共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法
Summary
目前的研究，介绍了胰腺人类胚胎干细胞分化诱导定向分化的方法。具有重要意义的发现，血管内皮细胞共培养介导派生为胰岛素表达细胞的胰腺祖细胞的人类胚胎干细胞的成熟。
Abstract
胚胎干细胞（ESC）上有两个主要特点是：它们可以无限期地在体外培植，在未分化的状态，他们是多能干细胞，因而有可能分化成多种细胞。这种特性使胚胎干细胞非常有吸引力的基于细胞治疗和再生治疗应用1。然而，对于充分发挥其潜力，实现细胞分化成成熟和功能的表型，这是一项艰巨的任务。诱导细胞分化的一个很有前途的方法是在体外设置紧密地模仿器官的路径。胰腺癌的发展被称为发生在特定的阶段，开始与内胚层，它可以发展成多个器官，包括肝脏和胰腺。内胚层诱导可以通过调制节点的通路，通过另外一个3激活素，多种生长因子结合4-7 </SUP>。明确的内胚层细胞，然后经过刺猬抑制实现由8环杷此外，它可以在体外抑制胰腺癌的承诺。是由几个平行的事件，包括抑制Notch信号通路介导胰腺成熟;胰腺祖细胞聚集成3维簇;诱导的血管;仅举几例。远远的ESC源性胰岛祖细胞体外成熟的最成功的已实现通过抑制Notch信号DAPT作用补充9。虽然成功了，这个结果中低产减少功能的成熟型。研究较少的地方，是内皮细胞在胰腺成熟细胞信号，这是越来越多地被作为一个重要的因素在体内胰岛成熟10,11赞赏的效果。
目前的研究，探讨内皮素等的影响人类ESC源性胰腺祖细胞的成熟胰岛素的胰岛样细胞胶质细胞信号。我们报告一个多阶段的人类胚胎干细胞首次对胚层诱导激活素抑制PI3K通路的一个定向分化协议。胰腺内胚层细胞的规范实现随着维甲酸诱导此外维甲酸抑制环杷刺猬信号。成熟的最后阶段被诱导内皮细胞共培养的配置实现信号。虽然一些内皮细胞已在共同的文化测试，在此我们提出我们的数据与大鼠心脏微血管内皮细胞（RHMVEC），主要是为便于分析。
Protocol
1。细胞维持 H1的人类胚胎干细胞（WiCell）均保持在合格的人类胚胎干细胞基底膜涂井mTeSR1媒体，与媒体每天都在变化。井被涂上稀释在25毫升的培养液中加入300μL的人类胚胎干细胞基底膜基底膜解决方案的准备，F12键。被添加到每个六孔板以及1毫升本matrigel的解决方案，或400μL加入到12孔板的每个井，并允许在室温下1小时大衣。机械代细胞刮，一旦他们达到了1至直径1.5mm的殖民地，在分?…
Discussion
胰腺发育期间，胰腺细胞的分化是在靠近主动脉内皮细胞;此外，胰岛细胞密集的血管，促进血糖和胰岛激素的迅速交流。鉴于这些事实，这并不奇怪，内皮细胞在胰腺器官的过程中发挥重要作用。虽然胰腺发育过程中血管内皮细胞的重要性日益受到重视，及其在胚胎干细胞体外分化中的作用研究。在我们前面的报告中，我们建立了小鼠胚胎干细胞的13对胰腺癌成熟内皮细胞?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们承认美国国立卫生研究院新的创新奖DP2 116520 ORAU拉尔夫·鲍威青年教师提升奖的支持。
Materials
| Name of the reagent | Company | Catalogue number | Concentrations |
| mTeSR1 (with supplement) | Stem cell Technologies | 5850 | |
| hESC qualified matrigel | BD Biosciences | 354277 | |
| DMEM:F12 | Invitrogen | 11330-032 | |
| MCDB-131 | Invitrogen | 10372019 | |
| MCDB-131 (Complete) | VEC Technologies | MCDB-131 | |
| B27 Supplement | Invitrogen | 17504044 | |
| Activin A | R&D | 338-AC | 100ng/ml |
| Wortmannin | Invitrogen | W3144 | 1μM |
| KAAD-Cyclopamie | Sigma-Aldrich | C4116 | 0.2μM |
| All-Trans Retinoic Acid | Sigma-Aldrich | R2625 | 2μM |
| DAPT | Sigma-Aldrich | D5942 | 30μM |
| Nicotinamide | Sigma-Aldrich | N0636 | 10 mM |
| Sodium Selenite | Sigma-Aldrich | S5261 | 30 nM |
| Insulin | Sigma-Aldrich | I1882 | 25 μg/ml |
| Transferrin | Sigma-Aldrich | T8158 | 50 μg/ml |
| EGF | R&D | 236-EG | 10ng/ml |
| EndoGro | VEC Technologies | ENDOGRO | 10mg |
| Heparin | Sigma-Aldrich | H3149 | 90μg/ml |
| Hydrocortisone | Sigma-Aldrich | H0888 | 1μg/ml |
| NucleoSpin RNA II | Macherey Nagel | 740955 | |
| ImProm II reverse transcription System | Promega | A3800 | |
| Brilliant II SYBR Green QPCR master mix | Straragene | 600548 | |
| Sox17 goat polyclonal IgG | Santa Cruz | sc-17355 | 1/500 |
| PDX1 goat polyclonal IgG | Santa Cruz | sc-14662 | 1/500 |
| C-Peptide Rabbit polyclonal | Cell Signaling | 4593 | 1/500 |
| Alexa Fluor 488 donkey anti-rabbit IgG | Invitrogen | A-21206 | 1/1000 |
| Alexa Fluor 647 donkey anti-goat IgG | Invitrogen | A-21447 | 1/1000 |
Table 2. Reagents and Kits.
