Embryoid body
{{short description|Three-dimensional aggregate of pluripotent stem cells}}
File:The-L-type-Ca2+-Channels-Blocker-Nifedipine-Represses-Mesodermal-Fate-Determination-in-Murine-pone.0053407.s007.ogv-expressing spontaneously beating cardiomyocytes.]]
Embryoid bodies (EBs) are three-dimensional aggregates formed by pluripotent stem cells. These include embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC)
EBs are differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. They mimic the characteristics seen in early-stage embryos. They are often used as a model system to conduct research on various aspects of developmental biology. They can also contribute to research focused on tissue engineering and regenerative medicine.
Background
The pluripotent cell types that comprise embryoid bodies include embryonic stem cells (ESCs) derived from the blastocyst stage of embryos from mouse (mESC),{{Cite journal
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}} Similar to ESCs cultured in monolayer formats, ESCs within embryoid bodies undergo differentiation and cell specification along the three germ lineages – endoderm, ectoderm, and mesoderm – which comprise all somatic cell types.{{Cite journal
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In contrast to monolayer cultures, however, the spheroid structures that are formed when ESCs aggregate enables the non-adherent culture of EBs in suspension, making EB cultures inherently scalable, which is useful for bioprocessing approaches, whereby large yields of cells can be produced for potential clinical applications.{{Cite journal
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}} Therefore, the three-dimensional structure, including the establishment of complex cell adhesions and paracrine signaling within the EB microenvironment,{{Cite journal
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Formation
EBs are formed by the homophilic binding of the Ca2+ dependent adhesion molecule E-cadherin, which is highly expressed on undifferentiated ESCs.{{Cite journal
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}} When cultured as single cells in the absence of anti-differentiation factors, ESCs spontaneously aggregate to form EBs.{{Cite journal
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}} 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 kinase (ROCK) pathway, including the small molecules Y-27632{{Cite journal
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}} 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 hydrodynamic forces imparted in mixed culture platforms increase the homogeneity of EB sizes when ESCs are inoculated within bulk suspensions.{{Cite journal
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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. 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.{{Cite journal
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Differentiation within EBs
Within the context of ESC differentiation protocols, EB formation is often used as a method for initiating spontaneous differentiation toward the three germ lineages. EB differentiation begins with the specification of the exterior cells toward the primitive endoderm phenotype.{{Cite journal
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| volume = 98
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| last1 = Li | first1 = X.
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| title = Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body
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}} similar to the composition and structure of basement membrane. In response to the ECM deposition, EBs often form a cystic cavity, whereby the cells in contact with the basement membrane remain viable and those at the interior undergo apoptosis, resulting in a fluid-filled cavity surrounded by cells.{{Cite journal
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| title = Signals for death and survival: A two-step mechanism for cavitation in the vertebrate embryo
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| title = Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation
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| title = Regulation of programmed cell death by basement membranes in embryonic development
| journal = The Journal of Cell Biology
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}} Subsequent differentiation proceeds to form derivatives of the three germ lineages. In the absence of supplements, the “default” differentiation of ESCs is largely toward ectoderm, and subsequent neural lineages.{{Cite book
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| chapter = Defined Conditions for Neural Commitment and Differentiation
| title = Differentiation of Embryonic Stem Cells
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}} However, alternative media compositions, including the use of fetal bovine serum as well as defined growth factor additives, have been developed to promote the differentiation toward mesoderm and endoderm lineages.{{Cite journal
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| title = Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture
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| doi = 10.1016/j.exphem.2008.04.003
| title = Analysis of the temporal and concentration-dependent effects of BMP-4, VEGF, and TPO on development of embryonic stem cell–derived mesoderm and blood progenitors in a defined, serum-free media
| journal = Experimental Hematology
| volume = 36
| issue = 9
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| year = 2008
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| title = Wnt, Activin, and BMP Signaling Regulate Distinct Stages in the Developmental Pathway from Embryonic Stem Cells to Blood
| doi = 10.1016/j.stem.2007.10.011
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| year = 2008
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}}
As a result of the three-dimensional EB structure, complex morphogenesis occurs during EB differentiation, including the appearance of both epithelial- and mesenchymal-like cell populations, as well as the appearance of markers associated with the epithelial-mesenchymal transition (EMT).{{Cite journal
| last1 = Ten Berge | first1 = D.
