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Semi-automatic Method for Ca2+ Imaging Data Analysis of Maturing Human Embryonic Stem Cells-Derived Retinal Pigment Epithelium

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Abstract

Ca2+ is a second messenger controlling vital cellular processes, including cell maturation. Changes in Ca2+ signaling during maturation of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have not been assessed previously. The aim of this study was to investigate maturation-dependent changes in transient intracellular Ca2+ ([Ca2+] i ) increases in hESC-RPE. For this, we developed image analysis tools to evaluate cell-specific Ca2+ signals from the entire field of view. Spontaneous and mechanically induced transient [Ca2+] i increases (STIs and MITIs) were analyzed in hESC-RPEs cultured for 9 or 28 days, altogether from more than 80,000 cells. Both cultures showed STIs: the longer culture time resulted in twofold increase of amount of cells with STIs. Mechanical stimulation induced intercellular Ca2+ waves in cells from both time points, but longer culture time reduced Ca2+ wave spreading. Depletion of intracellular Ca2+ stores decreased cell fraction with STIs and MITIs at both time points, and absence of extracellular Ca2+ had similar effect on cells with STIs. To conclude, hESC-RPE cells undergo significant Ca2+ signaling re-arrangements during a short maturation period increasing cell fraction with STIs, while decreasing coordinated cell response to mechanical stimulation. This knowledge and proposed analysis tools can be used for assessment of hESC-RPE maturation in vitro.

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Abbreviations

STI:

Spontaneous transient [Ca2+] i increase

MITI:

Mechanically induced transient [Ca2+] i increase

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Acknowledgments

This work was supported by the Finnish Funding Agency for Technology and Innovation, Health Research Council of the Academy of Finland (Grant Numbers 252225, 218050 and 137801) and Tampere University of Technology President’s Doctoral Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Outi Melin, Outi Heikkilä and Hanna Pekkanen are thanked for excellent technical assistance.

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Authors and Affiliations

  1. Department of Electronics and Communications Engineering, BioMediTech, Tampere University of Technology, Biokatu 6, FM-1, 33520, Tampere, Finland

    Amna E. Abu Khamidakh, Florentino Caetano dos Santos & Jari Hyttinen

  2. BioMediTech, University of Tampere, Biokatu 12, FM-5, 33520, Tampere, Finland

    Heli Skottman & Kati Juuti-Uusitalo

Authors
  1. Amna E. Abu Khamidakh
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  2. Florentino Caetano dos Santos
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  3. Heli Skottman
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  4. Kati Juuti-Uusitalo
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  5. Jari Hyttinen
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Correspondence to Amna E. Abu Khamidakh or Jari Hyttinen.

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Associate Editor Michael Gower oversaw the review of this article.

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Abu Khamidakh, A.E., dos Santos, F.C., Skottman, H. et al. Semi-automatic Method for Ca2+ Imaging Data Analysis of Maturing Human Embryonic Stem Cells-Derived Retinal Pigment Epithelium. Ann Biomed Eng 44, 3408–3420 (2016). https://doi.org/10.1007/s10439-016-1656-9

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  • DOI: https://doi.org/10.1007/s10439-016-1656-9

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