为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法
Summary
在这里，我们描述了该朝向为下游应用和进一步分化的改善的定形内胚层致力于分化的人类胚胎干细胞的纯化方法。
Abstract
多能干细胞，如胚胎干细胞（ESC）的分化能力允许对细胞替代疗法的潜在的治疗应用。终末分化的细胞类型可以用于各种退行性疾病的治疗。 在体外 ，这些细胞向肺，肝脏和胰腺的组织的分化需要作为第一步定型内胚层细胞的产生。这个步骤是限速为朝向末端成熟细胞类型，如产生胰岛素的β细胞，肝细胞或其它内胚层来源的细胞类型进一步分化。即朝向内胚层谱系定型细胞高度表达的转录因子如FOXA2，SOX17，HNF1B，该GATA家族的成员，且表面受体CXCR4多种。然而，分化的协议很少是100％的效率。这里，我们描述了一种用于一CXCR4阳性细胞群的分化后的纯化入DE通过使用磁微珠。这种净化此外删除不需要的谱系的细胞。平缓的纯化方法是快速，可靠的，并且可以用于改善下游应用和分化。
Introduction
多能干细胞，如胚胎干细胞（ESC）具有分化成几乎人体的任何细胞类型的能力。因此， 在体外分化协议可以被用于产生许多成人细胞类型，如心肌1，肝细胞2，β细胞3，肺上皮4或神经元细胞5。这使得胚胎干细胞对各种退行性疾病3的潜在治疗的有价值的工具。
胚胎干细胞的朝向肺，肝脏和胰腺的成人组织中体外分化需要一个伪原肠胚成让人想起定形内胚层（DE）6的细胞。因为向上述体细胞类型的下游分化是显著效率较低，最佳的内胚层分化被视为限速7。被朝内胚层系致力于细胞进行查在他们的基因表达谱racteristic变化。多能性主控调节基因被下调，而其他转录因子如FOXA2，SOX17，HNF1B，该GATA家族的成员和表面受体CXCR4被高度上调6，8，9。CXCR4已知由SMAD2被反式表达/ 3，交点/ TGF-β信号传导和SOX17的下游，由于在其启动子区域10特异性结合位点。因此，它是在一些报告6,8 11-13中使用的非常合适的标记。这些表达的变化反映了伪原肠事件，其中胚胎干细胞首先获取原始的条纹状的细胞群的特点，并随后提交到内胚层胚层6。
然而，分化的协议很少是100％有效，因为一些细胞可能会抵制分化过程或向其他意外谱系分化14。这些细胞可能负面influeNCE进一步分化。此外，剩余的未分化细胞海港供以后移植实验很大的风险，并可能导致畸胎瘤15-17。
除去这些不想要的细胞早期的表面标记物CXCR4的可用于该朝向DE 18致力于细胞的纯化。在这里，我们描述了从DE分化培养物的CXCR4 +细胞的阳性选择的方法。对于这一点，表面标志物CXCR4的是由再依次结合至磁微珠的抗体结合。不像FACS分选过程中的恶劣条件下，磁性标记DE样细胞然后可以很容易地在一个台式格式使用温和的纯化方法进行纯化。这个协议提供了用于抵制对DE分化过程中除去细胞群的一个简单的方法。
Protocol
1.人类ESC分化对定形内胚层培养在培养箱中的人胚胎干细胞（ESC），在37℃和5％ 的 CO 2。 涂覆新的6孔细胞培养板用1ml基底膜基质孵育培养餐具在RT至少30分钟。有关具体细节，请谈谈各自制造商的说明。 确认该培养的人胚胎干细胞已达到使用低倍率（ 例如，4X）在显微镜下的80％-90％汇合。吸通过吸走培养基，用无菌玻璃巴斯德吸管从空腔的介质。用?…
Representative Results
在分化时 胚胎干细胞进行基因和蛋白质表达的急剧变化。 图1描绘了可用于验证成功定形内胚层分化典型的标记基因。对于基因表达分析的主要目标是GSC，FOXA2和 SOX17在一个相对的基因表达分析尤其是FOXA2和SOX17相比未分化的胚胎干细胞时，增加了> 2000倍。GSC原条的形成过程中已经表示24小时内很早但它仍?…
Discussion
目前使用的分化协议很少导致100％的分化的细胞。对于仍然有要解决的原因，某些细胞抗蚀分化过程。取决于所用的分化方案的效率和ESC的倾向排队甚至分化成定形内胚层后，通常观察到残余的多能细胞的一定数量。这些残余细胞会削弱下游分化或进一步的分析，如转录组学，蛋白质组学和miRNA表达分析。残留的多能干细胞或其它不想要的谱系也可以表现出可与分化目标干扰旁分泌的影响。因此?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
茉莉Kresse的熟练技术援助深表感谢。
Materials
| Hues8 human embryonic stem cell line | Harvard Department of stem cell & regenerative biology | NA | Suitable cell line for endoderm generation |
| Hes3 human embryonic stem cell line | ES Cell International | NA | Suitable and robust cell line for endoderm generation |
| mTeSR1 | Stemcell Technologies | 5850 | ESC culture medium |
| FCS | Biowest | S1860 | |
| Advanced RPMI 1640 | Life Technologies | 12633012 | |
| CD184 (CXCR4)-APC, human | Miltenyi Biotec | 130-098-357 | |
