Differentiation Patterns of Embryonic Stem Cells in Two- versus Three-Dimensional Culture
Open accessAbstract
Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation. We compared mouse embryonic stem cells (ESCs) differentiated on collagen type I-coated surfaces (SLIDEs), embedded in collagen type I gels (GELs), and in suspension as embryoid bodies (EBs). For a systematic analysis in these studies, key parameters were kept identical to allow for direct comparison across culture configurations. We determined that all three configurations supported differentiation of ESCs and that the kinetics of differentiation differed greatly for cells cultured in 2D versus 3D. SLIDE cultures induced overall differentiation more quickly than 3D configurations, with earlier expression of cytoskeletal and extracellular matrix proteins. For 3D culture as GELs or EBs, cells clustered similarly, formed complex structures and promoted differentiation towards cardiovascular phenotypes. GEL culture, however, also allowed for contraction of the collagen matrix. For differentiation towards fibroblasts and smooth muscle cells which actively remodel their environment, GEL culture may be particularly beneficial. Overall, this study determined the effects of dimensionality on differentiation and helps in the rational design of protocols to generate phenotypes needed for tissue engineering and regenerative medicine.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In mouse embryonic stem cells, OCT4 levels decreased in both two-dimensional and three-dimensional cultures, indicating a loss of pluripotency, whereas NES and BRACHY-T markers increased.
“Pineda et al.(91) demonstrated that OCT4 in mouse ESCs decreased in both 2D and 3D cultures, showing a loss of pluripotency, whereas NES (ectoderm) and BRACHY-T (mesoderm) markers were shown to increase.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [91]
In Results, ‘Pluripotency and Germ Lineage Differentiation’ (page unavailable), OCT4 markedly decreased after four days in the 2D slide and 3D gel and embryoid-body cultures, indicating loss of pluripotency. The same section says NESTIN (NES) and BRACHY-T varied over time and were highest at day 4, rather than showing a general increase; ‘Functional Gene Groupings’ also reports that NES remained very low. (LLM, conf=0.85)
Experimental — accuracy not yet measuredFree EBs had more dynamic changes in genotypic expression levels of cytoskeletal genes over 12-day span compared with GEL cultures.
“Free EBs had more dynamic changes in the genotypic expression levels of cytoskeletal genes over the same 12-day span compared with the GEL cultures.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [91]
The Results section (Matrix Remodeling; page unavailable) reports that intermediate-filament gene expression rose in both EB and GEL cultures from day 4 to days 8 and 12, with higher levels in EBs at day 12. It does not establish more dynamic changes across cytoskeletal genes generally: the measured microtubule gene was unaffected by time or culture condition. (LLM, conf=0.82)
Experimental — accuracy not yet measuredA key difference in morphology of GEL cultures was presence of elongated masses along edges of gel form.
“A key difference in the morphology of GEL cultures was the presence of elongated masses along the edges of the gel form.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [91]
Pineda et al. found that GEL and unattached EB cultures produced similar cluster morphology, with defined boundaries and occasional cavities.
“GEL and unattached EB cultures produced similar cluster morphology, with defined boundaries and the occasional cavities.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [91]
Mouse embryonic stem cells grown on collagen type I-coated glass slides in two-dimensional culture differentiated more rapidly than cells in three-dimensional cultures (embryoid bodies and GELs).
“However, the ESCs cultured on glass slides (2D) coated with collagen type I had a faster rate of differentiation than in 3D cultures (EBs and GELs).”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [91]
By day 12, unattached EB cultures had higher expression levels of intermediate filaments lamin, keratin 8, and vimentin.
