Name: efficient generation of pancreas/duodenum homeobox protein 1+ posterior foregut/pancreatic progenitors from hpscs in adhesion cultures
Uploaded: 2019-03-27T12:30:00
Description: Watch this Scientific Journal Video about Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures at JoVE.com

这项工作得到了日本科学促进会 (JSPS) 通过科学研究 (C) (JPS KAKENHI Grant Number15K09385 和 18K08510) 至 t. t. 和 Jps 研究研究员补助金 (JPS KAKENGRANT 编号) 的资助。17J07622) 至 a. k. 和日本医学研究与发展机构 (AMED), 通过向 k. o 提供 "研究所细胞研究核心中心、再生医学实现研究中心网络" 的研究赠款。作者感谢彼得·卡拉吉安尼斯博士阅读了手稿。

京都大学医学系和医学研究生院伦理委员会批准了使用 Hpsc 的实验。
1. 材料的制备
请注意:准备所有培养基和试剂, 以便在无菌环境中进行细胞培养。使用前将基础培养基加热至室温 (RT)。在 RT 使用培养基, 在6小时内使用. 试剂的详情列于物料表。
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区别 (图 1 a)
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<li>阶段1A...

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PDX1+单元的生成由多个步骤组成;因此, 在适当的时间治疗细胞是至关重要的。在这些步骤中, 最终内皮的诱导效率在很大程度上影响最终的诱导效率, 可能是通过其他污染谱系细胞 (即中胚层和外胚层) 的干扰, 这些细胞可能会增殖和分泌一些因素, 而这些因素会产生。破坏特定的分化。如果 SOX17+细胞的比例在第4天 (第1 b 阶段) 低于 80%, 则对 pdx1+细胞的有效诱导可能会受到损?...

胰腺主要由外分泌细胞和内分泌细胞组成, 其功能障碍或超载导致胰腺炎、糖尿病和胰腺癌等多种疾病。为了阐明胰腺病的病因, 有必要对胰腺细胞的发育过程和功能进行分析。此外, 建立细胞组织补充治疗需要稳定的细胞供应和强大的质量。人类多能干细胞 (hpsc) 衍生的胰腺细胞是一个很有前途的细胞来源, 这是一个很有前途的细胞源, 和分化协议的胰腺细胞已深入研究1,2, 3, 4. 我的工作是什么？胰腺β细胞体外生成的新进展模仿了成人人β细胞的生成, 这些细胞在糖尿病模型小鼠2,3植入时显示出治疗效果。此外, 对健康和1型糖尿病患者捐献者诱导多能干细胞 (Ipsc) 产生的β细胞的分析显示, 包括在压力下 5, 没有功能差异.此外, 在与 6,7 患者相同的地点, 患者衍生的 ipsc 或 hpsc 存在基因突变, 导致胰腺细胞的疾病表型部分被复制。
为了从 Hpsc 中产生胰腺细胞, 采用了分步定向分化, 重述了发育过程。胰腺来源于早期胚胎的内皮层, 表达了性别决定性区域 y-盒 17 (SOX17) 和叉头盒 A2 (FOXA2)8。在小鼠研究的基础上, 内皮层形成原始的肠道管, 其特征是肝细胞核因子 1-beta (Hnf1β) 和肝细胞核因子 4-alpha (hnf4α) 的表达。原始的肠道管伸长并发展成呼吸系统、消化道和器官。伸长后, 后前部区成为推定胰腺区, 其特征是转录因子胰腺/十二指肠蛋白 1 (pdx1)8,9,10的表达。pdx1+肠道管的背侧和腹侧部分变厚形成胰芽, 其特征是胰腺转录因子1亚基α (ptf1a) 和 nk6 同源 box 1 (nkx6.1)8,11的共表达。这一表达标志着胰腺器官发生的形态开始。胰腺内真皮细胞是胰芽的组成部分, 形成一个分支管状的上皮结构网络 12,并最终分化为外分泌和内分泌细胞, 包括胰岛素分泌β细胞和葡萄糖分泌的α细胞。pdx1 的表达首先在推定胰腺区检测, 然后在整个胰腺发育过程中观察到, 并显示出β-和细胞9、13、14 的定位。虽然 Pdx1 + 细胞区不表达 Ptf1a或Nkx6.1 分化为胃狭窄, 十二指肠, 肝外胆管和一些肠道细胞在发育的中后期阶段在小鼠 9, pdx1+ 细胞在人类早期发育阶段被认为是胰腺的祖细胞。
在这里, 我们提出了一个详细的协议, 以区分 Hpsc 到 PDX1+细胞的胰腺谱系的生成。该方案通过播种离解的单细胞15、16、17来引发分化。一般情况下, 未分化的 Hpsc 在悬浮液或粘附中保持为菌落或细胞集体。因此, 大多数协议在传代后不久就开始分化。然而, 在菌落或聚集体的状态下, 细胞容易出现空间和转录异质性18、19、20、21、22, 这可能会阻碍向最终内皮的第一步分化, 然后是低效率的分化到 PDX1+细胞。本协议可以提供易于操作, 以提高和稳定一些 hPSC 线的分化效率, 这些线路以前被发现对内皮谱系和 PDX1+细胞23的分化效率很低,24,25岁

