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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

N-cadherin substrate reduces Rho/ROCK activation in ESC-/iPSC-derived neurospheres.ESCs and iPSCs were induced with CK/SB for 5 days and then plated on surface pre-coated with gelatin and N-cad-Fc for 48 h with and without ROCK inhibitor (Y-27632), RhoA activator (CN01) or FGF2-receptor antagonist (PD173074). (A) Bright-field images shows the enhancement of neurite extension on gelatin when cultured with Y-27632. Complete inhibition of neurite outgrowth was observed on N-cadherin substrate when cells were treated with CN01. The presence of neurite outgrowth was confirmed by staining with βIII-tubulin staining (red). (B) The degree of neurite extension on N-cadherin substrate was similar, regardless of the presence and absence of PD173074, as gauged by immunostianing of βIII-tubulin (red). (C) Pull-down assay of active RhoA protein in differentiated iPSCs. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
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pone.0135170.g004: N-cadherin substrate reduces Rho/ROCK activation in ESC-/iPSC-derived neurospheres.ESCs and iPSCs were induced with CK/SB for 5 days and then plated on surface pre-coated with gelatin and N-cad-Fc for 48 h with and without ROCK inhibitor (Y-27632), RhoA activator (CN01) or FGF2-receptor antagonist (PD173074). (A) Bright-field images shows the enhancement of neurite extension on gelatin when cultured with Y-27632. Complete inhibition of neurite outgrowth was observed on N-cadherin substrate when cells were treated with CN01. The presence of neurite outgrowth was confirmed by staining with βIII-tubulin staining (red). (B) The degree of neurite extension on N-cadherin substrate was similar, regardless of the presence and absence of PD173074, as gauged by immunostianing of βIII-tubulin (red). (C) Pull-down assay of active RhoA protein in differentiated iPSCs. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.

Mentions: An extensive body of research has demonstrated that FGFR2, β-catenin and RhoGTPase signaling pathways controlled by N-cadherin can stimulate axon growth in primary neural and glial cells [8,9,11,12,40]. To test if Rho/ROCK signaling is involved in stimulating neurite outgrowth, we cultured neurospheres for 48 h in the presence of RhoA activator (CN01) on N-cad-Fc and ROCK inhibitor (Y-27632) on gelatin. The neurite outgrowth was markedly increased on gelatin in the presence of Y-27632 and decreased on N-cad-Fc with CN01 (Fig 4Ai–4Aiv). Besides bright-field images, the neurite outgrowth was confirmed by Tuj staining (Fig 4Av–4Aviii). Treatment with PD173074, a specific antagonist of FGF2R, did not affect the formation and extension of neurites (Fig 4B). To provide the direct evidence on the involvement of Rho proteins in stimulating neurite outgrowth on N-cadherin substrate, the relative cellular concentrations of activated RhoA protein was determined using effector pull-down assay exploiting the RhoA binding domain Rhotekin. The activation of RhoA on N-cad-Fc was lower than cells cultured on gelatin-coated surface (Fig 4C).


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)

N-cadherin substrate reduces Rho/ROCK activation in ESC-/iPSC-derived neurospheres.ESCs and iPSCs were induced with CK/SB for 5 days and then plated on surface pre-coated with gelatin and N-cad-Fc for 48 h with and without ROCK inhibitor (Y-27632), RhoA activator (CN01) or FGF2-receptor antagonist (PD173074). (A) Bright-field images shows the enhancement of neurite extension on gelatin when cultured with Y-27632. Complete inhibition of neurite outgrowth was observed on N-cadherin substrate when cells were treated with CN01. The presence of neurite outgrowth was confirmed by staining with βIII-tubulin staining (red). (B) The degree of neurite extension on N-cadherin substrate was similar, regardless of the presence and absence of PD173074, as gauged by immunostianing of βIII-tubulin (red). (C) Pull-down assay of active RhoA protein in differentiated iPSCs. 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.g004: N-cadherin substrate reduces Rho/ROCK activation in ESC-/iPSC-derived neurospheres.ESCs and iPSCs were induced with CK/SB for 5 days and then plated on surface pre-coated with gelatin and N-cad-Fc for 48 h with and without ROCK inhibitor (Y-27632), RhoA activator (CN01) or FGF2-receptor antagonist (PD173074). (A) Bright-field images shows the enhancement of neurite extension on gelatin when cultured with Y-27632. Complete inhibition of neurite outgrowth was observed on N-cadherin substrate when cells were treated with CN01. The presence of neurite outgrowth was confirmed by staining with βIII-tubulin staining (red). (B) The degree of neurite extension on N-cadherin substrate was similar, regardless of the presence and absence of PD173074, as gauged by immunostianing of βIII-tubulin (red). (C) Pull-down assay of active RhoA protein in differentiated iPSCs. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
Mentions: An extensive body of research has demonstrated that FGFR2, β-catenin and RhoGTPase signaling pathways controlled by N-cadherin can stimulate axon growth in primary neural and glial cells [8,9,11,12,40]. To test if Rho/ROCK signaling is involved in stimulating neurite outgrowth, we cultured neurospheres for 48 h in the presence of RhoA activator (CN01) on N-cad-Fc and ROCK inhibitor (Y-27632) on gelatin. The neurite outgrowth was markedly increased on gelatin in the presence of Y-27632 and decreased on N-cad-Fc with CN01 (Fig 4Ai–4Aiv). Besides bright-field images, the neurite outgrowth was confirmed by Tuj staining (Fig 4Av–4Aviii). Treatment with PD173074, a specific antagonist of FGF2R, did not affect the formation and extension of neurites (Fig 4B). To provide the direct evidence on the involvement of Rho proteins in stimulating neurite outgrowth on N-cadherin substrate, the relative cellular concentrations of activated RhoA protein was determined using effector pull-down assay exploiting the RhoA binding domain Rhotekin. The activation of RhoA on N-cad-Fc was lower than cells cultured on gelatin-coated surface (Fig 4C).

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