牛诱导多能干细胞的建立及应用研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2022-1371

摘要/Abstract

摘要：

诱导性多能干细胞（induced pluripotent stem cells，iPSCs）是指通过导入特定的转录因子将终末分化的体细胞重编程为可以无限增殖更新并具有分化为三胚层多种细胞类型的一类干细胞系。目前对人与小鼠的iPSCs研究已经取得了很多重要成果，但其他动物，如牛等经济型有蹄类家畜iPSCs的研究始终没有突破性的进展。如何将外源转录因子通过重编程载体高效安全地导入体细胞中并持续表达是生产牛诱导多能干细胞（bovine induced pluripotent stem cells，biPSCs）的主要瓶颈。本文就biPSCs建立中重编程系统的选择、诱导因子的选择、小分子化合物的添加等方面进行综述，以期为进一步完善biPSCs及牛胚胎干细胞系的建立提供参考。

关键词: 牛诱导多能干细胞, 体细胞重编程, 培养体系, 小分子化合物

Abstract:

Induce pluripotent stem cells（iPSCs）refer to a class of stem cell lines that reprogram terminally differentiated somatic cells into infinite proliferation and regeneration and have multiple cell types differentiated into three germ layers by introducing specific transcription factors. At present, many important achievements have been made in the research of iPSCs in humans and mice, but no breakthrough has been made in the research of iPSCs in other animals, such as cattle and other economic ungulates. How to efficiently and safely introduce foreign transcription factors into somatic cells through reprogramming vectors and continuously express them is the main bottleneck in the production of bovine induced pluripotent stem cells. This article reviews the selection of reprogramming system, induction factors and small molecule compounds in the establishment of bovine iPSCs, aiming to provide reference for further improvement of biPSCs and the establishment of bovine embryonic stem cell lines.

Key words: bovine induced pluripotent stem cells, somatic cell reprogramming, cultivation system, small molecular compounds

曾虹, 曾睿琳, 付伟, 吉文汇, 兰道亮. 牛诱导多能干细胞的建立及应用研究进展[J]. 生物技术通报, 2023, 39(5): 130-141.

ZENG Hong, ZENG Rui-lin, FU Wei, JI Wen-hui, LAN Dao-liang. Research Progress in the Application and Establishment of Bovine Induced Pluripotent Stem Cells[J]. Biotechnology Bulletin, 2023, 39(5): 130-141.

