CSIRO Publishing blank image blank image blank image blank imageBooksblank image blank image blank image blank imageJournalsblank image blank image blank image blank imageAbout Usblank image blank image blank image blank imageShopping Cartblank image blank image blank image You are here: Journals > Reproduction, Fertility and Development   
Reproduction, Fertility and Development
  Vertebrate Reproductive Science & Technology
 
blank image Search
 
blank image blank image
blank image
 
  Advanced Search
   

Journal Home
About the Journal
Editorial Board
Contacts
Content
All Issues
Special Issues
Research Fronts
Sample Issue
For Authors
General Information
Instructions to Authors
Submit Article
Open Access
For Referees
Referee Guidelines
Review an Article
Annual Referee Index
For Subscribers
Subscription Prices
Customer Service
Print Publication Dates

blue arrow e-Alerts
blank image
Subscribe to our email Early Alert or RSS feeds for the latest journal papers.

red arrow Connect with us
blank image
facebook twitter youtube

red arrow Connect with SRB
blank image
facebook TwitterIcon

Affiliated Societies

RFD is the official journal of the International Embryo Transfer Society and the Society for Reproductive Biology.



Article << Previous     |     Next >>   Contents Vol 22(1)

392 EFFECTS OF TRICHOSTATIN A, AN EPIGENETIC MODIFIER AGENT, ON DIFFERENTIATION OF EMBRYONIC STEM CELLS INTO STRIATED MUSCLE CELLS

C. S. Oliveira A, N. Z. Saraiva A, Jasmin B, M. M. Souza A and T. A. D. Tetzner A

A DMVPRA-FCAV-São Paulo State University, Jaboticabal, SP, Brazil;
B IBCCF-Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
   

Abstract
Export Citation
Print
  


Abstract

In vitro generation of cardiomyocytes from embryonic stem cells (ES cells) is a promising approach to develop strategies for treatment of cardiac diseases. Epigenetic changes occur during ES cells differentiation, and by the first 5 days, the histone acetylation levels increase, promoting an improvement in gene expression. Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor and promotes histone hyperacetylation. In this study, we analyzed the effects of TSA treatment in ES cells differentiation into striated muscle cells. For that, murine ES cell line H106 was grown in hanging drops of 20 μL containing 2000 cells in DMEM medium supplemented with 15% FCS, 10 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 m L-glutamine, 10 mM nonessential amino acids, and 83.4 μg mL-1 amikacin. After 5 days, embryoid bodies were transferred individually to a 96-well plate treated with 0.1% swine gelatin. Trichostatin A treatment was performed during hanging drop culture (group 15 nM d0-5), at Day 5 for 24 h after transfer to adherent culture (groups 50 nM d5 and 100 nM d5), and at Day 13 for 24 h (groups 50 nM d13 and 100 nM d13). Area of embryoid bodies and apoptosis rate from control and 15 nM d0-5 groups were analyzed at Day 5. Analysis of contractile structures was carried out at Day 14. Imunnocitochemistry reactions for desmin and troponin I were performed at Day 7 and 17, respectively. Results of apoptosis, desmin, and troponin I cell rates (positive cells/total cells) were analyzed by chi-square test, with a significance level of 5%, on MINITAB Release 14.1. Areas of embryoid bodies were submitted to one-way ANOVA and Tukey’s post-test, with a significance level of 5%, using GraphPad software. Embryoid bodies developed on TSA supplemented medium presented smaller areas (15 nM d0-5: 6.75 ± 0.93 mm2; control: 15.84 ± 1.64 mm2) and greater apoptosis rates (15 nM d0-5: 29.53%; control: 20.18%). Contractile structures were greater on 50 nM d5 (90%c) and extremely less on the 15 nM d0-5 group (3.12%b). Groups 100 nM d5 (66.6%), 50 nM d13 (70.93%), and 100 nM d13 (80.7%a,c) were similar to the control group (68.25%a). Rate of desmin positive cells was greater on the 50 nM d5 group (31.53b) and less on the 100 nM d5 group (22.9c). The 15 nM d0-5 group (26.03a) was similar to control (25.25a). Rate of troponin I positive cells was greater on 50 nM d5 (8.65b) and 100 nM d13 (9.69b) and less on the 100 nM d5 group (2.63c). On the 15 nM d0-5 group, no positive cells were observed, and the 50 nM d13 group (6.67a) was similar to control (6.44a). In conclusion, the current study demonstrated that TSA improves striated muscle differentiation when supplemented at lesser concentrations at Day 5 (50 nM) and greater concentrations at Day 13 (100 nM) and promotes detrimental effects when used during embryoid body development, decreasing the area of structures and increasing apoptosis rate.


Acknowledgments are given to FAPESP 2007/55968-9 and 2008/58370-0.

Reproduction, Fertility and Development 22(5305) 352–353   http://dx.doi.org/10.1071/RDv22n1Ab392
Published online: 08 December 2009




 
Top  Email this page
 
   


Legal & Privacy | Contact Us | Help

CSIRO

© CSIRO 1996-2014