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Nov 2019
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Derivation of Induced Pluripotent Stem Cells from Human Fibroblasts Using a Non-integrative System in Feeder-free Conditions
无饲养层条件下利用非整合系统从人成纤维细胞中衍生诱导多能干细胞   

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Abstract

Induced pluripotent stem cells (iPSCs) are genetically reprogrammed somatic cells that exhibit features identical to those of embryonic stem cells (ESCs). Multiple approaches are available to derive iPSCs, among which the Sendai virus is the most effective at reprogramming different cell types. Here we describe a rapid, efficient, safe, and reliable approach to reprogram human fibroblasts into iPSCs that are compatible with future iPSCs uses such as genome editing and differentiation to a transplantable cell type.

Keywords: Feeder-free (无饲养层), Fibroblasts (成纤维细胞), Sendai virus (仙台病毒), Induced pluripotent stem cells (诱导多能干细胞), Transgene-free (非转基因)

Background

Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells that exhibit morphological and functional qualities remarkably similar to those of embryonic stem cells (ESCs) (Takahashi and Yamanaka, 2006; Yu et al., 2007). They offer a great opportunity not only for disease modeling, but for the development of therapeutic strategies for pathologies that involve tissue degeneration. Furthermore, the iPSCs promise relies on a safe replenishable cell source derived in chemically defined media and free of random DNA integration.

Reprogramming somatic cells into iPSCs requires the forced expression of transcription factors that support the pluripotent state, including OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN-28 (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007). Multiple approaches are available to deliver the transcription factors into the cells, including those that require integration into the host chromosomes (Takahashi and Yamanaka, 2006; Kane et al., 2010). Exogenous DNA integration can lead to unpredictable effects on the quality of the cells and safety after transplantation. Other approaches include DNA based vectors that exist episomally (Yu et al., 2011; Weltner et al., 2012) and thus, decrease the possibility of integration, and finally those that do not integrate into the host genome and are known as transgene-free. The transgene-free methods include mRNA (Warren and Wang, 2013), recombinant protein (Zhou et al., 2009) and Sendai virus (Fusaki et al., 2009). Delivering the pluripotency transcription factors as mRNA or recombinant protein is poorly effective and costly. In contrast, Sendai virus, is a highly effective RNA virus that efficiently reprogram different types of somatic cells.

Sendai is a cytoplasmic RNA replication incompetent virus (SeV) that safely and effectively delivers the reprogramming factors into somatic cells, and does not integrate into the genome or alter the genetic information of the cell (Li et al., 2000; Fusaki et al., 2009). Furthermore, the virus is cleared out from the cells after a few passages assuring zero footprint of both the vectors and transgenes. The Sendai reprogramming system is commercially available as CytoTune iPS 2.0 for research purposes and as CTS CytoTune iPS 2.1 for clinical use. Both systems contain vectors encoding the four Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC) optimized for generating iPSCs from human somatic cells, making Sendai virus the most rapid, efficient and cost-efficient method to generate transgene-free iPSCs.

Reliable and safe derivation of human iPSCs relies on the use of defined and qualified reagents that allow a smooth transition to downstream technologies and is compatible with GMP (Good Manufacture Practice) quality standards for large-scale cell production. Here we describe a reprogramming approach for human fibroblasts that uses a non-integrative system, chemically defined culture medium in feeder-free conditions. This approach enables rapid, efficient, safe and reliable derivation of iPSCs compatible with future uses including genome editing and differentiation to a transplantable cell type.

