Establishment of Tsc2‑deficient rat embryonic stem cells

Ito,Yoshitaka; Kawano,Haruna; Kanai,Fumio; Nakamura,Eri; Tada,Norihiro; Takai,Setsuo; Horie,Shigeo; Arai,Hajime; Kobayashi,Toshiyuki; Hino,Okio

doi:10.3892/ijo.2015.2913

Establishment of Tsc2‑deficient rat embryonic stem cells

Authors:
- Yoshitaka Ito
- Haruna Kawano
- Fumio Kanai
- Eri Nakamura
- Norihiro Tada
- Setsuo Takai
- Shigeo Horie
- Hajime Arai
- Toshiyuki Kobayashi
- Okio Hino
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Affiliations: Department of Neurosurgery, Juntendo University Graduate School of Medicine, Tokyo, Japan, Department of Molecular Pathogenesis, Juntendo University Graduate School of Medicine, Tokyo, Japan, Laboratory of Genome Research, Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan, Department of Clinical Radiology, Faculty of Health Sciences, Hiroshima International University, Hiroshima, Japan, Department of Urology, Juntendo University Graduate School of Medicine, Tokyo, Japan
Published online on: March 3, 2015 https://doi.org/10.3892/ijo.2015.2913
Pages: 1944-1952

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Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by TSC1 or TSC2 mutations. TSC causes the development of tumors in various organs such as the brain, skin, kidney, lung, and heart. The protein complex TSC1/2 has been reported to have an inhibitory function on mammalian target of rapamycin complex 1 (mTORC1). Treatment with mammalian target of rapamycin (mTOR) inhibitors has demonstrated tumor‑reducing effects in patients with TSC but is also associated with various adverse effects. In recent years, experiments involving in vivo differentiation of pluripotent stem cells have been reported as useful in elucidating mechanisms of pathogenesis and discovering new therapeutic targets for several diseases. To reveal the molecular basis of the pathogenesis caused by the Tsc2 mutation, we derived embryonic stem cells (ESCs) from Eker rats, which have the Tsc2 mutation and develop brain lesions and renal tumors. Although several studies have reported the necessity of Tsc1 and Tsc2 regulation to maintain ESCs and hematopoietic stem cells, we successfully established not only Tsc2+/+ and Tsc2+/- ESCs but also Tsc2-/- ESCs. We confirmed that these cells express pluripotency markers and retain the ability to differentiate into all three germ layers. Comprehensive gene expression analysis of Tsc2+/+ and Tsc2+/- ESCs revealed similar profiles, whereas the profile of Tsc2-/- ESCs was distinct from these two. In vitro differentiation experiments using these ESCs combined with in vivo experiments may reveal the mechanism of the tissue‑specific pathogenesis caused by the Tsc2 mutation and identify specific new therapeutic targets.

