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Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies

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Abstract

Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct trajectories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation.

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Acknowledgments

We thank the heart team and the hESC maintenance laboratory of Regea. We are grateful for the technical assistance of Ulla Jukarainen in immunocytochemistry. We thank Prof. Outi Hovatta for the HS lines. The study was funded by the Academy of Finland, the Finnish Heart Research Foundation, the Finnish Cultural Foundation, the Competitive Research Funding of Pirkanmaa Hospital District, the Kalle Kaihari Foundation and the Ida Montin Foundation.

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Authors and Affiliations

  1. Regea Institute for Regenerative Medicine, University of Tampere, Tampere University Hospital, Biokatu 12, 33520, Tampere, Finland

    Mari Pekkanen-Mattila, Ville Kujala, Riitta Suuronen, Heli Skottman, Katriina Aalto-Setälä & Erja Kerkelä

  2. Department of Developmental Biology, Medical School, University of Tampere, Tampere, Finland

    Markku Pelto-Huikko

  3. Laboratory of Atherosclerosis Genetics, Centre for Laboratory Medicine, Tampere University Hospital, Tampere, Finland

    Markku Pelto-Huikko

  4. Department of Biomedical Engineering, Tampere University of Technology, Tampere, Finland

    Riitta Suuronen

  5. Department of Eye, Ear and Oral Diseases, Tampere University Hospital, Tampere, Finland

    Riitta Suuronen

  6. Heart Center, Tampere University Hospital, Tampere, Finland

    Katriina Aalto-Setälä

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  1. Mari Pekkanen-Mattila
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Correspondence to Erja Kerkelä.

Electronic supplementary material

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418_2010_689_MOESM1_ESM.pdf

Supplementary Fig. 1. A tissue multi-array was prepared by punching 1 mm holes in 10% agarose. EBs were individually transferred to the wells filled with OCT compound, i.e. each hole contains replicates for individual samples from different hESC lines at different time points (PDF 238 kb)

418_2010_689_MOESM2_ESM.pdf

Supplementary Fig. 2. Average morphological changes and growth of EBs at the age of 1–20 days from four hESC lines. Scale bar 200 μm (PDF 623 kb)

418_2010_689_MOESM3_ESM.pdf

Supplementary Fig. 3. OCT3/4 shown to be strongly expressed at the protein level in undifferentiated hESC colonies (A). Infrequently single Brachyury T positive cells were seen (B), but the expression of SOX1 or AFP was never detected (C, D). Corresponding 4′,6-diamidino-2-phenylindole (DAPI) stainings (E–H). Scale bar 200 μm (PDF 301 kb)

418_2010_689_MOESM4_ESM.pdf

Supplementary Fig. 4. OCT3/4 protein expression in EBs from hESC lines HS181 and HS346. The expression of OCT3/4 is still quite high on day 4 and can be detected both in the middle of EBs and on the edges (A, B). A strongly OCT3/4 positive area in one 6-day-old EB from line HS181 (C) and more typical expression pattern in EB from HS346 (D). In general, the expression of OCT3/4 started to decrease from day 8 (E, G), although was still quite abundant in some EBs on day 8 (F) and day 10 (H). The site of inset is marked with black box showing the stained cells except in G, where no immunoreactivity was seen. Cavity formation is marked with asterisks in D. Scale bar 50 μm (PDF 289 kb)

418_2010_689_MOESM5_ESM.pdf

Supplementary Fig. 5. Beating aggregate consisting mainly of cardiomyocytes as almost all the cells stained positively with cardiac troponin T (A). Clear striated patterns in the cells can be seen in the inset (B) (arrows). Scale bar 50 μm (PDF 62.3 kb)

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Pekkanen-Mattila, M., Pelto-Huikko, M., Kujala, V. et al. Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies. Histochem Cell Biol 133, 595–606 (2010). https://doi.org/10.1007/s00418-010-0689-7

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