Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters
Tóm tắt
Traditional approaches for generating goat pluripotent stem cells (iPSCs) suffer from complexity and low preparation efficiency. Therefore, we tried to derive goat iPSCs with a new method by transfecting exogenous Oct4, Sox2, Klf4 and c-Myc mRNAs into goat embryonic fibroblasts (GEFs), and explore the mechanisms regarding the transcription regulation of the reprogramming factors in goat iPSCs induction. mRNAs of the four reprogramming factors were transfected into GEFs, and were localized in nucleus with approximately 90% transfection efficiency. After five consecutive transfections, GEFs tended to aggregate by day 10. Clones appeared on day 15–18, and typical embryonic stem cell -like clones formed on day 20. One thousand AKP staining positive clones were achieved in 104 GEFs, with approximately 1.0% induction efficiency. Immunofluorescence staining and qRT-PCR detection of the ESCs markers confirmed the properties of the goat iPSCs. The achieved goat iPSCs could be cultured to 22nd passage, which showed normal karyotype. The goat iPSCs were able to differentiate into embryoid bodies with three germ layers. qRT-PCR and western blot showed activated endogenous pluripotent factors expression in the later phase of mRNA-induced goat iPSCs induction. Epigenetic analysis of the endogenous pluripotent gene Nanog revealed its demethylation status in derived goat iPSCs. Core promoter regions of the four reprogramming factors were determined. Transcription factor binding sites, including Elf-1, AP-2, SP1, C/EBP and MZF1, were identified to be functional in the core promoter regions of these reprogramming genes. Demethylation and deacetylation of the promoters enhanced their transcription activities. We successfully generated goat iPSCs by transfection of Oct4, Sox2, Klf4 and c-Myc mRNAs into GEFs, which initiated the endogenous reprogramming network and altered the methylation status of pluripotent genes. Core promoter regions and functional transcription binding sites of the four reprogramming genes were identified. Epigenetic regulation was revealed to participate in mRNA induced iPSCs formation. Our study provides a safe and efficient approach for goat. iPSCs generation.
Tài liệu tham khảo
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–72.
Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292(292):154–6.
Wheeler MB. Development and validation of swine embryonic stem cells: a review. Reprod Fertil Dev. 1994;6(5):563–8.
Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A. 1995;92(17):7844–8.
Fernández M, Pirondi S, Chen BL, Del VG, Alessandri M, Farnedi A, et al. Isolation of rat embryonic stem-like cells: a tool for stem cell research and drug discovery. Dev Dyn. 2011;240(11):2482–94.
Kues WA, Niemann H. Advances in farm animal transgenesis. Prev Vet Med. 2011;102:146–56.
Deval PB, Lopez-Soria S, Nofrarias M, Martin M, Vordermeier HM, Villarreal-Ramos B, et al. Experimental model of tuberculosis in the domestic goat after endobronchial infection with Mycobacterium caprae. Clin Vaccine Immunol. 2011;18:1872–81.
Proffen BL, McElfresh M, Fleming BC, Murray MM. A comparative anatomical study of the human knee and six animal species. Knee. 2012;19:493–9.
Zhou K, Wu G, Li Y, Zhao L, Zhou R, Zhu Q, et al. Protective effects of indomethacin and dexamethasone in a goat model with intrauterine balloon aortic valvuloplasty. J Biomed Sci. 2012;19:74.
Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells. 2009;27(3):543–9.
Shu J, Wu C, Wu Y, Li Z, Shao S, Zhao W. Induction of pluripotency in mouse somatic cells with lineage specifiers. Cell. 2013;153(5):963–75.
Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mrna. Cell Stem Cell. 2010;7(5):618–30.
Rosa A, Brivanlou AH. Synthetic mrnas: powerful tools for reprogramming and differentiation of human cells. Cell Stem Cell. 2010;7(5):549–50.
Li C, Yu H, Ma Y, Shi G, Jiang J, Gu J, et al. Germline-competent mouse-induced pluripotent stem cell lines generated on human fibroblasts without exogenous leukemia inhibitory factor. PLoS One. 2009;4(8):e6724.
Zhang JD, Zhao LX, Wu BJ. Mouse induced pluripotent stem cells generated by piggybac. Scientia Sinica Vitae. 2012;42(7):553–61.
Anokye DF, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, et al. Highly efficient mirna-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell. 2011;8(4):376–88.
Liu H, Zhu F, Yong J, Zhang P, Hou P, Li H, et al. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell. 2009;3(6):587–90.
Zhang JN, Liu YB, Zhang JD, Jie SU, Yun X, Sun W, et al. (2011). Karyotyping of arab horse by g-banding technique. Acta Agriculturae Boreali-Sinica. 2011; 26(2):101-106.
Blelloch R, Venere M, Yen J, Ramalho-Santos M. Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell. 2011;1(3):245–7.
Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced pluripotent stem cells generated without viral integration. Hepatology. 2009;322(3):945–9.
Gump JM, Dowdy SF. Tat transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 2007;13(10):443–8.
Okita K, Nakagawa M, Hong H, Ichisaka T, Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Science. 2008;322(5903):949–53.
Angel M, Yanik MF. Innate immune suppression enables frequent transfection with rna encoding reprogramming proteins. PLoS One. 2010;5(7):e11756.
Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol. 2008;26(11):1276–84.
Arnold A, Naaldijk YM, Fabian C, Wirth H, Hans B, Guido N, et al. Reprogramming of human huntington fibroblasts using mrna. Isrn Cell Biology. 2011;2012(2012):1-12.
Heng BC, Heinimann K, Miny P, Iezzi G, Glatz K, Scherberich A, et al. Mrna transfection-based, feeder-free, induced pluripotent stem cells derived from adipose tissue of a 50-year-old patient. Metab Eng. 2013;18(1):9–24.