Materials and Reagents

  1. Falcon® 6-Well Flat-Bottom Plate, Tissue Culture-Treated (Falcon, catalog number: 38016 )
  2. Serological pipettes multiple sizes (Corning)
  3. Falcon 15 ml conical centrifuge tubes (Falcon, catalog number: 352097 )
  4. Falcon 50 ml conical centrifuge tubes (Falcon, catalog number: 352070 )
  5. Cells: Human fibroblast expanded from skin biopsies preferentially at passage 1 to passage 5. Early passage fibroblast can be obtained from a commercial source (i.e., ATCC), a repository (i.e., Corriell Institute) or expanded in the lab from skin biopsies (Vangipuram et al., 2013 describes the procedure in detail)
  6. Corning® Matrigel® hESC-Qualified Matrix (Corning, catalog number: 354277 )
  7. CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit (Thermo Scientific, catalog number: A16518 )
  8. DMEM/F-12 (Gibco, catalog number: 11320033 )
  9. Fetal Bovine Serum (Gibco, catalog number: 16000044 )
  10. MEM NEAA (Gibco, catalog number: 11140050 )
  11. Pen Strep (Gibco, catalog number: 15-140-122 )
  12. mTeSRTM 1 (STEMCELL Technologies, catalog number: 85850 )
  13. TrypLETM Express Enzyme (1x) (Gibco, catalog number: 12605036 )
  14. Anti-Human TRA-1-60, Mouse monoclonal (Thermo Fisher Scientific, catalog number: 41-1000 )
  15. Anti-Human OCT4, Rabbit polyclonal, IgG1 (Abcam, catalog number: ab19857 )
  16. Anti-Human SOX2 Antibody, Rabbit polyclonal IgG1 (Abcam, catalog number: ab97959 )
  17. STEMdiff Trilineage Differentiation kit, STEMCELL Technologies, catalog number: 0 5230 )
  18. Universal Mycoplasma Detection Kit (ATCC, catalog number: 30-1012K )
  19. Dulbecco’s PBS (DPBS) without Calcium and Magnesium (Gibco, catalog number: 14190136 )
  20. Polybrene Hexadimethrine Bromide (Sigma-Aldrich, catalog number: H9268 )
  21. 0.5 M EDTA (Gibco, catalog number: 15575020 )
  22. Fibroblast culture medium (see Recipes)
  23. 5 mM EDTA solution (see Recipes)
  24. 10 mM Y27632 stock solution (see Recipes)

Equipment

  1. Tissue culture incubator HeracellTM 150i (Thermo Fisher Scientific, catalog number: 51026283 )
  2. Beckman Avanti J-30I Refrigerated with plate adapters (Beckman Coulter, catalog number: 17039 )
  3. Tissue culture laminar flow hood (NuAire Class II Type A2 NU-540)
  4. Evos XL core microscope (Thermo Scientific, catalog number: AMEX1100 )
  5. Countess II FL Automated Cell Counter (Thermo Fisher Scientific, catalog number: AMQAF1000 )
  6. Water bath Isotemp (Fisher Scientific, catalog number: S35936 )
  7. Portable Pipet-aid (Drummond, model: DP-101 , catalog number: 4-000-101)
  8. Micro pipettes (Eppendorf)

Procedure

Day -1: Plate dermal fibroblast for reprogramming
Use low-passage (passage 1 to 5) human fibroblasts for reprogramming experiments. The reprogramming efficiency decreases with each passage. Hence, we don’t recommend reprogramming fibroblast after passage 6. A schematic timeline and representative pictures can be seen in Figure 1.

  1. Coat two wells of a 6 well plate with 2 ml of 5 mg/ml Matrigel and make sure it covers the entire well. Incubate at 37 °C for 30 min.
  2. Inspect the fibroblast culture for the desired confluency (more than 70%), aspirate the culture medium and rinse twice with 2 ml DPBS.
  3. Add 500 µl TrypLETM Express enzyme and incubate at 37 °C and 5% CO2 for 2 to 5 min or until fibroblasts have detached. TrypLETM is used because it has lower cell toxicity than standard Trypsin and it is an animal-free product.
  4. Add 1 ml fibroblast culture medium and transfer cell suspension to a 15-ml conical tube.
  5. Centrifuge at 200 x g for 5 min. Remove and discard supernatant.
  6. Resuspend cells in 1 ml of fresh fibroblast culture medium. Recipe available in the recipe section.
  7. Count fibroblasts using the desired method (e.g., Countess Automated Cell Counter), and plate 1 x 105 cells/well (10,000 cells/cm2) in the Matrigel-coated 6-well dish in 2 ml of fibroblast culture medium.
  8. Incubate 24 h at 37 °C with a humidified atmosphere and 5% CO2.


    Figure 1. Derivation of induced pluripotent stem cells from fibroblast using Sendai virus and chemically defined medium. A. Schematic view of general reprogramming procedure including culture medium used. B. Images of fibroblast morphology changes during the early stages of reprogramming. Scale bars: 650 μm.