“By day 12, unattached EB cultures had higher expression levels of the intermediate filaments lamin, keratin 8, and vimentin (91).”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [91]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Embryonic Stem Cell Lines Derived from Human Blastocysts
Thomson, J. A., Itskovitz‐Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., & Jones, J. M. (1998) · Science · doi:10.1126/science.282.5391.1145 · Cited by 15,947 · see citing works
Matrix elasticity directs stem cell lineage specification
Engler, A. J., Sen, S., Sweeney, H. L., & Discher, D. E. (2006) · Cell · doi:10.1016/j.cell.2006.06.044 · Cited by 13,915 · see citing works
Martin, G. R. (1981) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.78.12.7634 · Cited by 5,756 · see citing works
Lutolf, M. P., & Hubbell, J. A. (2005) · Nature Biotechnology · doi:10.1038/nbt1055 · Cited by 4,551 · see citing works
Adipose-derived stem cells for regenerative medicine
Gimble, J. M., Katz, A. J., & Bunnell, B. A. (2007) · Circulation Research · doi:10.1161/01.res.0000265074.83288.09 · Cited by 2,332 · see citing works
Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus
Wang, N., Tytell, J. D., & Ingber, D. E. (2008) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm2594 · Cited by 1,768 · see citing works
Extracellular-matrix tethering regulates stem-cell fate
Trappmann, B., Gautrot, J. E., Connelly, J. T., Strange, D. G. T., Li, Y., Oyen, M. L., Stuart, M. A. C., Boehm, H., Li, B., Viola Vogel, B., et al. (2012) · Nature Materials · doi:10.1038/nmat3339 · Cited by 1,570 · see citing works
Itskovitz‐Eldor, J., Schuldiner, M., Kenyagin‐Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., & Benvenisty, N. (2000) · Molecular Medicine · doi:10.1007/bf03401776 · Cited by 1,502 · see citing works
Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity
Hinz, B., Celetta, G., Tomasek, J. J., Gabbiani, G., & Chaponnier, C. (2001) · Molecular Biology of the Cell · doi:10.1091/mbc.12.9.2730 · Cited by 1,288 · see citing works
HNF-3β is essential for node and notochord formation in mouse development
Ang, S., & Rossant, J. (1994) · Cell · doi:10.1016/0092-8674(94)90522-3 · Cited by 981 · see citing works
Molecular basis of the effects of shear stress on vascular endothelial cells
Li, Y.-S. J., Haga, J. H., & Chien, S. (2005) · Journal of Biomechanics · doi:10.1016/j.jbiomech.2004.09.030 · Cited by 904 · see citing works
Nishikawa, S., Nishikawa, S., Hirashima, M., Matsuyoshi, N., & Kodama, H. (1998) · Development · doi:10.1242/dev.125.9.1747 · Cited by 829 · see citing works
Fibroblast biology in three-dimensional collagen matrices
Grinnell, F. (2003) · Trends in Cell Biology · doi:10.1016/s0962-8924(03)00057-6 · Cited by 819 · see citing works
Weinstein, D. C., Altaba, A. R. I., Chen, W. S., Hoodless, P. A., Prezioso, V. R., Jessell, T. M., & Darnell, J. E., Jr. (1994) · Cell · doi:10.1016/0092-8674(94)90523-1 · Cited by 783 · see citing works
Differentiation of Pluripotent Embryonic Stem Cells Into Cardiomyocytes
Boheler, K. R., Czyż, J., Tweedie, D., Yang, H., Anisimov, S. V., & Wobus, A. M. (2002) · Circulation Research · doi:10.1161/01.res.0000027865.61704.32 · Cited by 737 · see citing works
Gerecht, S., Burdick, J. A., Ferreira, L., Townsend, S. A., Langer, R., & Vunjak‐Novakovic, G. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0703723104 · Cited by 666 · see citing works
