Name: generation of 3d skin organoid from cord blood-derived induced pluripotent stem cells
Uploaded: 2019-04-18T14:30:00
Description: Watch this Scientific Journal Video about Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

这项工作得到了大韩民国卫生、福利和家庭事务部韩国保健技术 &Amp; 开发项目的赠款 (H16C2177、H18C1178) 的资助。

所有涉及动物的程序都是根据《实验动物福利法》、《实验动物护理和使用指南》以及机构动物护理机构提供的啮7号动物实验指南和政策进行的。韩国天主教大学医学院使用委员会 (IACUC)。研究议定书得到韩国天主教大学机构审查委员会的批准 (CUMC-2018-0191-01)。IACUC 和韩国天主教大学实验动物系 (DOLA) 松井校区于2017年认可了韩国食品药品监督管理局的韩国卓越动物实验室设施, 并获得了评估和动物...

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人类 Ipsc 被认为是个性化再生医学的新选择.患者派生的个性化 ipsc 反映了可用于疾病建模、药物筛选和自体移植18、19的患者特征。使用患者衍生的 ipsc 还可以克服原代细胞、缺乏足够的细胞数量和免疫反应5、17、19 的问题。然而, 由于时间、成本和劳动力限制, 生?...

皮肤覆盖身体最外层, 保护内脏。皮肤具有多种功能, 包括预防病原体、吸收和储存水、调节体温和排泄身体废物 2。皮肤移植可以根据皮肤来源进行分类;使用另一个捐献者皮肤的移植被称为同种异体移植, 使用患者自己的皮肤的移植是自体移植。虽然自体移植是首选的治疗方法, 因为它的低排斥风险, 皮肤活检是很难执行严重病变或皮肤细胞数量不足的患者。在严重烧伤的患者中, 覆盖大面积所需的皮肤细胞数量是其三倍。患者体内皮肤细胞的有限可用性导致需要同种异体移植的情况。同种异体移植被暂时使用, 直到可以进行自体移植, 因为它通常在大约 1周3后被宿主的免疫系统拒绝.为了克服患者免疫系统的排斥, 移植必须来自与患者4具有相同免疫身份的来源。
人的 Ipsc 是干细胞治疗的新兴细胞来源5。人类的 Ipsc 是由体细胞产生的, 使用的是 OCT4、SOX2、Klf4 和 c-Myc6 等重新编程因子。使用人的 ipsc 克服了胚胎干细胞 (esc) 的伦理和免疫学问题 7,8.人的 Ipsc 具有多能性, 可分为三个细菌层9。HLA 是再生医学中的一个关键因素, hla 的存在决定了免疫反应和排斥的可能性10。使用患者衍生的 Ipsc 解决了细胞源限制和免疫系统排斥的问题。Cbmc 也已成为再生医学的替代细胞来源11。强制 HLA 分型, 发生在 CBMC 银行业务, 可以很容易地用于研究和移植。此外, 纯合 hla 型 Ipsc 可广泛应用于各种患者12。Cbmc-ipsc 库是一种新的、有效的细胞治疗和同种异体再生医学12、13、14的策略。在这项研究中, 我们使用 Cbmc-ipsc, 分化为角质形成细胞和成纤维细胞, 并产生分层的三维皮肤层。这项研究的结果表明, Cbmcc-ipscc 衍生的3D 皮肤有机体是一种新的工具, 用于体外和体内皮肤科的研究。