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1	Kwiatkowski DJ, Whittemore VH and Thiele EA: Tuberous Sclerosis Complex: Genes, Clinical Features, and Therapeutics. Wiley-VCH Verlag GmbH & Co. KGaA; Weinheim: 2010, View Article : Google Scholar
2	European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell. 75:1305–1315. 1993. View Article : Google Scholar : PubMed/NCBI
3	van Slegtenhorst M, de Hoogt R, Hermans C, et al: Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science. 277:805–808. 1997. View Article : Google Scholar : PubMed/NCBI
4	Curatolo P, Bombardieri R and Jozwiak S: Tuberous sclerosis. Lancet. 372:657–668. 2008. View Article : Google Scholar : PubMed/NCBI
5	Laplante M and Sabatini DM: mTOR signaling in growth control and disease. Cell. 149:274–293. 2012. View Article : Google Scholar : PubMed/NCBI
6	Knudson AG Jr: Mutation and cancer: Statistical study of retino-blastoma. Proc Natl Acad Sci USA. 68:820–823. 1971. View Article : Google Scholar
7	Henske EP, Scheithauer BW, Short MP, Wollmann R, Nahmias J, Hornigold N, van Slegtenhorst M, Welsh CT and Kwiatkowski DJ: Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am J Hum Genet. 59:400–406. 1996.PubMed/NCBI
8	Au KS, Hebert AA, Roach ES and Northrup H: Complete inactivation of the TSC2 gene leads to formation of hamartomas. Am J Hum Genet. 65:1790–1795. 1999. View Article : Google Scholar : PubMed/NCBI
9	Tucker T and Friedman JM: Pathogenesis of hereditary tumors: Beyond the ‘two-hit’ hypothesis. Clin Genet. 62:345–357. 2002. View Article : Google Scholar : PubMed/NCBI
10	Chan JA, Zhang H, Roberts PS, Jozwiak S, Wieslawa G, Lewin-Kowalik J, Kotulska K and Kwiatkowski DJ: Pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: Biallelic inactivation of TSC1 or TSC2 leads to mTOR activation. J Neuropathol Exp Neurol. 63:1236–1242. 2004.PubMed/NCBI
11	Niida Y, Stemmer-Rachamimov AO, Logrip M, Tapon D, Perez R, Kwiatkowski DJ, Sims K, MacCollin M, Louis DN and Ramesh V: Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions. Am J Hum Genet. 69:493–503. 2001. View Article : Google Scholar : PubMed/NCBI
12	Onda H, Lueck A, Marks PW, Warren HB and Kwiatkowski DJ: Tsc2(+/−) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background. J Clin Invest. 104:687–695. 1999. View Article : Google Scholar : PubMed/NCBI
13	Kobayashi T, Minowa O, Kuno J, Mitani H, Hino O and Noda T: Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice. Cancer Res. 59:1206–1211. 1999.PubMed/NCBI
14	Kobayashi T, Minowa O, Sugitani Y, Takai S, Mitani H, Kobayashi E, Noda T and Hino O: A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice. Proc Natl Acad Sci USA. 98:8762–8767. 2001. View Article : Google Scholar : PubMed/NCBI
15	Meikle L, Pollizzi K, Egnor A, Kramvis I, Lane H, Sahin M and Kwiatkowski DJ: Response of a neuronal model of tuberous sclerosis to mammalian target of rapamycin (mTOR) inhibitors: Effects on mTORC1 and Akt signaling lead to improved survival and function. J Neurosci. 28:5422–5432. 2008. View Article : Google Scholar : PubMed/NCBI
16	Goorden SMI, van Woerden GM, van der Weerd L, Cheadle JP and Elgersma Y: Cognitive deficits in Tsc1^+/− mice in the absence of cerebral lesions and seizures. Ann Neurol. 62:648–655. 2007. View Article : Google Scholar : PubMed/NCBI
17	Ehninger D, Han S, Shilyansky C, Zhou Y, Li W, Kwiatkowski DJ, Ramesh V and Silva AJ: Reversal of learning deficits in a Tsc2^+/− mouse model of tuberous sclerosis. Nat Med. 14:843–848. 2008. View Article : Google Scholar : PubMed/NCBI
18	Eker R and Mossige J: A dominant gene for renal adenomas in the rat. Nature. 189:858–859. 1961. View Article : Google Scholar
19	Hino O, Mitani H and Knudson AG: Genetic predisposition to transplacentally induced renal cell carcinomas in the Eker rat. Cancer Res. 53:5856–5858. 1993.PubMed/NCBI
20	Kobayashi T, Hirayama Y, Kobayashi E, Kubo Y and Hino O: A germline insertion in the tuberous sclerosis (Tsc2) gene gives rise to the Eker rat model of dominantly inherited cancer. Nat Genet. 9:70–74. 1995. View Article : Google Scholar : PubMed/NCBI
21	Rennebeck G, Kleymenova EV, Anderson R, Yeung RS, Artzt K and Walker CL: Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development. Proc Natl Acad Sci USA. 95:15629–15634. 1998. View Article : Google Scholar : PubMed/NCBI
22	Mizuguchi M, Takashima S, Yamanouchi H, Nakazato Y, Mitani H and Hino O: Novel cerebral lesions in the Eker rat model of tuberous sclerosis: Cortical tuber and anaplastic ganglioglioma. J Neuropathol Exp Neurol. 59:188–196. 2000.PubMed/NCBI
23	Yeung RS, Katsetos CD and Klein-Szanto A: Subependymal astrocytic hamartomas in the Eker rat model of tuberous sclerosis. Am J Pathol. 151:1477–1486. 1997.PubMed/NCBI
24	Takahashi DK, Dinday MT, Barbaro NM and Baraban SC: Abnormal cortical cells and astrocytomas in the Eker rat model of tuberous sclerosis complex. Epilepsia. 45:1525–1530. 2004. View Article : Google Scholar : PubMed/NCBI
25	Prabhakar S, Goto J, Zhang X, et al: Stochastic model of Tsc1 lesions in mouse brain. PLoS One. 8:e642242013. View Article : Google Scholar : PubMed/NCBI
