体外 人体牙浆干细胞向胰腺血统的诱导
1Veterinary Stem Cell and Bioengineering Innovation Center (VSCBIC), Veterinary Pharmacology and Stem Cell Research Laboratory, Faculty of Veterinary Science,Chulalongkorn University, 2Department of Veterinary Anatomy, Faculty of Veterinary Medicine,Universitas Airlangga, 3Department of Anatomy, Faculty of Dentistry,Chulalongkorn University, 4Center of Excellence in Regenerative Dentistry, Faculty of Dentistry,Chulalongkorn University, 5Veterinary Stem Cell and Bioengineering Research Unit, Faculty of Veterinary Science,Chulalongkorn University, 6Department of Pharmacology, Faculty of Veterinary Science,Chulalongkorn University
Summary
本协议对区分人类牙浆干细胞（hDPSC）与 体外胰腺谱系的两种不同的诱导协议进行了比较：综合协议和非综合协议。综合协议产生更多的胰岛素产生细胞（IPCs）。
Abstract
截至2000年，使用埃德蒙顿协议治疗I型糖尿病胰岛移植的成功仍然面临一些障碍。其中包括数量有限的尸体胰腺捐献者和免疫抑制剂的长期使用。默森奇马尔干细胞（MSCs）被认为是胰岛细胞生成的替代来源的潜在候选者。我们以前的报告成功地说明了建立诱导协议，将人类牙髓干细胞（hDPSC）与胰岛素生成细胞（IPCs）区分。然而，上岗效率差别很大。本文通过综合（微环境和遗传操作）和非集成（微环境操作）诱导协议，演示了通过综合（微环境和遗传操作）来提供 hDPSC 衍生 IPC （hDPSC-IPCs） 的 hDPSC 胰腺感应效率的比较。结果表明，在多剂量葡萄糖挑战下，在三维菌落结构、产量、胰腺mRNA标记和功能特性方面，诱导方法具有明显的感应效率。这些发现将支持未来建立一个临床适用的IPC和胰腺血统生产平台。
Introduction
糖尿病是一个持续的全球关注。国际糖尿病联合会（IDF）的一份报告估计，全球糖尿病患病率将从2000年的1.51亿增加到2015年的4.15亿。最新的流行病学研究预测，全球糖尿病患病率将从2017年的4.51亿增加到2045年的6.93亿。胰岛移植利用埃德蒙顿协议的成功首次证明在2000年，当它被证明保持内源性胰岛素生产和稳定规范的血糖状况在I型糖尿病患者3。然而，埃德蒙顿协议的应用仍然面临瓶颈问题。尸体胰腺捐献者数量有限是主要问题，因为每个I型糖尿病患者至少需要2-4个小岛捐赠者。此外，长期使用免疫抑制剂可能会导致危及生命的副作用4，5。为了解决这个问题，在过去十年中，糖尿病的潜在疗法的开发主要侧重于从各种来源的干细胞6中产生有效的胰岛素生成细胞（IPCs）。
干细胞成为许多疾病的替代疗法，包括糖尿病I型，这是由β细胞的损失引起的。IPC移植是控制这些患者血糖的新方法。本文介绍了生成 IPC 的两种方法，即集成和非集成感应协议。诱导协议模仿自然胰腺发育过程，使成熟和功能IPC8，9。
在这项研究中，hDPSC的特点是MSC表面标记检测的流细胞学，多系分分潜力，和RT-qPCR，以确定干性特性和增殖基因标记（未显示的数据）8，9，10的表达。hDPSC被诱导走向明确的内皮，胰腺内皮，胰腺内分泌，胰腺β细胞或IPC（图1），分别7。为了诱导细胞，使用三步感应方法作为骨干协议。此协议称为非综合协议。在综合协议中，基本胰腺转录因子PDX1在 hDPSC 中表达过度，然后使用三步分化协议在 hDPSC 中引入过度表达PDX1。 非集成协议和集成协议之间的区别在于PDX1在集成协议中过度表达，而不是在非集成协议中过度表达。在这项研究中，胰腺分化被比较为综合和非综合协议。
Protocol
这项工作是根据《赫尔辛基宣言》进行的，并经朱拉隆功大学牙科学院人类研究伦理委员会批准。由于智齿问题，人类DPC（hDPSC）从从前摩尔和摩尔中提取的人类牙浆组织中被分离出来。根据批准的协议（HREC-DCU 2018/054）从患者那里获得知情同意。 1. 综合上岗协议 携带PDX1的扁豆载体的准备 使用人类胚胎肾（HEK）293FT细胞进行病毒包装。培养和维持这些细…
Representative Results
本文比较了两种上岗协议的结果。两个上岗协议的图表在图2A，C中都有说明。在这两个协议中，评估都是在光显微镜下进行的，图像是用 ImageJ 分析的。hDPSC 能够从上岗的第一天起在两个上岗协议中形成类似殖民地的结构。殖民地的形态是圆的和密集的，所有的殖民地漂浮在文化容器在整个上岗期间（图2B，D）。还确定了两个?…
Discussion
