使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生
Summary
通过使用非整合仙台病毒（SeV）载体介导的真皮成纤维细胞的重编程，实现人诱导的多能干细胞（hiPS）细胞的稳健衍生。使用重组人层粘连蛋白521（LN-521）基质和Essential E8（E8）培养基，使用无异种素和化学定义的培养条件进行hiPS细胞维持和克隆扩增。
Abstract
无性和完全定义的条件是稳定和可重复产生均质人诱导多能干细胞（hiPS）细胞的关键参数。饲养细胞或未定义基质上的hiPS细胞的维持易受批次差异，致病性污染和免疫原性风险的影响。使用定义的重组人层粘连蛋白521（LN-521）基质与无异种素和限定的培养基制剂组合减少变异性并允许hiPS细胞的一致产生。仙台病毒（SeV）载体是一种非整合的基于RNA的系统，因此规避了与基因组完整性整合载体可能具有的潜在破坏性影响相关的问题。此外，这些载体在真皮成纤维细胞的重新编程中已经表现出相对较高的效率。此外，细胞的酶促单细胞传代促进了hiPS细胞的均匀维持，而没有实质的茎干经验文化。在这里，我们描述了一个经过广泛测试和开发的方案，重点是重现性和易用性，为从成纤维细胞中产生定义和不含异种的人类hiPS细胞提供了一种可靠和实用的方法。
Introduction
由于Takahashi 等人首先推导出hiPS细胞系1,2 ，hiPS细胞为疾病建模，药物发现和用于在再生医学中产生细胞疗法的源材料提供了有用的工具3 。长期以来，HiPS细胞培养依赖于与成纤维细胞饲养细胞4,5或基质胶6和含有胎牛血清（FBS）的培养基配制物的共培养。批次差异是这些文化条件未定义性质的常见后果，导致不可预测的变化，这是这些协议不可靠性的主要原因7 。定义的培养基如基本8（E8） 8和定义的细胞培养基质例如LN-521 9的开发允许s用于建立高度可重复的方案，并有助于稳健的产生和维持均质的hiPS细胞7,8,9,10。
一体化免费重编程技术的发展已经跨越式发展。最初，重编程依赖于随机整合到基因组中的逆转录病毒载体，对基因组完整性具有破坏性影响11 。重编程方法的进展包括开发基于RNA的载体。 RNA载体相对于基于DNA的重编程方法具有优势，因为通过基因组重组的非预期整合是不可能的12 。 SeV载体通过没有DNA相11的单链RNA提供高和短暂的外源因子表达。由SeV交付的重编程载体在整个细胞扩增过程中被稀释，最终从培养物中脱落，提供了免打印方式的重编程。此后，多能性的维持依赖于多能性基因的内源性表达2 。
作为开创性的基于hiPS细胞的疗法正在开始进入临床试验，标准化批次，再现性和安全性的要求是解决13的重要问题。因此，应避免动物产地。例如，异种产品的使用与非人类病原体污染的风险相关。此外，在动物衍生的培养物组分存在下培养的细胞已经显示出将非人类硅酸掺入可能导致衍生细胞免疫原性的细胞膜中14 。因此，消除异种产品的需要对于任何未来的临床追求都是必要的。此协议适用于xe免疫和定义的培养在保持hiPS细胞移动细胞更接近临床依从性。
该协议描述了从成纤维细胞产生标准化的hiPS细胞的一致，高度可重现和易于使用的方法。它还提供了一种用户友好的文化系统，用于维护已建立的hiPS细胞。该协议已被用于在Karolinska机构的瑞典国家人类iPS核心设施中获得超过300个hiPS细胞系，其中一些线路之前已被描述为15,16 。
Protocol
Representative Results
Discussion
该协议的预期结果是成功生成几个克隆导出的hiPS细胞系。重要的是，这里描述的用于维持和扩增所建立的hiPS细胞的方法是可靠的，并且可以以少量干细胞培养的经验进行。已知使用ROCKi与LN-521基因进行单酶细胞传代，将细胞维持在核型正常，多能性和易于分化，同时避免诱导的异质性，基于集落的传代可刺激10,19,20。在LN-521上培养的hiPS细胞可以作为单细胞传代，而不添加ROCKi 10 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到SSF（B13-0074）和VINNOVA（2016-04134）资助者的支持。
Materials
| Dispase | Life Technologies | 171105-041 | Biopsy digestion |
| Collagenase | Sigma | C0130 | Biopsy digestion |
| Gelatin | Sigma | G1393 | Fibroblast matrix |
| IMDM | Life Technologies | 21980002-032 | Fibroblast medium |
| FBS | Invitrogen | 10270106 | Fibroblast medium |
| PenicillinStreptomycin | Life Technologies | 15070-063 | Antibiotic |
| Essential 8 media | ThermFisher Scientific | A1517001 | iPS cell culture media |
| LN-521 | Biolamina | LN521-03 | iPS cell culture matrix |
| TrypLE select 1X | ThermoFisher Scientific | 12563011 | Dissociation reagent |
| Rho-kinase inhibitor Y27632 | Millipore | SCM075 | Rho-kinase inhibitor (ROCKi) |
| CytoTune – iPS 2.0 reprogramming kit | Life Technologies | A1377801 | Sendai virus reprogramming vector |
| 35 mm tissue culture dish | Sarstedt | 83.3900.300 | Cell culture |
| 60 mm tissue culture dishes | Sarstedt | 83.3901.300 | Cell culture |
| 24 well tissue culture plates | Sarstedt | 83.3922.300 | Cell culture |
| T25 tissue culture flasks | VWR | 734-2311 | Cell culture |
| 15 ml tubes | Corning | 430791 | Centrifuge tubes |
| 1.5 ml tube | Eppendorf | 0030123.301 | 1.5 ml tube |
| CoolCell cell LX | Biocision | BCS-405 | Freezing container |
| DMSO | Sigma | D2650-100ml | Fibroblast freezing |
| Cryovials 1.8 ml | VWR | 479-6847 | Cryovials |
| PSC Cryopreservation Kit | Gibco | A2644601 | PSC CryomediumRevitaCell supplement |
| Trypsin-EDTA (0.05%) | ThermoFisherScientific | 25300054 | Fibroblast dissociation enzyme |
