从脐带血源性多能干细胞中提取三维皮肤有机细胞
1CiSTEM Laboratory, Catholic Induced Pluripotent Stem Cell (iPSC) Research Center, College of Medicine,The Catholic University of Korea, 2Department of Biomedicine & Health Science, Seoul St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, 3Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine,The Catholic University of Korea
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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…
Representative Results
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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Adenine | Sigma | A2786 | Component of differentiation medium for fibroblast |
| AggreWell Medium (EB formation medium) | STEMCELL | 05893 | EB formation |
| Anti-Fibronectin antibody | abcam | ab23750 | Fibroblast marker |
| Anti-KRT14 antibody | abcam | ab7800 | Keratinocyte marker |
| Anti-Loricrin antibody | abcam | ab85679 | Stratum corneum marker |
| Anti-p63 antibody | abcam | ab124762 | Keratinocyte marker |
| Anti-Vimentin antibody | Santa cruz | sc-7558 | Fibroblast marker |
| BAND AID FLEXIBLE FABRIC | Johnson & Johnson | – | Bandage |
| Basement membrane matrix (Matrigel) | BD | 354277 | Component of differentiation medium for fibroblast |
| BLACK SILK suture | AILEEE | SK617 | Skin graft |
| CaCl2 | Sigma | C5670 | Component of epithelial medium for 3D skin organoid |
| Collagen type I | BD | 354236 | 3D skin organoid |
| Collagen type IV | Santa-cruz | sc-29010 | Component of differentiation medium for keratinocyte |
| Defined keratinocyte-Serum Free Medium | Gibco | 10744-019 | Component of differentiation medium for keratinocyte |
| DMEM, high glucose | Gibco | 11995065 | Component of differentiation medium |
| DMEM/F12 Medium | Gibco | 11330-032 | Component of differentiation medium |
| Essential 8 medium | Gibco | A1517001 | iPSC medium |
| FBS, Qualified | Corning | 35-015-CV | Component of differentiation medium for fibroblast and keratinocyte |
| Glutamax Supplement | Gibco | 35050061 | Component of differentiation medium for fibroblast |
| Insulin | Invtrogen | 12585-014 | Component of differentiation medium for fibroblast and keratinocyte |
| Iris standard curved scissor | Professional | PC-02.10 | Surgical instrument |
| Keratinocyte Serum Free Medium | Gibco | 17005-042 | Component of differentiation medium for keratinocyte |
| L-ascorbic acid 2-phosphata sesquimagnesium salt hydrate | Sigma | A8960 | Component of differentiation medium for keratinocyte |
| MEM Non-Essential Amino Acid | Gibco | 1140050 | Component of differentiation medium for fibroblast |
| Meriam Forceps Thumb 16 cm | HIROSE | HC 2265-1 | Surgical instrument |
| NOD.CB17-Prkdc SCID/J | The Jackson Laboratory | 001303 | Mice strain for skin graft |
| Petri dish 90 mm | Hyundai Micro | H10090 | Plastic ware |
| Recombinant Human BMP-4 | R&D | 314-BP | Component of differentiation medium for keratinocyte |
| Recombinant human EGF protein | R&D | 236-EG | Component of differentiation medium for keratinocyte |
| Retinoic acid | Sigma | R2625 | Component of differentiation medium for keratinocyte |
| T/C Petridish 100 mm, 240/bx | TPP | 93100 | Plastic ware |
| Transferrin | Sigma | T3705 | Component of epithelial medium for 3D skin organoid |
| Transwell-COL collagen-coated membrane inserts | Corning | CLS3492 | Plastic ware for 3D skin organoid |
| Vitronectin | Life technologies | A14700 | iPSC culture |
| Y-27632 Dihydrochloride | peprotech | 1293823 | iPSC culture |
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Citar este artigo
Kim, Y., Ju, J. H. Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells. J. Vis. Exp. (146), e59297, doi:10.3791/59297 (2019).