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参考文献 86

[1]	邱佳慧, 谭季春. 胚胎干细胞与遗传病模型的构建[J]. 中国组织工程研究, 2018, 22(17): 2769-2774.
	Qiu JH, Tan JC. Embryonic stem cells and construction of a genetic disease model[J]. Chin J Tissue Eng Res, 2018, 22(17): 2769-2774.
[2]	Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell, 2006, 126(4): 663-676. doi: 10.1016/j.cell.2006.07.024 pmid: 16904174
[3]	Kim D, Roh S. Strategy to establish embryo-derived pluripotent stem cells in cattle[J]. Int J Mol Sci, 2021, 22(9): 5011. doi: 10.3390/ijms22095011 URL
[4]	Bogliotti YS, Wu J, Vilarino M, et al. Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts[J]. Proc Natl Acad Sci USA, 2018, 115(9): 2090-2095. doi: 10.1073/pnas.1716161115 pmid: 29440377
[5]	Talluri TR, Kumar D, Glage S, et al. Derivation and characterization of bovine induced pluripotent stem cells by transposon-mediated reprogramming[J]. Cell Reprogram, 2015, 17(2): 131-140. doi: 10.1089/cell.2014.0080 pmid: 25826726
[6]	Kawaguchi T, Tsukiyama T, Kimura K, et al. Generation of Naïve bovine induced pluripotent stem cells using PiggyBac transposition of doxycycline-inducible transcription factors[J]. PLoS One, 2015, 10(8): e0135403. doi: 10.1371/journal.pone.0135403 URL
[7]	Zhao LX, Wang ZX, Zhang JD, et al. Characterization of the single-cell derived bovine induced pluripotent stem cells[J]. Tissue Cell, 2017, 49(5): 521-527. doi: S0040-8166(17)30023-X pmid: 28720304
[8]	Bressan FF, Bassanezze V, de Figueiredo Pessôa LV, et al. Generation of induced pluripotent stem cells from large domestic animals[J]. Stem Cell Res Ther, 2020, 11(1): 247. doi: 10.1186/s13287-020-01716-5 pmid: 32586372
[9]	Pillai VV, Kei TG, Reddy SE, et al. Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance[J]. Anim Sci J, 2019, 90(9): 1149-1160. doi: 10.1111/asj.13272 pmid: 31322312
[10]	Tashiro K, Inamura M, Kawabata K, et al. Efficient adipocyte and osteoblast differentiation from mouse induced pluripotent stem cells by adenoviral transduction[J]. Stem Cells, 2009, 27(8): 1802-1811. doi: 10.1002/stem.108 pmid: 19544436
[11]	Choi IY, Lim H, Lee G. Efficient generation human induced pluripotent stem cells from human somatic cells with Sendai-virus[J]. J Vis Exp, 2014(86): 51406.
[12]	Kim D, Kim CH, Moon JI, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins[J]. Cell Stem Cell, 2009, 4(6): 472-476. doi: 10.1016/j.stem.2009.05.005 pmid: 19481515
[13]	Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA[J]. Cell Stem Cell, 2010, 7(5): 618-630. doi: 10.1016/j.stem.2010.08.012 pmid: 20888316
[14]	Desponts C, Ding S. Using small molecules to improve generation of induced pluripotent stem cells from somatic cells[J]. Methods Mol Biol, 2010, 636: 207-218. doi: 10.1007/978-1-60761-691-7_13 pmid: 20336525
[15]	Haridhasapavalan KK, Borgohain MP, Dey C, et al. An insight into non-integrative gene delivery approaches to generate transgene-free induced pluripotent stem cells[J]. Gene, 2019, 686: 146-159. doi: S0378-1119(18)31212-5 pmid: 30472380
[16]	Sumer H, Liu J, Malaver-Ortega LF, et al. NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts[J]. J Anim Sci, 2011, 89(9): 2708-2716. doi: 10.2527/jas.2010-3666 pmid: 21478453
[17]	Cao HG, Yang P, Pu Y, et al. Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins[J]. Int J Biol Sci, 2012, 8(4): 498-511. doi: 10.7150/ijbs.3723 pmid: 22457605
[18]	Deng YF, Liu QY, Luo C, et al. Generation of induced pluripotent stem cells from buffalo(Bubalus bubalis)fetal fibroblasts with buffalo defined factors[J]. Stem Cells Dev, 2012, 21(13): 2485-2494. doi: 10.1089/scd.2012.0018 URL
[19]	Zhao LX, Gao XF, Zheng YX, et al. Establishment of bovine expanded potential stem cells[J]. Proc Natl Acad Sci USA, 2021, 118(15): e2018505118. doi: 10.1073/pnas.2018505118 URL
[20]	Li LJ, Sun L, Gao FR, et al. Stk40 links the pluripotency factor Oct4 to the Erk/MAPK pathway and controls extraembryonic endoderm differentiation[J]. Proc Natl Acad Sci USA, 2010, 107(4): 1402-1407. doi: 10.1073/pnas.0905657107 pmid: 20080709
[21]	Zeineddine D, Papadimou E, Chebli K, et al. Oct-3/4 dose dependently regulates specification of embryonic stem cells toward a cardiac lineage and early heart development[J]. Dev Cell, 2006, 11(4): 535-546. pmid: 17011492