A hierarchical unsupervised growing neural network for clustering gene expression patterns
Herrero, J., Valencia, A., & Dopazo, J. (2001) · Bioinformatics · doi:10.1093/bioinformatics/17.2.126 · Cited by 622 · see citing works
Role of Integrins in Endothelial Mechanosensing of Shear Stress
Shyy, J. Y.-J., & Chien, S. (2002) · Circulation Research · doi:10.1161/01.res.0000038487.19924.18 · Cited by 550 · see citing works
Mechanotransduction at Cell-Matrix and Cell-Cell Contacts
Chen, C. S., Tan, J. L., & Tien, J. (2004) · Annual Review of Biomedical Engineering · doi:10.1146/annurev.bioeng.6.040803.140040 · Cited by 535 · see citing works
Bosnakovski, D., Mizuno, M., Kim, G., Takagi, S., Okumura, M., & FUJINAGA, T. (2006) · Biotechnology and Bioengineering · doi:10.1002/bit.20828 · Cited by 502 · see citing works
A microfabricated array bioreactor for perfused 3D liver culture
Powers, M. J., Domanský, K., Mofrad, M. R. K., Kalezi, A., Capitano, A. T., Upadhyaya, A., Kurzawski, P., Wack, K. E., Stolz, D. B., Kamm, R. D., et al. (2002) · Biotechnology and Bioengineering · doi:10.1002/bit.10143 · Cited by 464 · see citing works
Xu, Y., Zhu, X., Hahm, H. S., Wei, W., Hao, E., Hayek, A., & Ding, S. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1002024107 · Cited by 348 · see citing works
Dang, S. M., Kyba, M., Perlingeiro, R. C. R., Daley, G. Q., & Zandstra, P. W. (2002) · Biotechnology and Bioengineering · doi:10.1002/bit.10220 · Cited by 341 · see citing works
Yamamoto, K., Sokabe, T., Watabe, T., Miyazono, K., Yamashita, J. K., Obi, S., Ohura, N., Matsushita, A., Kamiya, A., & Ando, J. (2004) · American Journal of Physiology-Heart and Circulatory Physiology · doi:10.1152/ajpheart.00956.2004 · Cited by 338 · see citing works
Nostro, M. C., Cheng, X., Keller, G. M., & Gadue, P. J. (2008) · Cell stem cell · doi:10.1016/j.stem.2007.10.011 · Cited by 318 · see citing works
Rodríguez, J. P., González, M., Ríos, S., & Cambiazo, V. (2004) · Journal of Cellular Biochemistry · doi:10.1002/jcb.20234 · Cited by 276 · see citing works
Sumanasinghe, R. D., Bernacki, S. H., & Loboa, E. G. (2006) · Tissue Engineering · doi:10.1089/ten.2006.12.3459 · Cited by 227 · see citing works
Yamanaka, Y., Tamplin, O. J., Beckers, A., Gossler, A., & Rossant, J. (2007) · Developmental Cell · doi:10.1016/j.devcel.2007.10.016 · Cited by 182 · see citing works
Shinzato, S., Kobayashi, M., Mousa, W. F., Kamimura, M., Neo, M., Kitamura, Y., Kokubo, T., & Nakamura, T. (2000) · Journal of Biomedical Materials Research · doi:10.1002/(sici)1097-4636(200008)51:2 · Cited by 165 · see citing works
Inhibition of human embryonic stem cell differentiation by mechanical strain
Saha, S., Lin, J., de Pablo, J. J., & Palecek, S. P. (2005) · Journal of Cellular Physiology · doi:10.1002/jcp.20441 · Cited by 164 · see citing works
Differential Responses of Adult Cardiac Fibroblasts to in vitro Biaxial Strain Patterns
Lee, A. A., Delhaas, T., McCulloch, A. D., & Villarreal, F. J. (1999) · Journal of Molecular and Cellular Cardiology · doi:10.1006/jmcc.1999.1017 · Cited by 149 · see citing works
Liu, Y., Goldberg, A. J., Dennis, J. E., Gronowicz, G., & Kuhn, L. T. (2012) · PLoS ONE · doi:10.1371/journal.pone.0033225 · Cited by 147 · see citing works
Kanda, K., & Matsuda, T. (1994) · Cell Transplantation · doi:10.1177/096368979400300605 · Cited by 143 · see citing works
Carpenedo, R. L., Bratt-Leal, A. M., Marklein, R. A., Seaman, S. A., Bowen, N. J., McDonald, J. F., & McDevitt, T. C. (2009) · Biomaterials · doi:10.1016/j.biomaterials.2009.01.007 · Cited by 133 · see citing works
