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Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells
采用畸胎瘤形成分析法评估多能干细胞的多能性和致瘤性   

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Abstract

Pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) form teratomas when transplanted into immunodeficient mice. As teratomas contain all three germ layers (endoderm, mesoderm, ectoderm), teratoma formation assay is widely used as an index of pluripotency (Evans and Kaufman, 1981; Hentze et al., 2009; Gropp et al., 2012). On the other hand, teratoma-forming tumorigenicity also represents a major risk factor impeding potential clinical applications of pluripotent stem cells (Miura et al., 2009; Okano et al., 2013). Recently, we reported that iPSCs derived from naked mole-rat lack teratoma-forming tumorigenicity when engrafted into the testes of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice due to an ES cell-expressed Ras (ERAS) and Alternative reading frame (ARF)-dependent tumor-suppression mechanism specific to this species (Miyawaki et al., 2016). Here, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas for assessing the pluripotency and tumorigenicity.

Keywords: Pluripotent stem cells(多能干细胞), Teratoma(畸胎瘤), Tumorigenicity(致瘤性), NOD/SCID mice(NOD/SCID小鼠), Testis(睾丸)

Background

iPSCs and ESCs are exploited for applications in cell transplantation therapy for regenerative medicine. However, these cells form tumors called teratoma containing differentiated tissues when transplanted into immune-deficient mice. Therefore, the risk of their teratoma-forming tumorigenicity limits their clinical application. Several studies have reported the methods to overcome the risk of teratoma-forming tumorigeniticy (Itakura et al., 2017; Vazquez-Martin et al., 2012). Recently, we reported that iPSCs derived from naked mole-rats lack teratoma-forming tumorigenicity when engrafted into the testes of NOD/SCID mice due to species-specific activation of tumor-suppressor ARF and a disruption mutation of the oncogene ERAS (Miyawaki et al., 2016). In this protocol, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas. This approach can minimize the immune rejection due to the presence of the testicular–blood barrier (Cheng and Mruk, 2012). In addition, this approach is advantageous because transplanted cells are easily identified around the injection site even when they do not form tumors. Thus, the technique described herein is useful for assessing the pluripotency and tumorigenicity of pluripotent stem cells.

Materials and Reagents

  1. Falcon 15 ml conical tube (Corning, Falcon®, catalog number: 352196 )
  2. BIO-BIK 1.5 ml centrifuge tube (Ina-optika, catalog number: CF-0150 )
  3. 26-gauge needle (Terumo Medical, catalog number: NN-2613S )
  4. Laboratory wipe (Kimwipes) (KCWW, Kimberly-Clark, catalog number: 62011 )
  5. 0.22-μm filter unit (TPP Techno Plastic Products, catalog number: 99155 )
  6. 10 cm tissue culture dish (Corning, Falcon®, catalog number: 353046 )
  7. Pluripotent stem cells stably expressing a fluorescent marker, such as green fluorescent protein (GFP), for detection of injected cells
    Note: We used naked mole-rat (NMR) iPSCs (clones 24 and 27), mouse iPSCs (clones 20D17 and 38C2: Okita et al., 2007), and human iPSCs (clone 201B7: Takahashi et al., 2007), as described in our previous study (Miyawaki et al., 2016).
  8. Sterile phosphate-buffered saline (PBS) (NACALAI TESQUE, catalog number: 27575-31 )
  9. 0.1% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
  10. Fetal bovine serum (Biowest, catalog number: S1820-500 )
  11. Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D5796 )
  12. Penicillin/streptomycin (Wako Pure Chemical Industries, catalog number: 168-23191 )
  13. Non-essential amino acids (NACALAI TESQUE, catalog number: 06344-56 )
  14. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  15. L-Glutamine (NACALAI TESQUE, catalog number: 04260-64 )
  16. DMEM/F12 (Sigma-Aldrich, catalog number: D6421 )
  17. KnockOutTM Serum Replacement (Thermo Fisher Scientific, GibcoTM, catalog number: 10828028 )
  18. Fibroblast growth factor 2 (PeproTech, catalog number: 100-18B )
  19. Isoflurane (Wako Pure Chemical Industries, catalog number: 099-06571 )
  20. 70% ethanol
  21. Ampicillin (Wako Pure Chemical Industries, catalog number: 012-23303 )
  22. Paraformaldehyde (Junsei Chemical, catalog number: 58295-1201 )
  23. Sodium hydroxide (NACALAI TESQUE, catalog number: 31511-05 )
  24. Paraffin
  25. Hematoxylin and eosin (HE)
  26. iPS medium for mouse iPSCs (see Recipe 1)
  27. iPS medium for NMR and human iPSCs (see Recipe 2)
  28. 4% paraformaldehyde (PFA) (see Recipe 3)