Referências
- De Vos, J., Assou, S., Tondeur, S., Dijon, M., Hamamah, S. Les cellules souches embryonnaires humaines : de la transgression de l’embryon humain á la médecine régénératrice de demain. Gynécologie Obstétrique & Fertilité. 37, 620-626 (2009).
- Murtaugh, L. C., Melton, D. A. Genes, Signals, and Lineages in Pancreas Development. Annual Review of Cell and Developmental Biology. 19, 71-89 (2003).
- Kubo, A., Shinozaki, K., Shannon, J. M., Kouskoff, V., Kennedy, M., Woo, S., Fehling, H. J., Keller, G. Development of definitive endoderm from embryonic stem cells in culture. Development. 131, 1651-1662 (2004).
- D’Amour, K. A., Bang, A. G., Eliazer, S., Kelly, O. G., Agulnick, A. D., Smart, N. G., Moorman, M. A., Kroon, E., Carpenter, M. K., Baetge, E. E. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotech. 24, 1392-1401 (2006).
- Zhang, D., Jiang, W., Liu, M., Sui, X., Yin, X., Chen, S., Shi, Y., Deng, H. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res. 19, 429-438 (2009).
- Basma, H., Soto-Gutiérrez, A., Yannam, G. R., Liu, L., Ito, R., Yamamoto, T., Ellis, E., Carson, S. D., Sato, S., Chen, Y., Muirhead, D. Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes. Gastroenterology. 136, 990-999 (2009).
- Phillips, B. W., Hentze, H., Rust, W. L., Chen, Q. -. P., Chipperfield, H., Tan, E. -. K., Abraham, S., Sadasivam, A., Soong, P. L., Wang, S. T., Lim, R., Sun, W., Colman, A., Dunn, N. R. Directed Differentiation of Human Embryonic Stem Cells into the Pancreatic Endocrine Lineage. Stem Cells and Development. 16, 561-578 (2007).
- Kim, S. K., Melton, D. A. Pancreas development is promoted by cyclopamine, a Hedgehog signaling inhibitor. Proceedings of the National Academy of Sciences. 95, 13036-13041 (1998).
- Docherty, K. Growth and development of the islets of Langerhans: implications for the treatment of diabetes mellitus. Current Opinion in Pharmacology. 1, 641-649 (2001).
- Nikolova, G., Jabs, N., Konstantinova, I., Domogatskaya, A., Tryggvason, K., Sorokin, L., Fässler, R., Gu, G., Gerber, H. -. P., Ferrara, N., Melton, D. A. The Vascular Basement Membrane: A Niche for Insulin Gene Expression and [beta]> Cell Proliferation. Developmental Cell. 10, 397-405 (2006).
- Yoshitomi, H., Zaret, K. S. Endothelial cell interactions initiate dorsal pancreas development by selectively inducing the transcription factor Ptf1a. Development. 131, 807-817 (2004).
- Osafune, K., Caron, L., Borowiak, M., Martinez, R. J., Fitz-Gerald, C. S., Sato, Y., Cowan, C. A., Chien, K. R., Melton, D. A. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotech. 26, 313-315 (2008).
- Banerjee, I., Sharma, N., Yarmush, M. Impact of co-culture on pancreatic differentiation of embryonic stem cells. J. Tissue Eng. Regen. Med. 5, 313-323 (2010).
Tags
Endothelial Cell Co-cultureHuman Embryonic Stem CellsDirected Differentiation ApproachPancreatic Insulin Producing CellsEmbryonic Stem CellsPluripotentCell Based TherapyRegenerative TreatmentCellular DifferentiationOrganogenesisPancreas DevelopmentEndoderm InductionNodal Pathway ModulationActivin AGrowth FactorsSonic Hedgehog InhibitionCyclopaminePancreatic MaturationNotch Signaling InhibitionVascularization
Citar este artigo
Jaramillo, M., Banerjee, I. Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach. J. Vis. Exp. (61), e3759, doi:10.3791/3759 (2012).