Tensile Strain as a Regulator of Mesenchymal Stem Cell Osteogenesis
Kearney, E. M., Farrell, E., Prendergast, P. J., & Campbell, V. A. (2010) · Annals of Biomedical Engineering · doi:10.1007/s10439-010-9979-4 · Cited by 130 · see citing works
McMahon, L. A., Reid, A. J., Campbell, V. A., & Prendergast, P. J. (2007) · Annals of Biomedical Engineering · doi:10.1007/s10439-007-9416-5 · Cited by 129 · see citing works
Integrin-Mediated Collagen Matrix Reorganization by Cultured Human Vascular Smooth Muscle Cells
Lee, R. T., Berditchevski, F., Cheng, G. C., et al. (1995) · Circulation Research · doi:10.1161/01.res.76.2.209 · Cited by 124 · see citing works
Mechanics Regulates Fate Decisions of Human Embryonic Stem Cells
Sun, Y., Villa-Diaz, L. G., Lam, R. H. W., Chen, W., Krebsbach, P. H., & Fu, J. (2012) · PLoS ONE · doi:10.1371/journal.pone.0037178 · Cited by 118 · see citing works
Collagen IV Induces Trophoectoderm Differentiation of Mouse Embryonic Stem Cells
Schenke‐Layland, K., Angelis, E., Rhodes, K. E., et al. (2007) · Stem Cells · doi:10.1634/stemcells.2006-0729 · Cited by 110 · see citing works
Magic touch: how does cell–cell adhesion trigger actin assembly?
Bershadsky, A. D. (2004) · Trends in Cell Biology · doi:10.1016/j.tcb.2004.09.009 · Cited by 105 · see citing works
Hydrogels for in vivo‐like three‐dimensional cellular studies
DeVolder, R. J., & Kong, H. (2012) · WIREs Systems Biology and Medicine · doi:10.1002/wsbm.1174 · Cited by 102 · see citing works
Oh, S.-A., Lee, H., Lee, J. H., Kim, T.-H., Jang, J., Kim, H., & Wall, I. B. (2011) · Tissue Engineering Part A · doi:10.1089/ten.tea.2011.0360 · Cited by 85 · see citing works
Ahsan, T., & Nerem, R. M. (2010) · Tissue Engineering Part A · doi:10.1089/ten.tea.2010.0014 · Cited by 84 · see citing works
Self-citationCullen, D. K., Lessing, C., & LaPlaca, M. C. (2007) · Annals of Biomedical Engineering · doi:10.1007/s10439-007-9292-z · Cited by 81 · see citing works
A Simple Method for Forming Embryoid Body from Mouse Embryonic Stem Cells
Kurosawa, H., Imamura, T., Koike, M., Sasaki, K., & Amano, Y. (2003) · Journal of Bioscience and Bioengineering · doi:10.1016/s1389-1723(03)90148-4 · Cited by 79 · see citing works
Transcription factor regulation by mechanical stress
Mendez, M. G., & Janmey, P. A. (2012) · The International Journal of Biochemistry & Cell Biology · doi:10.1016/j.biocel.2012.02.003 · Cited by 78 · see citing works
Wang, H., Gilner, J. B., Bautch, V. L., Wang, D. Z., Wainwright, B. J., Kirby, S. L., & Patterson, C. (2006) · Journal of Biological Chemistry · doi:10.1074/jbc.m606610200 · Cited by 76 · see citing works
Nikmanesh, M., Shi, Z., & Tarbell, J. M. (2011) · Biotechnology and Bioengineering · doi:10.1002/bit.23302 · Cited by 64 · see citing works
Willems, E., & Leyns, L. (2008) · Differentiation · doi:10.1111/j.1432-0436.2007.00257.x · Cited by 59 · see citing works
Facile micropatterning of dual hydrogel systems for 3D models of neurite outgrowth
Curley, J. L., & Moore, M. J. (2011) · Journal of Biomedical Materials Research Part A · doi:10.1002/jbm.a.33195 · Cited by 54 · see citing works
Fuchs, C., Scheinast, M., Pasteiner, W., et al. (2011) · Cells Tissues Organs · doi:10.1159/000328712 · Cited by 50 · see citing works
Peister, A., Woodruff, M. A., Prince, J. J., Gray, D. P., Hutmacher, D. W., & Guldberg, R. E. (2011) · Stem Cell Research · doi:10.1016/j.scr.2011.03.001 · Cited by 48 · see citing works