Equipment

  1. Two humidified 5% CO2 cell culture incubators: 32 °C for NMR iPSCs, 37 °C for mouse and human iPSCs (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  2. Centrifuge (TOMY DIGITAL BIOLOGY, model: AX-501 )
  3. Coulter counter (Beckman Coulter, catalog number: 6605698 )
  4. Isoflurane vaporizer, chamber, and nose cone (Shinano, catalog number: SN-487-0T )
  5. Heating pad (Nissin, model: NHP-M30N )
  6. Operating scissors and tweezers (see Figure 1)
  7. 25 µl Hamilton syringe (Hamilton, model: 702 N, catalog number: 80400 )
  8. Microbalance (Shimadzu, model: ATX84 )
  9. Icebox


    Figure 1. Surgical instruments used in this protocol

Software

  1. GraphPad Prism software (GraphPad software, model: version 6.0)

Procedure

  1. Preparing cells for transplantation
    1. Maintain iPSCs according to the previous reports (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Miyawaki et al., 2016). It is necessary to prepare at least 2 x 106 cells. 
    2. Replace with fresh culture medium 1 h before dissociation.
    3. Aspirate culture medium and wash twice with PBS.
    4. Detach the cells using trypsin-EDTA (1 ml/10 cm dish).
    5. Stop the trypsin reaction with 15% fetal bovine serum-containing medium for mouse iPSCs (see Recipe 1) or trypsin inhibitor for NMR and human iPSCs (see Recipe 2).
    6. Collect cells in a 15 ml conical tube, and re-suspend cells in 4 ml of PBS.
    7. Count cells using a Coulter counter.
    8. Centrifuge cells at 200 x g for 5 min at room temperature.
    9. Aspirate supernatant and re-suspend iPSCs in an appropriate volume of PBS to yield a final concentration to 5 x 107 cells/ml (equal to 1 x 106 cells/20 μl).
    10. Transfer cells into a 1.5 ml tube.
    11. Place cells on ice until used for injection.

  2. Injection into mice
    1. (Optional) Administer ampicillin-containing water (1 g/L) orally to mice from 3 days before until 1 week after surgery.
    2. Place NOD/SCID mice on a heating pad and anesthetize with isoflurane.
    3. Wash surgical area with 70% ethanol and remove hair from the dorsal region.
    4. Make a 1 cm incision above the top of the preputial gland and open the abdomen.
    5. Carefully pull out the epididymal fat pad along with the testes (Figure 2A).
    6. Fill a Hamilton syringe with 20 µl (1 x 106 cells) of cell suspension.
    7. Cut the tunica vaginalis of the testis using a 26-gauge needle (Figure 2B).
    8. Use the Hamilton syringe to inject 20 μl of cell suspension (Figure 2C).
    9. Slowly remove the needle to avoid backflow of cells.
    10. Return the testes to their original location.
    11. Suture the wound.
    Note: Demonstration of the injection procedure can be viewed in Video 1.

    Video 1. Injection procedure. The cell suspension is blue stained for demonstration.

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  3. Defining pluripotency and tumorigenicity
    1. At 4, 10, 20, or 28 weeks after transplantation, anesthetize and sacrifice the mice in accordance with the Guide for the Care and Use of Laboratory Animals. Thereafter, dissect the tumors and testes (Figures 2D and 2E).
      Note: Tumors could be observed 4 weeks and 10 weeks after the injection of mouse iPSCs and human iPSCs, respectively.


      Figure 2. Injection iPSCs into testis of NOD/SCID mice. Transplant procedure (A-C). Representative photographs of teratoma (D) and testis without tumor formation (E). Arrowhead indicates teratoma formed by mouse iPSCs.

