Abstract
Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines.
The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation.
StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.
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References
Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227:271–278. https://doi.org/10.1006/dbio.2000.9912
Braam SR, Denning C, Matsa E, Young LE, Passier R, Mummery CL (2008) Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification. Nat Protoc 3:1435–1443. https://doi.org/10.1038/nprot.2008.140
Brimble SN, Zeng X, Weiler DA, Luo Y, Liu Y, Lyons IG, Freed WJ, Robins AJ, Rao MS, Schulz TC (2004) Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev 13:585–597. https://doi.org/10.1089/scd.2004.13.585
Carpenter MK, Rosler ES, Fisk GJ, Brandenberger R, Ares X, Miura T, Lucero M, Rao MS (2004) Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 229:243–258. https://doi.org/10.1002/dvdy.10431
Catalina P, Montes R, Ligero G, Sanchez L, de la Cueva T, Bueno C, Leone PE, Menendez P (2008) Human ESCs predisposition to karyotypic instability: is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties? Mol Cancer 7. https://doi.org/10.1186/1476-4598-7-76
Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429. https://doi.org/10.1038/nmeth.1593
Chin AC, Fong WJ, Goh LT, Philp R, Oh SK, Choo AB (2007) Identification of proteins from feeder conditioned medium that support human embryonic stem cells. J Biotechnol 130:320–328. https://doi.org/10.1016/j.jbiotec.2007.04.013
Dakhore S, Nayer B, Hasegawa K (2018) Human pluripotent stem cell culture: current status, challenges, and advancement. Stem Cells Int 2018:7396905. https://doi.org/10.1155/2018/7396905
Daniszewski M, Nguyen Q, Chy HS, Singh V, Crombie DE, Kulkarni T, Liang HH, Sivakumaran P et al (2018) Single-cell profiling identifies key pathways expressed by iPSCs cultured in different commercial media. iScience 7:30-39. https://doi.org/10.1016/j.isci.2018.08.016
Duggal G, Heindryckx B, Warrier S, Taelman J, Van der Jeught M, Deforce D, Chuva de Sousa Lopes S, De Sutter P (2015a) Exogenous supplementation of activin A enhances germ cell differentiation of human embryonic stem cells. Mol Hum Reprod 21:410–423. https://doi.org/10.1093/molehr/gav004
Duggal G, Warrier S, Ghimire S, Broekaert D, Van der Jeught M, Lierman S, Deroo T, Peelman L et al (2015b) Alternative routes to induce naive pluripotency in human embryonic stem cells. Stem Cells 33:2686–2698. https://doi.org/10.1002/stem.2071
Ghasemi-Dehkordi P, Allahbakhshian-Farsani M, Abdian N, Mirzaeian A, Saffari-Chaleshtori J, Heybati F, Mardani G, Karimi-Taghanaki A, Doosti A, Jami MS, Abolhasani M, Hashemzadeh-Chaleshtori M (2015) Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines. J Cell Commun Signal 9:233–246. https://doi.org/10.1007/s12079-015-0289-3
Granata A, Serrano F, Bernard WG, McNamara M, Low L, Sastry P, Sinha S (2016) An iPSC-derived vascular model of Marfan syndrome identifies key mediators of smooth muscle cell death. Nat Genet 49:97–109. https://doi.org/10.1038/ng.3723
Jozefczuk J, Drews K, Adjaye J (2012) Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J Vis Exp. https://doi.org/10.3791/3854
Lagarkova MA, Eremeev AV, Svetlakov AV, Rubtsov NB, Kiselev SL (2010) Human embryonic stem cell lines isolation, cultivation, and characterization. In Vitro Cell Dev Biol Anim 46:284–293. https://doi.org/10.1007/s11626-010-9282-6
Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA et al (2006) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24:185–187. https://doi.org/10.1038/nbt1177
Stover AE, Schwartz PH (2011) Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging. Methods Mol Biol 767:137–146. https://doi.org/10.1007/978-1-61779-201-4_10
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Van der Jeught M, Taelman J, Duggal G, Ghimire S, Lierman S, Chuva de Sousa Lopes SM, Deforce D, Deroo T et al (2015) Application of small molecules favoring naive pluripotency during human embryonic stem cell derivation. Cell Rep 17:170–180. https://doi.org/10.1089/cell.2014.0085
Warrier S, Popovic M, Van der Jeught M, Heindryckx B (2016) Establishment and characterization of naive pluripotency in human embryonic stem cells. Methods Mol Biol 1516:13–46. https://doi.org/10.1007/7651_2016_347
Warrier S, Van der Jeught M, Duggal G, Tilleman L, Sutherland E, Taelman J, Popovic M, Lierman S et al (2017) Direct comparison of distinct naive pluripotent states in human embryonic stem cells. Nat Commun 8:15055. https://doi.org/10.1038/ncomms15055
Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19:971–974. https://doi.org/10.1038/nbt1001-971
Yuan Y, Yang Y, Tian Y, Park J, Dai A, Roberts RM, Liu Y, Han X (2016) Efficient long-term cryopreservation of pluripotent stem cells at -80 degrees C. Sci Rep 6:34476. https://doi.org/10.1038/srep34476
Acknowledgements
We would like to thank Björn Menten (Center for Medical Genetics, Ghent University) for his invaluable practical expertise and assistance in the chromosomal analysis experiments of the present study.
Funding
B.H. and this research are supported by a Ghent University grant, Faculty Health and Medical Sciences; a research grant from Research Foundation—Flanders (FWO) (FWO/KAN/ 1507816N), the Agency for Innovation by Science and Technology (IWT, Project Number 150042) and by the Strategic Basic Research programme (SBO, Project Number B/14743/01). M.P. is supported by the Ghent University Special Research Fund (BOF01D08114). J.v.H. and her research are supported by the Special Research Fund of the Ghent University (BOF/STA 2016000401).
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Editor: Tetsuji Okamoto
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Aalders, J., Van den Vreken, N., Popovic, M. et al. Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells. In Vitro Cell.Dev.Biol.-Animal 55, 777–783 (2019). https://doi.org/10.1007/s11626-019-00413-9
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DOI: https://doi.org/10.1007/s11626-019-00413-9