This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.
最近,iPS细胞已经引起关注,因为细胞再生疗法的新来源。尽管用于产生iPSCs的初始方法依赖于通过侵入性活组织检查和转基因的逆转录病毒基因组的插入得到的皮肤成纤维细胞,已经出现了许多努力来避免这些缺点。人外周血T细胞是唯一的小区源用于产生iPSCs的。从T细胞衍生的iPSC含有T细胞受体(TCR)基因的重排,并且抗原特异性T细胞的来源。此外,在基因组中的T细胞受体的重排有标记单个细胞系和移植的和供体细胞区分的可能性。为iPS细胞的安全的临床应用中,它以最小化暴露新产生iPSCs的有害试剂的风险是重要的。虽然胎牛血清和饲养细胞已为多能干细胞培养必需的,最好是从培养系统中删除他们减少风险不可预知的致病性。为了解决这个问题,我们已经建立了一个协议,用于使用仙台病毒,以减少暴露的iPSC未定义的病原体的风险产生于人类外周T细胞的iPS细胞。虽然处理仙台病毒要求设备具备相应的生物安全水平,仙台病毒感染活化的T细胞无基因组插入,但效率很高。在这个协议中,我们展示了使用活化的T细胞培养和仙台病毒的组合从人外周血T细胞在无饲养条件iPS细胞的产生。
iPS细胞引起了极大关注,因为细胞在再生医学1-3一个突破性的来源。迄今为止,用于产生iPSCs的多样的方法已被报道4,5。其中,从人T细胞产生iPSCs的已经因为细胞采样6-8的较少创伤的方法的特别的兴趣。此外,从iPSCs的T细胞衍生的含有T细胞受体(TCR)基因重排,因而抗原特异性T细胞9,10的来源。因此,产生的T细胞衍生的iPS细胞安全是正在进行再生医学有用。
此方法是基于减少不可预知的致病性的风险的概念。为iPS细胞的临床应用安全是很重要的,以减少病原体11暴露的风险。先前在多能干细胞的许多培养系统,胎牛血清和饲养细胞已被用来作为基本试剂第 12条。然而,去除这两个从培养系统试剂是优选的iPSC产生,以减少不可预知的致病性的危险。
此外,此方法具有避免从患者和饲养细胞的制备费力侵袭性细胞采样的优点。因为T细胞衍生的iPSCs已经被成功地用于在疾病研究13,14,这种方法也适用和有益的,从患者产生疾病特异性的iPSC。
间T细胞重新编程的方法,使用仙台病毒(SeV载体)载体作为基因载体是能够产生iPSCs的高效率7,16的方法。此外,由于SeV载体是一个单链RNA病毒,并且不需要的DNA相对于复制,其在的iPSC代使用避免断宿主基因组17-19。因此,我们已经建立的方案,用于从人外周血T细胞的iPSC在无血清的FRee值,并使用基质胶,mTeSR培养基的组合无饲养条件下,和SeV载体。
We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation.
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger.
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.
The authors have nothing to disclose.
我们感谢三宅义子,早矢香Kanaami,奇哈纳藤田,美穗山口,夏子Henmi,和丽大野耐一从庆应义塾大学医学院提供技术援助。这部分工作是由R&D Systems的支持计划,以加速实际使用的健康研究成果,以及高速公路项目为再生医学的实现提供资金。
Ficoll-Paque PREMIUM | GE Healthcare | 17-5442-02 | |
Purified NA/LE mouse anti-human CD3 | BD Pharmingen | 555336 | |
KBM502 medium | KOHJIN BIO | 16025020 | Warm in 37 ℃ water bath before use |
Bovine albumin fraction V solution | Gibco | 15260-037 | |
BD Matrigel Matrix Growth Factor Reduced | BD Biosciences | 354230 | Thaw completely at 4℃ overnight and dilute it 50 times with Dulbecco's Modified Eagle's Medium before coating culture dishes |
mTeSR1 medium kit | STEM CELL | 5850 | Warm at room temperature before use |
Dissociation Solution | ReproCELL | RCHETP002 | |
D-PBS(–) | Wako | 045-29795 | |
SeV Vector kit CytoTune-iPS ver.1.0 | DNAVEC | DV-0303c | Thaw on ice before use |
100-mm tissue culture dish | Falcon | 353003 | |
96-well tissue culture plate | Falcon | 353078 | |
6-well tissue culture plate | Falcon | 353046 | |
15ml Centrifuge Tube | Greiner Bio-One | 188271 | |
50ml Centrifuge Tube | Greiner Bio-One | 227261 |