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			<td height="63" style="height:63px;width:223px;">3-Keto-N-aminoethyl-N&#8242;-aminocaproyldihydrocinnamoyl cyclopamine</td>
			<td style="width:100px;">Toronto Research Chemicals</td>
			<td style="width:123px;">K171000</td>
			<td style="width:172px;">CYC</td>
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			<td height="84" style="height:84px;width:223px;">4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">SC-203303</td>
			<td style="width:172px;">TTNPB</td>
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			<td height="63" style="height:63px;width:223px;">50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">339652</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Anti-CDX2 antibody [EPR2764Y]</td>
			<td style="width:100px;">Abcam</td>
			<td style="width:123px;">Ab76541</td>
			<td style="width:172px;">Anti-CDX2, &#215; 1/1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">B-27 Supplement (50 &#215;)</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">17504-044</td>
			<td style="width:172px;">Serum-free supplement</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">BD FACSAria II Cell Sorter</td>
			<td style="width:100px;">BD Biosciences</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For flow cytometry</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Biomedical freezer</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MDF-U538</td>
			<td>For -30 &#176;C storing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Cell Counting Slides for TC10/TC20 Cell Counter, Dual-Chamber</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450011</td>
			<td style="width:172px;">Counting slide glass</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CELL CULTURE MULTIWELL PLATE, 6 WELL, PS, CLEAR</td>
			<td style="width:100px;">Greiner bio-one</td>
			<td style="width:123px;">657165</td>
			<td style="width:172px;">For differentiation culture/6-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Centrifuge</td>
			<td style="width:100px;">TOMY</td>
			<td style="width:123px;">AX-310</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Centrifuge</td>
			<td style="width:100px;">TOMY</td>
			<td style="width:123px;">MX-305</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CHIR99021</td>
			<td style="width:100px;">Axon Medchem</td>
			<td style="width:123px;">Axon 1386</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CLEAN BENCH</td>
			<td style="width:100px;">SHOWA KAGAKU&#160;</td>
			<td style="width:123px;">S-1601PRV</td>
			<td style="width:172px;">Clean bench</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Corning CellBIND 6-well plate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">3335</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/6-well plate</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Corning Matrigel Basement Membrane Matrix Growth Factor Reduced</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">354230</td>
			<td style="width:172px;">Basement membrane matrix</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Corning Synthemax II-SC Substrate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">3535</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/synthetic surface material for hPSCs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Cryostat</td>
			<td style="width:100px;">Leica</td>
			<td style="width:123px;">Leica CM1510 S</td>
			<td style="width:172px;">For immunostaining of aggregates.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:223px;">Cytofix/Cytoperm Kit</td>
			<td style="width:100px;">Becton Dickinson</td>
			<td style="width:123px;">554714</td>
			<td style="width:172px;">Perm/Wash buffer is&#160; Permeabilization/Wash buffer. Cytofix/Cytoperm buffer is fixation and permeabilization buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Dako pen</td>
			<td style="width:100px;">Dako</td>
			<td style="width:123px;">S2002</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">dNTP mix (10 mM)</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td>18427-088</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A11055</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A10036</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A10040</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Donkey Serum</td>
			<td style="width:100px;">Merck Millipore</td>
			<td style="width:123px;">S30</td>
			<td style="width:172px;">Donkey serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">D-PBS(-) without Ca or Mg</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">14249-95</td>
			<td style="width:172px;">DPBS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Essential 8 Medium</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A1517001</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/hPSC maintenance medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Falcon 5mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">352235</td>
			<td style="width:172px;">5 mL round bottom polystyrene tube with cell strainer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 1000 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-1000S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 20 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-P20S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 200 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-P200S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Fluorescence Microscope</td>
			<td style="width:100px;">Keyence</td>
			<td style="width:123px;">BZ-X700</td>
			<td style="width:172px;">For immunostaining</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Forma Steri-Cycle CO2 incubator</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">370A</td>
			<td style="width:172px;">Incubator</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">HNF-1&#946; Antibody (C-20)</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">sc-7411</td>
			<td style="width:172px;">Anti-HNF1&#946;, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">HNF-4&#945; Antibody (H-171)</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">sc-8987</td>
			<td style="width:172px;">Anti-HNF4&#945;, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Hoechst 33342</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">H3570</td>
