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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

Enhancement of neurite outgrowth by substrate bound N-cadherin.Neurospheres generated in suspension for 5 days with and without CKI-7/SB-431542 were cultured on substrates pre-coated with N-cad-Fc, PLL, or gelatin for 48 hrs in the absence of growth factors. (A) N-cadherin substrate leads to upregulation of Ser473P (active) Akt in NPCs, as analyzed by western blot. (B) Cellular phenotype on different extracellular matrices was assessed by bright-field microscopy. (C) The degree of neurite outgrowth on the N-cad-Fc, gelatin, and PLL was quantified by neurite extension index, defined as the ratio of neurite outgrowth surface (Sn) and explant surface (Se). (D) The number of neurites per neurosphere was analyzed from bright-field images. Soluble N-cad-Fc was added in the growth medium of explants grown on gelatin substrate. (E) Immunofluorescence microscopy analysis of Nestin, βIII-tubulin, and MAP2 expression. DAPI shows total nuclei in the field of view. Data are mean ± SD, n = 3. Scale bar is equivalent to 50 μm unless otherwise mentioned. Abbreviation: PLL- poly-L-lysine; sol, soluble; Ncad, N-cad-Fc; Gel, gelatin.
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pone.0135170.g001: Enhancement of neurite outgrowth by substrate bound N-cadherin.Neurospheres generated in suspension for 5 days with and without CKI-7/SB-431542 were cultured on substrates pre-coated with N-cad-Fc, PLL, or gelatin for 48 hrs in the absence of growth factors. (A) N-cadherin substrate leads to upregulation of Ser473P (active) Akt in NPCs, as analyzed by western blot. (B) Cellular phenotype on different extracellular matrices was assessed by bright-field microscopy. (C) The degree of neurite outgrowth on the N-cad-Fc, gelatin, and PLL was quantified by neurite extension index, defined as the ratio of neurite outgrowth surface (Sn) and explant surface (Se). (D) The number of neurites per neurosphere was analyzed from bright-field images. Soluble N-cad-Fc was added in the growth medium of explants grown on gelatin substrate. (E) Immunofluorescence microscopy analysis of Nestin, βIII-tubulin, and MAP2 expression. DAPI shows total nuclei in the field of view. Data are mean ± SD, n = 3. Scale bar is equivalent to 50 μm unless otherwise mentioned. Abbreviation: PLL- poly-L-lysine; sol, soluble; Ncad, N-cad-Fc; Gel, gelatin.

Mentions: Studies imply that Akt activation by N-cadherin induces neuronal differentiation during cortical development [8]. To test the functionality of immobilized N-cadherin ectodomain through Akt activation, NPCs generated in the presence and absence of CK/SB for five days were plated in tissue culture dishes coated with N-cad-Fc and gelatin. As expected, cells seeded on N-cadherin showed increase in Ser473P Akt activation than gelatin controls (Fig 1A). We then assessed the effect of matrix tethered N-cadherin on neurite outgrowth. ESC/iPSC-derived neurospheres plated on N-cadherin-coated surface showed dramatic enhancement in neurite outgrowth than controls (Fig 1B). While it took at least 32 h to see any visible outgrowth on gelatin- or PLL-coated surfaces, neurospheres on N-cadherin substrate formed neurites within 18 h of culture, suggesting a faster growth rate on N-cadherin (data not shown). We have also used time-lapse video microscopy to confirm the growth of neurites on N-cadherin substrate (S1 Movie). These observations are supported by previous reports that local stimulation with extrinsic N-cadherin is sufficient for the activation of earliest signals involve in the formation of neurites [38]. The influence of N-cadherin was further analyzed quantitatively by previously reported neurite outgrowth assay [33] using a neurite extension index, defined as the ratio of neurite outgrowth surface (Sn) and explants surface (Se). Fig 1C shows that neurospheres cultured on N-cadherin substrate for 48 h formed 2- and 40-times longer neurites than spheres on poly-L-lysine (PLL) and gelatin, respectively. The neurite extension index on N-cad-Fc, PLL and gelatin were 4.20±0.084, 1.96±0.45 and 0.16±0.17, respectively. The differences in extension index were more dramatic while cells were cultured for additional days on these substrates. Compared to PLL and gelatin, the number of neurites on N-cad-Fc was also 4- and 60-fold higher, respectively (Fig 1D). With regard to the role of soluble N-cadherin ligand in axonal growth, evidence suggested both a stimulatory and a trivial effect on primary neural cells [39,40]. In this study, we did not observe any significant increase in neurite outgrowth when soluble N-cad-Fc was added in the differentiation medium of neurospheres grown on gelatin substrate (Fig 1D).


