Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment

Byung Sun Yoon, Seung Jun Yoo, Jeoung Eun Lee, Seungkwon You, Hoon Taek Lee, Hyun Soo Yoon

Research output: Contribution to journalArticle

132 Citations (Scopus)

Abstract

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 μM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 μM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Original languageEnglish
Pages (from-to)149-159
Number of pages11
JournalDifferentiation
Volume74
Issue number4
DOIs
Publication statusPublished - 2006 Apr 1

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Azacitidine
Cardiac Myocytes
Therapeutics
Human Embryonic Stem Cells
Cell- and Tissue-Based Therapy
Transmission Electron Microscopy
Fluorescence Microscopy
Confocal Microscopy
Methylation
Real-Time Polymerase Chain Reaction
Heart Diseases
Suspensions
Electron Microscopy
Cell Count
Polymerase Chain Reaction

Keywords

  • 5-azacytidine
  • Cardiomyocyte
  • Hanging drop culture
  • Human embryonic stem cell

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Developmental Biology
  • Cell Biology

Cite this

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. / Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo.

In: Differentiation, Vol. 74, No. 4, 01.04.2006, p. 149-159.

Research output: Contribution to journalArticle

Yoon, Byung Sun ; Yoo, Seung Jun ; Lee, Jeoung Eun ; You, Seungkwon ; Lee, Hoon Taek ; Yoon, Hyun Soo. / Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. In: Differentiation. 2006 ; Vol. 74, No. 4. pp. 149-159.
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