Limits...
An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors.

Haque A, Adnan N, Motazedian A, Akter F, Hossain S, Kutsuzawa K, Nag K, Kobatake E, Akaike T - PLoS ONE (2015)

Bottom Line: We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers.Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis.Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, Japan.

ABSTRACT
For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.

No MeSH data available.


Related in: MedlinePlus

Proposed pathway connecting N-cadherin, Rho/ROCK, β-catenin signaling in neural differentiation.The neurospheres were heterogeneous; harboring undifferentiated ESC/iPSCs might be due to irregular distribution of soluble factors. To achieve better homogeneity, these spheres were dissociated and expanded on N-cadherin-coated surface. The down-regulation of β-catenin and Rho/ROCK pathways inhibited dissociation-induced apoptosis and enhanced neural conversion. N-cadherin mediated homophilic interaction is also implicated in the enrichment of neural population by eliminating undifferentiated ESC/iPSCs under serum/growth factor-free differentiation condition.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526632&req=5

pone.0135170.g006: Proposed pathway connecting N-cadherin, Rho/ROCK, β-catenin signaling in neural differentiation.The neurospheres were heterogeneous; harboring undifferentiated ESC/iPSCs might be due to irregular distribution of soluble factors. To achieve better homogeneity, these spheres were dissociated and expanded on N-cadherin-coated surface. The down-regulation of β-catenin and Rho/ROCK pathways inhibited dissociation-induced apoptosis and enhanced neural conversion. N-cadherin mediated homophilic interaction is also implicated in the enrichment of neural population by eliminating undifferentiated ESC/iPSCs under serum/growth factor-free differentiation condition.

Mentions: In contrast to previously reported neural differentiation protocols which typically rely on the addition of exogenous soluble factors, this study demonstrates an alternative method using N-cadherin-based biomimetic substrate to promote the differentiation of ESC- and iPSC-derived NPCs. We also identified the mechanism underlying the enhanced differentiation process on N-cadherin coated cell culture plate and showed that N-cadherin adhesion couples to RhoGTPase and β-catenin to accelerate the process of neurite growth and suppress dissociation induced apoptosis even in serum/GF-free condition. We showed that binding selectivity of cadherins can be useful to purge remaining ESCs/iPSCs from differentiated NPCs. Such a biomimetic system allows a precise control of spatial presentation of ligand molecules to the cells, which is not possible in natural contacts where different types of adhesion proteins can coexist with the lack of lineage selectivity. The experimental findings are summarized in Fig 6.


An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors.

Haque A, Adnan N, Motazedian A, Akter F, Hossain S, Kutsuzawa K, Nag K, Kobatake E, Akaike T - PLoS ONE (2015)

Proposed pathway connecting N-cadherin, Rho/ROCK, β-catenin signaling in neural differentiation.The neurospheres were heterogeneous; harboring undifferentiated ESC/iPSCs might be due to irregular distribution of soluble factors. To achieve better homogeneity, these spheres were dissociated and expanded on N-cadherin-coated surface. The down-regulation of β-catenin and Rho/ROCK pathways inhibited dissociation-induced apoptosis and enhanced neural conversion. N-cadherin mediated homophilic interaction is also implicated in the enrichment of neural population by eliminating undifferentiated ESC/iPSCs under serum/growth factor-free differentiation condition.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526632&req=5

pone.0135170.g006: Proposed pathway connecting N-cadherin, Rho/ROCK, β-catenin signaling in neural differentiation.The neurospheres were heterogeneous; harboring undifferentiated ESC/iPSCs might be due to irregular distribution of soluble factors. To achieve better homogeneity, these spheres were dissociated and expanded on N-cadherin-coated surface. The down-regulation of β-catenin and Rho/ROCK pathways inhibited dissociation-induced apoptosis and enhanced neural conversion. N-cadherin mediated homophilic interaction is also implicated in the enrichment of neural population by eliminating undifferentiated ESC/iPSCs under serum/growth factor-free differentiation condition.
Mentions: In contrast to previously reported neural differentiation protocols which typically rely on the addition of exogenous soluble factors, this study demonstrates an alternative method using N-cadherin-based biomimetic substrate to promote the differentiation of ESC- and iPSC-derived NPCs. We also identified the mechanism underlying the enhanced differentiation process on N-cadherin coated cell culture plate and showed that N-cadherin adhesion couples to RhoGTPase and β-catenin to accelerate the process of neurite growth and suppress dissociation induced apoptosis even in serum/GF-free condition. We showed that binding selectivity of cadherins can be useful to purge remaining ESCs/iPSCs from differentiated NPCs. Such a biomimetic system allows a precise control of spatial presentation of ligand molecules to the cells, which is not possible in natural contacts where different types of adhesion proteins can coexist with the lack of lineage selectivity. The experimental findings are summarized in Fig 6.

Bottom Line: We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers.Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis.Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, Japan.

ABSTRACT
For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.

No MeSH data available.


Related in: MedlinePlus