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
1Program in Developmental Biology,Baylor College of Medicine, 2Center for Cell and Gene Therapy, Texas Children’s Hospital, and Houston Methodist Hospital,Baylor College of Medicine, 3Molecular and Cellular Biology Department,Baylor College of Medicine, 4Stem Cell and Regenerative Medicine Center,Baylor College of Medicine, 5McNair Medical Institute,Baylor College of Medicine
Summary
在这里, 我们提出了一种生成胰岛素表达3D 小鼠 pancreatoids 从游离漂浮 e10.5 分离的胰祖细胞和相关间质的协议。
Abstract
胰腺是由许多不同细胞类型组成的复杂器官, 它们协同工作来调节血糖的稳态和消化。这些细胞类型包括分泌酶的腺泡细胞, 一种 arborized 导管系统, 负责将酶输送到肠道, 以及产生激素的内分泌细胞。
内分泌β细胞是体内唯一的细胞类型, 它能产生胰岛素以降低血糖水平。糖尿病是一种以丧失或β细胞功能障碍为特征的疾病, 正达到流行的程度。因此, 必须建立一些协议来调查β细胞的发展, 它可以用于筛选目的, 以获得药物和细胞的治疗方法。虽然实验性研究老鼠的发展是必不可少的,在体内研究是费力和耗时的。培养细胞为筛查提供了更方便的平台;然而, 他们无法维持细胞多样性, 建筑组织, 和细胞之间的相互作用发现在体内.因此, 有必要开发新的工具来调查胰腺器官和生理。
胰腺上皮细胞在与间质的密切联系中发展, 从器官发生开始, 细胞组织并分化成复杂的、生理上有能力的成体器官。胰腺间质为内分泌的发展提供了重要信号, 其中许多还没有得到很好的理解, 因此在体外文化中很难重述。在这里, 我们描述一个协议, 文化三维, 细胞复杂的老鼠 organoids 保留间质, 称为 pancreatoids。e10.5 小鼠胰芽被解剖, 分离, 并培养在一个无支架的环境。这些漂浮细胞自组装与间质包络的发展 pancreatoid 和强健的数量的内分泌β细胞与腺泡和导管细胞一起发展。该系统可用于研究细胞的命运决定, 结构组织, 和形态发生, 细胞间的相互作用, 在器官发生, 或为药物, 小分子, 或基因筛选。
Introduction
界定正常发展和生理学的机制, 对于了解疾病病因, 最终培养治疗方法至关重要。虽然培养和区分干细胞能够快速和高通量地分析发展, 但它受现有的知识机构的限制, 这些机制调控细胞命运和人为概括发展在一个相对同构, 二维状态1,2。不仅是受外部影响影响的 “体内” 开发, 在利基和环境中提供分泌信号和组织支持的不同单元格类型也可用于引导器官, 但这些细胞的功能也依赖于其用于指导的环境3,4,5。鉴于这些外部线索的重要性, 差异化协议的局限性, 以及体内老鼠模型的费力性质, 需要新的系统来实验研究基本的发育过程和生理学。
生成三维复杂 organoids 的协议的出现, 提供了一个方便和一致的系统来研究器官发生、生理学、药物功效, 甚至发病机制.建立小鼠 organoids 为不同的系统, 如胃6和肠道7已经扩大了我们对器官的理解, 提供了一个工具来研究发展复杂性较少的限制比在体内和体外模型。由于这些进展在小鼠 organoid 形成和人类多潜能干细胞的出现, 人类肠道8, 视网膜9, 肾脏10,11, 和大脑12 organoids 已经产生, 这《汇辑》仅限于现有关于发展机制的知识。
特别感兴趣的是胰腺 organoids 的产生, 因为无数的疾病瘟疫不同的胰腺细胞类型, 包括腺泡细胞和导管在外分泌的胰腺功能不全13, 胰腺炎的腺泡细胞14, 并β细胞在糖尿病15。获得有关这些不同细胞类型发展的知识有助于理解其病理学, 也可以作为个性化药物筛选或移植的平台。以前, Greggio et开发了一种方法来创建小鼠胰腺 organoids, 它概括了体内形态发生并发展了由所有主要的胰腺上皮细胞组成的三维复杂结构。类型16,17。这是胰腺领域向前迈出的重要一步, 特别是使细胞体外能够使β细胞的生物研究得以发展。然而, 在本议定书中形成的内分泌细胞的匮乏, 除非 organoids 移植到组织, 在那里利基可以互动和提供指导提示17。间质构成利基的最大部分, 严重包裹发育上皮从器官的早期阶段到以后阶段, 包括内分泌分层和分化 3, 4, 18。间质与发展中的胰腺的相互作用又是外部信号的另一个例子, 也是维持体内细胞复杂性来研究器官的重要性.