    2. After removing moisture within the laboratory wipe, measure the weight of the tumor.
    3. Fix the tumors or testes overnight in PBS containing 4% PFA (see Recipe 3) for embedding in paraffin.
    4. Stain sections with hematoxylin and eosin (HE), or subject to immunohistochemical analysis, to detect expression of GFP and differentiation markers. The section of tumors and testes in our previous study are shown in Figure 3 (Miyawaki et al., 2016).


      Figure 3. Image of HE and immunohistochemical stained section of tumors and testis after transplantation of iPSCs. HE staining section of tumors and testis of mice transplanted with NMR iPSCs 10 weeks after transplantation (A and D), mouse iPSCs 4 weeks after transplantation (B) or human iPSCs 10 weeks after transplantation (C). Immunohistochemical analysis of GFP (E). Arrowheads indicate the transplanted GFP-labeled NMR iPSCs. Scale bar, 200 µm (A-C) and 1 cm (D and E).

Data analysis

The weights of tumors or testes were analyzed after logarithmic transformation (arbitrary units: 6 + log2). The Bartlett test was used to verify equal variances across populations. The statistical significance of the difference between experimental groups was determined by one-way analysis of variance or the Kruskal Wallis test followed by the Dunn’s method using GraphPad Prism 6 software. Statistical significance was considered when P < 0.05 and all data are displayed as mean ± SEM as in Miyawaki et al., 2016.
Weight of tumors and testes in our previous study are shown in Table 1 for reference (Miyawaki et al., 2016).

Table 1. Weight of each teratoma or testis, and mean weight

Recipes

  1. Mouse iPSCs medium
    Dulbecco’s modified Eagle’s medium (Sigma-Aldrich)
    15% fetal bovine serum (JRH or BioWest)
    1% penicillin/streptomycin (Wako Pure Chemical Industries)
    2 mM L-glutamine (NACALAI TESQUE)
    0.1 mM non-essential amino acids (NACALAI TESQUE)
    0.1 mM β-mercaptoethanol (Sigma-Aldrich)
    Human recombinant leukemia inhibitory factor (NACALAI TESQUE)
    Filtered by 0.22-μm filter unit, stored at 4 °C
  2. NMR and human iPSCs medium
    DMEM/F12 (Sigma-Aldrich)
    20% KnockOut Serum Replacement (Thermo Fisher Scientific)
    1% penicillin/streptomycin (Wako Pure Chemical Industries)
    2 mM L-glutamine (NACALAI TESQUE)
    0.1 mM non-essential amino acids (NACALAI TESQUE)
    0.1 mM β-mercaptoethanol (Sigma-Aldrich)
    4 ng/ml fibroblast growth factor 2 (PeproTech)
    Filtered by 0.22-μm filter unit, stored at 4 °C
  3. 4% PFA
    4 g PFA
    100 ml of PBS
    Heat on hot plate stirrer
    Add 20 μl of 1 M sodium hydroxide to dissolve residual PFA
    Aliquot and freeze at -20 °C for storage
    Note: The investigator must wear a facemask, gloves, lab coat and eye protection during PFA preparation.

Acknowledgments

This work was supported in part by PRESTO of the Japan Science and Technology Agency, Grants-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Grant-in-Aid for Scientific Research on Innovative Areas ‘Oxygen Biology: a new criterion for integrated understanding of life’ from the MEXT to K.M., and K.M. and S.M. were Research Fellows of the Japanese Society for the Promotion of Science.

