Name: in vitro induction of human dental pulp stem cells toward pancreatic lineages
Uploaded: 2021-09-25T14:30:00
Description: Watch this Scientific Journal Video about In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages at JoVE.com

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。

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
<ol><li>携带PDX1的扁豆载体的准备
	<ol>
<li>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...

<ol>
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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，?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific Corporation, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#174; 60 mm TC-treated Culture Dish</td>
			<td>Corning&#174;</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>12800017</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#8482;</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) powder, pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>P3813-10PAK</td>
			<td>One pack is used for preparing 1 L of PBS solution with sterile DDI</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral Vector Carrying PDX1 Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon&#174; Ultra-15 Centrifugal Filter</td>
			<td>Merck Millipore, USA</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>Human pWPT-PDX1 plasmid</td>
			<td>Addgene</td>
			<td>12256</td>
			<td>Gift from Didier Trono; http://n2t.net/addgene:12256; RRID: Addgene_12256</td>
		</tr>
		<tr>
			<td>Millex-HV Syringe Filter Unit, 0.45&#160;&#181;m</td>
			<td>Merck Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>Gift from Didier Trono; http://n2t.net/addgene:12259; RRID: Addgene_12259</td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Merck Millipore</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2 plasmid</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>Gift from Didier Trono; http://n2t.net/addgene:12260; RRID: Addgene_12260</td>
		</tr>
		<tr>
			<td>Three-step Induction Protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A Recombinant Human Protein</td>
			<td>Merck Millipore</td>
			<td>GF300</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA, Cohn fraction V, fatty acid free)</td>
			<td>Sigma-Aldrich</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucagon-like peptide (GLP)-1</td>
			<td>Sigma-Aldrich</td>
			<td>G3265</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids (NEAAs)</td>
			<td>Thermo Fisher Scientific Corporation</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-treated cell culture dish, 60mm</td>
			<td>Eppendorf</td>
			<td>30701011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium butyrate</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro induction of human dental pulp stem cells toward pancreatic lineages

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。

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?...

Watch this Scientific Journal Video about In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages at JoVE.com

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

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）。

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

这段视频展示了人类牙髓干细胞向胰腺谱系的体外诱导。干细胞在许多疾病中提供替代疗法，包括导致β细胞生物拉伤的I型糖尿病。胰岛素生成细胞的移植将是导致终生的新方法。在本视频中介绍了产生胰岛素生成细胞的两种方法，包括集成和非综合感应协议。在这部分，解释了从人类牙髓干细胞中诱导胰岛素产生细胞的整合协议。因为诱导协议是混合自然胰腺发育过程。获得天然和功能胰岛素产生细胞。用于从牙髓干细胞等中性干细胞生成胰岛素生成细胞，采用多步骤诱导协议。间质干细胞分别用于"确定内皮"、胰腺内分泌、胰腺内分泌，然后分别用于胰腺β细胞或胰岛素生成细胞。为了诱导细胞，使用三步感应方法作为骨干协议。综合方案采用了人类牙髓干细胞对基本胰腺转录因子PDX-1的过期。然后，PDX-1超过表达的干细胞将进一步诱导使用三步分化协议。胰腺分化潜力，由综合协议，将比较非综合协议，如动画中所示。这个想法是根据朱拉隆功大学牙科学院人类研究伦理委员会批准的协议，征得患者知情同意的。此协议以 PDX-1 过期开始。2至5个部分的人体牙浆干细胞，70-80%的汇合，用于转导。细胞被尝试化和计数。然后，100万个细胞被安置在转染容器上，并在一夜之间孵育。24小时后，根据所需的感染倍数，将携带PDX-1基因的新鲜制备的杀毒软件添加到培养容器中。然后，转染的细胞在孵化器中保存24小时。然后，新鲜培养介质被改变并再维持48个时期。在使用进一步诱导步骤之前，先检查转染细胞的形态。人类牙髓干细胞与PDX-1过期后，然后尝试和悬浮在第一个胰腺感应介质，所以所谓的介质 A.In 这一步，低附件培养容器被使用。PDX-1的形态在观察到中等A诱导后的第3天超过表达的人类牙髓干细胞。然后，具有良好形态的细胞将被用于进一步诱导步骤。下一步，第二感应介质，所谓的介质B被添加到培养容器中。PDX-1在观察到中等诱导后的第二天超过表达的人类牙髓干细胞的形态。然后，具有良好形态的细胞将被用于进一步诱导步骤。下一步，第三个感应介质，所谓的介质C被添加到培养容器中。5天后观察到细胞形态。收集细胞群落以作进一步分析。包括菌落形态和大小、胰腺基因标记表达和有关葡萄糖刺激的C-肽分泌物或GSCS检测的功能特性。对于非综合感应方法，采用了前面提到的三步感应协议。一百万个细胞在中A中重新悬浮，并坐在低附件培养容器上。然后培养了三天。在形态检查后，分别在第三天和第五天将包括中B和C在内的一系列感应介质添加到培养容器中。在第10天，观察细胞形态。收集细胞群落以作进一步分析，包括菌落形态和大小、胰腺基因标记表达以及有关葡萄糖刺激的C-肽分泌物或GSCS检测的功能特性。两个上岗协议的结果在两个协议中进行比较。人类牙浆干细胞能够形成殖民地般的结构，因为第一天的诱导。殖民地的外观是圆和密集的。在整个上岗期间，所有的殖民地都漂浮在文化容器中。综合诱导协议的总菌落计数比非综合诱导的菌落总数高一点。殖民地大小百分比下的进化显示出一种有益的趋势，从小到中小的殖民地形成后综合诱导，这对于减少殖民地内的坏死核心具有重要意义。胰腺基因标记分析表明，综合电感协议使菌落的胰腺标记（包括 MAP-A、GLUT-2、胰岛素和 GLP-1R）过期率较高，表明与测量胰岛素生成细胞的分化。对功能葡萄糖C肽分泌很重要。结果表明，这两种诱导协议都使菌落对葡萄糖挑战有良好的反应。通过这项研究，两种胰腺诱导方案都能够在胰腺标记期满和功能特性方面在体外生成胰岛素生成细胞，这些协议显示了胰岛素产生细胞形成的更好质量。建议人类间质干细胞的趋势，如人类牙髓干细胞在干细胞为基础的糖尿病治疗中应用在不远的将来。

Research

JoVE Journal

Bioengineering

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

Use of Human Perivascular Stem Cells for Bone Regeneration

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

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 ...

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 ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

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 ...

体外 人体牙浆干细胞向胰腺血统的诱导

体外人体牙浆干细胞向胰腺血统的诱导