小鼠胚胎干细胞的无血清单层培养神经细胞分化
Summary
This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.
Abstract
分化的小鼠胚胎干细胞的能力（ESC）的神经祖细胞允许控制神经规范以及产生成熟的神经细胞类型的进一步研究的机制的研究。在这个协议中，我们描述了ESC的使用无血清，单层培养神经祖细胞分化的方法。该方法具有可扩展性，高效率和结果在4内生产70％的神经祖细胞〜 – 6天。它可以从在各种条件下生长的各种菌株施加到ESC。神经祖细胞可以允许进一步分化为功能性神经元和神经胶质细胞或分析用显微镜，流式细胞仪或分子技术。分化过程是适合于延时显微镜，并且可以与使用记者行监测神经规范过程进行组合。我们提供培养基制备和细胞密度优化的详细说明，使该过程为bë施加到最ESC线和各种细胞培养容器。
Introduction
胚胎干细胞是从早期胚胎与体外无限增殖的能力，同时保留分化成以下再引入适当阶段胚胎所有成年细胞类型（通过形成嵌合体的能力），注射到同基因或免疫妥协的宿主衍生的多能细胞（通过形成畸胎瘤）或体外受到适当的线索1。 在体外分化的小鼠胚胎干细胞分化为神经谱系的于1995年首次被描述和所涉及的形成多细胞悬浮聚集体（胚体，胚）在补充有形态发生维甲酸2-4含血清介质。自那时以来，各种协议已被开发以允许神经分化5。许多人仍利用聚合，更为共培养与诱导细胞类型和几个涉及使用无血清介质。所有的协议有优势第二缺点和神经的确切性质或神经细胞产生的也有所不同，根据使用的协议。
理想的协议将是健壮的，可扩展和使用完全确定的培养基和基板，是适合于非侵入性监控分化过程的，并导致在神经祖细胞的纯群体的产生能够由外部线索和分化来图案成所有的神经元和神经胶质亚型具有高效率和产率在相当短的时间。在过去的十几年，我们一直在使用，用于从小鼠ESC神经祖细胞和神经元密度低，无血清贴壁单层培养6-10的方法。这个协议来完成许多上文所列的标准：在我们手中分化的效率已经相当一致多年来和各种细胞系，它可被放大或缩小（我们成功地使用从96孔板的船只直径15厘米迪畲族）和所使用的介质是良好定义的。这个过程是适合于缩时显微术为分化的监测和各种图案形成线索可加入以诱导不同类型的神经元亚型的的产生（ 例如，Shh和FGF8为多巴胺能神经元6）。
尽管如此，还是有一些挑战这一协议的成功应用。其中一个重要方面是精心准备的媒体。我们始终准备介质内部尽管商业替代品的可用性。其中所使用的添加剂的（N2;见协议 ）有修改过的标准的商用版本。最后，这种方法的成功应用的最重要的步骤之一是在电镀的最优的细胞密度。这主要是因为，而诱导信号中的一个（FGF4 11）的自分泌性质要 求足够细胞存在，以允许最佳viabil性和分化，在太高的密度分化受损（可能部分是由于自分泌生产LIF 12）。因此重要的是，这两个介质制备和细胞电镀仔细进行和一致，以确保最佳的效果。
Protocol
Representative Results
Discussion
单层神经分化协议已经使用了十多年6。该协议是高效率的，由确定的培养基，并在单层系统，使该系统更适用于临床前完成（ 例如，药物筛选）使用。然而，也有确定分化效率一些关键的因素。本文指出的那些因素，并针对每个障碍物的溶液。
电镀在分化条件后密度的细胞可能是最关键的因素。在太高的密度铺板细胞减少分化的效率，同时在太低的密度电镀?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Work was funded by a Development and Promotion of Science and Technology scholarship from the Thai Ministry for Education to W.W. and grants from Tenovus and the Anonymous Trust to M.P.S.
