在常规免疫细胞化学鉴定无血清条件下人类iPS细胞的小规模的传播
Summary
Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.
Abstract
There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.
Introduction
重新编程人成年体细胞到诱导多能干细胞（iPS细胞）提供了一种方法，以获得患者特异性的细胞的潜在无限供给到研究疾病1,2。扼要体外的疾病表型将使得可信检验与疾病相关的细胞和分子机制，增强药物发现和个性化药物3。此外，人iPS细胞（iPS细胞）提供导出其可以用作唯一的资源来替换死亡或功能失调的细胞，并在几个障碍4的范围内，恢复功能的特定细胞类型的可能性。
以使用上述应用程序的iPSC的重要前提是，确保在培养膨胀过程中他们的多能性和分化状态被维持。通常情况下，techniq的UE，如流式细胞术，Western印迹，聚合酶链反应和功能分析，这需要大量的细胞和专用设备，用于IPSC的多能性6，7，8，9，10的详细的分析。然而，iPS细胞“未分化状态的常规评估可能有效地通过这些细胞的有限传播专门为免疫细胞化学（ICC）来实现，因此，涉及减少时间和资源。
最新进展允许的iPSC的所限定的无血清条件下生长的，它是在需要的鼠成纤维细胞饲养层和含血清介质常规培养系统一个显著改善。然而，目前的文献不包括描述如何尽快过渡馈线iPSCs的逐步明确协议层到无饲养系统。
在此背景下，本协议系统详细说明了如何生长在辐照的小鼠胚胎成纤维细胞人iPS细胞（IMEF）饲养层可以是（1），适于在无血清培养基中传播，和（2）在小规模培养的特别支持稳健免疫细胞化学分析。总体而言，这种方法代表了用于传播在无血清条件人类iPS细胞用于确认使用免疫细胞化学在常规基础上它们的多能及时和成本有效的方法。
Protocol
Representative Results
Discussion
这里介绍的系统协议提供了一个节省时间和成本效益的方法，在一个按比例缩小的培养技术的形式，专门通过免疫组化来支持有效的多能性的分析。
是所描述的方法的主要优点如下。需要传统上超过3至4个通道，以过渡从饲养层的iPSC馈线-自由培养条件，以消除使用散装解离技术后剩余的残余的iMEFs和典型单层形态出现12,13。与此相?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Funding Sources: The University of Arizona, The Jim Himelic Foundation, and the Arizona Center for the Biology of Complex Diseases.
Materials
| DMEM-F12/HEPES | Life Technologies | 11330032 | |
| Knockout Serum Replacement | Life Technologies | 10828028 | |
| L-Glutamine | Life Technologies | 25030081 | |
| MEM-NEAA | Life Technologies | 11140050 | |
| 2-mercaptoethanol | Life Technologies | 21985023 | |
| Recombinant Human FGF-Basic | Cell Sciences | CRF001B | |
| Y-27632 ROCK Inhibitor | R&D | 1254 | |
| Collagenase Type IV | Life Technologies | 17104019 | |
| Matrigel hESC-qualified Matrix | Corning | 354277 | |
| mTeSR1 Basal Medium | StemCell Technologies | 05850 | |
| mTeSR1 5X Supplement | StemCell Technologies | 05850 | |
| Gentle Cell Dissociation Buffer | StemCell Technologies | 07174 | |
| 0.1M PO4 Buffer | In-House | n/a | |
| Paraformaldeyde, prill | Electron Microscopy Sciences | 19202 | |
| 1X Phoshate Buffered Saline | n/a | n/a | |
| Normal Goat Serum | Life Technologies | 16210072 | |
| Bovine Serum Albumin | Sigma-Aldrich | A2153 | |
| Triton-X-100 | Sigma-Aldrich | X100 | |
| Oct-4A (C30A3) Rabbit mAb, Sox2 (D6D9) Rabbit mAb, SSEA4 (MC813) Mouse mAb, TRA-1-60(S) (TRA-1-60(S)) Mouse mAb |
Cell Signaling Cell Signaling Cell Signaling Cell Signaling |
2840 3579 4755 4746 |
Alternatively, a combination of 6 pluripotency primary antibodies can be purchased together as a kit in Catalog #9656 |
| Goat anti-Ms IgM Alexa Fluor 488 | Life Technologies | A21042 | |
| Goat anti-Ms IgG3 Alexa Fluor 488 | Life Technologies | A21151 | |
| Goat anti-Rb IgG Alexa Fluor 594 | Life Technologies | A11037 | |
| Multiwell Cell Culture Plates | Fisher Scientific | 0720080/0720081 | Available in 6, 12, 24, 48, 96 well sizes |
| Chamber Slides | Fisher Scientific | 12 565 21 | Available in Glass or Permanox Plastic in 1, 2, 4, 8, 16 well sizes |
| Coverglass for growth | Fisher Scientific | 12 545 82 | Available in 12, 15, 18, 22 and 25mm sizes |
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