Editing Embryoid body (section)

==Formation==
EBs are formed by the [[cell junction|homophilic binding]] of the Ca2+ dependent adhesion molecule [[E-cadherin]], which is highly expressed on undifferentiated ESCs.<ref name="ReferenceD">{{Cite journal 
| last1 = Kurosawa | first1 = H. 
| title = Methods for inducing embryoid body formation: In vitro differentiation system of embryonic stem cells 
| doi = 10.1263/jbb.103.389 
| journal = Journal of Bioscience and Bioengineering 
| volume = 103 
| issue = 5 
| pages = 389–398 
| year = 2007 
| pmid = 17609152 
| pmc = 
}}</ref><ref>{{Cite journal 
| last1 = Larue | first1 = L. 
| last2 = Antos | first2 = C. 
| last3 = Butz | first3 = S. 
| last4 = Huber | first4 = O. 
| last5 = Delmas | first5 = V. 
| last6 = Dominis | first6 = M. 
| last7 = Kemler | first7 = R. 
| title = A role for cadherins in tissue formation 
| journal = Development 
| volume = 122 
| issue = 10 
| pages = 3185–3194 
| year = 1996 
| doi = 10.1242/dev.122.10.3185 
| pmid = 8898231
}}</ref><ref name="ReferenceE">{{Cite journal 
| last1 = Yoon | first1 = B. S. 
| last2 = Yoo | first2 = S. J. 
| last3 = Lee | first3 = J. E. 
| last4 = You | first4 = S. 
| last5 = Lee | first5 = H. T. 
| last6 = Yoon | first6 = H. S. 
| doi = 10.1111/j.1432-0436.2006.00063.x 
| title = Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment 
| journal = Differentiation 
| volume = 74 
| issue = 4 
| pages = 149–159 
| year = 2006 
| pmid = 16683985 
| pmc = 
}}</ref>  When cultured as single cells in the absence of anti-differentiation factors, ESCs spontaneously aggregate to form EBs.<ref name="ReferenceD"/><ref>{{Cite journal 
| last1 = Park | first1 = J. H. 
| last2 = Kim | first2 = S. J. 
| last3 = Oh | first3 = E. J. 
| last4 = Moon | first4 = S. Y. 
| last5 = Roh | first5 = S. I. 
| last6 = Kim | first6 = C. G. 
| last7 = Yoon | first7 = H. S. 
| title = Establishment and Maintenance of Human Embryonic Stem Cells on STO, a Permanently Growing Cell Line 
| doi = 10.1095/biolreprod.103.017467 
| journal = Biology of Reproduction 
| volume = 69 
| issue = 6 
| pages = 2007–2014 
| year = 2003 
| pmid = 12930726 
| pmc = 
| doi-access = free 
}}</ref><ref>{{Cite journal 
| last1 = Williams | first1 = R. L. 
| last2 = Hilton | first2 = D. J. 
| last3 = Pease | first3 = S. 
| last4 = Willson | first4 = T. A. 
| last5 = Stewart | first5 = C. L. 
| last6 = Gearing | first6 = D. P. 
| last7 = Wagner | first7 = E. F. 
| last8 = Metcalf | first8 = D. 
| last9 = Nicola | first9 = N. A. 
| doi = 10.1038/336684a0 
| last10 = Gough | first10 = N. M. 
| title = Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells 
| journal = Nature 
| volume = 336 
| issue = 6200 
| pages = 684–687 
| year = 1988 
| pmid = 3143916 
| pmc = 
| bibcode = 1988Natur.336..684W 
| s2cid = 4346252 
}}</ref><ref>{{Cite journal 
| last1 = Ludwig | first1 = T. E. 
| last2 = Levenstein | first2 = M. E. 
| last3 = Jones | first3 = J. M. 
| last4 = Berggren | first4 = W. T. 
| last5 = Mitchen | first5 = E. R. 
| last6 = Frane | first6 = J. L. 
| last7 = Crandall | first7 = L. J. 
| last8 = Daigh | first8 = C. A. 
| last9 = Conard | first9 = K. R. 
| last10 = Piekarczyk 
| doi = 10.1038/nbt1177 | first10 = M. S. 
| last11 = Llanas | first11 = R. A. 
| last12 = Thomson | first12 = J. A. 
