Animal Reproduction (AR)
Animal Reproduction (AR)
Conference papers

Adding a dimension to cell fate

Tiziana A.L. Brevini, Elena F.M. Manzoni, Sharon Arcuri, Fulvio Gandolfi

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Cell fate specification, gene expression and spatial restriction are process finely tuned by epigenetic regulatory mechanisms. At the same time, mechanical forces have been shown to be crucial to drive cell plasticity and boost differentiation. Indeed, several studies have demonstrated that transitions along different specification states are strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, that can modulate both cell potency and differentiation, through the activation of specific mechanosensing-related pathways. An overview of small molecule ability to modulate cell plasticity and define cell fate is here presented and results, showing the possibility to erase the epigenetic signature of adult dermal fibroblasts and convert them into insulin-producing cells (EpiCC) are described. The beneficial effects exerted on such processes, when cells are homed on an adequate substrate, that shows “in vivo” tissue-like stiffness are also discussed and the contribution of the Hippo signalling mechano-transduction pathway as one of the mechanisms involved is examined. In addition, results obtained using a genetically modified fibroblast cell line, expressing the enhanced green fluorescent protein (eGFP) under the control of the porcine insulin gene (INS) promoter (INS-eGFP transgenic pigs), are reported. This model offers the advantage to monitor the progression of cell conversion in real time mode. All these observations have a main role in order to allow a swift scale-up culture procedure, essential for cell therapy and tissue engineering applied to human regenerative medicine, and fundamental to ensure an efficient translation process from the results obtained at the laboratory bench to the patient bedside. Moreover, the creation of reliable in vitro model represents a key point to ensure the development of more physiological models that, in turn, may reduce the number of animals used, implementing non-invasive investigations and animal welfare and protection.


3D culture, mechano-sensing, epigenetic conversion.


Agostini C, Zambello R, Facco M, Perin A, Piazza F, Siviero M, Basso U, Bortolin M, Trentin L, Semenzato G. 1999. CD8 T-cell infiltration in extravascular tissues of patients with human immunodeficiency virus infection. Interleukin-15 upmodulates costimulatory pathways involved in the antigen-presenting cells-T-cell interaction. Blood, 93:1277-1286.

Beyer TA, Weiss A, Khomchuk Y, Huang K, Ogunjimi AA, Varelas X, Wrana JL. 2013. Switch enhancers interpret TGF-beta and Hippo signaling to control cell fate in human embryonic stem cells. Cell Rep, 5:1611-1624.

Brevini TA, Pennarossa G, Rahman M M, Paffoni A, Antonini S, Ragni G, deEguileor M, Tettamanti G, Gandolfi F. 2014. Morphological and molecular changes of human granulosa cells exposed to 5-Azacytidine and addressed toward muscular differentiation. Stem Cell Rev, 10:633-642.

Brevini TA, Pennarossa G, Maffei S, Gandolfi F. 2015. Phenotype switching through epigenetic conversion. Reprod Fertil Dev, 27:776-783.

Brevini TA, Pennarossa G, Acocella F, Brizzola S, Zenobi A, Gandolfi F. 2016. Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells. Vet J, 211:52-56

Brevini TAL, Pennarossa G, Manzoni EFM, Gandolfi F. 2018. Safety and efficacy of epigenetically converted human fibroblasts into insulin-secreting cells: a preclinical study. Adv Exp Med Biol, 1079:151-162.

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green fluorescent protein as a marker for gene expression. Science, 263:802-805.

Chandrakanthan V, Yeola A, Kwan JC, Oliver RA, Qiao Q, Kang YC, Zarzour P, Beck D, Boelen L, Unnikrishnan A, Villanueva JE, Nunez AC, Knezevic K, Palu C, Nasrallah R, Carnell M, Macmillan A, Whan R, Yu Y, Hardy P, Grey S T, Gladbach A, Delerue F, Ittner L, Mobbs R, Walkley CR, Purton LE, Ward RL, Wong JW, Hesson LB, Walsh W, Pimanda JE. 2016. PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells. Proc Natl Acad Sci U S A, 113:E2306-2315

Chowdhury F, Li Y, Poh YC, Yokohama-Tamaki T, Wang N, Tanaka TS. 2010. Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions. PLoS ONE, 5:e15655.