References

  1. Cheng, C. Y. and Mruk, D. D. (2012). The blood-testis barrier and its implications for male contraception. Pharmacol Rev 64(1): 16-64.
  2. Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292(5819): 154-156.
  3. Gropp, M., Shilo, V., Vainer, G., Gov, M., Gil, Y., Khaner, H., Matzrafi, L., Idelson, M., Kopolovic, J., Zak, N. B. and Reubinoff, B. E. (2012). Standardization of the teratoma assay for analysis of pluripotency of human ES cells and biosafety of their differentiated progeny. PLoS One 7(9): e45532.
  4. Hentze, H., Soong, P. L., Wang, S. T., Phillips, B. W., Putti, T. C. and Dunn, N. R. (2009). Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. Stem Cell Res 2(3): 198-210.
  5. Itakura, G., Kawabata, S., Ando, M., Nishiyama, Y., Sugai, K., Ozaki, M., Iida, T., Ookubo, T., Kojima, K., Kashiwagi, R., Yasutake, K., Nakauchi, H., Miyoshi, H., Nagoshi, N., Kohyama, J., Iwanami, A., Matsumoto, M., Nakamura, M. and Okano, H. (2017). Fail-safe system against potential tumorigenicity after transplantation of iPSC derivatives. Stem Cell Reports 8(3): 673-684.
  6. Miura, K., Okada, Y., Aoi, T., Okada, A., Takahashi, K., Okita, K., Nakagawa, M., Koyanagi, M., Tanabe, K., Ohnuki, M., Ogawa, D., Ikeda, E., Okano, H. and Yamanaka, S. (2009). Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 27(8): 743-745.
  7. Miyawaki, S., Kawamura, Y., Oiwa, Y., Shimizu, A., Hachiya, T., Bono, H., Koya, I., Okada, Y., Kimura, T., Tsuchiya, Y., Suzuki, S., Onishi, N., Kuzumaki, N., Matsuzaki, Y., Narita, M., Ikeda, E., Okanoya, K., Seino, K., Saya, H., Okano, H. and Miura, K. (2016). Tumour resistance in induced pluripotent stem cells derived from naked mole-rats. Nat Commun 7: 11471.
  8. Okano, H., Nakamura, M., Yoshida, K., Okada, Y., Tsuji, O., Nori, S., Ikeda, E., Yamanaka, S. and Miura, K. (2013). Steps toward safe cell therapy using induced pluripotent stem cells. Circ Res 112(3): 523-533.
  9. Okita, K., Ichisaka, T. and Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature 448(7151): 313-317.
  10. 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.
  11. 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.
  12. Vazquez-Martin, A., Cufi, S., Lopez-Bonet, E., Corominas-Faja, B., Oliveras-Ferraros, C., Martin-Castillo, B. and Menendez, J. A. (2012). Metformin limits the tumourigenicity of iPS cells without affecting their pluripotency. Sci Rep 2: 964.

简介

多能干细胞,如诱导多能干细胞(iPSCs)和胚胎干细胞(ESC),当移植到免疫缺陷小鼠时,形成畸胎瘤。由于畸胎瘤包含所有三个胚层(内胚层,中胚层,外胚层),畸胎瘤形成测定被广泛用作多能性的指标(Evans和Kaufman,1981; Hentze等,2009; Gropp等,2012)。另一方面,畸胎瘤形成致瘤性也是阻碍多能干细胞潜在临床应用的主要危险因素(Miura et al。,2009; Okano等,2013)。最近,我们报道了由于ES细胞表达的Ras(ERAS)和替代物,嫁接到非肥胖型糖尿病/严重联合免疫缺陷型(NOD / SCID)小鼠的睾丸中,从裸鼠睾丸衍生的iPSC缺乏畸胎瘤形成致瘤性阅读框(ARF)依赖于该物种特异性的肿瘤抑制机制(Miyawaki等,2016)。在这里,我们描述了将多能干细胞移植到NOD / SCID小鼠的睾丸中以产生用于评估多能性和致瘤性的畸胎瘤的方法。
【背景】iPSCs和ESC用于再生医学细胞移植治疗中的应用。然而,当移植到免疫缺陷小鼠中时,这些细胞形成称为含有分化组织的畸胎瘤的肿瘤。因此,其畸胎瘤形成致瘤性的风险限制了其临床应用。几项研究报道了克服畸胎瘤形成肿瘤发生风险的方法(Itakura et al。,2017; Vazquez-Martin et al。,2012)。最近,我们报道,由于由裸鼠睾丸造成的iPSC由于肿瘤抑制因子ARF的物种特异性激活和致癌基因ERAS的破坏突变(Miyawaki et al),因此在嫁接到NOD / SCID小鼠的睾丸中时,缺乏畸胎瘤形成致瘤性。,2016)。在该方案中,我们描述了将多能干细胞移植到NOD / SCID小鼠的睾丸中以产生畸胎瘤的方法。这种方法可以减少由于睾丸 - 血液屏障的存在引起的免疫排斥(Cheng和Mruk,2012)。此外,这种方法是有利的,因为即使不形成肿瘤,移植细胞也容易在注射部位周围识别。因此,本文所述的技术可用于评估多能干细胞的多能性和致瘤性。