在这里, 我们描述如何产生三维胰腺 organoids, 称为 pancreatoids, 从分离 e10.5 小鼠胰腺祖细胞。这些 pancreatoids 保留本机间质, 自组装在自由漂浮条件, 并产生所有主要的胰腺细胞类型, 包括强健的数量的内分泌β细胞 19.这种方法最适合分析内分泌的发展, 因为以前的协议缺乏强健的内分泌分化。然而, 使用 Greggio et所描述的胰 organoids 的协议, 更适合于分析胰腺上皮分支和形态发生, 因为 pancreatoids 中分支更受限制。
Protocol
该方法所描述的所有动物实验均经贝勒医学院机构动物护理和使用委员会批准。 1. 小鼠胚胎日10.5 胰祖细胞的制备 注: 本协议不需要在无菌条件下遵循, 直到步骤 2, 但它是最佳的消毒解剖工具和喷雾与70% 乙醇之前使用。 为解剖, 填充冰桶和放置一个容器磷酸盐缓冲盐水 (PBS) 在冰。清洁两个细尖钳和解剖剪刀与70% 乙醇。设置在解剖区附近, 一个容?…
Representative Results
仔细解剖小鼠胚胎在 e10.5 从子宫角应该产生未损坏的胚胎在 PBS 进一步解剖 (图 1A)。胃肠道可以有效地从胚胎中移除 (图 1B), 允许辨别肠道和胃交界处的背胰芽 (图 1C-F)。e10.5 的胰芽以前有特征;祖祖先应表达Pdx1、Sox9、Ptf1a、和Hes120?…
Discussion
细胞培养模型的进展对于正确的模型开发, 产生临床相关的细胞类型, 测试药物疗效, 甚至移植到病人来说是至关重要的。然而, 人工综述在菜肴中的发展是有挑战性的, 因为我们还远未了解器官和生理的机制在体内.因此,体外细胞的生成效率低下, 不能充分发挥作用, 不能长时间维持, 或使体内可比细胞的其他异常发生。这是因为许多不同的细胞类型相互作用, 而复杂的形态发生变化影?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢 Jolanta Chmielowiec 就该协议和手稿提供了有益的讨论。我们还感谢本杰明 Arenkiel 获得共聚焦显微镜。这项工作得到了 NIH (P30-DK079638 到手机和 T32HL092332-13 到 M.A.S. 和手机)、捷尔医学基金会 (手机) 以及在医疗知识和发展残疾研究中心的共焦核心 (nih U54 HD083092 的支持)。肯尼迪国家儿童健康和人类发展研究所)。
Materials
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| Aspirator Tube Assemblies for Calibrated Microcapillary Pipettes | Sigma-Aldrich | A5177 | |
| BarnStead NanoPure Nuclease-free water | ThermoFisher | D119 | |
| Borosilicate Capillary Tubes | Sutter Instruments | GB1007515 | O.D. 1mm, I.D. 0.75mm, 1.5cm length |
| CaCl2 | Sigma-Aldrich | C5080 | |
| Cell-Repellent 96-Well Microplate | Greiner Bio-One | 650970 | U-bottom |
| Centrifuge 5424 R | Eppendorf | 5401000013 | |
| Chloroform | Sigma-Aldrich | 233306 | |
| Chromogranin-A antibody | Abcam | ab15160 | |
| Compact, Modular Stereo Microscope M60 | Leica | ||
| Countess Automated Cell Counter | Invitrogen | C10310 | |
| Countess Cell Counter Slides | Invitrogen | C10312 | |
| CryoStar NX70 | ThermoFisher | 957000L | |
| D-(+)-Glucose | Sigma-Aldrich | G7528 | |
| DAPI (4',6-Diamidine-2'-phenylindole-dihydrochloride) | Roche | 10 236 276 001 | Powder |
| DBA antibody | Vector Lab | RL-1032 | |
| Dispase II, Powder | Gibco | 17105041 | |
| DMEM/F-12, HEPES | Gibco | 11330032 | |
| Dnase I | Invitrogen | 18068-015 | |
| Dumont #5 Forceps | Fine Science Tools | 11251-10 | 0.05 x 0.02 mm; Titanium; Biology tip |
| EGF (Epidermal growth factor) | Sigma-Aldrich | E9644 | |
| Ethanol, 200 Proof | Decon Laboratories | 2716 | |
| Forma Steri Cycle CO2 Incubators | ThermoFisher | 370 | |
| Fluoromount-G | Southern Biotech | OB10001 | |
| Heparin sodium salt from porcine intestinal mucosa | Sigma-Aldrich | H3149-10KU | |
| INSM1 Antibody | Santa Cruz BioTechnology | sc-271408 | Polyclonal Mouse IgG |
| Isopropanol | Fisher | a4164 | |
| Isothesia Isoflurane, USP | Henry Schein | 11695-6776-2 | |
| Insulin Antibody | Dako | A056401 | Polyclonal Guinea Pig |