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)

Enhancement of neurite outgrowth by substrate bound N-cadherin.Neurospheres generated in suspension for 5 days with and without CKI-7/SB-431542 were cultured on substrates pre-coated with N-cad-Fc, PLL, or gelatin for 48 hrs in the absence of growth factors. (A) N-cadherin substrate leads to upregulation of Ser473P (active) Akt in NPCs, as analyzed by western blot. (B) Cellular phenotype on different extracellular matrices was assessed by bright-field microscopy. (C) The degree of neurite outgrowth on the N-cad-Fc, gelatin, and PLL was quantified by neurite extension index, defined as the ratio of neurite outgrowth surface (Sn) and explant surface (Se). (D) The number of neurites per neurosphere was analyzed from bright-field images. Soluble N-cad-Fc was added in the growth medium of explants grown on gelatin substrate. (E) Immunofluorescence microscopy analysis of Nestin, βIII-tubulin, and MAP2 expression. DAPI shows total nuclei in the field of view. Data are mean ± SD, n = 3. Scale bar is equivalent to 50 μm unless otherwise mentioned. Abbreviation: PLL- poly-L-lysine; sol, soluble; Ncad, N-cad-Fc; Gel, gelatin.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135170.g001: Enhancement of neurite outgrowth by substrate bound N-cadherin.Neurospheres generated in suspension for 5 days with and without CKI-7/SB-431542 were cultured on substrates pre-coated with N-cad-Fc, PLL, or gelatin for 48 hrs in the absence of growth factors. (A) N-cadherin substrate leads to upregulation of Ser473P (active) Akt in NPCs, as analyzed by western blot. (B) Cellular phenotype on different extracellular matrices was assessed by bright-field microscopy. (C) The degree of neurite outgrowth on the N-cad-Fc, gelatin, and PLL was quantified by neurite extension index, defined as the ratio of neurite outgrowth surface (Sn) and explant surface (Se). (D) The number of neurites per neurosphere was analyzed from bright-field images. Soluble N-cad-Fc was added in the growth medium of explants grown on gelatin substrate. (E) Immunofluorescence microscopy analysis of Nestin, βIII-tubulin, and MAP2 expression. DAPI shows total nuclei in the field of view. Data are mean ± SD, n = 3. Scale bar is equivalent to 50 μm unless otherwise mentioned. Abbreviation: PLL- poly-L-lysine; sol, soluble; Ncad, N-cad-Fc; Gel, gelatin.
Mentions: Studies imply that Akt activation by N-cadherin induces neuronal differentiation during cortical development [8]. To test the functionality of immobilized N-cadherin ectodomain through Akt activation, NPCs generated in the presence and absence of CK/SB for five days were plated in tissue culture dishes coated with N-cad-Fc and gelatin. As expected, cells seeded on N-cadherin showed increase in Ser473P Akt activation than gelatin controls (Fig 1A). We then assessed the effect of matrix tethered N-cadherin on neurite outgrowth. ESC/iPSC-derived neurospheres plated on N-cadherin-coated surface showed dramatic enhancement in neurite outgrowth than controls (Fig 1B). While it took at least 32 h to see any visible outgrowth on gelatin- or PLL-coated surfaces, neurospheres on N-cadherin substrate formed neurites within 18 h of culture, suggesting a faster growth rate on N-cadherin (data not shown). We have also used time-lapse video microscopy to confirm the growth of neurites on N-cadherin substrate (S1 Movie). These observations are supported by previous reports that local stimulation with extrinsic N-cadherin is sufficient for the activation of earliest signals involve in the formation of neurites [38]. The influence of N-cadherin was further analyzed quantitatively by previously reported neurite outgrowth assay [33] using a neurite extension index, defined as the ratio of neurite outgrowth surface (Sn) and explants surface (Se). Fig 1C shows that neurospheres cultured on N-cadherin substrate for 48 h formed 2- and 40-times longer neurites than spheres on poly-L-lysine (PLL) and gelatin, respectively. The neurite extension index on N-cad-Fc, PLL and gelatin were 4.20±0.084, 1.96±0.45 and 0.16±0.17, respectively. The differences in extension index were more dramatic while cells were cultured for additional days on these substrates. Compared to PLL and gelatin, the number of neurites on N-cad-Fc was also 4- and 60-fold higher, respectively (Fig 1D). With regard to the role of soluble N-cadherin ligand in axonal growth, evidence suggested both a stimulatory and a trivial effect on primary neural cells [39,40]. In this study, we did not observe any significant increase in neurite outgrowth when soluble N-cad-Fc was added in the differentiation medium of neurospheres grown on gelatin substrate (Fig 1D).

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