| DMEM/F12 | Life Technologies | 31331-028 | Digestive enzymes dilutant |
| DPBS | Life Technologies | 14190-094 | PBS |
| HBSS | ThermoFIsher Scientific | 14025-050 | Biopsy preparation |
| Haemocytometer | Sigma | BRAND, 718920 | Cell counting |
References
- Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131 (5), 861-872 (2007).
- Jin, Z. B., Okamoto, S., Xiang, P., Takahashi, M. Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling. Stem Cells Transl Med. 1 (6), 503-509 (2012).
- Zhou, H., Ding, S. Evolution of induced pluripotent stem cell technology. Curr Opin Hematol. 17 (4), 276-280 (2010).
- Thomson, J. A., et al. Embryonic stem cell lines derived from human blastocysts. Science. 282 (5391), 1145-1147 (1998).
- Hovatta, O., et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod. 18 (7), 1404-1409 (2003).
- Ludwig, T. E., et al. Feeder-independent culture of human embryonic stem cells. Nat Methods. 3 (8), 637-646 (2006).
- Rodin, S., et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat Biotech. 28 (6), 611-615 (2010).
- Chen, G., et al. Chemically defined conditions for human iPSC derivation and culture. Nat Meth. 8 (5), 424-429 (2011).
- Rodin, S., Antonsson, L., Hovatta, O., Tryggvason, K. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions. Nat Protoc. 9 (10), 2354-2368 (2014).
- Rodin, S., et al. Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nat Commun. 5, 3195 (2014).
- Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci. 85 (8), 348-362 (2009).
- Warren, L., et al. Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell. 7 (5), 618-630 (2010).
- Unger, C., Skottman, H., Blomberg, P., Dilber, M. S., Hovatta, O. Good manufacturing practice and clinical-grade human embryonic stem cell lines. Hum Mol Genet. 17 (R1), R48-R53 (2008).
- Martin, M. J., Muotri, A., Gage, F., Varki, A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med. 11 (2), 228-232 (2005).
- Kele, M., et al. Generation of human iPS cell line CTL07-II from human fibroblasts, under defined and xeno-free conditions. Stem Cell Research. 17 (3), 474-478 (2016).
- Uhlin, E., et al. Derivation of human iPS cell lines from monozygotic twins in defined and xeno free conditions. Stem Cell Research. 18, 22-25 (2017).
- . Biosafety in Microbiological and Biomedical Laboratories Available from: https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf (2009)
- Itskovitz-Eldor, J., et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med. 6 (2), 88-95 (2000).
- Chen, K. G., Mallon, B. S., McKay, R. D., Robey, P. G. Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell. 14 (1), 13-26 (2014).
- Jacobs, K., et al. Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability. Stem Cell Reports. 6 (3), 330-341 (2016).
- Schlaeger, T. M., et al. A comparison of non-integrating reprogramming methods. Nat Biotech. 33 (1), 58-63 (2015).
- . Ideal Xeno-Free Culture Conditions For Human Fibroblasts That Facilitate Efficient Reprogramming Available from: https://tools.thermofisher.com/content/sfs/posters/xeno-free-culture-conditions-fibroblasts-poster.pdf (2016)
- Trokovic, R., Weltner, J., Noisa, P., Raivio, T., Otonkoski, T. Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells. Stem Cell Res. 15 (1), 254-262 (2015).