| anti-APC MicroBeads | Miltenyi Biotec | 130-090-855 | |
| OctoMACS Separator | Miltenyi Biotec | 130-042-109 | magnetic field |
| Y-27632 | Selleck Chemicals | S1049 | ROCK inhibitor |
| CHIR-99021 | Tocris Bioscience | 4423 | |
| Activin A | Peprotech | 120-14 | |
| Gentle Cell Dissociation Reagent | Stemcell Technologies | 7174 | Enzyme-free passaging solution, alternative: Trypsin/EDTA |
| Matrigel* | Corning | 354277 | basement membrane matrix * solve and store in aliquots at -80 °C as outlined in the suppliers manual. Upon use, thaw on ice, dilute in 25 ml ice-cold knockout DMEM/F-12. Add 1 ml to each well of a 6-well plate and incubate for 45 min at room temperature. Remove the matrigel and use immediately. |
| MS Columns | Miltenyi Biotec | 30-042-201 | |
| MACS Separator | Miltenyi Biotec | 130-042-302 | |
| Human FOXA2 FW gggagcggtgaagatgga |
Life Technologies | NA | |
| Human FOXA2 REV tcatgttgctcacggaggagta |
Life Technologies | NA | |
| Human GSC FW gaggagaaagtggaggtctggtt |
Life Technologies | NA | |
| Human GSC REV ctctgatgaggaccgcttctg |
Life Technologies | NA | |
| SOX17 TaqMan assay | Applied Biosystems | Hs00751752_s1 | |
| Human SOX7 FW gatgctgggaaagtcgtggaagg |
Life Technologies | NA | |
| Human SOX7 REV tgcgcggccggtacttgtag |
Life Technologies | NA | |
| Human POU5F1 FW cttgctgcagaagtgggtggagg |
Life Technologies | NA | |
| Human POU5F1 REV ctgcagtgtgggtttcgggca |
Life Technologies | NA | |
| Human Nanog FW ccgagggcagacatcatcc |
Life Technologies | NA | |
| Human Nanog REV ccatccactgccacatcttct |
Life Technologies | NA | |
| Human TBP FW caa cag cct gcc acc tta cgc tc |
Life Technologies | NA | |
| Human TBP REV agg ctg tgg ggt cag tcc agt g |
Life Technologies | NA | |
| Human TUBA1A FW ggc agt gtt tgt aga ctt gga acc c |
Life Technologies | NA | |
| Human TUBA1A REV tgt gat aag ttg ctc agg gtg gaa g |
Life Technologies | NA | |
| Human G6PD FW agg ccg tca cca aga aca ttc a |
Life Technologies | NA | |
| Human G6PD REV cga tga tgc ggt tcc agc cta t |
Life Technologies | NA | |
| Anti-SOX2 | Santa Cruz Biotechnology | sc-17320 | |
| Anti-FOXA2 | MerckMillipore | 07-633 | |
| Anti-SOX17 | R&D Systems | AF1924 | |
| NA = not applicable |
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Cite This Article
Davenport, C., Diekmann, U., Naujok, O. A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells. J. Vis. Exp. (109), e53655, doi:10.3791/53655 (2016).