关键字:多能干细胞, 畸胎瘤, 致瘤性, NOD/SCID小鼠, 睾丸

材料和试剂

  1. Falcon 15 ml锥形管(Corning,Falcon ®,目录号:352196)
  2. BIO-BIK 1.5ml离心管(Ina-optika,目录号:CF-0150)
  3. 26号针(Terumo Medical,目录号:NN-2613S)
  4. 实验室擦拭(Kimwipes)(KCWW,Kimberly-Clark,目录号:62011)
  5. 0.22μm过滤器(TPP Techno Plastic Products,目录号:99155)
  6. 10厘米组织培养皿(Corning,Falcon ®,目录号:353046)
  7. 稳定表达荧光标记物(如绿色荧光蛋白(GFP))的多能干细胞用于检测注射细胞
    注意:我们使用赤裸鼠(NMR)iPSC(克隆24和27),小鼠iPSC(克隆20D17和38C2:Okita等人,2007)和人iPSC(克隆201B7:Takahashi等人, 2007),如我们以前的研究(Miyawaki等人,2016)所述。
  8. 无菌磷酸盐缓冲盐水(PBS)(NACALAI TESQUE,目录号:27575-31)
  9. 0.1%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:15090046)
  10. 胎牛血清(Biowest,目录号:S1820-500)
  11. Dulbecco改良的Eagle's培养基(Sigma-Aldrich,目录号:D5796)
  12. 青霉素/链霉素(Wako Pure Chemical Industries,目录号:168-23191)
  13. 非必需氨基酸(NACALAI TESQUE,目录号:06344-56)
  14. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  15. L-谷氨酰胺(NACALAI TESQUE,目录号:04260-64)
  16. DMEM / F12(Sigma-Aldrich,目录号:D6421)
  17. KnockOut TM 血清置换(Thermo Fisher Scientific,Gibco TM,目录号:10828028)
  18. 成纤维细胞生长因子2(PeproTech,目录号:100-18B)
  19. 异氟烷(Wako Pure Chemical Industries,目录号:099-06571)
  20. 70%乙醇
  21. 氨苄青霉素(Wako Pure Chemical Industries,目录号:012-23303)
  22. 多聚甲醛(Junsei Chemical,目录号:58295-1201)
  23. 氢氧化钠(NACALAI TESQUE,目录号:31511-05)
  24. 石蜡
  25. 苏木精和曙红(HE)
  26. 用于iPSC鼠标的iPS介质(参见配方1)
  27. 用于核磁共振和人类iPSCs的iPS介质(参见方法2)
  28. 4%多聚甲醛(PFA)(见配方3)

设备

  1. 两个加湿的5%CO 2细胞培养箱:用于NMR iPSC的32℃,用于小鼠和人iPSC的37℃(Thermo Fisher Scientific,Thermo Scientific&amp; Heracell TM 150i)
  2. 离心机(TOMY DIGITAL BIOLOGY,型号:AX-501)
  3. 库尔特计数器(Beckman Coulter,目录号:6605698)
  4. 异氟烷蒸发器,腔室和鼻锥(Shinano,目录号:SN-487-0T)
  5. 加热垫(Nissin,型号:NHP-M30N)
  6. 操作剪刀和镊子(见图1)
  7. 25μlHamilton注射器(Hamilton,型号:702N,目录号:80400)
  8. 微量天平(Shimadzu,型号:ATX84)
  9. 冰箱