26	Kobayashi T, Adachi H, Mitani H, Hirayama Y and Hino O: Toward chemotherapy for Tsc2-mutant renal tumor. Proc Jpn Acad. 79:22–25. 2003. View Article : Google Scholar
27	Kenerson HL, Aicher LD, True LD and Yeung RS: Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res. 62:5645–5650. 2002.PubMed/NCBI
28	Lee L, Sudentas P, Donohue B, et al: Efficacy of a rapamycin analog (CCI-779) and IFN-γ in tuberous sclerosis mouse models. Genes Chromosomes Cancer. 42:213–227. 2005. View Article : Google Scholar
29	McCormack FX, Inoue Y, Moss J, et al: National Institutes of Health Rare Lung Diseases Consortium; MILES Trial Group: Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N Engl J Med. 364:1595–1606. 2011. View Article : Google Scholar : PubMed/NCBI
30	Franz DN, Belousova E, Sparagana S, et al: Efficacy and safety of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis complex (EXIST-1): A multicentre, randomised, placebo-controlled phase 3 trial. Lancet. 381:125–132. 2013. View Article : Google Scholar
31	Bissler JJ, Kingswood JC, Radzikowska E, et al: Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): A multicentre, randomised, double-blind, placebo-controlled trial. Lancet. 381:817–824. 2013. View Article : Google Scholar : PubMed/NCBI
32	Mizuguchi M: Abnormal giant cells in the cerebral lesions of tuberous sclerosis complex. Congenit Anom (Kyoto). 47:2–8. 2007. View Article : Google Scholar
33	Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying QL and Smith A: Capture of authentic embryonic stem cells from rat blastocysts. Cell. 135:1287–1298. 2008. View Article : Google Scholar : PubMed/NCBI
34	Li P, Tong C, Mehrian-Shai R, et al: Germline competent embryonic stem cells derived from rat blastocysts. Cell. 135:1299–1310. 2008. View Article : Google Scholar : PubMed/NCBI
35	Evans MJ and Kaufman MH: Establishment in culture of pluripotential cells from mouse embryos. Nature. 292:154–156. 1981. View Article : Google Scholar : PubMed/NCBI
36	Martin GR: Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA. 78:7634–7638. 1981. View Article : Google Scholar : PubMed/NCBI
37	Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS and Jones JM: Embryonic stem cell lines derived from human blastocysts. Science. 282:1145–1147. 1998. View Article : Google Scholar : PubMed/NCBI
38	Easley CA IV, Ben-Yehudah A, Redinger CJ, Oliver SL, Varum ST, Eisinger VM, Carlisle DL, Donovan PJ and Schatten GP: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells. Cell Reprogram. 12:263–273. 2010. View Article : Google Scholar : PubMed/NCBI
39	Gan B, Sahin E, Jiang S, Sanchez-Aguilera A, Scott KL, Chin L, Williams DA, Kwiatkowski DJ and DePinho RA: mTORC1-dependent and -independent regulation of stem cell renewal, differentiation, and mobilization. Proc Natl Acad Sci USA. 105:19384–19389. 2008. View Article : Google Scholar : PubMed/NCBI
40	He J, Kang L, Wu T, et al: An elaborate regulation of Mammalian target of rapamycin activity is required for somatic cell reprogramming induced by defined transcription factors. Stem Cells Dev. 21:2630–2641. 2012. View Article : Google Scholar : PubMed/NCBI
41	Shiono M, Kobayashi T, Takahashi R, et al: The G1556S-type tuberin variant suppresses tumor formation in tuberous sclerosis 2 mutant (Eker) rats despite its deficiency in mTOR inhibition. Oncogene. 27:6690–6697. 2008. View Article : Google Scholar : PubMed/NCBI
42	Benvenuto G, Li S, Brown SJ, Braverman R, Vass WC, Cheadle JP, Halley DJ, Sampson JR, Wienecke R and DeClue JE: The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination. Oncogene. 19:6306–6316. 2000. View Article : Google Scholar
43	Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ and Smith A: Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell. 153:335–347. 2013. View Article : Google Scholar : PubMed/NCBI
44	Li S, Thangapazham RL, Wang JA, Rajesh S, Kao TC, Sperling L, Moss J and Darling TN: Human TSC2-null fibroblast-like cells induce hair follicle neogenesis and hamartoma morphogenesis. Nat Commun. 2:2352011. View Article : Google Scholar : PubMed/NCBI
45	Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA and Svendsen CN: Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature. 457:277–280. 2009. View Article : Google Scholar :
46	Lee G, Papapetrou EP, Kim H, et al: Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature. 461:402–406. 2009. View Article : Google Scholar : PubMed/NCBI
47	Yang YM, Gupta SK, Kim KJ, et al: A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell Stem Cell. 12:713–726. 2013. View Article : Google Scholar : PubMed/NCBI
48	Williams BO, Schmitt EM, Remington L, Bronson RT, Albert DM, Weinberg RA and Jacks T: Extensive contribution of Rb-deficient cells to adult chimeric mice with limited histopathological consequences. EMBO J. 13:4251–4259. 1994.PubMed/NCBI
49	Kawamata M and Ochiya T: Two distinct knockout approaches highlight a critical role for p53 in rat development. Sci Rep. 2:9452012. View Article : Google Scholar : PubMed/NCBI
50	Kielman MF, Rindapää M, Gaspar C, van Poppel N, Breukel C, van Leeuwen S, Taketo MM, Roberts S, Smits R and Fodde R: Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling. Nat Genet. 32:594–605. 2002. View Article : Google Scholar : PubMed/NCBI