Establishment of human induced pluripotent stem cell lines

doi:10.3969/j.issn.2095-4344.2015.06.009

Abstract

Abstract:

BACKGROUND: Induced pluripotent stem cells have a generating process similar to cancer stem cells and possess stem cell characteristics extremely close to human embryonic stem cells. Therefore, studies on induced pluripotent stem cells are in favor of an increased awareness and understanding of human development and tumor occurrence.
OBJECTIVE: To master the technology of establishing induced pluripotent stem cell lines in order to build the technology platform for reprogramming specific disease cells, thus to explore the pathogenesis of the disease using reprogramming technology.
METHODS: Retroviruses containing Oct4, Sox2, Klf-4, and c-Myc4 transcription factors were used to transfect human skin fibroblasts (HS27 cells), and then the cells were induced to differentiate to human embryonic stem cell-like clones in the human embryonic stem cell culture conditions. The cells were picked and further amplified. After observation of clone morphology and alkaline phosphatase activity test, immunofluorescence detection was used to detect whether human embryonic stem cell markers Oct4, Sox2, c-Myc, Klf-4 expressed, the pendant-drop method was used to detect the ability of HS27-derived clones to form teratomas and verify the differentiation into three germ layers.
RESULTS AND CONCLUSION: After viral infection, embryonic stem cell-like clones formed and were negative for green fluorescent proteins. These clones were similar to human embryonic stem cell clones in cell morphology. After further amplification, they were positive for alkaline phosphatase. Immunofluorescence results showed that the clones expressed Oct4, Sox2, c-Myc, Klf-4, and HS27 cells-derived clones injected into immunodeficient mice could form teratomas and had the ability to differentiate into the three germ layers under hematoxylin-eosin staining. In this experiment, induced pluripotent stem cell lines were successfully constructed, laying a good experimental foundation for disease-specific cell reprogramming research.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Induced Pluripotent Stem Cells, Embryonic Stem Cells, Teratoma

CLC Number:

R394.2

Shi Ying-xu, Han Yan-qiu, Xie Yin-liang, Du Hua. Establishment of human induced pluripotent stem cell lines[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(6): 868-875.

Figures/Tables 4

2.1 反转录病毒生产与滴度测定结果携带Oct4、Sox2、Klf4、c-Myc 基因的反转录病毒载体与病毒包装质粒共转染293T细胞，48 h后倒置荧光显微镜下可见绿色荧光，表明转染成功(图1)；收集病毒上清经过滤浓缩后进行滴度测定，病毒滴度值都在1×106 U/mL以上(结果未显示)。 2.2 HS27细胞在体外诱导为诱导性多潜能干细胞及功能鉴定 HS27细胞在体外重编程为诱导性多潜能干细胞，显微镜下观察可见HS27细胞逐渐转变并出现人胚胎干细胞样的克隆形态。经含有携带Oct4、Sox2、Klf4、c-Myc基因包装的反转录病毒感染HS27细胞，在病毒感染第7，8天可见到HS27细胞形态由长梭形渐变为圆形并出现聚集现象，在病毒感染第14天可见人胚胎干细胞样克隆出现，第20天左右挑取的克隆中具有典型的人胚胎干细胞克隆。挑取的人胚胎干细胞样克隆经传代培养后，可见有多个克隆的细胞仍能够保持未分化状态，选取两三株疑似克隆进行鉴定，其他克隆冻存备用。疑似克隆经AP染色后呈阳性，克隆为紫红色，而饲养层细胞不着色，这一结果表明疑似克隆具有较高的碱性磷酸酶活性(图2)，其命名为诱导性多潜能干细胞1。诱导性多潜能干细胞1在体外可长期传代培养，仍具有典型的人胚胎干细胞克隆样形态。当HS27细胞在体外重编程为人胚胎干细胞样克隆后，克隆所具有的绿色荧光逐渐减弱，预示当HS27细胞被重编程转化为诱导性多潜能干细胞后，有部分增强型绿色荧光蛋白基因沉默(图3)。免疫细胞化学检测显示，诱导性多潜能干细胞1能够表达人胚胎干细胞所特有的全能性的标志基因(即Oct4、SSAE-4、TRA-1-60、TRA-1-81)(图4-7)，而不表达SSAE-1。诱导性多潜能干细胞1具有在体外分化形成囊性拟胚体的能力(图8)。并且HS27细胞来源的克隆注入免疫缺陷小鼠体内可以形成畸胎瘤并经苏木精-伊红染色显示具有向三胚层分化能力(图9)。"