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| doi = 10.1016/j.stem.2008.09.013
| title = Wnt Signaling Mediates Self-Organization and Axis Formation in Embryoid Bodies
| journal = Cell Stem Cell
| volume = 3
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| pages = 508–518
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| pmc =2683270
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| last8 = McDevitt | first8 = T. C.
| doi = 10.1369/jhc.2009.954826
| title = Synthesis and Organization of Hyaluronan and Versican by Embryonic Stem Cells Undergoing Embryoid Body Differentiation
| journal = Journal of Histochemistry and Cytochemistry
| volume = 58
| issue = 4
| pages = 345–358
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| pmid = 20026669
| pmc =2842597
}} Additionally, the inductive effects resulting from signaling between cell populations in EBs results in spatially and temporally defined changes, which promote complex morphogenesis.{{Cite journal
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| s2cid = 22010083
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| title = Geometric Control of Cardiomyogenic Induction in Human Pluripotent Stem Cells
| journal = Tissue Engineering Part A
| volume = 17
| issue = 15–16
| pages = 1901–1909
| year = 2011
| pmid = 21417693
| pmc =
| hdl = 1807/33799
| hdl-access = free
}} Tissue-like structures are often exhibited within EBs, including the appearance of blood islands reminiscent of early blood vessel structures in the developing embryo, as well as the patterning of neurite extensions (indicative of neuron organization) and spontaneous contractile activity (indicative of cardiomyocyte differentiation) when EBs are plated onto adhesive substrates such as gelatin. More recently, complex structures, including optic cup-like structures were created in vitro resulting from EB differentiation.{{Cite journal
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| title = Self-organizing optic-cup morphogenesis in three-dimensional culture
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}}
Parallels with embryonic development
{{Main|embryogenesis}}
Much of the research central to embryonic stem cell differentiation and morphogenesis is derived from studies in developmental biology and mammalian embryogenesis. For example, immediately after the blastocyst stage of development (from which ESCs are derived), the embryo undergoes differentiation, whereby cell specification of the inner cell mass results in the formation of the hypoblast and epiblast.{{Cite journal
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| last3 = Wilson | first3 = D. B.
| title = Distinct roles for visceral endoderm during embryonic mouse development
| journal = The International Journal of Developmental Biology
| volume = 43
| issue = 3
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}} Later on in postimplantation development, the anterior-posterior axis is formed and the embryo develops a transient structure known as the primitive streak.{{Cite journal
| last1 = Burdsal | first1 = C. A.
| last2 = Damsky | first2 = C. H.
| last3 = Pedersen | first3 = R. A.
| title = The role of E-cadherin and integrins in mesoderm differentiation and migration at the mammalian primitive streak
| journal = Development
| volume = 118
| issue = 3
| pages = 829–844
| year = 1993
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| pmid = 7521282
}} Much of the spatial patterning that occurs during the formation and migration of the primitive streak results from the secretion of agonists and antagonists by various cell populations, including the growth factors from the Wnt and transforming growth factor β (TGFβ) families (Lefty 1, Nodal), as well as repressors of the same molecules (Dkk-1, Sfrp1, Sfrp5).{{Cite journal
| last1 = Finley | first1 = K. R.
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| title = The mouse secreted frizzled-related protein 5 gene is expressed in the anterior visceral endoderm and foregut endoderm during early post-implantation development
| journal = Gene Expression Patterns
| volume = 3
| issue = 5
| pages = 681–684
| year = 2003
| pmid = 12972006
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| last1 = Kemp | first1 = C.
| last2 = Willems | first2 = E.
| last3 = Abdo | first3 = S.
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| last5 = Leyns | first5 = L.
| title = Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development
| doi = 10.1002/dvdy.20408
| journal = Developmental Dynamics
| volume = 233
| issue = 3
| pages = 1064–1075
| year = 2005
| pmid = 15880404
| pmc =
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| last1 = Rivera-Pérez | first1 = J. A.
| last2 = Magnuson | first2 = T.
| doi = 10.1016/j.ydbio.2005.09.012
| title = Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3
| journal = Developmental Biology
| volume = 288
| issue = 2
| pages = 363–371
| year = 2005
| pmid = 16289026
| pmc =
| doi-access = free
}} Due to the similarities between embryogenesis and ESC differentiation, many of the same growth factors are central to directed differentiation approaches.