Day 0: Transduction
  1. Inspect the fibroblast culture, proceed with transduction if cell density is approximately 40-50% confluent, and cells have fully adhered and extended. If those parameters are not meet, wait another 24 h.
  2. Warm 1 ml of fibroblast culture medium in a water bath for each well to be transduced calculate the volume of each premade virus needed to reach a multiplicity of infection of (MOI) of 5:5:3 (KOS MOI = 5, hc-Myc MOI = 5, hKlf4 MOI = 3) using the live cell count from the day of the seeding, and the titer information on the CoA (calculate based on the lot number, the CoA can be download from thermofisher.com/cytotune). Calculate the volume of virus using the formula:

    CIU = Cell infectious units
    For example, to calculate the volume of hKOS virus with a titter of 1.1 x 108 CIU;


  3. Thaw one set of CytoTuneTM 2.0 Sendai aliquots from -80 °C and briefly centrifuge the tube. Place it immediately on ice until ready to use.
  4. Add the calculated volumes of each of the three CytoTuneTM 2.0 Sendai virus to 1 ml of pre-warmed fibroblast culture medium.
  5. OPTIONAL: Add 4 μg/ml of polybrene to the medium. Ensure that the solution of virus and polybrene is thoroughly mixed by pipetting the mixture gently up and down. Complete the next step within 5 min.Polybrene increases the transduction efficiency of the virus, however, it can be toxic for some cell types. It neutralizes the charge repulsion between the virus and the cell surface increasing the overall transduction efficiency.
  6. Aspirate the fibroblast culture medium from the cells, and add the reprogramming virus and polybrene mixture to the well containing the cells.
  7. Close the 6-well dish, securely place on the plate adaptor, and centrifuge at 1,200 x g for 45 min at room temperature. Once the centrifugation is complete, add an additional 1 ml of prewarm fibroblast culture medium.
  8. Incubate the plate overnight in a 37 °C incubator with a humidified atmosphere of 5% CO2.

Day 1: Remove virus
After 24 h, aspirate medium with viruses and add 2 ml fresh fibroblast culture medium. Expect to see cytotoxicity 24-48 h post-transduction, this is an indication of high uptake of the virus. See Figure 2.


Figure 2. Images of morphological changes seventy-two hours after transduction with Sendai virus. Scale bars: 650 μm.


Days 2 to 3
Remove 2 ml of used medium and add 2 ml of fibroblast culture medium. Expect to see changes in cell morphology.

Day 4: Transition to defined medium
There are many different chemically defined culture medium commercially available for deriving and maintaining iPSCs. We have used mTeSR1 medium (Stem Cell Technologies), STEMFLEX (Life technologies) and mTeSR plus (Stem Cell Technologies) agnostically.
  1. Remove 500 μl of fibroblast culture medium and add 500 μl of fresh mTeSR1.

Day 5
  1. Remove 1 ml of culture medium and add 1 ml of fresh mTeSR1.

Day 6
  1. Remove 1.5 ml of culture medium and add 1.5 ml of fresh mTeSR1.

Day 7
Remove 2 ml of medium culture medium and add the same volume of fresh mTeSR1.

Days 8-20
Feed cells daily with 2 ml until colonies are ready to be passaged. Remove partially reprogrammed and differentiated colonies by scraping them before medium change. Fully reprogrammed colonies have round shape with well-defined borders, cells display identical morphology with a high ratio of nucleus to cytoplasm and prominent nucleoli. In contrast, partially reprogrammed colonies have undefined borders, an amorphous shape, and are composed of different types of cells.

Day 21: First passage
  1. When colonies are ready to pick, prepare one Matrigel coated dish per colony to be passage, and incubate at 37 °C for at least 30 min.
  2. Aspirate Matrigel from plates and add 2 ml mTeSR1 + 10 µM ROCK inhibitor.
  3. Use a 22 gauge needle or a pulled glass pipette to cut colonies in a grid-like pattern into small fragments. See Figure 3.
  4. Use a 100 µl pipettor to scrape fragments and collect them. Immediately transfer the fragments to the Matrigel coated plates with mTeSR1 + 10 µM ROCK inhibitor.
  5. Rock plate back-and-forth and side-to-side to evenly distribute the cell fragments and incubate overnight at 37 °C with a humidified atmosphere of 5% CO2.
  6. Feed cells daily until ready to passage (usually every 6 to 7 days).
  7. Repeat Steps 1 to 10 for 7 passages. We found that > 95% of the iPSCs clones at passage 7, are transgene free.


    Figure 3. Images of colonies cut in a grid-like pattern into small fragments. Scale bars: 650 μm.