Riha, G. M., Wang, X., Wang, H., Chai, H., Mu, H. H., Lin, P. H., Lumsden, A. B., Yao, Q. C., & Chen, C. (2006) · Surgery · doi:10.1016/j.surg.2006.07.043 · Cited by 46 · see citing works
Effects of shear stress on germ lineage specification of embryonic stem cells
Wolfe, R. P., Leleux, J. A., Nerem, R. M., & Ahsan, T. (2012) · Integrative Biology · doi:10.1039/c2ib20040f · Cited by 46 · see citing works
Self-citationEpiblast stem cells contribute new insight into pluripotency and gastrulation
Chenoweth, J. G., McKay, R. D. G., & Tesar, P. J. (2010) · Development Growth & Differentiation · doi:10.1111/j.1440-169x.2010.01171.x · Cited by 46 · see citing works
Krawetz, R. J., Taiani, J. T., Wu, E. Y., et al. (2011) · Tissue Engineering Part A · doi:10.1089/ten.tea.2011.0174 · Cited by 29 · see citing works
Human Mesenchymal Stem Cells Form Multicellular Structures in Response to Applied Cyclic Strain
Doyle, A. M., Nerem, R. M., & Ahsan, T. (2009) · Annals of Biomedical Engineering · doi:10.1007/s10439-009-9644-y · Cited by 22 · see citing works
Self-citation
Flagged first, then by citations
Research works printed but not linked: 1
- Elisseeff J McIntosh W Anseth K Riley S Ragan P Langer R 2000 Photoencapsulation of chondrocytes in poly(ethylene oxide)-based semi-interpenetrating networks J Biomed Mater Res 51 2 164 171 10825215
Cited by
Sorted by citations
Modeling physiological events in 2D vs. 3D cell culture
Duval, K., Grover, H., Han, L.-H., Mou, Y., Pegoraro, A. F., Fredberg, J., & Chen, Z. (2017) · Physiology · doi:10.1152/physiol.00036.2016 · Cited by 1,849 · see citing works
3D Bioprinting of Tumor Models for Cancer Research
Kang, Y., Datta, P., Shanmughapriya, S., & Ozbolat, I. T. (2020) · ACS Applied Bio Materials · doi:10.1021/acsabm.0c00791 · Cited by 112 · see citing works
- Authors
- Pineda, E. T., Nerem, R. M., & Ahsan, T.
- Affiliations
- Tulane University (US)
- Georgia Institute of Technology (US)
- ORCID
- Ahsan, Tabassum
- Venue
- Cells Tissues Organs
- Publisher
- S. Karger AG
- Published
- 2013
- Volume
- 197
- Issue
- 5
- Pages
- 399-410
- ISSN
- 1422-6405 (ISSN-L)
- 1422-6421
- PMC
- PMC3732738
- OpenAlex
- W1987602592
- Cited by
- 77
- References
- 57
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 4 · 2023: 4 · 2022: 16 · 2021: 5 · 2020: 14 · 2019: 2 · 2018: 7 · 2017: 4 · 2016: 5 · 2015: 2 · 2014: 5 · 2013: 4
- FWCI
- 2.39
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Cells Tissues Organs (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Georgia Institute of Technology
- Tulane University
- National Institutes of Health
- National Institute of General Medical Sciences (P20GM103629)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
- Health Sciences › Medicine › Genetics › Mesenchymal stem cell research
- Keywords
- mouse embryonic stem cells, directed differentiation, monolayer culture, 3D cell culture, type I collagen, embryoid bodies, collagen gel, slides, cardiovascular differentiation, fibroblast differentiation, smooth muscle cells, extracellular matrix, collagen matrix contraction, cell clustering, tissue engineering
- MeSH terms
- Animals, Cell Differentiation, Collagen, Cytoskeleton, Extracellular Matrix, Gels, Gene Expression Regulation, Germ Cells, Biomarkers, Cluster Analysis, Cell Culture Techniques, Cell Lineage, Pluripotent Stem Cells, Cell Proliferation, Mice, Embryonic Stem Cells
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