[22]	Chew JL, Loh YH, Zhang WS, et al. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells[J]. Mol Cell Biol, 2005, 25(14): 6031-6046. doi: 10.1128/MCB.25.14.6031-6046.2005 URL
[23]	Nakagawa M, Takizawa N, Narita M, et al. Promotion of direct reprogramming by transformation-deficient Myc[J]. Proc Natl Acad Sci USA, 2010, 107(32): 14152-14157. doi: 10.1073/pnas.1009374107 pmid: 20660764
[24]	Pessôa LVF, Bressan FF, Freude KK. Induced pluripotent stem cells throughout the animal Kingdom: availability and applications[J]. World J Stem Cells, 2019, 11(8): 491-505. doi: 10.4252/wjsc.v11.i8.491 pmid: 31523369
[25]	Kinoshita M, Barber M, Mansfield W, et al. Capture of mouse and human stem cells with features of formative pluripotency[J]. Cell Stem Cell, 2021, 28(3): 453-471.e8. doi: 10.1016/j.stem.2020.11.005 URL
[26]	Han XP, Han JY, Ding FR, et al. Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells[J]. Cell Res, 2011, 21(10): 1509-1512. doi: 10.1038/cr.2011.125 pmid: 21826109
[27]	才文道力玛, 伊敏娜, 王希生, 等. 大动物多能干细胞建立研究进展[J]. 中国实验动物学报, 2020, 28(5): 695-701.
	Caiwendaolima, Yiminna, Wang XS, et al. Research progress on the establishment of pluripotent stem cells in large animals[J]. Acta Lab Animalis Sci Sin, 2020, 28(5): 695-701.
[28]	Wang B, Wu LL, Li DW, et al. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Jdp2-Jhdm1b-Mkk6-Glis1-nanog-essrb-Sall4[J]. Cell Rep, 2019, 27(12): 3473-3485.e5. doi: S2211-1247(19)30697-7 pmid: 31216469
[29]	Pillai VV, Koganti PP, Kei TG, et al. Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells[J]. Biol Open, 2021, 10(10): bio058756. doi: 10.1242/bio.058756 URL
[30]	Canizo JR, Vazquez Echegaray C, Klisch D, et al. Exogenous human OKSM factors maintain pluripotency gene expression of bovine and porcine iPS-like cells obtained with STEMCCA delivery system[J]. BMC Res Notes, 2018, 11(1): 509. doi: 10.1186/s13104-018-3627-8 pmid: 30053877
[31]	Wang SW, Wu DC, et al. Androgen receptor-mediated apoptosis in bovine testicular induced pluripotent stem cells in response to phthalate esters[J]. Cell Death Dis, 2013, 4(11): e907. doi: 10.1038/cddis.2013.420
[32]	Cravero D, Martignani E, Miretti S, et al. Generation of induced pluripotent stem cells from bovine epithelial cells and partial redirection toward a mammary phenotype in vitro[J]. Cell Reprogram, 2015, 17(3): 211-220. doi: 10.1089/cell.2014.0087 pmid: 26053520
[33]	Bai CY, Li XC, Gao YH, et al. Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells[J]. J Pineal Res, 2016, 61(2): 154-167. doi: 10.1111/jpi.12334 pmid: 27090494
[34]	Polo JM, Liu S, Figueroa ME, et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells[J]. Nat Biotechnol, 2010, 28(8): 848-855. doi: 10.1038/nbt.1667 pmid: 20644536
[35]	Poetsch MS, Strano A, Guan KM. Human induced pluripotent stem cells: from cell origin, genomic stability, and epigenetic memory to translational medicine[J]. Stem Cells, 2022, 40(6): 546-555. doi: 10.1093/stmcls/sxac020 URL
[36]	Scesa G, Adami R, Bottai D. iPSC preparation and epigenetic memory: does the tissue origin matter?[J]. Cells, 2021, 10(6): 1470. doi: 10.3390/cells10061470 URL
[37]	Recchia K, Pessôa LVF, Pieri NCG, et al. Recchia K, Pessôa LVF, Pieri NCG, et al.. Influence of cell type in in vitro induced reprogramming in cattle[J]. Life(Basel), 2022, 12(8): 1139.
[38]	Hou PP, Li YQ, Zhang X, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds[J]. Science, 2013, 341(6146): 651-654. doi: 10.1126/science.1239278 pmid: 23868920
[39]	Ho R, Papp B, Hoffman JA, et al. Stage-specific regulation of reprogramming to induced pluripotent stem cells by Wnt signaling and T cell factor proteins[J]. Cell Rep, 2013, 3(6): 2113-2126. doi: 10.1016/j.celrep.2013.05.015 pmid: 23791530
[40]	Ring DB, Johnson KW, Henriksen EJ, et al. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo[J]. Diabetes, 2003, 52(3): 588-595. doi: 10.2337/diabetes.52.3.588 URL
[41]	Mao J, Zhang Q, Deng W, et al. Epigenetic modifiers facilitate induction and pluripotency of Porcine iPSCs[J]. Stem Cell Rep, 2017, 8(1): 11-20. doi: 10.1016/j.stemcr.2016.11.013 URL
[42]	Onder TT, Kara N, Cherry A, et al. Chromatin-modifying enzymes as modulators of reprogramming[J]. Nature, 2012, 483(7391): 598-602. doi: 10.1038/nature10953
