Abrogation of E-Cadherin-Mediated Cellular Aggregation Allows Proliferation of Pluripotent Mouse Embryonic Stem Cells in Shake Flask Bioreactors

Abstract

BackgroundA fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times.Methodology/Principal FindingsHere we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers.Conclusions/SignificanceThis is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.

P120-catenin regulates REST/CoREST, and modulates mouse embryonic stem cell differentiation

“…Although mESCs share similarities with hESCs, they differ in several major aspects (Hirai et al, 2011). This includes that stemness in mESCs is insensitive to cell-cell dissociation (Mohamet et al, 2010). p120-catenin depletion destabilizes Ecadherin (Davis et al, 2003), often resulting in altered cell-cell interactions.…”

The Function of E-cadherin in ES Cell Pluripotency

“…However, our data in E-cadherin-/-ES cells demonstrated that these cells could be cultured in static suspension culture and maintain pluripotent marker expression over 30d (Mohamet et al, 2010). Furthermore, we have recently demonstrated that wild type mES cells can be cultured as a near single cell suspension over prolonged periods in scalable shake flasks in the absence of additional media supplements (Mohamet et al, 2010). Using the E-cadherin neutralising antibody DECMA-1 we have shown that wtES cells exhibit doubling times of 15.6h±4.7 and mean-fold increase in viable cell numbers over 48h of 16±0.9.…”

“…E-cadherin has been demonstrated to be the cause of aggregation of ES cells in suspension culture (Fok and Zandstra, 2005) and several groups have suggested that abrogation of E-cadherin in ES cells results in low cell viability (Fok and Zandstra, 2005) and could adversely affect the pluripotent status of the cells when cultured in bioreactors (Dang et al, 2004). However, our data in E-cadherin-/-ES cells demonstrated that these cells could be cultured in static suspension culture and maintain pluripotent marker expression over 30d (Mohamet et al, 2010). Furthermore, we have recently demonstrated that wild type mES cells can be cultured as a near single cell suspension over prolonged periods in scalable shake flasks in the absence of additional media supplements (Mohamet et al, 2010).…”

“…The key difference between loss of E-cadherin expression in hES and mES cells, therefore, appears to be cellular disaggregation in the absence of a substratum. For example, whilst hES cells maintain viability in adherent culture following treatment with the E-cadherin neutralising antibody SHE78.7, the majority of the population die within 24h when cultured in suspension (Mohamet et al, 2010). In summary, E-cadherin exhibits a range of functions in ES cells which result in stabilisation of epithelial integrity and associated signalling pathways.…”

“…Under these conditions, wtES cells could be cultured for 15d whilst maintaining expression of pluripotency markers and high cell viability. In addition, the cells exhibited a normal karyotype and, subsequently, were able to differentiate to cells representative of the three primary germ layers (Mohamet et al, 2010). Culture of ES cells in shake flasks provides a useful cost-effective method which significantly decreases the requirement for technical input and plastic consumables associated with current adherent methods.…”