<table border="0" cellpadding="0" cellspacing="0" style="width:965px;" width="964">
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		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Adenine</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:136px;">A2786</td>
			<td style="width:401px;">Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AggreWell Medium (EB formation medium)</td>
			<td>STEMCELL</td>
			<td>05893</td>
			<td>EB formation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Fibronectin antibody</td>
			<td>abcam</td>
			<td>ab23750</td>
			<td>Fibroblast marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-KRT14 antibody</td>
			<td>abcam</td>
			<td>ab7800</td>
			<td>Keratinocyte marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Loricrin antibody</td>
			<td>abcam</td>
			<td>ab85679</td>
			<td>Stratum corneum marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-p63 antibody</td>
			<td>abcam</td>
			<td>ab124762</td>
			<td>Keratinocyte marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Vimentin antibody</td>
			<td>Santa cruz</td>
			<td>sc-7558</td>
			<td>Fibroblast marker</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BAND AID FLEXIBLE FABRIC</td>
			<td>Johnson &#38; Johnson</td>
			<td>-</td>
			<td>Bandage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Basement membrane matrix (Matrigel)</td>
			<td>BD</td>
			<td>354277</td>
			<td>Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BLACK SILK suture</td>
			<td>AILEEE</td>
			<td>SK617</td>
			<td>Skin graft</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CaCl2</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td>Component of epithelial medium for 3D skin organoid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagen type I</td>
			<td>BD</td>
			<td>354236</td>
			<td>3D skin organoid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagen type IV</td>
			<td>Santa-cruz</td>
			<td>sc-29010</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Defined keratinocyte-Serum Free Medium</td>
			<td>Gibco</td>
			<td>10744-019</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11995065</td>
			<td>Component of differentiation medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM/F12 Medium</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>Component of differentiation medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Essential 8 medium</td>
			<td>Gibco</td>
			<td>A1517001</td>
			<td>iPSC medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FBS, Qualified</td>
			<td>Corning</td>
			<td>35-015-CV</td>
			<td>Component of differentiation medium for fibroblast and keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax Supplement&#160;</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Insulin</td>
			<td>Invtrogen</td>
			<td>12585-014</td>
			<td>Component of differentiation medium for fibroblast and keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Iris standard curved scissor</td>
			<td>Professional</td>
			<td>PC-02.10</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Keratinocyte Serum Free Medium</td>
			<td>Gibco</td>
			<td>17005-042</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">L-ascorbic acid 2-phosphata sesquimagnesium salt hydrate</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM Non-Essential Amino Acid</td>
			<td>Gibco</td>
			<td>1140050</td>
			<td>Component of differentiation medium for fibroblast</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Meriam Forceps Thumb 16 cm</td>
			<td>HIROSE</td>
			<td>HC 2265-1</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">NOD.CB17-Prkdc SCID/J</td>
			<td>The Jackson Laboratory</td>
			<td>001303</td>
			<td>Mice strain for skin graft</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Petri dish 90 mm</td>
			<td>Hyundai Micro</td>
			<td>H10090</td>
			<td>Plastic ware</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Recombinant Human BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant human EGF protein</td>
			<td>R&#38;D</td>
			<td>236-EG</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Component of differentiation medium for keratinocyte</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">T/C Petridish 100 mm, 240/bx</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Plastic ware</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Transferrin</td>
			<td>Sigma</td>
			<td>T3705</td>
			<td>Component of epithelial medium for 3D skin organoid</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Transwell-COL collagen-coated membrane inserts&#160;</td>
			<td>Corning</td>
			<td>CLS3492</td>
			<td>Plastic ware for 3D skin organoid&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Vitronectin</td>
			<td>Life technologies</td>
			<td>A14700</td>
			<td>iPSC culture</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Y-27632 Dihydrochloride</td>
			<td>peprotech</td>
			<td>1293823</td>
			<td>iPSC culture</td>
		</tr>
	</tbody>
</table>

generation of 3d skin organoid from cord blood-derived induced pluripotent stem cells