			<td style="width:172px;">For nucleus staining, &#215; 1/200 dilution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Human Pancreas Total RNA</td>
			<td style="width:100px;">Ambion</td>
			<td style="width:123px;">AM7954</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human PDX-1/IPF1 Antibody</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">AF2419</td>
			<td style="width:172px;">Anti-PDX1, goat IgG, &#215; 1/200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human SOX17 Antibody</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">AF1924</td>
			<td style="width:172px;">Anti-SOX17, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Improved MEM Zinc Option medium</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">10373-017</td>
			<td style="width:172px;">iMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Incubation chamber</td>
			<td style="width:100px;">Cosmo Bio</td>
			<td style="width:123px;">10DO</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Latex Examination Gloves</td>
			<td style="width:100px;">Adachi</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">MAS coated slide glass</td>
			<td style="width:100px;">Matsunami Glass</td>
			<td>83-1881</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">MicroAmp Fast 96-well Reaction Plate</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;">4346907</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Microscope</td>
			<td style="width:100px;">Olympus</td>
			<td style="width:123px;">CKX41N-31PHP</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Microtube</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">131-515CS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Monoclonal Anti-&#945;-Fetoprotein</td>
			<td style="width:100px;">SIGMA</td>
			<td style="width:123px;">A8452</td>
			<td style="width:172px;">Anti-AFP, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Nanodeop 8000</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Oligo dT</td>
			<td style="width:100px;">FASMAC</td>
			<td style="width:123px;">Custom made Oligo&#160;</td>
			<td style="width:172px;">For RT-qPCR of sequence is &#34;TTTTTTTTTTTTTTTTTTTT&#34;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Paraformaldehyde, powder</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">26126-54</td>
			<td style="width:172px;">PFA, fixative, diluted in DPBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Pharmaceutical refrigerator</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MPR-514</td>
			<td>For 4 &#176;C storing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">PIPETMAN P&#160;</td>
			<td style="width:100px;">GILSON</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">Pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Recombinant Human KGF/FGF-7</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">251-KG</td>
			<td style="width:172px;">KGF</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Recombinant Human Noggin</td>
			<td style="width:100px;">PeproTech</td>
			<td style="width:123px;">120-10C</td>
			<td style="width:172px;">NOGGIN</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Recombinant Human/Mouse/Rat Activin A</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">338-AC</td>
			<td style="width:172px;">Activin A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">ReverTra Ace (100 U/&#956;L)</td>
			<td style="width:100px;">TOYOBO</td>
			<td style="width:123px;">TRT-101</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Rnase-Free Dnase Set (50)</td>
			<td style="width:100px;">QIAGEN</td>
			<td style="width:123px;">79254</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Rneasy Mini Kit</td>
			<td style="width:100px;">QIAGEN</td>
			<td style="width:123px;">74104</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">RPMI 1640 with L-Gln</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">30264-85</td>
			<td style="width:172px;">RPMI 1640</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sealing Film for Real Time</td>
			<td style="width:100px;">Takara</td>
			<td style="width:123px;">NJ500</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 10 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td style="width:123px;">4488</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 25 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td>4489</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 5 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td style="width:123px;">4487</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sox2 (D6D9) XP Rabbit mAb</td>
			<td style="width:100px;">Cell signaling</td>
			<td style="width:123px;">3579S</td>
			<td style="width:172px;">Anti-SOX2, &#215; 1/200 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">StepOnePlus</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sucrose</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td>30406-25</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">TB Green 
			Premix Ex Taq II&#160;</td>
			<td style="width:100px;">Takara</td>
			<td>RR820B</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">TC20 Automated Cell Counter</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450101J1</td>
			<td style="width:172px;">Automatic cell counter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek OCT compound 4583&#160;</td>
			<td style="width:100px;">Sakura Finetechnical</td>
			<td>4583</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek Cryomold Molds/Adapters</td>
			<td style="width:100px;">Sakura Finetechnical</td>
			<td>4566</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Triton X-100</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">35501-15</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Trypan Blue</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450021</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Ultracold freezer</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MDF-U33V</td>
			<td>For -80 &#176;C storing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">15575-038</td>
			<td style="width:172px;">Dilute with DPBS to prepare 0.5 mM EDTA</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Veriti Thermal Cycler</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Y-27632</td>
			<td style="width:100px;">Wako</td>
			<td style="width:123px;">251-00514</td>
			<td style="width:172px;"></td>
		</tr>
	</tbody>
</table>

