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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 homophilic interaction enrich neuronal cells by eliminating undifferentiated ESCs/iPSCs.(A) The presence of Nanog expression in ESC- and iPSC-derived differentiated cells at 20 days of differentiation, as analyzed by qRT-PCR. Nanog expression was detectable regardless of the presence and absence of CK/SB. (B) qRT-PCR analysis for the expression of Nanog in differentiated cells cultured on N-cad-Fc and gelatin at day 20. (C) Nanog-GFP expressing iPSCs-derived neurospheres generated in suspension with CK/SB were seeded on N-cad-Fc shows the presence of Nanog (green) and Nestin (red) at 7 days of differentiation. (D) iPSC-derived neurosperes grown in suspension with CK/SB were enzymatically dissociated and plated on N-cadherin substrate for 48 h. Fluorescence images shows the absence of Nanog-GFP and the presence of Tuj (red). (E) Different from neurosphere-based differentiation, qRT-PCR analysis shows the absence of Nanog in dissociated cells cultured on N-cadherin for 48 h (total 7 days of differentiation). (F) mRNA expression analysis of Nestin, Ngn1 and Tuj expression in iPSC-derived progeny derived with or without CK/SB. Dissociated cells on N-cadherin substrate were harvested for analysis on 7 days of differentiation. (G, H) FACS analysis of Nanog-GFP expressing iPSCs. Neurospheres on day 5 were dissociated and cultured on N-cad-Fc and gelatin substrate for 48 h before analysis. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
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pone.0135170.g003: N-cadherin homophilic interaction enrich neuronal cells by eliminating undifferentiated ESCs/iPSCs.(A) The presence of Nanog expression in ESC- and iPSC-derived differentiated cells at 20 days of differentiation, as analyzed by qRT-PCR. Nanog expression was detectable regardless of the presence and absence of CK/SB. (B) qRT-PCR analysis for the expression of Nanog in differentiated cells cultured on N-cad-Fc and gelatin at day 20. (C) Nanog-GFP expressing iPSCs-derived neurospheres generated in suspension with CK/SB were seeded on N-cad-Fc shows the presence of Nanog (green) and Nestin (red) at 7 days of differentiation. (D) iPSC-derived neurosperes grown in suspension with CK/SB were enzymatically dissociated and plated on N-cadherin substrate for 48 h. Fluorescence images shows the absence of Nanog-GFP and the presence of Tuj (red). (E) Different from neurosphere-based differentiation, qRT-PCR analysis shows the absence of Nanog in dissociated cells cultured on N-cadherin for 48 h (total 7 days of differentiation). (F) mRNA expression analysis of Nestin, Ngn1 and Tuj expression in iPSC-derived progeny derived with or without CK/SB. Dissociated cells on N-cadherin substrate were harvested for analysis on 7 days of differentiation. (G, H) FACS analysis of Nanog-GFP expressing iPSCs. Neurospheres on day 5 were dissociated and cultured on N-cad-Fc and gelatin substrate for 48 h before analysis. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.

Mentions: At this stage rather than separating subtype specific neuronal and glial cells, which act synergistically to maintain physiological function [46], we put the focused on the isolation of neural/glial cells from heterogeneous cell populations by analyzing the markers for pluripotency (Nanog), mesoderm (Brachyury, Bra), and endoderm (albumin, Alb) lineages. Compared to mouse brain as negative control, the expression of markers for mesoderm and endoderm lineages was almost in differentiated cells cultured with and without CK/SB (S3 Fig). We infer from this and other studies that the removal of serum that contains poorly defined endoderm- and mesoderm-inducing factors together with early differentiation induction under adhesion-free condition preferentially induced neuronal lineages [47]. However, we cannot oversee the cumulative data suggesting the obvious presence of residual undifferentiated cells associated with neurosphere- or embryoid body-based differentiation protocol [42,48]. Not surprisingly, the expression of Nanog was detectable in all conditions regardless of the presence and absence of CK/SB (Fig 3A). Compared to N-cadherin substrate, Nanog expression was higher in cells on gelatin substrate. While CK/SB was not added into the differentiation media the expression of Nanog was increased by a factor of 2 and 30 for N-cadherin and gelatin substrates, respectively (Fig 3A and 3B). These data further confirm the neuro-inductive effect of substrate bound N-cadherin. We also checked the presence of Nanog-GFP expressing cells at day 7 in order to localize remaining undifferentiated cells. Interestingly, a cluster of cells expressing Nanog-GFP was observed at the innermost layer of the spheres, which was clearly distinguishable by their physical separation from nestin-expressing cells (Fig 3C). This data is not surprising considering the notion that some pluripotent cells escape differentiation and remain undifferentiated over months under neural differentiation condition [48]. Regardless, we found that the neural differentiation on gelatin was more heterogeneous than N-cad-Fc substrate and the presence of remaining undifferentiated cells was inevitable, irrespective of the addition of exogenous soluble neuro-inductive signals.