| KAPA SYBR FAST Universal | KAPA Biosystems | KK4618 | |
| KCl | KaryoMax | 10575090 | |
| KnockOut Serum Replacement | Invitrogen | 10828028 | |
| Leica TCS SPE High-Resolution Spectral Confocal | Leica | ||
| MgCl2 | Sigma-Aldrich | 442615 | |
| Mouse C-Peptide ELISA | ALPCO | 80-CPTMS-E01 | |
| Mouse Ultrasensitive Insulin ELISA | ALPCO | 80-INSMSU-E01 | |
| MX35 Microtome Blades | ThermoFisher | 3052835 | |
| NaCl | Sigma-Aldrich | S7653 | |
| NaHCO3 | Sigma-Aldrich | S3817 | |
| NaH2PO4 | Sigma-Aldrich | ||
| Normal Donkey Serum | Jackson Immuno Research | 017-000-121 | |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| PBS 1X | Corning | 21-040-CV | |
| Pdx1 antibody | DSHB | F6A11 | Monoclonal Mouse MIgG1 |
| Peel-A-Way Disposable Embedding Molds | VWR | 15160-157 | |
| Penicillin-Streptomycin Solution | Corning | MT30002CI | |
| PMA (Phorbol 12-Myristate 13-Acetate) | Sigma-Aldrich | P1585 | |
| Protein LoBind Microcentrifuge Tubes | Eppendorf | 22431081 | 1.5mL Capacity |
| Recombinant Human FGF-10 Protein | R&D Systems | 345-FG | |
| Recombinant Human FGF-Acidic | Peprotech | 100-17A | |
| Recombinant Human R-Spondin I Protein | R&D Systems | 4546-RS | |
| BenchRocker 2D | Benchmark | BR2000 | |
| Sucrose 500g | Sigma-Aldrich | S0389 | |
| SuperFrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
| Super Pap Pen | Electron Microscopy Sciences | 71310 | |
| Thermomixer R | Eppendorf | 05-412-401 | |
| Tissue Tek O.C.T. Compound | Sakura | 4583 | |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| TRIzol Reagent | Invitrogen | 15596018 | |
| TrypLE Express | Invitrogen | 12604039 | (1x), no Phenol Red |
| Trypan Blue Stain | Invitrogen | 15250061 | For cell counting slides |
| Trypsin-EDTA (0.05%) | Corning | 25-052-CI | |
| Trypsin-EDTA (0.25%) | Gibco | 25200072 | Phenol Red |
| Ultra-Low Attachment 24-Well Plate | Corning | 3473 | |
| Ultra-Low Attachment Spheroid Plate 96-Well | Corning | 4520 | |
| Vimentin Antibody | EMD Millipore | AB5733 | Polyclonal Chicken IgY |
| Vortex Genie | BioExpres | S-7350-1 | |
| Y-27632 Dihydrochloride | R&D Systems | 1254 | Also known as ROCK inhibitor |
| Zeiss 710 Confocal Microscope | Zeiss |
Referências
- Clevers, H. Modeling Development and Disease with Organoids. Cell. 165 (7), 1586-1597 (2016).
- Akkerman, N., Defize, L. H. Dawn of the organoid era: 3D tissue and organ cultures revolutionize the study of development, disease, and regeneration. Bioessays. 39 (4), (2017).
- Guo, T., Landsman, L., Li, N., Hebrok, M. Factors expressed by murine embryonic pancreatic mesenchyme enhance generation of insulin-producing cells from hESCs. Diabetes. 62 (5), 1581-1592 (2013).
- Landsman, L., et al. Pancreatic mesenchyme regulates epithelial organogenesis throughout development. PLoS Biology. 9 (9), 1001143 (2011).
