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
Summary
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。
Abstract
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。
Introduction
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
Protocol
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。 区别 (图 1 a) 阶段1A…
Representative Results
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…
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 3-Keto-N-aminoethyl-N′-aminocaproyldihydrocinnamoyl cyclopamine | Toronto Research Chemicals | K171000 | CYC |
| 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid | Santa Cruz Biotechnology | SC-203303 | TTNPB |
| 50 mL Conical Sterile Polypropylene Centrifuge Tubes | Thermo Fisher Scientific | 339652 | |
| Anti-CDX2 antibody [EPR2764Y] | Abcam | Ab76541 | Anti-CDX2, × 1/1000 dilution |
| B-27 Supplement (50 ×) | Thermo Fisher Scientific | 17504-044 | Serum-free supplement |
| BD FACSAria II Cell Sorter | BD Biosciences | For flow cytometry | |
| Biomedical freezer | SANYO | MDF-U538 | For -30 °C storing |
| Cell Counting Slides for TC10/TC20 Cell Counter, Dual-Chamber | BIO-RAD | 1450011 | Counting slide glass |
| CELL CULTURE MULTIWELL PLATE, 6 WELL, PS, CLEAR | Greiner bio-one | 657165 | For differentiation culture/6-well plate |
| Centrifuge | TOMY | AX-310 | For cell culturing |
| Centrifuge | TOMY | MX-305 | For RT-qPCR |
| CHIR99021 | Axon Medchem | Axon 1386 | |
| CLEAN BENCH | SHOWA KAGAKU | S-1601PRV | Clean bench |
| Corning CellBIND 6-well plate | Corning | 3335 | For feeder-free culture of hPSCs/6-well plate |
| Corning Matrigel Basement Membrane Matrix Growth Factor Reduced | Corning | 354230 | Basement membrane matrix |
| Corning Synthemax II-SC Substrate | Corning | 3535 | For feeder-free culture of hPSCs/synthetic surface material for hPSCs |
| Cryostat | Leica | Leica CM1510 S | For immunostaining of aggregates. |
| Cytofix/Cytoperm Kit | Becton Dickinson | 554714 | Perm/Wash buffer is Permeabilization/Wash buffer. Cytofix/Cytoperm buffer is fixation and permeabilization buffer. |
| Dako pen | Dako | S2002 | For immunostaining of aggregates |
| dNTP mix (10 mM) | Thermo Fisher Scientific | 18427-088 | For RT-qPCR |
| Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | A11055 | Secondary antibody, × 1/500 dilution |
| Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 | Thermo Fisher Scientific | A10036 | Secondary antibody, × 1/500 dilution |
| Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 | Thermo Fisher Scientific | A10040 | Secondary antibody, × 1/500 dilution |
| Donkey Serum | Merck Millipore | S30 | Donkey serum |
| D-PBS(-) without Ca or Mg | Nacalai tesque | 14249-95 | DPBS |
| Essential 8 Medium | Thermo Fisher Scientific | A1517001 | For feeder-free culture of hPSCs/hPSC maintenance medium |
| Falcon 5mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | Corning | 352235 | 5 mL round bottom polystyrene tube with cell strainer |
| Filter Tip, 1000 µL | Watoson | 124-1000S | Use together with pipettes |
| Filter Tip, 20 µL | Watoson | 124-P20S | Use together with pipettes |
| Filter Tip, 200 µL | Watoson | 124-P200S | Use together with pipettes |
| Fluorescence Microscope | Keyence | BZ-X700 | For immunostaining |
| Forma Steri-Cycle CO2 incubator | Thermo Fisher Scientific | 370A | Incubator |
