Abstract
Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 × 105 cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.
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References
Chen M, Lin YQ, Xie SL, Wu HF, Wang JF (2010) Enrichment of cardiac differentiation of mouse embryonic stem cells by optimizing the hanging drop method. Biotechnol Lett
Dang SM, Kyba M, Perlingeiro R, Daley GQ, Zandstra PW (2002) Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol Bioeng 78:442–453
Dobrovolskaia-Zavadskaia N (1927) Sur la mortification spontanée chez la souris nouveau-née et sur l’existence d’un caractère (facteur) hereditaire, non-viable. Crit Rev Soc Biol 97:114–119
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156
Gerecht-Nir S, Cohen S, Itskovitz-Eldor J (2004) Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation. Biotechnol Bioeng 86:493–502
Höpfl G, Gassmann M, Desbaillets I (2004) Differentiating embryonic stem cells into embryoid bodies. Methods Mol Biol 254:79–98
Hwang YS, Cho J, Tay F, Heng JY, Ho R, Kazarian SG, Williams DR, Boccaccini AR, Polak JM, Mantalaris A (2009) The use of murine embryonic stem cells, alginate encapsulation and rotary microgravity bioreactor in bone tissue engineering. Biomaterials 30(4):499–507
Kobolak J, Kiss K, Polgar Z, Mamo S, Rogel-Gaillard C, Tancos Z, Bock I, Baji AG, Tar K, Pirity MK, Dinnyes A (2009) Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos. BMC Mol Biol 10:88–100
Lü S, Liu S, He W, Duan C, Li Y, Liu Z, Zhang Y, Hao T, Wang Y, Li D, Wang C, Gao S (2008) Bioreactor cultivation enhances NTEB formation and differentiation of NTES cells into cardiomyocytes. Cloning Stem Cells 10:363–370
Magin TM, McWhir J, Melton DW (1992) A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Res 20:3795–3796
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:7634–7638
Ranganayakulu G, Elliott DA, Harvey RP, Olson EN (1998) Divergent roles for NK-2 class homeobox genes in cardiogenesis in flies and mice. Development 125(16):3037–3048
Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 97:11307–11312
Schwartz RJ, Olson EN (1999) Building the heart piece by piece: modularity of cis-elements regulating Nkx2-5 transcription. Development 126:4187–4192
Shamblott MJ, Bugg EM, Lawler AM, Gearhart JD (2002) Craniofacial abnormalities resulting from targeted disruption of the murine Sim2 gene. Dev Dyn 224:373–380
Yirme G, Amit M, Laevsky I, Osenberg S, Itskovitz-Eldor J (2008) Establishing a dynamic process for the formation, propagation, and differentiation of human embryoid bodies. Stem Cells Dev 17:1227–1241
Acknowledgments
This study was supported by EU Seventh Framework Programme (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485; Resolve, FP7-HEALTH-F4-2008-202047); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205 and NKTH/KPI (NKFP_07_1-ES2HEART-HU OM-00202-2007), CHE-TRF senior scholarship No. RTA 5080010 (M.T.) and the Thailand Commission on Higher Education (CHE-PhD-SW-2005-100 (S.R.), CHE-PhD-SW-2007 (N.K.)).
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Rungarunlert, S., Klincumhom, N., Bock, I. et al. Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor. Biotechnol Lett 33, 1565–1573 (2011). https://doi.org/10.1007/s10529-011-0614-8
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DOI: https://doi.org/10.1007/s10529-011-0614-8