[43]	Chen JK, Liu H, Liu J, et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs[J]. Nat Genet, 2013, 45(1): 34-42. doi: 10.1038/ng.2491 pmid: 23202127
[44]	Su Y, Wang L, Fan ZQ, et al. Establishment of bovine-induced pluripotent stem cells[J]. Int J Mol Sci, 2021, 22(19): 10489. doi: 10.3390/ijms221910489 URL
[45]	Huangfu DW, Maehr R, Guo WJ, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds[J]. Nat Biotechnol, 2008, 26(7): 795-797. doi: 10.1038/nbt1418 pmid: 18568017
[46]	Mahapatra PS, Singh R, Kumar K, et al. Valproic acid assisted reprogramming of fibroblasts for generation of pluripotent stem cells in buffalo(Bubalus bubalis)[J]. Int J Dev Biol, 2017, 61(1-2): 81-88. doi: 10.1387/ijdb.160006sb URL
[47]	Kuo HH, Gao XZ, DeKeyser JM, et al. Negligible-cost and weekend-free chemically defined human iPSC culture[J]. Stem Cell Reports, 2020, 14(2): 256-270. doi: 10.1016/j.stemcr.2019.12.007 URL
[48]	Miura T, Yuasa N, Ota H, et al. Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions[J]. Biochem Biophys Res Commun, 2019, 518(3): 506-512. doi: 10.1016/j.bbrc.2019.08.082 URL
[49]	Saunders A, Faiola F, Wang JL. Concise review: pursuing self-renewal and pluripotency with the stem cell factor Nanog[J]. Stem Cells, 2013, 31(7): 1227-1236. doi: 10.1002/stem.1384 pmid: 23653415
[50]	Paynter JM, Chen J, Liu XD, et al. Propagation and maintenance of mouse embryonic stem cells[J]. Methods Mol Biol, 2019, 1940: 33-45. doi: 10.1007/978-1-4939-9086-3_3 pmid: 30788816
[51]	Gafni O, Weinberger L, Mansour AA, et al. Derivation of novel human ground state naive pluripotent stem cells[J]. Nature, 2013, 504(7479): 282-286. doi: 10.1038/nature12745
[52]	Heo YT, Quan XY, Xu YN, et al. CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells[J]. Stem Cells Dev, 2015, 24(3): 393-402. doi: 10.1089/scd.2014.0278 pmid: 25209165
[53]	Botigelli RC, Pieri NCG, Bessi BW, et al. Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells[J]. Front Cell Dev Biol, 2022, 10: 938709. doi: 10.3389/fcell.2022.938709 URL
[54]	Weinberger L, Ayyash M, Novershtern N, et al. Dynamic stem cell states: naive to primed pluripotency in rodents and humans[J]. Nat Rev Mol Cell Biol, 2016, 17(3): 155-169. doi: 10.1038/nrm.2015.28
[55]	Hassani SN, Totonchi M, Gourabi H, et al. Signaling roadmap modulating naive and primed pluripotency[J]. Stem Cells Dev, 2014, 23(3): 193-208. doi: 10.1089/scd.2013.0368 URL
[56]	王肖肖, 柴小青, 叶守东. 人胚胎干细胞Naïve多能性状态建立的研究进展[J]. 生命的化学, 2017, 37(2): 187-192.
	Wang XX, Chai XQ, Ye SD. Research progress of the establishment of Naïve pluripotent state of human embryonic stem cells[J]. Chem Life, 2017, 37(2): 187-192.
[57]	Nichols J, Smith A. Naive and primed pluripotent states[J]. Cell Stem Cell, 2009, 4(6): 487-492. doi: 10.1016/j.stem.2009.05.015 pmid: 19497275
[58]	Heard E. Recent advances in X-chromosome inactivation[J]. Curr Opin Cell Biol, 2004, 16(3): 247-255. pmid: 15145348
[59]	Smith A. Formative pluripotency: the executive phase in a developmental continuum[J]. Development, 2017, 144(3): 365-373. doi: 10.1242/dev.142679 pmid: 28143843
[60]	Xiang JZ, Wang HN, Zhang YY, et al. LCDM medium supports the derivation of bovine extended pluripotent stem cells with embryonic and extraembryonic potency in bovine-mouse chimeras from iPSCs and bovine fetal fibroblasts[J]. FEBS J, 2021, 288(14): 4394-4411. doi: 10.1111/febs.15744 pmid: 33524211
[61]	Soto DA, Navarro M, Zheng CB, et al. Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells[J]. Sci Rep, 2021, 11(1): 11045. doi: 10.1038/s41598-021-90422-0 pmid: 34040070
[62]	Liu K, Wang F, Ye XY, et al. KSR-based medium improves the generation of high-quality mouse iPS cells[J]. PLoS One, 2014, 9(8): e105309. doi: 10.1371/journal.pone.0105309 URL
[63]	Okada M, Oka M, Yoneda Y. Effective culture conditions for the induction of pluripotent stem cells[J]. Biochim Biophys Acta, 2010, 1800(9): 956-963. doi: 10.1016/j.bbagen.2010.04.004 pmid: 20417254
[64]	Rawat N, Singh MK, Sharma T, et al. Media switching at different time periods affects the reprogramming efficiency of buffalo fetal fibroblasts[J]. Anim Biotechnol, 2021, 32(2): 155-168. doi: 10.1080/10495398.2019.1671435 URL