| title = Derivation of human embryonic stem cells in defined conditions 
| journal = Nature Biotechnology 
| volume = 24 
| issue = 2 
| pages = 185–187 
| year = 2006 
| pmid = 16388305 
| pmc = 
| s2cid = 11484871 
}}</ref> Such spontaneous formation is often accomplished in bulk suspension cultures whereby the dish is coated with non-adhesive materials, such as [[agar]] or hydrophilic polymers, to promote the preferential adhesion between single cells, rather than to the culture substrate.  As hESC undergo apoptosis when cultured as single cells, EB formation often necessitates the use of inhibitors of the [[rho-associated protein kinase|rho associated kinase]] (ROCK) pathway, including the small molecules Y-27632<ref>{{Cite journal 
| last1 = Watanabe | first1 = K. 
| last2 = Ueno | first2 = M. 
| last3 = Kamiya | first3 = D. 
| last4 = Nishiyama | first4 = A. 
| last5 = Matsumura | first5 = M. 
| last6 = Wataya | first6 = T. 
| last7 = Takahashi | first7 = J. B. 
| last8 = Nishikawa | first8 = S. 
| last9 = Nishikawa | first9 = S. I. 
| last10 = Muguruma 
| doi = 10.1038/nbt1310 | first10 = K. 
| last11 = Sasai | first11 = Y. 
| title = A ROCK inhibitor permits survival of dissociated human embryonic stem cells 
| journal = Nature Biotechnology 
| volume = 25 
| issue = 6 
| pages = 681–686 
| year = 2007 
| pmid = 17529971 
| pmc = 
| s2cid = 8213725 
}}</ref> and 2,4 disubstituted thiazole (Thiazovivin/Tzv).<ref>{{Cite journal 
| last1 = Xu | first1 = Y. 
| last2 = Zhu | first2 = X. 
| last3 = Hahm | first3 = H. S. 
| last4 = Wei | first4 = W. 
| last5 = Hao | first5 = E. 
| last6 = Hayek | first6 = A. 
| last7 = Ding | first7 = S. 
| doi = 10.1073/pnas.1002024107 
| title = Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules 
| journal = Proceedings of the National Academy of Sciences 
| volume = 107 
| issue = 18 
| pages = 8129–8134 
| year = 2010 
| pmid = 20406903 
| pmc =2889586 
| bibcode = 2010PNAS..107.8129X 
| doi-access = free 
}}</ref>  Alternatively, to avoid dissociation into single cells, EBs can be formed from hESCs by manual separation of adherent colonies (or regions of colonies) and subsequently cultured in suspension.  Formation of EBs in suspension is amenable to the formation of large quantities of EBs, but provides little control over the size of the resulting aggregates, often leading to large, irregularly shaped EBs.  As an alternative, the [[fluid dynamics|hydrodynamic]] forces imparted in mixed culture platforms increase the homogeneity of EB sizes when ESCs are inoculated within bulk suspensions.<ref>{{Cite journal 
| last1 = Carpenedo | first1 = R. L. 
| last2 = Sargent | first2 = C. Y. 
| last3 = McDevitt | first3 = T. C. 
| doi = 10.1634/stemcells.2006-0523 
| title = Rotary Suspension Culture Enhances the Efficiency, Yield, and Homogeneity of Embryoid Body Differentiation 
| journal = Stem Cells 
| volume = 25 
| issue = 9 
| pages = 2224–2234 
| year = 2007 
| pmid = 17585171 
| pmc = 
| s2cid = 25461651 
| doi-access = free 
}}</ref>

Formation of EBs can also be more precisely controlled by the inoculation of known cell densities within single drops (10-20 μL) suspended from the lid of a Petri dish, known as hanging drops.<ref name="ReferenceE"/>  While this method enables control of EB size by altering the number of cells per drop, the formation of hanging drops is labor-intensive and not easily amenable to scalable cultures.  Additionally, the media can not be easily exchanged within the traditional hanging drop format, necessitating the transfer of hanging drops into bulk suspension cultures after 2–3 days of formation, whereby individual EBs tend to agglomerate.  Recently, new technologies have been developed to enable media exchange within a modified hanging drop format.<ref>{{Cite journal 
| last1 = Tung | first1 = Y. C. 
| last2 = Hsiao | first2 = A. Y. 
| last3 = Allen | first3 = S. G. 
| last4 = Torisawa | first4 = Y. S. 
| last5 = Ho | first5 = M. 
| last6 = Takayama | first6 = S. 