IPSCs cloning and expansion
After passage 7, IPSCs are passage with EDTA buffer, as follow:
  1. Coat 6 wells of a 6-well tissue culture plate with Matrigel and place in incubate at 37 °C for 30 min.
  2. Prior to use, allow the Matrigel-coated plate to equilibrate to room temperature for at least 1 h.
  3. Just before dissociating cells for passaging, aspirate the liquid Matrigel solution from the wells and replace with 1 ml of mTeSR1 + 10mM Y27632 cell culture media per well. Set aside.
  4. Wash the cells with 2 ml 0.5 mM EDTA, aspirate.
  5. Add 2 ml of room temperature 0.5 mM EDTA solution to the cells.
  6. Incubate the culture at 37 °C for 3 to 5 min, or until cells begin to separate uniformly throughout the entire colony. Do not allow the cultures detach in the EDTA solution.
  7. As soon as the cells appear rounded and uniform separation is seen throughout the colonies, carefully aspirate the EDTA solution from the well. Do not rinse.
  8. Immediately add 1 ml of mTeSR1 + 10mM Y27632. With a 5 ml pipet, take up the 1 ml of media from the well, and very gently dispense it against the culture surface to dissociate the cells from the dish. Repeat 1 to 2 more times, if needed.
  9. Be careful not to over-pipet the cell suspension.
  10. Dispense the cells gently into the 15 ml conical tube containing an additional 3 ml of pre-warmed media.
  11. Pipet the solution very gently 1 time to mix, and dispense 1 ml of the cell suspension drop-wise into each of the 6 new Matrigel-coated wells, and immediately rock plate back-and-forth and side-to-side to evenly distribute the colony pieces across the well.
  12. Incubate undisturbed at 37 °C and 5% CO2 overnight.
  13. Replace cell culture media every day with 2.5 ml of fresh mTeSR1, warmed to room temperature.
  14. Monitor cells daily and passage as needed.

Characterization and cryopreservation
Cells are expanded for cryopreservation at passage 10. Then, iPSCs are characterized (Figure 4) and prepare for downstream usage in genome engineering experiments or differentiation into specific cell types. Characterization includes:
  1. Immunofluorescence staining of cell surface markers such Anti-Human TRA-1-60, Mouse monoclonal and transcription factors such as anti-Human OCT4, Rabbit polyclonal, IgG1 and anti-Human SOX2 Antibody, Rabbit polyclonal IgG1.
  2. Pluripotency and trilineage differentiation capabilities (using the STEMdiff Trilineage Differentiation kit, and the Taqman hPSC Score card Panel.
  3. Test for mycoplasma contamination using commercially available kits such as The Universal Mycoplasma Detection Kit.
  4. G-banding Karyotype.


    Figure 4. Characterization of iPSC derived from fibroblast. A. Bright field images of iPSCs, B. Immunofluorescence staining for pluripotency markers Tra-1-60 and OCT4. C. Taqman ScoreCard assay for pluripotent iPSCs (left panel) and iPSCs differentiated into endoderm, mesoderm and ectoderm (right panel). D. G-banding karyotype.

Recipes

  1. Fibroblast culture medium
    The following recipe is to prepare 500 ml of fibroblast culture medium. Aseptically mix the following:
    445 ml high glucose DMEM
    50 ml heat-inactivated fetal bovine serum (FBS)
    5 ml non-essential amino acids
  2. 5 mM EDTA solution
    Dilute 0.5 ml of 0.5 M EDTA in 500 ml DPBS without Calcium and Magnesium. Store at room temperature
  3. 10 mM Y27632 stock solution
    Aseptically add 3.122 ml of sterile water to 10 mg of Y27632
    Mix thoroughly by pipetting, aliquot in Eppendorf’s and store at -80 °C
    Avoid repeated freezing and thawing

Acknowledgments

This protocol was developed as part of standard operations procedures of the Human Pluripotent Stem Cell core facility at UNC-CH. This work is supported by the UNC Office of Research.

Competing interests

The authors do not have any financial or non-financial interest in the subject matter or materials discussed in this manuscript.

Ethics

This research was carried out in according with the UNC-CH institutional guidelines.

