The effects of Fndc5 overexpression on characteristics of mouse embryonic stem cells and the expression of three germ layer markers and mitochondrial genes

Farzaneh Rabiee 1,2 , Mahboobeh Forouzanfar 2 , Somayeh Tanhaei 2 , Majid Motavali-bashi 1 , Kamran Ghaedi 1,2 , Mohammad Hossein Nasr Esfahani 2 1 Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran.

2 Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

Objectives

Fndc5 formerly known as peroxisomal protein, is suggested to be a PGC1-alpha-dependent myokine and is secreted as Irisin , responsible for browning of white fat tissues. In adult mouse, mRNA expression level of Fndc5 is high in heart, skeletal muscle and brain. Our previous studies have revealed a significant increase in Fndc5 mRNA when mouse embryonic stem cells were differentiated into beating bodies and neural precursor cells . As a step closer to clarify the function of Fndc5 on embryonic stem cell differentiation we have overexpressed Fndc5 in embryonic stem cell to analyze the expression of three germ layers marker and mitochondrial genes.

Methods

Stably transformed overexpressing Fndc5 mouse embryonic stem cells (Rb20-Fndc5) after induction by doxycyclin were cultured for three days. Then, 5 × 10 5 of treated mESCs were suspended in a non-adhesive dish by the medium without LIF,and small molecules SB and PD (for stemness maintenance) for six days in the absence of Fndc5 overexpression. Media were changed every 2 days. Total RNA was extracted from cultured cells . cDNA synthesis was carried out with cDNA Synthesis Kit . Real-time PCR was performed to estimate the mRNA expression levels of target genes.

Results

The results depicted that expression of mesodermal and endodermal genes such as Brachyury,Dkk1, Isl1 and Foxa2 was higher in those cells with overexpressing Fndc5 compare with the control. furthermore there were not significant difference in the expression levels of ectodermal and mitochondrial markers in overexpressing Fndc5 group and control.

References Conclusions

In conclusion , Fndc5 may be a master gene , whose product would modulate the expression of endodermal and mesodermal genes. However it has not any effect on mitochondrial and ectodermal genes. Further experiments are required for clarification the function of Fndc5.

Fig- 1: (aggregation protocole)