[65]	Yoshida Y, Takahashi K, Okita K, et al. Hypoxia enhances the generation of induced pluripotent stem cells[J]. Cell Stem Cell, 2009, 5(3): 237-241. doi: 10.1016/j.stem.2009.08.001 pmid: 19716359
[66]	Bessi BW, Botigelli RC, Pieri NCG, et al. Cattle in vitro induced pluripotent stem cells generated and maintained in 5 or 20% oxygen and different supplementation[J]. Cells, 2021, 10(6): 1531. doi: 10.3390/cells10061531 URL
[67]	Lim JWE, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells[J]. Proteomics, 2002, 2(9): 1187-1203. pmid: 12362336
[68]	Cong S, Cao GF, Liu DJ. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro[J]. Cytotechnology, 2014, 66(6): 995-1005. doi: 10.1007/s10616-013-9653-4 URL
[69]	Xu WQ, Hao RF, Wang J, et al. Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells[J]. Sci Rep, 2022, 12(1): 9177. doi: 10.1038/s41598-022-13249-3 pmid: 35654935
[70]	Xu W, Hao R, Wang J, et al. Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells[J]. Sci Rep, 2022, 12(1): 9177. doi: 10.1038/s41598-022-13249-3 pmid: 35654935
[71]	Souralova T, Holubcova Z, Kyjovska D, et al. Xeno- and feeder-free derivation of two sex-discordant sibling lines of human embryonic stem cells[J]. Stem Cell Res, 2021, 57: 102574. doi: 10.1016/j.scr.2021.102574 URL
[72]	Seo JH, Jeon YJ. Global proteomic analysis of mesenchymal stem cells derived from human embryonic stem cells via connective tissue growth factor treatment under chemically defined feeder-free culture conditions[J]. J Microbiol Biotechnol, 2022, 32(1): 126-140. doi: 10.4014/jmb.2110.10032 URL
[73]	Guo R, Ye X, Yang J, et al. Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells[J]. Nat Commun, 2018, 9(1): 2620. doi: 10.1038/s41467-018-05038-2 pmid: 29976922
[74]	Soto DA, Ross PJ. Pluripotent stem cells and livestock genetic engineering[J]. Transgenic Res, 2016, 25(3): 289-306. doi: 10.1007/s11248-016-9929-5 pmid: 26894405
[75]	Kumar D, Talluri TR, Selokar NL, et al. Perspectives of pluripotent stem cells in livestock[J]. World J Stem Cells, 2021, 13(1): 1-29. doi: 10.4252/wjsc.v13.i1.1 pmid: 33584977
[76]	Selvaraj V, Wildt DE, Pukazhenthi BS. Induced pluripotent stem cells for conserving endangered species?[J]. Nat Methods, 2011, 8(10): 805-807. doi: 10.1038/nmeth.1715 pmid: 21959133
[77]	Comizzoli P, Holt WV. Recent advances and prospects in germplasm preservation of rare and endangered species[J]. Adv Exp Med Biol, 2014, 753: 331-356. doi: 10.1007/978-1-4939-0820-2_14 pmid: 25091916
[78]	Hildebrandt TB, Hermes R, Colleoni S, et al. Embryos and embryonic stem cells from the white rhinoceros[J]. Nat Commun, 2018, 9(1): 2589. doi: 10.1038/s41467-018-04959-2 pmid: 29973581
[79]	Plews JR, Gu MX, Longaker MT, et al. Large animal induced pluripotent stem cells as pre-clinical models for studying human disease[J]. J Cell Mol Med, 2012, 16(6): 1196-1202. doi: 10.1111/j.1582-4934.2012.01521.x pmid: 22212700
[80]	Bassols A, Costa C, Eckersall PD, et al. The pig as an animal model for human pathologies: a proteomics perspective[J]. Proteomics Clin Appl, 2014, 8(9-10): 715-731. doi: 10.1002/prca.201300099 URL
[81]	Cong XQ, Zhang SM, Ellis MW, et al. Large animal models for the clinical application of human induced pluripotent stem cells[J]. Stem Cells Dev, 2019, 28(19): 1288-1298. doi: 10.1089/scd.2019.0136 pmid: 31359827
[82]	Gün G, Kues WA. Current progress of genetically engineered pig models for biomedical research[J]. Biores Open Access, 2014, 3(6): 255-264. doi: 10.1089/biores.2014.0039 pmid: 25469311
[83]	Herath S, Dobson H, Bryant CE, et al. Use of the cow as a large animal model of uterine infection and immunity[J]. J Reprod Immunol, 2006, 69(1): 13-22. doi: 10.1016/j.jri.2005.09.007 pmid: 16386311
[84]	Yapura J, Mapletoft RJ, Pierson R, et al. A bovine model for examining the effects of an aromatase inhibitor on ovarian function in women[J]. Fertil Steril, 2011, 96(2): 434-438.e3. doi: 10.1016/j.fertnstert.2011.05.038 pmid: 21696721
[85]	Moradi S, Mahdizadeh H, Šarić T, et al. Research and therapy with induced pluripotent stem cells(iPSCs): social, legal, and ethical considerations[J]. Stem Cell Res Ther, 2019, 10(1): 341. doi: 10.1186/s13287-019-1455-y
[86]	Post MJ. Cultured meat from stem cells: challenges and prospects[J]. Meat Sci, 2012, 92(3): 297-301. doi: 10.1016/j.meatsci.2012.04.008 pmid: 22543115