efficient generation of pancreas/duodenum homeobox protein 1+ posterior foregut/pancreatic progenitors from hpscs in adhesion cultures

人多能干细胞 (hPSC) 衍生胰腺细胞是再生医学中一种很有前途的细胞来源, 也是研究人类发育过程的平台。分步定向分化, 重述发育过程是产生胰腺细胞的主要方法之一, 包括胰十二指肠蛋白同源蛋白 1+ (pdx1+) 胰腺祖细胞。传统的协议在通过后不久就开始与小菌落进行分化。然而, 在菌落或聚集的状态下, 细胞容易发生异质性, 这可能会阻碍对 PDX1+细胞的分化。在这里, 我们提出了一个详细的协议, 以区分 Hpsc 到 PDX1+细胞。该方案由四个步骤组成, 通过播种离解的单细胞启动分化。在诱导 SOX17+确定内皮细胞后, 将两个原始肠道管标记 HNF1Β和 hnf4α表达, 并最终分化为 pdx1+细胞。本协议提供了简单的处理, 并可能提高和稳定一些 hPSC 线的分化效率, 以前发现, 这些线路不有效地分化为内皮谱系或 PDX1+细胞。

传播的 hpsc (585a129,30) 被浓缩并形成一个适合分化的均匀单层 (图 1b)。未分化的 Hpsc (第0阶段) 在低细胞密度 (1-1. 1.5 x10 5 细胞 2) 时分离并重新播种为单细胞.在1小时内, 细胞连接到板上, 开始显示突起。在第一天, 细胞增殖, 分布良好, 覆盖80% 至90% 的表面积。在第一阶段 b, 由于死细胞, 介质出现多云。去除死细胞对于...

Watch this Scientific Journal Video about Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures at JoVE.com

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

在这里, 我们提出了一个详细的协议, 以区分人类多能干细胞 (Hpsc) 到胰十二指肠蛋白 1+ (pdx1+) 细胞的产生的非群体类型单层生长的基础上, 胰腺谱系离解的单细胞。该方法适用于同源 hPSC-derived 衍生细胞的产生、遗传操作和筛选。