| s2cid = 35415772 
| doi = 10.1039/c0an00609b 
| title = High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array 
| journal = The Analyst 
| volume = 136 
| issue = 3 
| pages = 473–478 
| year = 2011 
| pmid = 20967331 
| pmc =7454010 
| bibcode = 2011Ana...136..473T 
}}</ref>  In addition, technologies have also been developed to physically separate cells by forced aggregation of ESCs within individual wells or confined on adhesive substrates,<ref>{{Cite journal 
| last1 = Park | first1 = J. 
| last2 = Cho | first2 = C. H. 
| last3 = Parashurama | first3 = N. 
| last4 = Li | first4 = Y. 
| last5 = Berthiaume | first5 = F. O. 
| last6 = Toner | first6 = M. 
| last7 = Tilles | first7 = A. W. 
| last8 = Yarmush | first8 = M. L. 
| doi = 10.1039/b704739h 
| title = Microfabrication-based modulation of embryonic stem cell differentiation 
| journal = Lab on a Chip 
| volume = 7 
| issue = 8 
| pages = 1018–1028 
| year = 2007 
| pmid = 17653344 
| pmc = 
| url = http://oasis.postech.ac.kr/handle/2014.oak/11320 
}}</ref><ref>{{Cite journal 
| last1 = Mohr | first1 = J. C. 
| last2 = De Pablo | first2 = J. J. 
| last3 = Palecek | first3 = S. P. 
| doi = 10.1016/j.biomaterials.2006.07.012 
| title = 3-D microwell culture of human embryonic stem cells 
| journal = Biomaterials 
| volume = 27 
| issue = 36 
| pages = 6032–6042 
| year = 2006 
| pmid = 16884768 
| pmc = 
}}</ref><ref name="ReferenceF">{{Cite journal 
| last1 = Hwang | first1 = Y. -S. 
| last2 = Chung | first2 = B. G. 
| last3 = Ortmann | first3 = D. 
| last4 = Hattori | first4 = N. 
| last5 = Moeller | first5 = H. -C. 
| last6 = Khademhosseini | first6 = A. 
| doi = 10.1073/pnas.0905550106 
| title = Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11 
| journal = Proceedings of the National Academy of Sciences 
| volume = 106 
| issue = 40 
| pages = 16978–16983 
| year = 2009 
| pmid = 19805103 
| pmc =2761314 
| bibcode = 2009PNAS..10616978H 
| doi-access = free 
}}</ref><ref>{{Cite journal 
| last1 = Ungrin | first1 = M. D. 
| last2 = Joshi | first2 = C. 
| last3 = Nica | first3 = A. 
| last4 = Bauwens | first4 = C. L. 
| last5 = Zandstra | first5 = P. W. 
| editor1-last = Callaerts 
| editor1-first = Patrick 
| title = Reproducible, Ultra High-Throughput Formation of Multicellular Organization from Single Cell Suspension-Derived Human Embryonic Stem Cell Aggregates 
| doi = 10.1371/journal.pone.0001565 
| journal = PLOS ONE 
| volume = 3 
| issue = 2 
| pages = e1565 
| year = 2008 
| pmid = 18270562 
| pmc =2215775 
| bibcode = 2008PLoSO...3.1565U 
| doi-access = free 
}}</ref> which enables increased throughput, controlled formation of EBs.  Ultimately, the methods used for EB formation may impact the heterogeneity of EB populations, in terms of aggregation kinetics, EB size and yield, as well as differentiation trajectories.<ref name="ReferenceF"/><ref>{{Cite journal 
| last1 = Sargent | first1 = C. Y. 
| last2 = Berguig | first2 = G. Y. 
| last3 = McDevitt | first3 = T. C. 
| doi = 10.1089/ten.tea.2008.0145 
| title = Cardiomyogenic Differentiation of Embryoid Bodies is Promoted by Rotary Orbital Suspension Culture 
| journal = Tissue Engineering Part A 
| volume = 15 
| issue = 2 
| pages = 331–342 
| year = 2009 
| pmid = 19193130 
| pmc = 
}}</ref><ref>{{Cite journal 
| last1 = Bauwens | first1 = C. L. L. 
| last2 = Peerani | first2 = R. 
| last3 = Niebruegge | first3 = S. 
| last4 = Woodhouse | first4 = K. A. 
| last5 = Kumacheva | first5 = E. 
| last6 = Husain | first6 = M. 
| last7 = Zandstra | first7 = P. W. 
| doi = 10.1634/stemcells.2008-0183 
| title = Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories 
| journal = Stem Cells 
| volume = 26 
| issue = 9 
| pages = 2300–2310 
| year = 2008 
| pmid = 18583540 
| pmc = 
| doi-access = free 
}}</ref>