实现 MSC 的 IPC 产量提高在糖尿病治疗中起着至关重要的作用。集成协议的关键步骤依赖于用于转导的细胞质量和转导细胞的质量。一些细胞要求，应该检查成功的转导是确保细胞健康，细胞银行管理和细胞处于间歇活动状态。此外，监测转导细胞的生存能力也起着重要作用。转导不太成功是由于受刺激细胞12的生存能力差造成的。对于非综合协议，形态外观和浮动菌落应实现，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SK、WR 和 QDL 得到了朱拉隆功大学兽医干细胞和生物工程研究组、拉查达菲塞克松博特捐赠基金的支持。托和人民党得到了朱拉隆功学术进步到其2 世纪项目的支持。CS得到了兽医科学学院、朱拉隆功学术促进二世纪项目 、兽医干细胞和生物工程研究组、拉查达菲塞克松博特捐赠基金、朱拉隆功大学和政府研究基金的支持。
Materials
| Cell Culture | |||
| Antibiotic-Antimycotic | Thermo Fisher Scientific Corporation, USA | 15240062 | |
| Corning® 60 mm TC-treated Culture Dish | Corning® | 430166 | |
| Dulbecco’s Modified Eagle Medium (DMEM) | Thermo Fisher Scientific Corporation | 12800017 | |
| Fetal bovine serum (FBS) | Thermo Fisher Scientific Corporation | 10270106 | |
| GlutaMAX™ | Thermo Fisher Scientific Corporation | 35050061 | |
| Phosphate buffered saline (PBS) powder, pH 7.4 | Sigma-Aldrich | P3813-10PAK | One pack is used for preparing 1 L of PBS solution with sterile DDI |
| Trypsin-EDTA (0.25%) | Thermo Fisher Scientific Corporation | 25200072 | |
| Lentiviral Vector Carrying PDX1 Preparation | |||
| Amicon® Ultra-15 Centrifugal Filter | Merck Millipore, USA | UFC910024 | |
| Human pWPT-PDX1 plasmid | Addgene | 12256 | Gift from Didier Trono; http://n2t.net/addgene:12256; RRID: Addgene_12256 |
| Millex-HV Syringe Filter Unit, 0.45 µm | Merck Millipore | SLHV033RB | |
| pMD2.G plasmid | Addgene | 12259 | Gift from Didier Trono; http://n2t.net/addgene:12259; RRID: Addgene_12259 |
| Polybrene Infection / Transfection Reagent | Merck Millipore | TR-1003-G | |
| psPAX2 plasmid | Addgene | 12260 | Gift from Didier Trono; http://n2t.net/addgene:12260; RRID: Addgene_12260 |
| Three-step Induction Protocol | |||
| Activin A Recombinant Human Protein | Merck Millipore | GF300 | |
| Beta-mercaptoethanol | Thermo Fisher Scientific Corporation | 21985-023 | |
| Bovine serum albumin (BSA, Cohn fraction V, fatty acid free) | Sigma-Aldrich | A6003 | |
| Glucagon-like peptide (GLP)-1 | Sigma-Aldrich | G3265 | |
| Insulin-Transferrin-Selenium (ITS) | Invitrogen | 41400-045 | |
| Nicotinamide | Sigma-Aldrich | N0636 | |
| Non-Essential Amino Acids (NEAAs) | Thermo Fisher Scientific Corporation | 11140-050 | |
| Non-treated cell culture dish, 60mm | Eppendorf | 30701011 | |
| Sodium butyrate | Sigma-Aldrich | B5887 | |
| Taurine | Sigma-Aldrich | T0625 |
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Cite This Article
Kuncorojakti, S., Rodprasert, W., Le, Q. D., Osathanon, T., Pavasant, P., Sawangmake, C. In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages. J. Vis. Exp. (175), e62497, doi:10.3791/62497 (2021).