皮肤是人体最大的器官, 有许多功能。皮肤作为身体的屏障和保护者, 调节身体功能。仿性主义是模仿自然的模型、系统和元素, 目的是解决复杂的人类问题1。皮肤仿生学是体外疾病研究和体内再生医学的有用工具。人体诱导多能干细胞具有无限增殖和分化为三种生殖层的能力。人类 Ipsc 是由各种原代细胞产生的, 如血细胞、角质形成细胞、成纤维细胞等。其中, 脐带血单个核细胞 (Cbmc) 已从同种异体再生医学的角度成为替代细胞来源。Cbmc 在再生医学中很有用, 因为人类白细胞抗原 (HLA) 分型对细胞银行系统至关重要。我们提供了一种方法, 分化为角质形成细胞和成纤维细胞, 并产生三维皮肤有机体。Cbmc-ipscc 衍生角质形成细胞和成纤维细胞具有类似于原代细胞系的特征。3D 皮肤有机体是通过将表皮层覆盖在真皮层而产生的。通过移植这种3D 皮肤有机体, 生成了一个人性化的小鼠模型。本研究表明, 三维人体 ipsc 衍生皮肤有机体可能是一种新的, 替代工具的皮肤科研究在体外和体内。

皮肤在很大程度上是由表皮和真皮组成的。角质形成细胞是表皮的主要细胞类型, 成纤维细胞是真皮的主要细胞类型。角质形成细胞分化的方案如图 1a 所示。Cbmcc-ipcsc 保存在一个玻璃体涂膜盘中 (图 1b)。在这项研究中, 我们区分 Cbmc-ipsc 到角质形成细胞和成纤维细胞使用 EB 形成。我们使用悬挂下降法生成了 Eb, 以确保角质形成?...

Watch this Scientific Journal Video about Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

从脐带血源性多能干细胞中提取三维皮肤有机细胞

我们提出了一个协议, 说明如何区分诱导多能干细胞衍生角质形成细胞和成纤维细胞, 并产生三维皮肤有机体, 使用这些角质形成细胞和成纤维细胞。该协议包含生成人性化小鼠模型的附加步骤。这里介绍的技术将改进皮肤科的研究。