    图1.本协议中使用的手术仪器

软件

  1. GraphPad PRISM软件(GraphPad软件,型号:6.0版)

程序

  1. 准备移植细胞
    1. 根据先前的报告(Takahashi和Yamanaka,2006; Takahashi等人,2007; Miyawaki等人,2016)保持iPSC。有必要准备至少2×10 6个单元格。
    2. 在解离前1小时更换新鲜培养基
    3. 吸出培养基,用PBS洗涤两次
    4. 使用胰蛋白酶-EDTA(1ml / 10cm皿)分离细胞。
    5. 停止与用于小鼠iPSC的15%胎牛血清培养基(见配方1)或用于NMR和人iPSC的胰蛋白酶抑制剂的胰蛋白酶反应(参见方法2)。
    6. 将细胞收集在15ml锥形管中,并将细胞重新悬浮在4ml PBS中
    7. 使用库尔特计数器计数单元格。
    8. 在室温下将细胞以200×g离心5分钟。
    9. 吸出上清液并将iPSC再次悬浮在适当体积的PBS中,以终浓度至5×10 7细胞/ ml(等于1×10 6细胞/ 20 μl)
    10. 将细胞转移到1.5ml管中
    11. 将细胞置于冰上直至注射使用。

  2. 注射入小鼠
    1. (可选)在手术前3天直到手术后1周,向小鼠口服氨苄青霉素含水(1 g / L)。
    2. 将NOD / SCID小鼠放在加热垫上,用异氟烷麻醉
    3. 用70%乙醇清洗手术区域,并从背部区域去除头发
    4. 在注射腺顶部上方切开1厘米的切口并打开腹部。
    5. 仔细拔出睾丸附睾脂肪垫(图2A)。
    6. 用20μl(1×10 6个细胞)的细胞悬液填充汉密尔顿注射器。
    7. 使用26号针切割睾丸阴道分泌物(图2B)
    8. 使用汉密尔顿注射器注射20μl细胞悬浮液(图2C)
    9. 慢慢取下针头以避免细胞回流。
    10. 将睾丸返回原来的位置。
    11. 缝合伤口。
    注意:可以在视频1中查看注射过程的演示。

    Video 1. Injection procedure. The cell suspension is blue stained for demonstration.

    To play the video, you need to install a newer version of Adobe Flash Player.

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  3. 定义多能性和致瘤性
    1. 移植后4,10,20或28周,按照“实验动物护理和使用指南”麻醉并处死小鼠。此后,解剖肿瘤和睾丸(图2D和2E) 注意:分别注射小鼠iPSC和人iPSC后4周和10周可观察到肿瘤。


      图2.将iPSCs注射到NOD / SCID小鼠的睾丸中。 移植手术(A-C)。畸胎瘤(D)和无肿瘤形成的睾丸的代表性照片(E)。箭头表示由小鼠iPSC形成的畸胎瘤。

    2. 除去实验室擦拭物中的水分后,测量肿瘤的重量
    3. 在含有4%PFA的PBS(见方案3)中将肿瘤或睾丸固定过夜,以便包埋在石蜡中。
    4. 用苏木精和伊红(HE)进行染色切片,或进行免疫组织化学分析,检测GFP和分化标记物的表达。我们以前研究中的肿瘤和睾丸部分如图3所示(Miyawaki等人,2016)。


      图3.移植后iPSCs肿瘤和睾丸HE和免疫组织化学染色切片的图像。移植后10周,用iPSCs移植的小鼠的肿瘤和睾丸的HE染色切片(A和D),小鼠iPSCs移植后4周(B)或人iPSCs移植后10周(C)。 GFP(E)的免疫组织化学分析。箭头表示移植的GFP标记的NMR iPSC。刻度棒,200μm(A-C)和1cm(D和E)。

数据分析

对数变换(任意单位:6 + log2)分析肿瘤或睾丸的重量。巴特利特测试用于验证人口中的相等差异。实验组之间差异的统计学意义通过单因素方差分析或Kruskal Wallis检验确定,然后使用GraphPad Prism 6软件进行Dunn方法。当P 时考虑统计学意义0.05,并且所有数据以均匀±SEM显示,如Miyawaki等人,2016年。
我们以前的研究中肿瘤和睾丸的重量显示在表1中供参考(Miyawaki等人,2016)。