- Lammert, E., Cleaver, O., Melton, D. Induction of pancreatic differentiation by signals from blood vessels. Science. 294 (5542), 564-567 (2001).
- Barker, N., et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell. 6 (1), 25-36 (2010).
- Sato, T., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 459 (7244), 262-265 (2009).
- Spence, J. R., et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature. 470 (7332), 105-109 (2011).
- Eiraku, M., et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature. 472 (7341), 51-56 (2011).
- Xia, Y., et al. The generation of kidney organoids by differentiation of human pluripotent cells to ureteric bud progenitor-like cells. Nature Protocols. 9 (11), 2693-2704 (2014).
- Takasato, M., et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature. 526 (7574), 564-568 (2015).
- Lancaster, M. A., et al. Cerebral organoids model human brain development and microcephaly. Nature. 501 (7467), 373-379 (2013).
- Loftus, S. K., et al. Acinar cell apoptosis in Serpini2-deficient mice models pancreatic insufficiency. PLoS Genetics. 1 (3), 38 (2005).
- Kleeff, J., et al. Chronic pancreatitis. Nat Rev Dis Primers. 3, 17060 (2017).
- Murtaugh, L. C., Melton, D. A. Genes, signals, and lineages in pancreas development. Annual Review of Cell and Developmental Biology. 19, 71-89 (2003).
- Greggio, C., De Franceschi, F., Figueiredo-Larsen, M., Grapin-Botton, A. In vitro pancreas organogenesis from dispersed mouse embryonic progenitors. Journal of Visualized Experiments. (89), 51725 (2014).
- Greggio, C., et al. Artificial three-dimensional niches deconstruct pancreas development in vitro. Development. 140 (21), 4452-4462 (2013).
- Sneddon, J. B., Borowiak, M., Melton, D. A. Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme. Nature. 491 (7426), 765-768 (2012).
- Scavuzzo, M. A., Yang, D., Borowiak, M. Organotypic pancreatoids with native mesenchyme develop Insulin producing endocrine cells. Scientific Reports. 7 (1), 10810 (2017).
- Murtaugh, L. C., Stanger, B. Z., Kwan, K. M., Melton, D. A. Notch signaling controls multiple steps of pancreatic differentiation. Proceedings of the National Academy of Sciences of the United States of America. 100 (25), 14920-14925 (2003).
- Nelson, S. B., Schaffer, A. E., Sander, M. The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells. Development. 134 (13), 2491-2500 (2007).
- Seymour, P. A., et al. SOX9 is required for maintenance of the pancreatic progenitor cell pool. Proceedings of the National Academy of Sciences of the United States of America. 104 (6), 1865-1870 (2007).
- Kawaguchi, Y., et al. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nature Genetics. 32 (1), 128-134 (2002).
- Hara, M., et al. Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells. American Journal of Physiology: Endocrinology and Metabolism. 284 (1), 177-183 (2003).
- Holthofer, H., Schulte, B. A., Spicer, S. S. Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney. Cell and Tissue Research. 249 (3), 481-485 (1987).
- Reichert, M., et al. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells. Nature Protocols. 8 (7), 1354-1365 (2013).
- Winkler, H., Fischer-Colbrie, R. The chromogranins A and B: the first 25 years and future perspectives. Neurociência. 49 (3), 497-528 (1992).
- Burcelin, R., Knauf, C., Cani, P. D. Pancreatic alpha-cell dysfunction in diabetes. Diabetes and Metabolism. 34, 49-55 (2008).
- Del Prato, S., Marchetti, P. Beta- and alpha-cell dysfunction in type 2 diabetes. Hormone and Metabolic Research. 36 (11-12), 775-781 (2004).
- Piciucchi, M., et al. Exocrine pancreatic insufficiency in diabetic patients: prevalence, mechanisms, and treatment. International Journal of Endocrinology. 2015, 595649 (2015).
- Campbell-Thompson, M., Rodriguez-Calvo, T., Battaglia, M. Abnormalities of the Exocrine Pancreas in Type 1 Diabetes. Current Diabetes Reports. 15 (10), 79 (2015).
- Shivaprasad, C., Pulikkal, A. A., Kumar, K. M. Pancreatic exocrine insufficiency in type 1 and type 2 diabetics of Indian origin. Pancreatology. 15 (6), 616-619 (2015).
Citar este artigo
Scavuzzo, M. A., Teaw, J., Yang, D., Borowiak, M. Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro. J. Vis. Exp. (136), e57599, doi:10.3791/57599 (2018).