Abstract
Pluripotent human embryonic stem cell (hESC) lines are a promising model system in developmental and tissue regeneration research. Differentiation of hESCs towards the three germ layers and finally tissue specific cell types is often performed through the formation of embryoid bodies (EBs) in suspension or hanging droplet culture systems. However, these systems are inefficient regarding embryoid body (EB) formation, structural support to the EB and long term differentiation capacity. The present study investigates if agarose, as a semi solid matrix, can facilitate EB formation and support differentiation of hESC lines. The results showed that agarose culture is able to enhance EB formation efficiency with 10% and increase EB growth by 300%. The agarose culture system was able to maintain expression of the three germ layers over 8 weeks of culture. All of the four hESC lines tested developed EBs in the agarose system although with a histological heterogeneity between cell lines as well as within cell lines. In conclusion, a 3-D agarose culture of spherical hESC colonies improves EB formation and growth in a cost effective, stable and non-laborious technique.
Abbreviations
- hESC:
-
Human embryonic stem cell
- EB:
-
Embryoid body
- EBs:
-
Embryoid bodies
- SCID:
-
Severe combined immunodeficiency
- 3-D:
-
Three dimensional
- MEF:
-
Mouse embryonic fibroblast
- PBS:
-
Phosphate buffered saline
- AFP:
-
Alpha fetoprotein
- DAPI:
-
4′,6-diamidino-2-phenylindole
- Bfgf:
-
Basic fibroblast growth factor
- PPD11:
-
p-Phenylenediamine anti-fade solution
- IgG:
-
Immuno globulin G
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Acknowledgments
The study was funded by: Swedish Research Council grant no. 2005-7544, The ALF/LUA research grant from the Sahlgrenska University Hospital. The Inga Britt and Arne Lundberg Research Foundation.
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All work with the human embryonic stem cells was conducted according to the ISSCR Guidelines for the Conduct of Human Embryonic Stem Cell Research and with the approval of the local ethics committees at the University of Gothenburg and the University of Uppsala.
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Stenberg, J., Elovsson, M., Strehl, R. et al. Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells. Cytotechnology 63, 227–237 (2011). https://doi.org/10.1007/s10616-011-9344-y
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DOI: https://doi.org/10.1007/s10616-011-9344-y