Editing Embryoid body (section)

==Challenges to directing differentiation==
In contrast to the differentiation of ESCs in monolayer cultures, whereby the addition of soluble morphogens and the extracellular microenvironment can be precisely and homogeneously controlled, the three-dimensional structure of EBs poses challenges to directed differentiation.<ref name="ReferenceC"/><ref name="TEB 2011">{{Cite journal 
| last1 = Kinney | first1 = M. A. 
| last2 = Sargent | first2 = C. Y. 
| last3 = McDevitt | first3 = T. C. 
| doi = 10.1089/ten.TEB.2011.0040 
| title = The Multiparametric Effects of Hydrodynamic Environments on Stem Cell Culture 
| journal = Tissue Engineering Part B: Reviews 
| volume = 17 
| issue = 4 
| pages = 249–262 
| year = 2011 
| pmid = 21491967 
| pmc =3142632 
}}</ref>  For example, the visceral endoderm population which forms the exterior of EBs, creates an exterior “shell” consisting of tightly connected [[epithelial]]-like cells, as well as a dense [[extracellular matrix|ECM]].<ref name="ReferenceG">{{Cite journal 
| last1 = Carpenedo | first1 = R. L. 
| last2 = Bratt-Leal | first2 = A. S. M. 
| last3 = Marklein | first3 = R. A. 
| last4 = Seaman | first4 = S. A. 
| last5 = Bowen | first5 = N. J. 
| last6 = McDonald | first6 = J. F. 
| last7 = McDevitt | first7 = T. C. 
| doi = 10.1016/j.biomaterials.2009.01.007 
| title = Homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules 
| journal = Biomaterials 
| volume = 30 
| issue = 13 
| pages = 2507–2515 
| year = 2009 
| pmid = 19162317 
| pmc =2921510 
}}</ref><ref>{{Cite journal 
| last1 = Sachlos | first1 = E. 
| last2 = Auguste | first2 = D. T. 
| doi = 10.1016/j.biomaterials.2008.08.012 
| title = Embryoid body morphology influences diffusive transport of inductive biochemicals: A strategy for stem cell differentiation 
| journal = Biomaterials 
| volume = 29 
| issue = 34 
| pages = 4471–4480 
| year = 2008 
| pmid = 18793799 
| pmc = 
}}</ref>  Due to such physical restrictions, in combination with EB size, [[transport phenomena|transport]] limitations occur within EBs, creating gradients of morphogens, metabolites, and nutrients.<ref name="TEB 2011"/>  It has been estimated that oxygen transport is limited in cell aggregates larger than approximately 300&nbsp;μm in diameter;<ref>{{Cite journal 
| last1 = Van Winkle | first1 = A. P. 
| last2 = Gates | first2 = I. D. 
| last3 = Kallos | first3 = M. S. 
| doi = 10.1159/000330691 
| title = Mass Transfer Limitations in Embryoid Bodies during Human Embryonic Stem Cell Differentiation 
| journal = Cells Tissues Organs 
| year = 2012 
| pmid = 22249133 
| pmc =
 | volume=196
 | issue=1
 | pages=34–47
| s2cid = 42754482 
}}</ref> however, the development of such gradients are also impacted by molecule size and cell uptake rates.  Therefore, the delivery of morphogens to EBs results in increased heterogeneity and decreased efficiency of differentiated cell populations compared to monolayer cultures.  One method of addressing transport limitations within EBs has been through polymeric delivery of morphogens from within the EB structure.<ref name="ReferenceG"/><ref>{{Cite journal 
| last1 = Bratt-Leal | first1 = A. S. M. 
| last2 = Carpenedo | first2 = R. L. 
| last3 = Ungrin | first3 = M. D. 
| last4 = Zandstra | first4 = P. W. 
| last5 = McDevitt | first5 = T. C. 
| title = Incorporation of biomaterials in multicellular aggregates modulates pluripotent stem cell differentiation 
| doi = 10.1016/j.biomaterials.2010.08.113 
| journal = Biomaterials 
| volume = 32 
| issue = 1 
| pages = 48–56 
| year = 2011 
| pmid = 20864164 
| pmc =2987521 
}}</ref><ref>{{Cite journal 
| last1 = Purpura | first1 = K. A. 
| last2 = Bratt-Leal | first2 = A. S. M. 
| last3 = Hammersmith | first3 = K. A. 
| last4 = McDevitt | first4 = T. C. 
| last5 = Zandstra | first5 = P. W. 
| title = Systematic engineering of 3D pluripotent stem cell niches to guide blood development 
| doi = 10.1016/j.biomaterials.2011.10.051 
| journal = Biomaterials 
| volume = 33 
| issue = 5 
| pages = 1271–1280 
| year = 2012 
| pmid = 22079776 
| pmc =  4280365}}</ref> Additionally, EBs can be cultured as individual microtissues and subsequently assembled into larger structures for tissue engineering applications.<ref>{{Cite journal 
| last1 = Bratt-Leal | first1 = A. S. M. 
| last2 = Kepple | first2 = K. L. 
| last3 = Carpenedo | first3 = R. L. 
| last4 = Cooke | first4 = M. T. 
| last5 = McDevitt | first5 = T. C. 
| title = Magnetic manipulation and spatial patterning of multi-cellular stem cell aggregates 
| doi = 10.1039/c1ib00064k 
| journal = Integrative Biology 
| volume = 3 
| issue = 12 
| pages = 1224–1232 
| year = 2011 
| pmid = 22076329 
| pmc =  4633527}}</ref>  Although the complexity resulting from the three-dimensional adhesions and signaling may recapitulate more native tissue structures,<ref>{{Cite journal 
| last1 = Akins | first1 = R. E. 
| last2 = Rockwood | first2 = D. 
| last3 = Robinson | first3 = K. G. 
| last4 = Sandusky | first4 = D. 
| last5 = Rabolt | first5 = J. 
| last6 = Pizarro | first6 = C. 
| doi = 10.1089/ten.tea.2009.0458 
| title = Three-Dimensional Culture Alters Primary Cardiac Cell Phenotype 
| journal = Tissue Engineering Part A 
| volume = 16 
| issue = 2 
| pages = 629–641 
| year = 2010 
| pmid = 20001738 
| pmc =2813151 
}}</ref><ref>{{Cite journal 
| last1 = Chang | first1 = T. T. 
| last2 = Hughes-Fulford | first2 = M. 
| doi = 10.1089/ten.tea.2007.0434 
| title = Monolayer and Spheroid Culture of Human Liver Hepatocellular Carcinoma Cell Line Cells Demonstrate Distinct Global Gene Expression Patterns and Functional Phenotypes 
| journal = Tissue Engineering Part A 
| volume = 15 
| issue = 3 
| pages = 559–567 
| year = 2009 
| pmid = 18724832 
| pmc =6468949 
}}</ref> it also creates challenges for understanding the relative contributions of mechanical, chemical, and physical signals to the resulting cell phenotypes and morphogenesis.