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 homophilic interaction enrich neuronal cells by eliminating undifferentiated ESCs/iPSCs.(A) The presence of Nanog expression in ESC- and iPSC-derived differentiated cells at 20 days of differentiation, as analyzed by qRT-PCR. Nanog expression was detectable regardless of the presence and absence of CK/SB. (B) qRT-PCR analysis for the expression of Nanog in differentiated cells cultured on N-cad-Fc and gelatin at day 20. (C) Nanog-GFP expressing iPSCs-derived neurospheres generated in suspension with CK/SB were seeded on N-cad-Fc shows the presence of Nanog (green) and Nestin (red) at 7 days of differentiation. (D) iPSC-derived neurosperes grown in suspension with CK/SB were enzymatically dissociated and plated on N-cadherin substrate for 48 h. Fluorescence images shows the absence of Nanog-GFP and the presence of Tuj (red). (E) Different from neurosphere-based differentiation, qRT-PCR analysis shows the absence of Nanog in dissociated cells cultured on N-cadherin for 48 h (total 7 days of differentiation). (F) mRNA expression analysis of Nestin, Ngn1 and Tuj expression in iPSC-derived progeny derived with or without CK/SB. Dissociated cells on N-cadherin substrate were harvested for analysis on 7 days of differentiation. (G, H) FACS analysis of Nanog-GFP expressing iPSCs. Neurospheres on day 5 were dissociated and cultured on N-cad-Fc and gelatin substrate for 48 h before analysis. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135170.g003: N-cadherin homophilic interaction enrich neuronal cells by eliminating undifferentiated ESCs/iPSCs.(A) The presence of Nanog expression in ESC- and iPSC-derived differentiated cells at 20 days of differentiation, as analyzed by qRT-PCR. Nanog expression was detectable regardless of the presence and absence of CK/SB. (B) qRT-PCR analysis for the expression of Nanog in differentiated cells cultured on N-cad-Fc and gelatin at day 20. (C) Nanog-GFP expressing iPSCs-derived neurospheres generated in suspension with CK/SB were seeded on N-cad-Fc shows the presence of Nanog (green) and Nestin (red) at 7 days of differentiation. (D) iPSC-derived neurosperes grown in suspension with CK/SB were enzymatically dissociated and plated on N-cadherin substrate for 48 h. Fluorescence images shows the absence of Nanog-GFP and the presence of Tuj (red). (E) Different from neurosphere-based differentiation, qRT-PCR analysis shows the absence of Nanog in dissociated cells cultured on N-cadherin for 48 h (total 7 days of differentiation). (F) mRNA expression analysis of Nestin, Ngn1 and Tuj expression in iPSC-derived progeny derived with or without CK/SB. Dissociated cells on N-cadherin substrate were harvested for analysis on 7 days of differentiation. (G, H) FACS analysis of Nanog-GFP expressing iPSCs. Neurospheres on day 5 were dissociated and cultured on N-cad-Fc and gelatin substrate for 48 h before analysis. DAPI shows total nuclei in the field of view. Scale bar: 50 μm.
Mentions: At this stage rather than separating subtype specific neuronal and glial cells, which act synergistically to maintain physiological function [46], we put the focused on the isolation of neural/glial cells from heterogeneous cell populations by analyzing the markers for pluripotency (Nanog), mesoderm (Brachyury, Bra), and endoderm (albumin, Alb) lineages. Compared to mouse brain as negative control, the expression of markers for mesoderm and endoderm lineages was almost in differentiated cells cultured with and without CK/SB (S3 Fig). We infer from this and other studies that the removal of serum that contains poorly defined endoderm- and mesoderm-inducing factors together with early differentiation induction under adhesion-free condition preferentially induced neuronal lineages [47]. However, we cannot oversee the cumulative data suggesting the obvious presence of residual undifferentiated cells associated with neurosphere- or embryoid body-based differentiation protocol [42,48]. Not surprisingly, the expression of Nanog was detectable in all conditions regardless of the presence and absence of CK/SB (Fig 3A). Compared to N-cadherin substrate, Nanog expression was higher in cells on gelatin substrate. While CK/SB was not added into the differentiation media the expression of Nanog was increased by a factor of 2 and 30 for N-cadherin and gelatin substrates, respectively (Fig 3A and 3B). These data further confirm the neuro-inductive effect of substrate bound N-cadherin. We also checked the presence of Nanog-GFP expressing cells at day 7 in order to localize remaining undifferentiated cells. Interestingly, a cluster of cells expressing Nanog-GFP was observed at the innermost layer of the spheres, which was clearly distinguishable by their physical separation from nestin-expressing cells (Fig 3C). This data is not surprising considering the notion that some pluripotent cells escape differentiation and remain undifferentiated over months under neural differentiation condition [48]. Regardless, we found that the neural differentiation on gelatin was more heterogeneous than N-cad-Fc substrate and the presence of remaining undifferentiated cells was inevitable, irrespective of the addition of exogenous soluble neuro-inductive signals.

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