Materials
| Fetal bovine serum | Life Technologies | 10270-106 | |
| DMEM/F12 | Life Technologies | 11320-074 | |
| Neurobasal medium | Life Technologies | 21103-049 | |
| StemPro Accutase Cell Dissociation Reagent | Life Technologies | A11105-01 | |
| B-27 Supplement, serum free | Life Technologies | 17504-044 | |
| Insulin | Sigma | I6634 | Reconstitute with sterile 0.01M HCl |
| Apo-transferrin | Sigma | T1147 | Reconstitute with sterile water |
| Progesterone | Sigma | P8783 | Reconstitute with ethanol |
| Putrescine | Sigma | P5780 | Reconstitute with sterile water |
| Sodium selenite | Sigma | S5261 | Reconstitute with sterile water |
| Bovine albumin fraction V | Life Technologies | 15260-037 | |
| L-Glutamine | Life Technologies | 25030-081 | Make sure it is completely dissolved before use as glutamine is usually sedimented |
| Gelatine | Sigma | G1890 | |
| 6-well tissue culture dish | Thermo Scientific | 140675 | |
| 24-well tissue culture dish | Thermo Scientific | 142475 | |
| 96-well tissue culture dish | Thermo Scientific | 167008 | |
| GMEM | Life Technologies | 11710-035 | |
| Fetal bovine serum | Life Technologies | 10270-106 | |
| MEM Non-essential amino acids solution | Life Technologies | 11140-050 | |
| Sodium pyruvate | Life Technologies | 11360-070 | |
| 2-mercaptoethanol | Sigma | M7522 | |
| Leukaemia inhibitory factor | prepared in-house as in Smith 1991 Journal of Tissue Culture Methods | ||
| Tween-20 | Sigma | P1379 | |
| 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) | Sigma | D9542 | Protect from light |
| Mouse anti betaIII tubulin IgG antibody | Covance | MMS-435P | |
| Fluorescence-labelled anti-Mouse IgG antibody | Life Technologies | A31571 | Protect from light |
| 25cm2 tissue culture flask | Thermo Scientific | 156367 | |
References
- Smith, A. G. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol. 17, 435-462 (2001).
- Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342-357 (1995).
- Fraichard, A., et al. In vitro. differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 108, 3181-3188 (1995).
- Strubing, C., et al. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech. Dev. 53, 275-287 (1995).
- Stavridis, M. P., Smith, A. G. Neural differentiation of mouse embryonic stem cells. Biochem Soc Trans. 31, 45-49 (2003).
- Ying, Q. L., Stavridis, M., Griffiths, D., Li, M., Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol. 21, 183-186 (2003).
- Ying, Q. L., Smith, A. G. Defined conditions for neural commitment and differentiation. Methods Enzymol. 365, 327-341 (2003).
- Stavridis, M. P., Lunn, J. S., Collins, B. J., Storey, K. G. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. Development. 134, 2889-2894 (2007).
- Stavridis, M. P., Collins, B. J., Storey, K. G. Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation. Development. 137, 881-890 (2010).
- Speakman, C. M., et al. Elevated O-GlcNAc Levels Activate Epigenetically Repressed Genes and Delay Mouse ESC Differentiation Without Affecting Naive to Primed Cell Transition. Stem Cells. 32, 2605-2615 (2014).
- Kunath, T., et al. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development. 134, 2895-2902 (2007).
- Dani, C., et al. Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway. Dev Biol. 203, 149-162 (1998).
- Smith, A. G. Culture and differentiation of embryonic stem cells. Journal of Tissue Culture Methods. 13, 89-94 (1991).
- Aubert, J., et al. Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice. Proc. Natl. Acad. Sci. U. S. A. 100, 11836-11841 (2003).
- Niwa, H., Burdon, T., Chambers, I., Smith, A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048-2060 (1998).
- Silva, J., Smith, A. Capturing pluripotency. Cell. 132, 532-536 (2008).