非整合型方法 Approach of non-integrating	优点 Pros	缺点 Cons
腺病毒	基因组整合频率低，载体容量大	重编程效率低，具有免疫原性，对某些细胞需多次感染
仙台病毒	无基因组整合，载体容量大，转染效率高，稳定转染，重编程效率高	多个转录因子需多个病毒携带，具有免疫原性，需传代10次以上才可完全清除病毒基因组，只能加工RNA
质粒转染	基因组整合频率低，操作简单，免疫原性低，无病毒组分	不能自我复制，重编程效率极低，需要多次转染
转座子	不涉及病毒，可精确切除，只需一次转染，载体容量大，免疫原性低	可能导致突变和染色体重排，重编程效率低，需要用转座酶进行切除
微环DNA载体	基因组整合频率低，操作简单，转染效率高，免疫原性极低	不能自我复制，需多次转染，重编程效率极低
小分子化合物	具有瞬时控制能力，易操作，能促进iPSCs的生成	对毒性效率存在争议，确定或非特异的效果具有不确定性或非特异性

非整合型方法 Approach of non-integrating	优点 Pros	缺点 Cons
腺病毒	基因组整合频率低，载体容量大	重编程效率低，具有免疫原性，对某些细胞需多次感染
仙台病毒	无基因组整合，载体容量大，转染效率高，稳定转染，重编程效率高	多个转录因子需多个病毒携带，具有免疫原性，需传代10次以上才可完全清除病毒基因组，只能加工RNA
质粒转染	基因组整合频率低，操作简单，免疫原性低，无病毒组分	不能自我复制，重编程效率极低，需要多次转染
转座子	不涉及病毒，可精确切除，只需一次转染，载体容量大，免疫原性低	可能导致突变和染色体重排，重编程效率低，需要用转座酶进行切除
微环DNA载体	基因组整合频率低，操作简单，转染效率高，免疫原性极低	不能自我复制，需多次转染，重编程效率极低
小分子化合物	具有瞬时控制能力，易操作，能促进iPSCs的生成	对毒性效率存在争议，确定或非特异的效果具有不确定性或非特异性