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

这种技术的主要优点是，在刺激之前，细胞均匀地分散，然后在串联细胞粘附中均匀地刺激。演示这个程序的将是来自我们实验室的研究生AzumaKimura。要开始此过程，将六毫升 RPMI 1640 转移到冰面上冷却到摄氏四度的管中。使用冷却的一毫升移液器尖端，添加两毫克的地下室膜基质。通过温和移液混合，使其成为浓度为每毫升0.33毫克的基底膜基质。接下来，使用移液器将稀释的地下膜基质的两毫升转移到六孔细胞培养板的每个孔中。在37摄氏度下孵育板60至90分钟。在此之后，将板在室温下保持长达三个小时，直到准备好使用。首先，吸气使用介质，并使用移液器将两毫升 0.5 毫摩尔 EDTA 添加到每井中，以清洗六井板中培养的 hPCS。然后，从井中吸出EDTA，并在每口井中加入两毫升新鲜0.5毫摩尔EDTA。在37摄氏度下孵育板5分钟。在此之后，从每个井中吸出 EDTA。在室温下加入一毫升的hPSC维护介质，每井补充10微摩尔Y27632。移液器轻轻地，但快速吹掉板上附加的细胞，并分离任何团状细胞到单个细胞。将细胞悬浮液转移到含有4毫升hPSC维护介质的50毫升离心管中，每井补充10微摩尔Y27632，并通过移液混合。接下来，在管中混合15微升细胞悬浮液和15微升的试盘蓝色。使用 10 微升移液器将稀释的细胞悬浮液的 10 微升转移到重复的细胞计数幻灯片中。使用自动单元格计数器，计算单元格数并计算单元格悬浮液中的细胞密度。将悬浮液分配到50毫升管中，密度为1至50万个细胞，用于每一个井。在200倍G和室温下离心管5分钟。在此之后，使用移液器吸升液，并使用每井1A阶段介质的一毫升重新悬浮细胞。轻轻移液细胞悬浮液，然后每井再加入一毫升的1A级介质。使用1000微升移液器，从先前准备的细胞培养板的孔中吸出稀释的基底膜基质。立即轻轻移液管中的悬浮液，将两毫升移入六井板的每个井中。用铝箔盖住板，防止其光线，并在室温下将板放在干净的长凳上 10 到 15 分钟。之后，小心地将板转移到37摄氏度的孵化器中，用5%的二氧化碳和24小时的加湿气氛。首先，轻轻摇动板，并使用移液器吸气使用介质。然后，向每井添加两毫升 DPBS。再次轻轻摇动板，吸气用过的 DPBS。在每个井中加入四毫升的1B级介质，预热至37摄氏度。小心地将板在37摄氏度下用5%的二氧化碳和加湿大气转移到培养器上48小时。在此之后，轻轻摇动板并吸气用过的介质。在每个井中加入两毫升的 DPBS。重复此愿望并再增加 DPBS 一次。轻轻摇动板并吸气用过的 DPBS。在每个井中加入四毫升的1B级介质，预热至37摄氏度。小心地将盘子转移到孵化器，在37摄氏度下，用5%的二氧化碳和加湿的大气培养24小时。首先，轻轻摇动板，吸走用过的介质。在六井板的每个井中加入两毫升的 DPBS。然后，轻轻摇动板，吸气用过的 DPBS。将二期介质的四毫升，预热至37摄氏度，到每井。小心地将盘子转移到37摄氏度的孵化器中，将5%的二氧化碳和加湿大气转移到培养器上四天。轻轻摇动板，吸气用过的介质。在每个井中加入两毫升的 DPBS。重复此吸气和添加 DPPS 的过程。在此之后，轻轻摇动板并吸气用过的 DPBS。在每个井中加入四毫升的三期介质，预热至37摄氏度。小心地将盘子转移到37摄氏度的孵化器中，将5%的二氧化碳和加湿的大气转移到培养三天。本研究中，对繁殖的HIPSEs进行浓缩，形成一种适合分化的同质单层。未分化的hIPSEs在低细胞密度下分离并重新播种为单细胞。在一小时内，细胞连接到板，并开始显示突起。第一天，细胞被扩散和分布良好，覆盖80%至90%的表面区域。第三天和第四天，细胞形成一个同质的单层表，可以描述为鹅卵石外观。此时，大多数细胞停止表达 SOX2，这是未分化细胞的标记，而是以超过 90% 的 SOX17 表示一个明确的内端标记，SOX17。然后细胞开始表达原始的 goptupe 标记，HNF1-beta 和 HNF4-alpha，并最终在超过 90% 的后前前脑胰蛋白酶系标记，PDX1，PDX1 阳性细胞诱导可在另一条 hIPSE 线 1231A3 和 hESE 线 KhES-3 中重现。阶段标记的MRNA表达QRTPCR结果与免疫污染一致，PDX1的MRNA表达在第三阶段明显，之后会显著增加。由于未分化的ESIPS细胞作为菌落生长，单个细胞处于不同的状态。该协议提供了一种方法，以均匀地刺激细胞为当选的分化。

Research

JoVE Journal

Developmental Biology

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

科研

发育生物学

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

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 ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...