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Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells

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Abstract

Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 μA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in β-MHC without increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation.

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Acknowledgments

We would like to thank R. Hollis Whittington for his role in developing the stimulation microelectrode arrays, and Omer Inan, Mozziyar Etemadi, and Richard Wiard for their help in developing the electrical stimulation hardware. This work was supported in part by the California Institute for Regenerative Medicine (CIRM) through cooperative agreement RS1-00232-1 (GTAK), by the National Institutes of Health (NIH) grants R21HL089027, R21HL091453, R33HL089027, RC1HL100490 (JCW), the Burroughs Wellcome Fund Career Award for Medical Scientists (BWF CAMS; JCW), and by the National Science Foundation Graduate Student Research Fellowship (NSF-GSRF; MQC).

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Authors and Affiliations

  1. Department of Bioengineering, Stanford University, 330 Serra Mall, CISX-206X, Stanford, CA, 94305, USA

    Michael Q. Chen

  2. Department of Radiology, Stanford University School of Medicine, Edwards Building R354, Stanford, CA, 94305, USA

    Xiaoyan Xie, Ning Sun & Joseph C. Wu

  3. Department of Bioengineering, Stanford University, Edwards Building R354, Stanford, CA, 94305, USA

    Kitchener D. Wilson

  4. Department of Medicine, Stanford University School of Medicine, Edwards Building R354, Stanford, CA, 94305, USA

    Joseph C. Wu

  5. Department of Electrical Engineering, Stanford University, 330 Serra Mall, CISX-202X, Stanford, CA, 94305, USA

    Laurent Giovangrandi & Gregory T. A. Kovacs

  6. Department of Medicine, Stanford University School of Medicine, 330 Serra Mall, CISX-202X, Stanford, CA, 94305, USA

    Gregory T. A. Kovacs

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  1. Michael Q. Chen
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  2. Xiaoyan Xie
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  3. Kitchener D. Wilson
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  4. Ning Sun
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  6. Laurent Giovangrandi
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  7. Gregory T. A. Kovacs
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Correspondence to Michael Q. Chen.

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The first two authors have contributed equally to this work.

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12195_2009_96_MOESM4_ESM.tif

Immunostaining of stimulated cells. Intermediate Stage ES cells stimulated at 30 μA for 4 days were trypsinized, lightly re-plated onto glass chamber slides, and imaged on a fluorescent microscope. Cardiomyocyte marker troponin-T, gap junction Cx43, and ES marker Oct4 were stained in green while nuclei were stained in blue (TIFF 2705 kb)

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Chen, M.Q., Xie, X., Wilson, K.D. et al. Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells. Cel. Mol. Bioeng. 2, 625–635 (2009). https://doi.org/10.1007/s12195-009-0096-0

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  • DOI: https://doi.org/10.1007/s12195-009-0096-0

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