组别 Group	Naïve状态 Naïve state	Primed状态 Primed state
培养条件	LIF/血清或2i	Activin A/bFGF或CHIR/IWR1
FGF2依赖性	不依赖	依赖
LIF依赖性	依赖	不依赖
BMP4依赖性	依赖	不依赖
Wnt激活	自我更新	分化
克隆形态	隆起	扁平
XX失活情况	XaXa	XaXi
Nanog表达	高	低
Fgf5表达	低	高
OCT4增强子	远端	近端
H3K24me3表达	低	高
嵌合小鼠胚胎	能	不能

组别 Group	Naïve状态 Naïve state	Primed状态 Primed state
培养条件	LIF/血清或2i	Activin A/bFGF或CHIR/IWR1
FGF2依赖性	不依赖	依赖
LIF依赖性	依赖	不依赖
BMP4依赖性	依赖	不依赖
Wnt激活	自我更新	分化
克隆形态	隆起	扁平
XX失活情况	XaXa	XaXi
Nanog表达	高	低
Fgf5表达	低	高
OCT4增强子	远端	近端
H3K24me3表达	低	高
嵌合小鼠胚胎	能	不能

培养基 Medium	主要添加成分 Main component	参考文献 Reference
CTRF	TeSR1培养基（无FGF2和TGFb）、FGF2和IWR1	[4]
LCDM	hLIF、CHIR99021、（S）-（+）-dimethindene maleate、minocycline hydrochloride	[60]
TiF培养基	mTeSR plus培养基、PD0325901、CHIR-99021、hLIF、IWR1、iDOT1L、Forskolin	[44]
bEPSC培养基	mTeSR1培养基、CHIR99021、WH-4-023、XAV939、IWR-1、维生素C、LIF、Activin A	[19]