References

  1. Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. and Hasegawa, M. (2009). 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.
  2. Kane, N. M., Nowrouzi, A., Mukherjee, S., Blundell, M. P., Greig, J. A., Lee, W. K., Houslay, M. D., Milligan, G., Mountford, J. C., von Kalle, C., Schmidt, M., Thrasher, A. J. and Baker, A. H. (2010). Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors. Mol Ther 18(12): 2139-2145.
  3. Li, H. O., Zhu, Y. F., Asakawa, M., Kuma, H., Hirata, T., Ueda, Y., Lee, Y. S., Fukumura, M., Iida, A., Kato, A., Nagai, Y. and Hasegawa, M. (2000). A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression. J Virol 74(14): 6564-6569.
  4. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5): 861-872.
  5. Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4): 663-676.
  6. Vangipuram, M., Ting, D., Kim, S., Diaz, R. and Schüle, B. (2013). Affiliations expand Skin punch biopsy explant culture for derivation of primary human fibroblasts. J Vis Exp (77): e3779.
  7. Warren, L. and Wang, J. (2013). Feeder-free reprogramming of human fibroblasts with messenger RNA. Curr Protoc Stem Cell Biol 27: Unit 4A 6.
  8. Weltner, J., Anisimov, A., Alitalo, K., Otonkoski, T. and Trokovic, R. (2012). Induced pluripotent stem cell clones reprogrammed via recombinant adeno-associated virus-mediated transduction contain integrated vector sequences. J Virol 86(8): 4463-4467.
  9. Yu, J., Chau, K. F., Vodyanik, M. A., Jiang, J. and Jiang, Y. (2011). Efficient feeder-free episomal reprogramming with small molecules. PLoS One 6(3): e17557.
  10. Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, II and Thomson, J. A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858): 1917-1920.
  11. Zhou, H., Wu, S., Joo, J. Y., Zhu, S., Han, D. W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Scholer, H. R., Duan, L. and Ding, S. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4(5): 381-384.

简介

[摘要] 诱导多能干细胞(iPSCs)是一种经过基因重组的体细胞,具有与胚胎干细胞(ESCs)相同的特性。有多种方法可以获得iPSCs,其中仙台病毒是最有效的重编程不同的细胞类型。在这里,我们描述了一种快速、高效、安全、可靠的方法,将人类成纤维细胞重新编程为与将来iPSCs相兼容的iPSCs,如基因组编辑和分化为可移植细胞类型。

[背景] 诱导多能干细胞(iPSCs)是经过基因重组的成体细胞,其形态和功能特性与胚胎干细胞(esc)非常相似(Takahashi和Yamanaka,2006;Yu等人,2007)。它们不仅为疾病建模提供了一个很好的机会,而且为涉及组织退化的病理学的治疗策略的发展提供了一个很好的机会。此外,iPSCs的承诺依赖于一个安全的可补充的细胞源,来源于化学定义的培养基中,并且没有随机的DNA整合。
将体细胞重编程为iPSCs需要强制表达支持多潜能状态的转录因子,包括OCT4、SOX2、KLF4、c-MYC、NANOG和LIN-28(Takahashi和Yamanaka,2006;Takahashi等人,2007;Yu等人,2007)。有多种方法可以将转录因子传递到细胞中,包括那些需要整合到宿主染色体中的方法(Takahashi and Yamanaka,2006;Kane等人,2010)。外源性DNA整合会对移植后细胞的质量和安全性产生不可预测的影响。其他方法包括以DNA为基础的载体(Yu等人,2011年;Weltner等人,2012年),因此减少了整合的可能性,最后是那些不整合到宿主基因组中的,被称为无转基因的载体。无转基因方法包括mRNA(Warren and Wang,2013)、重组蛋白(Zhou et al.,2009)和仙台病毒(Fusaki et al.,2009)。以mRNA或重组蛋白的形式传递多能性转录因子是一种低效且昂贵的方法。相比之下,仙台病毒是一种高效的RNA病毒,能有效地对不同类型的体细胞进行重组。
仙台病毒(Sendai)是一种胞质RNA复制不全病毒(SeV),它安全有效地将重编程因子传递到体细胞中,不会整合到基因组或改变细胞的遗传信息(Li等人,2000;Fusaki等人,2009)。此外,病毒在经过几次传代后被清除出细胞,保证了载体和转基因的零足迹。仙台重编程系统的市面上可作为CytoTune iPS 2.0进行研究,也可作为CTS CytoTune iPS 2.1用于临床应用。这两种系统都包含编码四种山中因子(OCT4、SOX2、KLF4和c-MYC)的载体,这些因子被优化用于从人类体细胞生成iPSCs,这使得仙台病毒成为产生无转基因iPSCs的最快速、最有效和最具成本效益的方法。
人类iPSCs的可靠和安全的衍生依赖于所定义和合格的试剂的使用,这些试剂允许顺利过渡到下游技术,并且符合大规模细胞生产的GMP(良好制造规范)质量标准。在这里,我们描述了一种人类成纤维细胞的重编程方法,它使用非整合系统,化学定义的培养基,在无饲养条件下。这种方法能够快速、高效、安全、可靠地衍生出与未来应用兼容的iPSCs,包括基因组编辑和向可移植细胞类型分化。