表1.每个畸胎瘤或睾丸的重量,平均体重

食谱

  1. 鼠标iPSC中等
    Dulbecco改良的Eagle's培养基(Sigma-Aldrich)
    15%胎牛血清(JRH或BioWest)
    1%青霉素/链霉素(和光纯药)
    2mM L-谷氨酰胺(NACALAI TESQUE)
    0.1mM非必需氨基酸(NACALAI TESQUE)
    0.1mMβ-巯基乙醇(Sigma-Aldrich)
    人重组白血病抑制因子(NACALAI TESQUE)
    用0.22μm过滤器过滤,在4°C下储存
  2. NMR和人类iPSCs培养基
    DMEM / F12(Sigma-Aldrich)
    20%KnockOut血清替代品(Thermo Fisher Scientific)
    1%青霉素/链霉素(和光纯药)
    2mM L-谷氨酰胺(NACALAI TESQUE)
    0.1mM非必需氨基酸(NACALAI TESQUE)
    0.1mMβ-巯基乙醇(Sigma-Aldrich)
    4ng / ml成纤维细胞生长因子2(PeproTech)
    用0.22μm过滤器过滤,在4°C下储存
  3. 4%PFA
    4克PFA
    100毫升PBS
    热板搅拌器加热
    加入20μl1M氢氧化钠溶解残留的PFA 在-20°C下等分并冷冻保存 注意:调查人员必须在准备PFA期间佩戴面罩,手套,实验衣和眼睛保护。

致谢

这项工作得到日本科技部PRESTO,日本教育,文化,体育科技部科学促进会科学研究资助基金的支持,创新型地区“氧气生物学科学研究资助计划”:从MEXT到KM和KM的综合了解生活的新标准和S.M.是日本科学促进会的研究员。

参考

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  2. Evans,MJ和Kaufman,MH(1981)。&nbsp; 建立在来自小鼠胚胎的多能细胞的培养中。自然 292(5819):154-156。
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  5. Itakura,G.,Kawabata,S.,Ando,M.,Nishiyama,Y.,Sugai,K.,Ozaki,M.,Iida,T.,Ookubo,T.,Kojima,K.,Kashiwagi, Yasutake,K.,Nakauchi,H.,Miyoshi,H.,Nagoshi,N.,Kohyama,J.,Iwanami,A.,Matsumoto,M.,Nakamura,M。和Okano,H。(2017) 防止移植iPSC衍生物后发生潜在致瘤性的失败安全系统。 / a> 干细胞报告 8(3):673-684。
  6. Miura,K.,Okada,Y.,Aoi,T.,Okada,A.,Takahashi,K.,Okita,K.,Nakagawa,M.,Koyanagi,M.,Tanabe,K.,Ohnuki, Ogawa,D.,Ikeda,E.,Okano,H.and Yamanaka,S。(2009)。诱导多能干细胞系的安全性的变化 Nat Biotechnol 27(8):743-745。
  7. Miyawaki,S.,Kawamura,Y.,Oiwa,Y.,Shimizu,A.,Hachiya,T.,Bono,H.,Koya,I.,Okada,Y.,Kimura,T.,Tsuchiya, Suzuki,S.,Onishi,N.,Kuzumaki,N.,Matsuzaki,Y.,Narita,M.,Ikeda,E.,Okanoya,K.,Seino,K.,Saya,H.,Okano, Miura,K.(2016)。感染中的抗肿瘤性来自裸体鼹鼠的多能干细胞。 7:11471.
  8. Okano,H.,Nakamura,M.,Yoshida,K.,Okada,Y.,Tsuji,O.,Nori,S.,Ikeda,E.,Yamanaka,S。和Miura,K。(2013)。 使用诱导性多能干细胞进行安全细胞治疗的步骤。 Circ Res 112(3):523-533。
  9. Okita,K.,Ichisaka,T.和Yamanaka,S。(2007)。产生种系感受态诱导多能干细胞。自然 448(7151):313-317。
  10. Takahashi,K.,Tanabe,K.,Ohnuki,M.,Narita,M.,Ichisaka,T.,Tomoda,K.and Yamanaka,S。(2007)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/18035408”target =“_ blank”>通过确定的因素从成年人成纤维细胞诱导多能干细胞。细胞 131(5):861-872。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Miyawaki, S., Okada, Y., Okano, H. and Miura, K. (2017). Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells. Bio-protocol 7(16): e2518. DOI: 10.21769/BioProtoc.2518.
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