Proceeding of Veterinary and Animal Science Days 2017, 6th- 8th June, Milan, Italy 

HAF © 2013 

Vol. IV, No. 1s   ISSN: 2283-3927 

   

 

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Keywords 

Epigenetic conversion; 

Pancreatic Differentiation, 

Matrices; 3-D culture systems; 

Oxygen tension 

 

CORRESPONDING AUTHOR 

Alessandro Zenobi  

alessandro.zenobi@unimi.it 

 

JOURNAL HOME PAGE 

riviste.unimi.it/index.php/haf 

Matrix stiffness and oxygen tension  

modulate epigenetic conversion of mouse 

dermal fibroblasts into insulin  

producing cells 

Alessandro Zenobi*1, Fulvio Gandolfi2 and Tiziana A.L. Brevini1 

 

 

1University of Milan, Department of Health, Animal Science and Food Safety, Italy 

2University of Milan, Department of Agricultural and Environmental Sciences - Production,  
Landscape, Agroenergy, Italy 

 
Abstract 

In vivo, cells are surrounded by a three-dimensional (3-D) organization of supporting matrix, 

neighboring cells and a gradient of chemical and mechanical signals (Antoni, et al., 2015). However, 

the present understanding of many biological processes is mainly based on two-dimensional (2-D) 

systems that typically provides a static environment. In the present study, we tested two different 3-

D culture systems and apply them to the epigenetic conversion of mouse dermal fibroblasts into 

insulin producing-cells (Pennarossa, et al., 2013; Brevini, et al., 2015), combining also the use of two 

oxygen tensions. In particular, cells were differentiated using the Polytetrafluoroethylene micro-

bioreactor (PTFE) and the Polyacrylamide (PAA) gels with different stiffness (1 kPa; 4 kPa), maintained 

either in the standard 20% or in the more physiological 5% oxygen tensions. Standard differentiation 

performed on plastic substrates was assessed as a control. Cell morphology (Fig.1A), insulin 

expression and release were analyzed to evaluate the role of both stiffness and oxygen tension in the 

process. The results obtained showed that 1 kPa PAA gel and PTFE system induced a significantly 

higher insulin expression and release than plastic and 4 kPa PAA gel, especially in low oxygen 

condition (Fig.1B). Furthermore, comparing the efficiency of the two systems tested, 1 kPa PAA gel 

ensured a higher insulin transcription than PTFE (Fig.1C). Recent studies show the direct influence of 

substrates on lineage commitment and cell differentiation (Engler, et al., 2006; Evans, et al., 2009). 

The evidence here presented confirm that the use of an appropriate stiffness (similar to the pancreatic 

tissue), combined with a physiological oxygen tension, promote β-cell differentiation, with beneficial 

effects on cell functional activity and insulin release. The present results highlight the importance of 

3-D cell rearrangement and oxigen tension to promote in vitro epigenetic conversion of mouse 

fibroblasts into insulin-producing cells.  

 

Acknowledgments:  

The research was funded by Carraresi Foundation. 

Authors are members of the COST Actions BM1308, CM1406 and CA16119. 

http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en


Proceeding of Veterinary and Animal Science Days 2017, 6th- 8th June, Milan, Italy 

HAF © 2013 

Vol. IV, No. 1s   ISSN: 2283-3927 

 

 

 

 

 

 

 

 

 

 

 

 
Fig.1: A) representative image of cells differentiated in PTFE (20% and 5% oxygen) and on PAA gels (20% and 5% 
oxygen); B) insulin release in all samples after hyperglycemic stimulation with 20 mM of glucose. Different 
superscripts indicate statistical differences among the samples (SPSS software, p≤0,05). Insulin release is 
expressed as mean value ± SD; C) insulin expression in the more efficient experimental groups (PTFE 5% O2; 1 kPa 
5% O2). Different superscripts indicate statistical differences between the samples (SPSS software, p≤0,05). Gene 
expression levels are reported with the highest expression set to 1 and the other relative to this. 

 

 

References 

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vivo. International Journal of Molecular Sciences, 16(3): 5517–5527; 

Brevini, T.A.L., Pennarossa, G., Maffei, S., Zenobi, A., Gandolfi, F., 2015. Epigenetic conversion as a safe and simple 

method to obtain insulin-secreting cells from adult skin fibroblasts. JoVe, 109, e53880; 

Engler, A.J., Sen, S., Sweeney, H.L. and Discher D.E., 2006. Matrix elasticity directs stem cell lineage specification. 

Cell, 126(4): p. 677-89; 

Evans, N.D., Minelli, C., Gentleman, E., LaPointe, V., Patankar, S.N., Kallivretaki, M., Chen, X., Roberts, C.J. and 

Stevens, M.M., 2009. Substrate stiffness affects early differentiation events in embryonic stem cells. European 

Cells & Materials, 18: p. 1-13; discussion 13-4; 

Pennarossa, G., Maffei, S., Campagnol, M., Tarantini, L., Gandolfi, F., Brevini, T.A.L. 2013. Brief demethylation 

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National Academy of Sciences, 110(22), 8948–8953. 

http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en