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.

Enabling stem cell therapies through synthetic stem cell–niche engineering

“…As an example, mouse ES cells have been immobilized onto surfaces in clusters or as single cells to demonstrate that homotypic ES cell interactions depress colony-forming capacity in a manner associated with E-cadherin ligation (64). In addition, bow tie-shaped microwells, designed to limit the contact area between cells, have been used to demonstrate that homotypic ES cell interactions regulate specification toward neuroectoderm (possibly via connexin 43 ligation modulation) (65). In a very elegant non-stem cell example, stromal cells and hepatocytes have been cultured on micromachined silicon substrates with mobile parts, in order to dynamically modulate their interactions (66).…”

Connexin43 Is Required for the Maintenance of Multipotency in Skin-Derived Stem Cells

“…They have been shown to allow the movement of molecules smaller than 1 kD between the cytoplasm of two cells [1]. Gap junctions have been shown to play a critical role in many developmental events such as cellular proliferation, apoptosis, differentiation, and organogenesis [2][3][4][5]. Connexin43 (Cx43) is considered the most widely expressed connexin and has been shown to play critical roles in many organ systems including folliculogenesis in the ovary, development and function of the heart, and osteoblast differentiation in bone development, among others [5][6][7][8][9][10].…”

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

“…The presence of all three germ layers demonstrates that EB culture is a crude but effective means of recapitulating early stages of embryogenesis in vitro Itskovitz-Eldor et al 2000). Theoretically, embryonic stem cells can differentiate to any cell type and many examples of in vitro differentiation exist including generation of skeletal and brain tissue (Bielby et al 2004;Parekkadan et al 2008). Consequently, ES cells provide a renewable source of cellular material for tissue engineering and regenerative medicine (Ungrin et al 2008).…”