In addition, advancements of EB culture resulted in the development of embryonic organoids (Gastruloids) which show remarkable parallels to embryonic development{{Cite bioRxiv|last1=Turner|first1=David|last2=Alonso-Crisostomo|first2=Luz|last3=Girgin|first3=Mehmet|last4=Baillie-Johnson|first4=Peter|last5=Glodowski|first5=Cherise R.|last6=Hayward|first6=Penelope C.|last7=Collignon|first7=Jérôme|last8=Gustavsen|first8=Carsten|last9=Serup|first9=Palle|date=2017-01-31|title=Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling|biorxiv=10.1101/104539}}{{Cite bioRxiv|last1=Turner|first1=David Andrew|last2=Glodowski|first2=Cherise R.|last3=Luz|first3=Alonso-Crisostomo|last4=Baillie-Johnson|first4=Peter|last5=Hayward|first5=Penny C.|last6=Collignon|first6=Jérôme|last7=Gustavsen|first7=Carsten|last8=Serup|first8=Palle|last9=Schröter|first9=Christian|date=2016-05-13|title=Interactions between Nodal and Wnt signalling Drive Robust Symmetry Breaking and Axial Organisation in Gastruloids (Embryonic Organoids)|biorxiv=10.1101/051722}}{{Cite journal|last1=Baillie-Johnson|first1=Peter|last2=Brink|first2=Susanne Carina van den|last3=Balayo|first3=Tina|last4=Turner|first4=David Andrew|last5=Arias|first5=Alfonso Martinez|date=2015-11-24|title=Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro|url=http://www.jove.com/video/53252/generation-aggregates-mouse-embryonic-stem-cells-that-show-symmetry|journal=Journal of Visualized Experiments|issue=105|pages=e53252|doi=10.3791/53252|issn=1940-087X|pmc=4692741|pmid=26650833}}{{Cite journal|last1=Brink|first1=Susanne C. van den|last2=Baillie-Johnson|first2=Peter|last3=Balayo|first3=Tina|last4=Hadjantonakis|first4=Anna-Katerina|last5=Nowotschin|first5=Sonja|last6=Turner|first6=David A.|last7=Arias|first7=Alfonso Martinez|date=2014-11-15|title=Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells|journal=Development|language=en|volume=141|issue=22|pages=4231–4242|doi=10.1242/dev.113001|issn=0950-1991|pmc=4302915|pmid=25371360}}{{Cite journal|last1=Turner|first1=David A.|last2=Hayward|first2=Penelope C.|last3=Baillie-Johnson|first3=Peter|last4=Rué|first4=Pau|last5=Broome|first5=Rebecca|last6=Faunes|first6=Fernando|last7=Arias|first7=Alfonso Martinez|date=2014-11-15|title=Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells|journal=Development|language=en|volume=141|issue=22|pages=4243–4253|doi=10.1242/dev.112979|issn=0950-1991|pmc=4302903|pmid=25371361}} such as symmetry-breaking, localised brachyury expression, the formation of the embryonic axes (anteroposterior, dorsoventral and Left-Right) and gastrulation-like movements.
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.{{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
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}} 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 ECM.{{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
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| 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 =
}} Due to such physical restrictions, in combination with EB size, transport limitations occur within EBs, creating gradients of morphogens, metabolites, and nutrients. It has been estimated that oxygen transport is limited in cell aggregates larger than approximately 300 μm in diameter;{{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
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| s2cid = 42754482
}} 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.{{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
| 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}} Additionally, EBs can be cultured as individual microtissues and subsequently assembled into larger structures for tissue engineering applications.{{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}} Although the complexity resulting from the three-dimensional adhesions and signaling may recapitulate more native tissue structures,{{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
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| 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
}} it also creates challenges for understanding the relative contributions of mechanical, chemical, and physical signals to the resulting cell phenotypes and morphogenesis.