Christman JK. 2002. 5-Azacytidine and 5-aza-2[prime]-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene, 21:5483-5495.

Constantinides PG, Jones PA, Gevers W. 1977. Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment. Nature, 267:364-366.

Darmon M, Nicolas JF, Lamblin D. 1984. 5-Azacytidine is able to induce the conversion of teratocarcinoma-derived mesenchymal cells into epithelia cells. EMBO J, 3:961-967.

Demirkaya E, Lanni S, Bovis F, Galasso R, Ravelli A, Palmisani E, Consolaro A, Pederzoli S, Marafon D, Simianer S, Martini A, Ruperto N, Pistorio A. 2016. A Meta-Analysis to estimate the placebo effect in randomized controlled trials in juvenile idiopathic arthritis. Arthritis Rheumatol, 68:1540-1550.

Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S. 2011. Role of YAP/TAZ in mechanotransduction. Nature, 474:179-183.

Engler AJ, Sen S, Sweeney HL, Discher DE. 2006. Matrix elasticity directs stem cell lineage specification. Cell, 126:677-689.

Evans ND, Minelli C, Gentleman E, LaPointe V, Patankar S N, Kallivretaki M, Chen X, Roberts CJ, Stevens MM. 2009. Substrate stiffness affects early differentiation events in embryonic stem cells. Eur Cell Mater, 18:1-13; discussion 13-14.

Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, Nguyen NK, Thrun S, Lutolf MP, Blau HM. 2010. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science, 329:1078-1081.

Harrison SE, Sozen B, Christodoulou N, Kyprianou C, Zernicka-Goetz M. 2017. Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science, 356:pii: eaal1810.

Heise J, Liu Z, Stock KF, Rensing S, Reinhardt F. Simianer H. 2016. The genetic structure of longevity in dairy cows. J Dairy Sci, 99:1253-1265.

Hemberger M, Dean W, Reik W. 2009. Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington's canal. Nat Rev Mol Cell Biol, 10:526-537.

Higuchi S, Watanabe TM, Kawauchi K, Ichimura T, Fujita H. 2014. Culturing of mouse and human cells on soft substrates promote the expression of stem cell markers. J Biosci Bioeng, 117:749-755.

Huebsch N, Arany PR, Mao AS, Shvartsman D, Ali OA, Bencherif SA, Rivera-Feliciano J, Mooney DJ. 2010. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat Mater, 9:518-526.

Jiang T, Xing B, Rao J. 2008. Recent developments of biological reporter technology for detecting gene expression. Biotechnol Genet Eng Rev, 25:41-75.

Jones PA. 1985. Altering gene expression with 5-azacytidine. Cell, 40:485-486.

Jost D. 2014. Bifurcation in epigenetics: implications in development, proliferation, and diseases. Phys Rev E Stat Nonlin Soft Matter Phys, 89:010701.

Kaneko T, Masuda I, Horie N, Shimoyama T. 2012. New bone formation in nongrafted sinus lifting with space-maintaining management: a novel technique using a titanium bone fixation device. J Oral Maxillofac Surg, 70:e217-224.

Laschke MW, Korbel C, Rudzitis-Auth J, Gashaw I, Reinhardt M, Hauff P, Zollner TM, Menger MD. 2010. High-resolution ultrasound imaging: a novel technique for the noninvasive in vivo analysis of endometriotic lesion and cyst formation in small animal models. Am J Pathol, 176, 585-593.

