞在培养中，直到它们达到75%的汇合后，重复三次尝试性，以获得细胞，通过四。然后，将 I-DMEM 替换为 S-DMEM，在 37 摄氏度下用 5% 的二氧化碳孵育 48 小时，使细胞同步。在细胞同步过程中，为成纤维细胞免费编程准备成纤维细胞膨胀介质和完全未修改RNA重新编程鸡尾酒。为了简化9管未改性RNA等分的制备，这些等分管被分成36个，我们习惯了一个附着在罩壁上的方案。通过将 100 微升的地下室膜基质解冻到每孔，将 24 孔板均匀地覆盖底部，使 24 孔板涂覆。在37摄氏度下孵育至少一小时，然后播种细胞。用培养中的细胞从板中吸出介质，并在 1X PBS 中快速清洗。重复尝试性，以获得通过五的细胞。吸制上一样物，并在适当体积的成纤维细胞膨胀介质中重新暂停颗粒。用 70% 酒精准备和清洁血细胞计。然后，在1.5毫升管中轻轻混合10微升的尝试蓝色和10微升的细胞悬浮液，在室温下孵育一至两分钟，以备2分钟之用。将溶液的10微升移液放入血细胞计中，并放在倒置相对比显微镜的舞台上进行计数。不可行的细胞是蓝色的，而活的细胞是无污染的。数血细胞计室至少两个正方形中的细胞。进行稀释，获得每500微升成纤维细胞膨胀介质的2.5倍至第四细胞的密度，并重新暂停细胞颗粒。移液 500 微升的细胞悬浮到 24 井板的每个井中。在37摄氏度和5%的二氧化碳下孵育细胞过夜。早晨，从每一个井中去除旧介质，代之以500微升预扎的无因素培养基。在37%摄氏度下孵育，在5%二氧化碳下至少6小时。在室温下解冻五个 15.4 微升共 nmRNA 重编程鸡尾酒，然后将它们放在冰中。将234.6微升的减少血清介质加入到每种分体中，轻轻移液三至五次，并将每根都标为管A.Label五个无菌无RNase 0.5毫升管作为管B，并将6微升合成小干扰RNA转染试剂与244微升的减量血清介质混合。然后，使用移液器将每个管B的内容添加到管 A 滴明智。通过敲击管的底部混合。在室温下孵育15分钟。之后，将125微升的nmRNA转染复合溶液从5个等分到每一个井，将板和移液器向下倾斜到介质中。轻轻摇动，将 20 孔中的内容混合。使用其余四个孔作为参考。在37摄氏度和5%的二氧化碳下孵育板15小时。第二天，吸媒体。在无菌罩下，重复转染程序，如第一天，每天三天以上。第五天，吸气介质，并在无菌罩下与新鲜、预热的 XF/FF 培养介质进行交流。在37摄氏度和5%的二氧化碳下孵育板。每天通过相位对比显微镜监测细胞培养，直到观察IPC菌落的形成。该协议的目的是使用非集成重新编程方法重新编程从腹部皮肤分离的皮肤纤维细胞。人类成纤维细胞在培养后一周内从腹部皮肤样本中脱光，在两周内达到85%的汇合率。细胞的特点是塑料培养皿上的粘合剂生长，其形态从拉长和主轴形状到扁平和星形。成纤维细胞的形态和排列及培养在BMM上播种后发生显著变化，它们获得了拉长形态，并排列成细枝形结构。小菌落在第一天从第一次转染开始在文化中已经可见，其规模随着时间的推移而逐渐增大，而它们的数量增加到第七天，然后在第七天至第14天之间保持稳定，这可能是由于较小的菌落合并形成更大的菌落。由于重新编程程序是使用无抗生素介质进行的，在无菌条件得不到保证的情况下，微生物污染成为一个主要问题。增殖率和电镀密度影响重新编程效率，因此根据细胞增殖率规划细胞传通和确定电镀密度十分重要。该协议将动物源试剂替代为适当的无异种试剂，允许在非临床用途的完全无异种培养环境中将成年人类成纤维细胞重新编程到 IPS 细胞中。人体细胞是血液传播病原体的潜在感染源，因此，在整个过程中应佩戴个人防护设备。

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

An Improved Method for the Preparation of Type I Collagen From Skin

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Generation of Self-assembled Vascularized Human Skin Equivalents

自组装血管化人体皮肤等价物的生成

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...