title: Cell-cell interaction modulates neuroectodermal specification of embryonic stem cells.
abstract: The controlled differentiation of embryonic stem (ES) cells is of utmost interest to their clinical, biotechnological, and basic science use. Many investigators have combinatorially assessed the role of specific soluble factors and extracellular matrices in guiding ES cell fate, yet the interaction between neighboring cells in these heterogeneous cultures has been poorly defined due to a lack of conventional tools to specifically uncouple these variables. Herein, we explored the role of cell-cell interactions during neuroectodermal specification of ES cells using a microfabricated cell pair array. We tracked differentiation events in situ, using an ES cell line expressing green fluorescent protein (GFP) under the regulation of the Sox1 gene promoter, an early marker of neuroectodermal germ cell commitment in the adult forebrain. We observed that a previously specified Sox1-GFP+ cell could induce the specification of an undifferentiated ES cell. This induction was modulated by the two cells being in contact and was dependent on the age of previously specified cell prior to coculture. A screen of candidate cell adhesion molecules revealed that the expression of connexin (Cx)-43 correlated with the age-dependent effect of cell contact in cell pair experiments. ES cells deficient in Cx-43 showed aberrant neuroectodermal specification and lineage commitment, highlighting the importance of gap junctional signaling in the development of this germ layer. Moreover, this study demonstrates the integration of microscale culture techniques to explore the biology of ES cells and gain insight into relevant developmental processes otherwise undefined due to bulk culture methods.
authors.affiliation: Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, MA, USA.
authors.forename: Biju
authors.initials: B
authors.lastname: Parekkadan
authors.forename: Yevgeny
authors.initials: Y
authors.lastname: Berdichevsky
authors.forename: Daniel
authors.initials: D
authors.lastname: Irimia
authors.forename: Avrum
authors.initials: A
authors.lastname: Leeder
authors.forename: Gabriel
authors.initials: G
authors.lastname: Yarmush
authors.forename: Mehmet
authors.initials: M
authors.lastname: Toner
authors.forename: John B
authors.initials: JB
authors.lastname: Levine
authors.forename: Martin L
authors.initials: ML
authors.lastname: Yarmush
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002450:Cell Communication / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics / Q000502:physiology; D002490:Central Nervous System / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D018920:Coculture Techniques; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004475:Ectoderm / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D047108:Embryonic Development / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D055748:SOXB1 Transcription Factors; D015398:Signal Transduction / Q000502:physiology
chemical_list: D018031:Connexin 43; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D019006:Neural Cell Adhesion Molecules; D055748:SOXB1 Transcription Factors; C098693:Sox1 protein, mouse; D049452:Green Fluorescent Proteins