Lian I, Kim J, Okazawa H, Zhao J, Zhao B, Yu J, Chinnaiyan A, Israel M A, Goldstein LS, Abujarour R, Ding S, Guan KL. 2010. The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev, 24:1106-1118.

Manzoni EF, Pennarossa G, deEguileor M, Tettamanti G, Gandolfi F, Brevini TA. 2016. 5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts. Sci Rep, 6:37017.

Mirakhori F, Zeynali B, Kiani S, Baharvand H. 2015. Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells. Cell J, 17:153-158.

Palii SS, Van Emburgh BO, Sankpal UT, Brown KD, Robertson KD. 2008. DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B. Mol Cell Biol, 28:752-771.

Pennarossa G, Maffei S, Campagnol M, Tarantini L, Gandolfi F, Brevini TA. 2013. Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc Natl Acad Sci U S A, 110:8948-8953.

Pennarossa G, Maffei S, Campagnol M, Rahman MM, Brevini TA, Gandolfi F. 2014. Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine. Stem Cell Rev, 10:31-43.

Pennarossa G, Santoro R, Manzoni EFM, Pesce M, Gandolfi F, Brevini TAL. 2018. Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate. Stem Cell Rev, 14:398-411.

Piccolo S, Dupont S, Cordenonsi M. 2014. The biology of YAP/TAZ: hippo signaling and beyond. Physiol Rev, 94:1287-1312.

Rivron NC, Frias-Aldeguer J, Vrij EJ, Boisset JC, Korving J, Vivie J, Truckenmuller RK, van Oudenaarden A, van Blitterswijk CA, Geijsen N. 2018. Blastocyst-like structures generated solely from stem cells. Nature, 557:106-111.

Schellenberg A, Joussen S, Moser K, Hampe N, Hersch N, Hemeda H, Schnitker J, Denecke B, Lin Q, Pallua N, Zenke M, Merkel R, Hoffmann B, Wagner, W. 2014. Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells. Biomaterials, 35:6351-6358.

Shipony Z, Mukamel Z, Cohen NM, Landan G, Chomsky E, Zeliger SR, Fried YC, Ainbinder E, Friedman N, Tanay A. 2014. Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells. Nature, 513:115-119.

Swain PS, Elowitz MB, Siggia ED. 2002. Intrinsic and extrinsic contributions to stochasticity in gene expression. Proc Natl Acad Sci U S A, 99:12795-12800.

Tan SJ, Fang JY, Wu Y, Yang Z, Liang G, Han B. 2015. Muscle tissue engineering and regeneration through epigenetic reprogramming and scaffold manipulation. Sci Rep, 5:16333.

Taylor SM, Constantinides PA, Jones PA. 1984. 5-Azacytidine, DNA methylation, and differentiation. Curr Top Microbiol Immunol, 108:115-127.

Wang KC, Yeh YT, Nguyen P, Limqueco E, Lopez J, Thorossian S, Guan KL, Li YJ, Chien S. 2016. Flow-dependent YAP/TAZ activities regulate endothelial phenotypes and atherosclerosis. Proc Natl Acad Sci U S A, 113:11525-11530.

Yamamoto K, Hayashi S, Nakabori T, Shibuya M, Ichiba M, Inada M. 2012. Endoscopic submucosal dissection using endoclips to assist in mucosal flap formation (novel technique: "clip flap method"). Endoscopy, 44(Suppl 2 UCTN):E334-335.

Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S. 2009. Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell, 5:237-241.

Young RA. 2011. Control of the embryonic stem cell state. Cell, 144:940-954.

Zhu J, Adli M, Zou J Y, Verstappen G, Coyne M, Zhang X, Durham T, Miri M, Deshpande V, De Jager P L, Bennett DA, Houmard JA, Muoio D M, Onder TT, Camahort R, Cowan CA, Meissner A, Epstein C B, Shoresh N, Bernstein BE. 2013. Genome-wide chromatin state transitions associated with developmental and environmental cues. Cell, 152:642-654.

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