| HNF-1β Antibody (C-20) | Santa Cruz Biotechnology | sc-7411 | Anti-HNF1β, × 1/200 dilution |
| HNF-4α Antibody (H-171) | Santa Cruz Biotechnology | sc-8987 | Anti-HNF4α, × 1/200 dilution |
| Hoechst 33342 | Thermo Fisher Scientific | H3570 | For nucleus staining, × 1/200 dilution |
| Human Pancreas Total RNA | Ambion | AM7954 | For RT-qPCR |
| Human PDX-1/IPF1 Antibody | R&D Systems | AF2419 | Anti-PDX1, goat IgG, × 1/200 dilution |
| Human SOX17 Antibody | R&D Systems | AF1924 | Anti-SOX17, × 1/200 dilution |
| Improved MEM Zinc Option medium | Thermo Fisher Scientific | 10373-017 | iMEM |
| Incubation chamber | Cosmo Bio | 10DO | For immunostaining of aggregates |
| Latex Examination Gloves | Adachi | ||
| MAS coated slide glass | Matsunami Glass | 83-1881 | For immunostaining of aggregates |
| MicroAmp Fast 96-well Reaction Plate | Applied Biosystems/Thermo Fisher Scientific | 4346907 | For RT-qPCR |
| Microscope | Olympus | CKX41N-31PHP | For cell culturing |
| Microtube | Watoson | 131-515CS | |
| Monoclonal Anti-α-Fetoprotein | SIGMA | A8452 | Anti-AFP, × 1/200 dilution |
| Nanodeop 8000 | Thermo Fisher Scientific | For RT-qPCR | |
| Oligo dT | FASMAC | Custom made Oligo | For RT-qPCR of sequence is "TTTTTTTTTTTTTTTTTTTT" |
| Paraformaldehyde, powder | Nacalai tesque | 26126-54 | PFA, fixative, diluted in DPBS |
| Pharmaceutical refrigerator | SANYO | MPR-514 | For 4 °C storing |
| PIPETMAN P | GILSON | Pipette | |
| Recombinant Human KGF/FGF-7 | R&D Systems | 251-KG | KGF |
| Recombinant Human Noggin | PeproTech | 120-10C | NOGGIN |
| Recombinant Human/Mouse/Rat Activin A | R&D Systems | 338-AC | Activin A |
| ReverTra Ace (100 U/μL) | TOYOBO | TRT-101 | For RT-qPCR |
| Rnase-Free Dnase Set (50) | QIAGEN | 79254 | For RT-qPCR |
| Rneasy Mini Kit | QIAGEN | 74104 | For RT-qPCR |
| RPMI 1640 with L-Gln | Nacalai tesque | 30264-85 | RPMI 1640 |
| Sealing Film for Real Time | Takara | NJ500 | For RT-qPCR |
| Serological pipettes 10 mL | Costar/Corning | 4488 | For cell culturing |
| Serological pipettes 25 mL | Costar/Corning | 4489 | For cell culturing |
| Serological pipettes 5 mL | Costar/Corning | 4487 | For cell culturing |
| Sox2 (D6D9) XP Rabbit mAb | Cell signaling | 3579S | Anti-SOX2, × 1/200 dilution |
| StepOnePlus | Applied Biosystems/Thermo Fisher Scientific | For RT-qPCR | |
| Sucrose | Nacalai tesque | 30406-25 | For immunostaining of aggregates |
| TB Green Premix Ex Taq II |
Takara | RR820B | For RT-qPCR |
| TC20 Automated Cell Counter | BIO-RAD | 1450101J1 | Automatic cell counter |
| Tissue-Tek OCT compound 4583 | Sakura Finetechnical | 4583 | For immunostaining of aggregates |
| Tissue-Tek Cryomold Molds/Adapters | Sakura Finetechnical | 4566 | For immunostaining of aggregates |
| Triton X-100 | Nacalai tesque | 35501-15 | |
| Trypan Blue | BIO-RAD | 1450021 | |
| Ultracold freezer | SANYO | MDF-U33V | For -80 °C storing |
| UltraPure 0.5M EDTA, pH 8.0 | Thermo Fisher Scientific | 15575-038 | Dilute with DPBS to prepare 0.5 mM EDTA |
| Veriti Thermal Cycler | Applied Biosystems/Thermo Fisher Scientific | For RT-qPCR | |
| Y-27632 | Wako | 251-00514 |
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Citer Cet Article
Toyoda, T., Kimura, A., Tanaka, H., Osafune, K. Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures. J. Vis. Exp. (145), e57641, doi:10.3791/57641 (2019).