关键字:无饲养层, 成纤维细胞, 仙台病毒, 诱导多能干细胞, 非转基因

 材料和试剂
 
1.    Falcon®6孔平板,组织培养处理(Falcon,目录号:38016)
2.    多种尺寸的血清学移液管(康宁)
3.    Falcon 15毫升锥形离心管(Falcon,目录号:352097)
4.    Falcon 50毫升锥形离心管(Falcon,目录号:352070)
5.    细胞:人成纤维细胞在第1代到第5代从皮肤活检组织中优先扩增。早期传代成纤维细胞可从商业来源(即ATCC)、储存库(即Corriell Institute)获得,或在实验室从皮肤活检中扩增(Vangipuram等人,2013年详细描述了该过程)
6.    康宁®Matrigel®hESC合格矩阵(康宁,目录号:354277)
7.    CytoTuneTM iPS 2.0仙台重编程套件(Thermo Scientific,目录号:A16518)
8.    DMEM/F-12(Gibco,目录号:11320033)
9.    16000044牛血清(目录号:GIB44)
10.  MEM NEAA(Gibco,目录号:11140050)
11.  Pen Strep(Gibco,目录号:15-140-122)
12.  mTeSRTM 1(STEMCELL Technologies,目录号:85850)
13.  胰蛋白酶表达酶(1x)(Gibco,目录号:12605036)
14.  抗人TRA-1-60,小鼠单克隆抗体(Thermo Fisher Scientific,目录号:41-1000)
15.  抗人OCT4,兔多克隆,IgG1(Abcam,目录号:ab19857)
16.  抗人SOX2抗体,兔多克隆IgG1(Abcam,目录号:ab97959)
17.  STEMdiff Trilineage Differentication kit,STEMCELL Technologies,目录号:05230)
18.  通用支原体检测试剂盒(ATCC,目录号:30-1012K)
19.  不含钙和镁的Dulbecco PBS(DPBS)(Gibco,目录号:14190136)
20.  聚布伦-六甲基溴化铵(Sigma-Aldrich,目录号:H9268)
21.  0.5 M EDTA(Gibco,目录号:15575020)
22.  成纤维细胞培养基(见配方)
23.  5 mM EDTA溶液(见配方)
24.  10 mM Y27632原液(见配方)
 
设备
 
1.     组织培养培养箱HeracellTM 150i(赛默飞世尔科技公司,目录号:51026283)
2.     Beckman Avanti J-30I冷藏带板适配器(Beckman Coulter,目录号:17039)
3.     组织培养层流罩(NuAire II类A2 NU-540)
4.     Evos XL核心显微镜(Thermo Scientific,目录号:AMEX1100)
5.     Countess II FL自动电池计数器(Thermo Fisher Scientific,目录号:AMQAF1000)
6.     水浴恒温(Fisher Scientific,目录号:S35936)
7.     便携式移液管辅助器(Drummond,型号:DP-101,目录号:4-000-101)
8.     微量移液管(Eppendorf)
 
程序
 
第1天:培养真皮成纤维细胞进行重组
用低代(第1~5代)人成纤维细胞进行重编程实验。重编程效率随着每一段的增加而降低。因此,我们不建议在第6代之后重新编程成纤维细胞。图1显示了时间轴示意图和代表性图片。
1.    在6孔板的两个孔上涂抹2 ml 5 mg/ml的基质凝胶,并确保其覆盖整个孔。在37°C下培养30分钟。
2.    检查成纤维细胞培养物是否达到所需的融合度(大于70%),抽吸培养基并用2 ml DPBS冲洗两次。
3.    添加500µl胰蛋白酶,在37°C和5%CO2条件下培养2至5分钟或直到成纤维细胞分离。之所以使用胰蛋白酶,是因为它比标准胰蛋白酶具有更低的细胞毒性,而且是一种动物自由产品。
4.    加入1ml成纤维细胞培养基,将细胞悬液转移到15ml锥形管中。
5.    以200 x g离心5分钟。去除并丢弃上清液。
6.    在1ml新鲜成纤维细胞培养基中复苏细胞。配方部分提供的配方
7.    使用所需的方法(例如,Countess自动细胞计数器)计数成纤维细胞,并将1 x 105个细胞/孔(10000个细胞/cm2)置于2 ml成纤维细胞培养基中的基质凝胶涂层6孔培养皿中。
8.    在37°C和加湿空气和5%CO2下培养24小时。
 