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

脐带血单核细胞（CBMC）已成为再生医学的潜在细胞来源，因为人类白细胞抗原类型对细胞库系统至关重要。CBMC诱导的多能干细胞，或CMBC-iPSC，可以分化成角蛋白细胞和成纤维细胞。为了产生3D皮肤器官，我们使用皮肤学研究。首先在37摄氏度和10%的二氧化碳下将CBMC-iPSC培养到镀维特龙素涂层的100毫升板上。当细胞达到80%的汇合时，用PBS洗涤培养物，每盘一毫升一毫升EDTA，在37摄氏度和5%的二氧化碳下治疗两分钟。当细胞分离后，停止每板三毫升E8介质的反应，通过离心收集细胞。将颗粒重新悬浮在五毫升E8介质中进行计数，将一次-10至六分之六的细胞转移到15毫升锥形管中进行离心。用2.5毫升的胚胎体形成介质重新悬浮转移的细胞，辅以10微摩尔罗相关激酶，或 ROCK抑制剂。使用移液器将 125 微升的细胞液滴转移到未涂覆的培养板盖上。放置所有液滴后，将盘子翻过来，让液滴在37摄氏度和5%的二氧化碳下从盖子上悬挂24小时。第二天，用新鲜的E8介质清洗盖子，将胚胎体溶液转移到50毫升锥形管中。允许胚胎体在室温下沉淀一分钟，然后吸上一清液，用新鲜E8培养的胚胎体在37摄氏度的90毫米培养皿中重新悬浮，直到其分化。对于CBMC-iPSC角蛋白分化，用新鲜的E8培养基取代胚胎体培养物的E8培养基，辅以每毫升骨质原蛋白4的一毫微克，在37摄氏度和5%的二氧化碳下24小时孵育。第二天，将胚胎身体收获成50毫升的圆锥管，让胚胎在室温下沉淀一分钟。接下来，用六毫升角蛋白细胞分化介质 1 或 KDM1 替换上一代，辅以 10 微摩尔 ROCK 抑制剂，将胚胎体转移到四型胶原蛋白涂层的 100 毫米培养皿中。在培养的天零到八天，每隔一天用新鲜的KDM1代替介质，辅以三微摩尔视网膜酸和每毫升25毫微克，每毫升骨形态遗传蛋白4和表皮生长因子。第9天至12天，每隔一天用KDM2代替介质，辅以三微摩尔视网膜酸，每毫升骨形态遗传蛋白25毫微克4，每毫升表皮生长因子20毫微克。第13天至30天，每隔一天将介质更改为KDM3，每毫升骨形态遗传蛋白4，每毫升20毫微克，每毫升表皮生长因子20毫微克。对于CBMC-iPSC成纤维细胞分化，在6毫升成纤维细胞分化介质1中重新悬浮胚胎体培养，辅以10微摩尔 ROCK抑制剂，将胚胎体悬浮转移到地下室膜基质涂层的100毫米培养皿中。经过三天的孵育，用0.5纳米摩尔骨形态遗传蛋白4治疗培养物4天，然后将培养基改变为成纤维细胞分化介质2。在文化的第七天，每隔一天将介质更改为 FDM2 一周。在培养的第14天，分离细胞与一毫摩尔EDTA如证明，并收集细胞在三毫升的成纤维细胞分化介质2离心。将细胞重新悬浮在五毫升成纤维细胞分化介质1中进行计数，并转移两次至十至六分之六的细胞至非涂层培养皿。在培养的第21天，将培养的两次-10至6-6细胞转移到新鲜成纤维细胞分化介质1的一型胶原蛋白涂层100毫米培养皿。在培养的第28天，将两次-10至6个 iPSC 衍生的成纤维细胞转移到一个新的非涂层菜。对于三维皮肤器官生成，从培养中收获 iPSC 衍生的成纤维细胞，将两次-10 到 50 细胞转移到 15 毫升锥形管中进行离心。将成纤维细胞颗粒重新悬浮在1.5毫升的成纤维细胞分化介质1和1.5毫升的中和 I 型胶原蛋白溶液中。在室温下，在六孔微孔板中孵育刀片上的细胞30分钟。当溶液凝固后，在刀片顶部加入两毫升介质，在井底加入三毫升。然后将成纤维细胞基质放入培养箱中五到七天，直到凝胶完成，基质不再收缩。在孵育结束时，收获 iPSC 衍生的角蛋白细胞，将一次-10 至 6 次细胞转移到 15 毫升锥形管中进行离心。将颗粒重新悬浮在50至100微升的低钙上皮介质1中，用角蛋白细胞溶液取代成纤维细胞基质的介质。在37摄氏度下15分钟后，用两毫升的上皮介质1替换刀片顶部的介质，用三毫升的上皮介质将刀片的介质更换，并将板返回孵化器。两天后，用正常的钙上皮介质2替换插入物中的培养，再进行两天的培养。在培养的第四天，去除所有的培养，并添加三毫升的玉米化介质到底部井只，以产生一个空气-液体接口。然后将板返回孵化器长达 14 天，然后收获 3D 皮肤器官进行下游分析。CBMC-iPSC衍生的角蛋白细胞具有类似于原发性角蛋白细胞的形态，在这些细胞中增加了角蛋白细胞标记的表达。CBMC-iPSC衍生的成纤维细胞具有类似于原发性成纤维细胞的形态，多能干细胞标记10月4日的表现受到低调节，而成纤维细胞标记则向上调节。3D皮肤器官的厚度在3D培养过程中增加，确认3D皮肤器官是由 iPSC 衍生的角蛋白细胞和成纤维细胞产生的。两周后，移植的3D皮肤器官移植有效整合到受体小鼠皮肤，HNE分析证实。此外，角蛋白细胞成熟和表皮分化标记在CBMC-iPSC衍生的3D皮肤器官中表达，表现出功能分化，有效的移植，并有效愈合小鼠皮肤缺陷。我们过去使用高钙介质，诱导分层的3D皮肤器官层模仿皮肤附近。分析表明皮肤是分层的。CBMC-iPSC 是皮肤移植的潜在细胞来源，CBMC-iPSC 衍生的 3D 皮肤器官可用于与皮肤科、美容筛查和再生医学相关的研究。

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...