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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

Suppression of Rho/ROCK and β-catenin signaling pathways by N-cadherin substrate in singly dissociated cells.iPSC-derived neurosperes grown in suspension with and without CK/SB were enzymatically dissociated at day 5 and cultured on N-cadherin and gelatin substrate for 48 h. (A) In contrast to N-cadherin substrate, cells on gelatin failed to form neurites in the absence of Y-27632, as analyzed by bright-field images. Cellular phenotype on N-cadherin substrate was similar irrespective of the presence and absence CK/SB. (B) Compered to spheres seeded on N-cadherin substrate, pull-down assay shows down-regulation of active RhoA in dissociated cells. (C) β-catenin expression was analyzed by western-blotting. Immunostaining images for the expression of β-catenin (red) in shepres plated on (D) gelatin, and (E) N-cad-Fc substrate. β-catenin (red) expression is higher in Nanog expressing cells (green). (F) β-catenin expression was undetectable in dissociated cells plated on surfaces pre-coated with N-cad-Fc. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
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pone.0135170.g005: Suppression of Rho/ROCK and β-catenin signaling pathways by N-cadherin substrate in singly dissociated cells.iPSC-derived neurosperes grown in suspension with and without CK/SB were enzymatically dissociated at day 5 and cultured on N-cadherin and gelatin substrate for 48 h. (A) In contrast to N-cadherin substrate, cells on gelatin failed to form neurites in the absence of Y-27632, as analyzed by bright-field images. Cellular phenotype on N-cadherin substrate was similar irrespective of the presence and absence CK/SB. (B) Compered to spheres seeded on N-cadherin substrate, pull-down assay shows down-regulation of active RhoA in dissociated cells. (C) β-catenin expression was analyzed by western-blotting. Immunostaining images for the expression of β-catenin (red) in shepres plated on (D) gelatin, and (E) N-cad-Fc substrate. β-catenin (red) expression is higher in Nanog expressing cells (green). (F) β-catenin expression was undetectable in dissociated cells plated on surfaces pre-coated with N-cad-Fc. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.

Mentions: We also analyzed the effect of N-cadherin on Rho/ROCK signal activation in dissociated cells cultured on N-cadherin for 48 h without growth factors. As shown in Fig 5A, cells on gelatin were able to form neurites outgrowth in the presence of Y-27632. However, the differentiated cells on gelatin never achieved a true confluent culture nor do they form a homogeneous monolayer. In contrast, dissociated cells cultured on N-cadherin-coated surface attached well and formed a monolayer of cells with extended neurites in the absence of Y-27632. Similar effect was observed in cells induced with and without CK/SB. There were no differences with regard to cell shapes and neurite growth between cells growing on N-cadherin in the absence or presence of Y-27632 (data not shown). Pull-down assay also shows a significant down-regulation of RhoA activity in dissociated cells cultured on N-cadherin, irrespective of the addition of CK/SB in the early stages of differentiation (Fig 5B). These data suggest that N-cadherin stimulates neurite outgrowth by suppressing Rho/ROCK signaling.


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)

Suppression of Rho/ROCK and β-catenin signaling pathways by N-cadherin substrate in singly dissociated cells.iPSC-derived neurosperes grown in suspension with and without CK/SB were enzymatically dissociated at day 5 and cultured on N-cadherin and gelatin substrate for 48 h. (A) In contrast to N-cadherin substrate, cells on gelatin failed to form neurites in the absence of Y-27632, as analyzed by bright-field images. Cellular phenotype on N-cadherin substrate was similar irrespective of the presence and absence CK/SB. (B) Compered to spheres seeded on N-cadherin substrate, pull-down assay shows down-regulation of active RhoA in dissociated cells. (C) β-catenin expression was analyzed by western-blotting. Immunostaining images for the expression of β-catenin (red) in shepres plated on (D) gelatin, and (E) N-cad-Fc substrate. β-catenin (red) expression is higher in Nanog expressing cells (green). (F) β-catenin expression was undetectable in dissociated cells plated on surfaces pre-coated with N-cad-Fc. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135170.g005: Suppression of Rho/ROCK and β-catenin signaling pathways by N-cadherin substrate in singly dissociated cells.iPSC-derived neurosperes grown in suspension with and without CK/SB were enzymatically dissociated at day 5 and cultured on N-cadherin and gelatin substrate for 48 h. (A) In contrast to N-cadherin substrate, cells on gelatin failed to form neurites in the absence of Y-27632, as analyzed by bright-field images. Cellular phenotype on N-cadherin substrate was similar irrespective of the presence and absence CK/SB. (B) Compered to spheres seeded on N-cadherin substrate, pull-down assay shows down-regulation of active RhoA in dissociated cells. (C) β-catenin expression was analyzed by western-blotting. Immunostaining images for the expression of β-catenin (red) in shepres plated on (D) gelatin, and (E) N-cad-Fc substrate. β-catenin (red) expression is higher in Nanog expressing cells (green). (F) β-catenin expression was undetectable in dissociated cells plated on surfaces pre-coated with N-cad-Fc. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
Mentions: We also analyzed the effect of N-cadherin on Rho/ROCK signal activation in dissociated cells cultured on N-cadherin for 48 h without growth factors. As shown in Fig 5A, cells on gelatin were able to form neurites outgrowth in the presence of Y-27632. However, the differentiated cells on gelatin never achieved a true confluent culture nor do they form a homogeneous monolayer. In contrast, dissociated cells cultured on N-cadherin-coated surface attached well and formed a monolayer of cells with extended neurites in the absence of Y-27632. Similar effect was observed in cells induced with and without CK/SB. There were no differences with regard to cell shapes and neurite growth between cells growing on N-cadherin in the absence or presence of Y-27632 (data not shown). Pull-down assay also shows a significant down-regulation of RhoA activity in dissociated cells cultured on N-cadherin, irrespective of the addition of CK/SB in the early stages of differentiation (Fig 5B). These data suggest that N-cadherin stimulates neurite outgrowth by suppressing Rho/ROCK signaling.

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