 
图1。用仙台病毒和化学培养基从成纤维细胞中获得诱导多能干细胞。A、 一般重编程程序的示意图,包括所使用的培养基。B、 重组早期成纤维细胞形态学改变的图像。比例尺:650米。μ
 
第0天:转导
1.    检查成纤维细胞培养,如果细胞密度约为40-50%汇合,且细胞已完全粘附和伸展,则继续进行转导。如果这些参数不满足,请再等待24小时。
2.    在水浴中加热1ml成纤维细胞培养基,对于每个要转化的井,使用播种当天的活细胞计数,计算达到5:5:3(KOS-MOI=5,hc-Myc-MOI=5,hKlf4-MOI=3)的多重感染所需的每个预制病毒的体积,以及CoA上的效价信息(根据批号计算,CoA可从thermofisher.com/cytotune). 使用以下公式计算病毒体积:
 
 
 
细胞感染单位
例如,以1.1x108ciu的滴度计算hKOS病毒的体积;
 
 
 
3.    将一组CytoTuneTM 2.0仙台小份样品从-80℃解冻,并对试管进行短暂离心。立即将其放在冰上,直到可以使用。
4.    将三种CytoTuneTM 2.0仙台病毒的计算体积加入1毫升预热的成纤维细胞培养基中。
5.    可选择的聚丁二烯/ml添加到培养基中。用移液管轻轻上下移动,确保病毒和聚布伦的溶液完全混合。在5分钟内完成下一步。聚布伦可提高病毒的转导效率,但对某些细胞类型可能有毒。它能中和病毒与细胞表面的电荷排斥作用,提高整体转导效率。
6.    从细胞中吸取成纤维细胞培养基,将重编程病毒和聚布伦混合物加入含有细胞的孔中。
7.    关闭6孔培养皿,安全地放置在板适配器上,并在室温下以1200 x g离心45分钟。离心分离完成后,再加入1毫升预热的成纤维细胞培养基。
8.    将培养皿在37°C的培养箱中培养一晚,培养箱的湿度为5%CO2。
 
第一天:清除病毒
24h后,用病毒抽吸培养基,加入2ml新鲜成纤维细胞培养基。预期在转导后24-48小时出现细胞毒性,这是病毒高摄取的迹象。见图2。
 
 
图2。仙台病毒转导72小时后形态学改变图像。 比例尺:650μm。
 
第2-3天
取出2ml用过的培养基,加入2ml成纤维细胞培养基。希望能看到细胞形态的变化。
 
第4天:过渡到指定介质
有许多不同的化学定义的培养基商业上可用于衍生和维持iPSCs。我们使用了mTeSR1培养基(干细胞技术)、STEMFLEX(生命技术)和mTeSR plus(干细胞技术)。
1.     取出500μl成纤维细胞培养基,加入500μl新鲜mTeSR1。
 
 
第5天
1.     取出1毫升培养基,并加入1毫升新鲜的mTeSR1。
 
第6天
2.     取出1.5毫升培养基,并加入1.5毫升新鲜的mTeSR1。
 
第7天
取出2ml培养基,加入等量的新鲜mTeSR1。
 
第8-20天
每天喂细胞2ml,直到菌落准备好传代。在更换培养基之前,通过刮除部分重编程和分化的菌落。完全重编程的菌落呈圆形,边界清晰,细胞形态一致,核质比例高,核仁明显。相反,部分重编程的菌落边界不明确,呈无定形,由不同类型的细胞组成。
 
第21天:第一次通过
1.    当菌落准备好采摘时,为每个菌落准备一个基质凝胶包被的培养皿,并在37°C下培养至少30分钟。
2.    从板中吸取基质凝胶,并添加2 ml mTeSR1+10µM岩石抑制剂。
3.    用一根22号的针或一根玻璃吸管把网格状的菌落切成小块。见图3。
4.    使用100µl移液管刮取碎片并收集。立即将碎片转移到带有mTeSR1+10µM岩石抑制剂的基质凝胶涂层板上。
9.    前后摇动平板,使细胞碎片均匀分布,并在37°C和5%CO2的增湿空气中孵育过夜。
10.  每天给细胞喂食直到准备好传代(通常每6到7天)。
11.  对7个通道重复步骤1到10。我们发现在第7代,95%以上的iPSCs克隆是无转基因的。
 