References

[1] Takahashi K, Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006;126(4):663-676.
[2] Takahashi K, Tanabe K, Ohnuki M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007;131(5):861-872.
[3] Yu J, Vodyanik MA, Smuga-Otto K,et al.Induced pluripotent stem cell lines derived from human somatic cells.Science. 2007;318(5858):1917-1920.
[4] Geurts AM, Cost GJ, Freyvert Y,et al.Knockout rats via embryo microinjection of zinc-finger nucleases.Science. 2009; 325(5939):433.
[5] Liu H, Zhu F, Yong J,et al.Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts.Cell Stem Cell. 2008;3(6):587-590.
[6] Wu Z, Chen J, Ren J,et al.Generation of pig induced pluripotent stem cells with a drug-inducible system.J Mol Cell Biol. 2009;1(1):46-54.
[7] Huangfu D, Maehr R, Guo W,et al.Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.Nat Biotechnol. 2008;26(7): 795-797.
[8] Rhee YH, Ko JY, Chang MY,et al.Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.J Clin Invest. 2011;121(6):2326-2335.
[9] Jia F, Wilson KD, Sun N,et al.A nonviral minicircle vector for deriving human iPS cells.Nat Methods. 2010;7(3):197-199.
[10] Fukushima S, Ihn H, Nishimura Y,et al.Cancer immunotherapy by utilizing dedritic cells derived from pluripotent stem cells.Nihon Rinsho Meneki Gakkai Kaishi. 2011;34(3):113-120.
[11] Hou P, Li Y, Zhang X, et al.Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds.Science. 2013;341(6146):651-654.
[12] Kohandel M, Haselwandter CA, Kardar M,et al. Quantitative model for efficient temporal targeting of tumor cells and neovasculature.Comput Math Methods Med. 2011;2011: 790721.
[13] Allegrucci C, Rushton MD, Dixon JE,et al.Epigenetic reprogramming of breast cancer cells with oocyte extracts. Mol Cancer. 2011;10(1):7.
[14] Shah M, Allegrucci C.Stem cell plasticity in development and cancer: epigenetic origin of cancer stem cells.Subcell Biochem. 2013;61:545-565.
[15] Takahashi K, Okita K, Nakagawa M,et al. Induction of pluripotent stem cells from fibroblast cultures.Nat Protoc. 2007;2(12):3081-3089.
[16] Kutner RH, Zhang XY, Reiser J.Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.Nat Protoc. 2009;4(4):495-505.
[17] Tiscornia G, Singer O, Verma IM.Production and purification of lentiviral vectors.Nat Protoc. 2006;1(1):241-245.
[18] Takahashi K, Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006;126(4):663-676.
[19] Shu J, Wu C, Wu Y, et al.Induction of pluripotency in mouse somatic cells with lineage specifiers.Cell. 2013;153(5): 963-975.
[20] Hanna JH, Saha K, Jaenisch R.Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues.Cell. 2010;143(4):508-525.
[21] Warren L, Manos PD, Ahfeldt T,et al.Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.Cell Stem Cell. 2010;7(5):618-630.
[22] Gurdon JB, Melton DA.Nuclear reprogramming in cells. Science. 2008;322(5909):1811-1815.
[23] Costa FF, Seftor EA, Bischof JM,et al.Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment.Epigenomics. 2009;1(2):387-398.
[24] Allegrucci C, Rushton MD, Dixon JE,et al.Epigenetic reprogramming of breast cancer cells with oocyte extracts. Mol Cancer. 2011;10(1):7.
[25] Lin SL, Chang DC, Chang-Lin S,et al.Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state.RNA. 2008;14(10):2115-2124.
[26] Utikal J, Maherali N, Kulalert W,et al.Sox2 is dispensable for the reprogramming of melanocytes and melanoma cells into induced pluripotent stem cells.J Cell Sci. 2009;122(Pt 19): 3502-3510.
[27] Carette JE, Pruszak J, Varadarajan M,et al.Generation of iPSCs from cultured human malignant cells.Blood. 2010; 115(20):4039-4042.