 
图3。蜂群的图像被切割成网格状的小块。 比例尺:650μm。
 
 
IPSCs克隆和扩展
第7段为通道,EDTA后为缓冲区:
1.     在6孔组织培养板的6孔处涂上基质凝胶,并置于37°C下培养30分钟。
2.     在使用前,让基质凝胶涂层板在室温下平衡至少1小时。
3.     在分离细胞传代前,从孔中吸取液体基质凝胶溶液,每孔换成1mL mTeSR1+10mM Y27632细胞培养基。放在一边。
4.     用2ml 0.5mmEDTA清洗细胞,抽吸。
5.     向试管中添加2毫升室温0.5毫米EDTA溶液。
6.     将培养物在37℃孵育3-5分钟,或直到细胞开始在整个菌落中均匀分离。不允许EDTA溶液中的培养基分离。摄氏度
7.     一旦细胞呈圆形,并且在菌落中观察到均匀的分离,小心地从孔中吸取EDTA溶液。不要冲洗。
8.     立即添加1ml mTeSR1+10mM Y27632。用5毫升移液管从培养皿中取出1毫升培养基,轻轻地将其贴在培养表面上,使细胞与培养皿分离。如果需要,再重复1到2次。
9.     小心不要过度吸管细胞悬浮液。
10.  将细胞轻轻地分配到含有3毫升预热培养基的15毫升锥形管中。
11.  用移液管轻轻移取1次混合,并将1ml细胞悬浮液逐滴分配到6个新的基质凝胶涂层孔中,然后立即来回摇动平板,使菌落碎片均匀分布在孔内。
12.  在37°C和5%CO2的条件下静置培养过夜。
13.  每天用2.5 ml新鲜mTeSR1更换细胞培养基,加热至室温。
14.  每天监测细胞并根据需要传代。
 
表征和低温保存
在第10代时,细胞被扩大用于冷冻保存。然后,对iPSCs进行表征(图4),并为下游用于基因组工程实验或分化成特定的细胞类型做好准备。特征包括:
1.     细胞表面标记物如抗人TRA-1-60、小鼠单克隆和转录因子如抗人OCT4、兔多克隆、IgG1和抗人SOX2抗体、兔多克隆IgG1的免疫荧光染色。
2.     多潜能和三系分化能力(使用STEMdiff三系分化工具包和Taqman hPSC记分卡面板)。
3.     使用商用试剂盒(如通用支原体检测试剂盒)检测支原体污染。
4.     G显带核型。
 
 
图4。成纤维细胞来源iPSC的特性研究。A、 iPSCs的明亮视野图像,B。多能性标记物Tra-1-60和OCT4的免疫荧光染色。C、 Taqman记分卡法检测多潜能iPSCs(左图)和分化为内胚层、中胚层和外胚层的iPSCs(右图)。D、 核型。
 
食谱
 
1成纤维细胞1成纤维细胞培养基
以下配方为制备500ml成纤维细胞培养基。无菌混合以下物质:
445毫升高糖DMEM 50毫升热灭活胎牛血清(FBS)5毫升非必需氨基酸
2.    5 mM EDTA溶液
将0.5 ml 0.5 M EDTA稀释到500 ml DPBS中,不含钙和镁。室温下储存
3.    10 mM Y27632原液
无菌地将Y2122.ml加入10 mg无菌水
通过移液管彻底混合,将小份分份放入Eppendorf's中,并在-80°C下储存
避免反复冻融
 
致谢
 
该方案是作为UNC-CH人类多能干细胞核心设施标准操作程序的一部分而制定的。这项工作得到了UNC研究办公室的支持。
 
 
相互竞争的利益
 
作者对本手稿中讨论的主题或材料没有任何经济或非经济利益。
 
伦理学
 
这项研究是根据UNC-CH机构指南进行的。
 
工具书类
 
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Copyright Beltran et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Beltran, A. A., Molina, S. G. and Beltran, A. (2020). Derivation of Induced Pluripotent Stem Cells from Human Fibroblasts Using a Non-integrative System in Feeder-free Conditions. Bio-protocol 10(20): e3788. DOI: 10.21769/BioProtoc.3788.
  2. Battaglia, R. A., Beltran, A. S., Delic, S., Dumitru, R., Robinson, J. A., Kabiraj, P., Herring, L. E., Madden, V. J., Ravinder, N., Willems, E., Newman, R. A., Quinlan, R. A., Goldman, J. E., Perng, M.-D., Inagaki, M. and Snider, N. T. (2019). Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity. Elife: e47789.
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