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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

Neuronal differentiation on N-cadherin substrate without exogenous growth factors or inhibitors.(A-B) ESCs cultured in suspension for 5 days with and without CKI-7 and SB-431542 (CK/SB) was further induced on N-cadherin substrate for 48 h in the absence of exogenous growth factors. The degree of neural conversion was analyzed by (A) neurite extension index and (B) qRT-PCR for relative mRNA expression of nestin, neurogenin 1 (Ngn1), and βIII-tubulin (Tuj). (C) Cellular phenotype of differentiated cells on N-cad-Fc and gelatin substrates at 20 days of differentiation was assessed by bright-field images. Scale bar: 200 μm. (D) Characterization of neural cells at 20 days of differentiation by the expression of neuronal progenitor cell markers, including N-cad, Nestin, Ngn1, Mitf and Tuj. (E) The relative mRNA expression level of neurotransmitter and subtype-specific genes (Nurr, CHAT, SLC, GAD2 and GFAP) in ESC- and iPSC-derived neuronal cells differentiated for 20 days on N-cadherin substrate. Abbreviation: ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; Ngn1- Neurogenin 1; Mitf- Microphthalmia-associated transcription factor; N-cad, N-cadherin; Nurr- nuclear receptor-related factor; CHAT- choline acetyltransferase; SLC- solute carrier; GAD- glutamate decarboxylase; GFAP- glial fibrillary acidic protein.
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pone.0135170.g002: Neuronal differentiation on N-cadherin substrate without exogenous growth factors or inhibitors.(A-B) ESCs cultured in suspension for 5 days with and without CKI-7 and SB-431542 (CK/SB) was further induced on N-cadherin substrate for 48 h in the absence of exogenous growth factors. The degree of neural conversion was analyzed by (A) neurite extension index and (B) qRT-PCR for relative mRNA expression of nestin, neurogenin 1 (Ngn1), and βIII-tubulin (Tuj). (C) Cellular phenotype of differentiated cells on N-cad-Fc and gelatin substrates at 20 days of differentiation was assessed by bright-field images. Scale bar: 200 μm. (D) Characterization of neural cells at 20 days of differentiation by the expression of neuronal progenitor cell markers, including N-cad, Nestin, Ngn1, Mitf and Tuj. (E) The relative mRNA expression level of neurotransmitter and subtype-specific genes (Nurr, CHAT, SLC, GAD2 and GFAP) in ESC- and iPSC-derived neuronal cells differentiated for 20 days on N-cadherin substrate. Abbreviation: ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; Ngn1- Neurogenin 1; Mitf- Microphthalmia-associated transcription factor; N-cad, N-cadherin; Nurr- nuclear receptor-related factor; CHAT- choline acetyltransferase; SLC- solute carrier; GAD- glutamate decarboxylase; GFAP- glial fibrillary acidic protein.

Mentions: Default differentiation of mouse ESC to neuroectoderm occurs when culturing cells in serum-free medium and in the absence of any exogenous GFs [37,42]. Previous reports have also shown a vital role of endogenous signals in inducing mesoderm and endoderm lineages when differentiated without exogenous GFs [43,44]. Therefore, we sought to investigate if the effects of N-cadherin substrate on neurite growth and neuronal conversion could be replicated without exogenously added soluble GFs and inhibitors. Compared to CK/SB-free differentiation, a significant enhancement in neurite outgrowth was observed when differentiations were performed under the guidance of CK/SB (Fig 2A). Surprisingly, the expression level of well described markers for NPCs including nestin, neurogenin 1 (Ngn1), and βIII-tubulin (Tuj) was similar at day 7 in both conditions, as analyzed by qRT-PCR (Fig 2B).


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)

Neuronal differentiation on N-cadherin substrate without exogenous growth factors or inhibitors.(A-B) ESCs cultured in suspension for 5 days with and without CKI-7 and SB-431542 (CK/SB) was further induced on N-cadherin substrate for 48 h in the absence of exogenous growth factors. The degree of neural conversion was analyzed by (A) neurite extension index and (B) qRT-PCR for relative mRNA expression of nestin, neurogenin 1 (Ngn1), and βIII-tubulin (Tuj). (C) Cellular phenotype of differentiated cells on N-cad-Fc and gelatin substrates at 20 days of differentiation was assessed by bright-field images. Scale bar: 200 μm. (D) Characterization of neural cells at 20 days of differentiation by the expression of neuronal progenitor cell markers, including N-cad, Nestin, Ngn1, Mitf and Tuj. (E) The relative mRNA expression level of neurotransmitter and subtype-specific genes (Nurr, CHAT, SLC, GAD2 and GFAP) in ESC- and iPSC-derived neuronal cells differentiated for 20 days on N-cadherin substrate. Abbreviation: ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; Ngn1- Neurogenin 1; Mitf- Microphthalmia-associated transcription factor; N-cad, N-cadherin; Nurr- nuclear receptor-related factor; CHAT- choline acetyltransferase; SLC- solute carrier; GAD- glutamate decarboxylase; GFAP- glial fibrillary acidic protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135170.g002: Neuronal differentiation on N-cadherin substrate without exogenous growth factors or inhibitors.(A-B) ESCs cultured in suspension for 5 days with and without CKI-7 and SB-431542 (CK/SB) was further induced on N-cadherin substrate for 48 h in the absence of exogenous growth factors. The degree of neural conversion was analyzed by (A) neurite extension index and (B) qRT-PCR for relative mRNA expression of nestin, neurogenin 1 (Ngn1), and βIII-tubulin (Tuj). (C) Cellular phenotype of differentiated cells on N-cad-Fc and gelatin substrates at 20 days of differentiation was assessed by bright-field images. Scale bar: 200 μm. (D) Characterization of neural cells at 20 days of differentiation by the expression of neuronal progenitor cell markers, including N-cad, Nestin, Ngn1, Mitf and Tuj. (E) The relative mRNA expression level of neurotransmitter and subtype-specific genes (Nurr, CHAT, SLC, GAD2 and GFAP) in ESC- and iPSC-derived neuronal cells differentiated for 20 days on N-cadherin substrate. Abbreviation: ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; Ngn1- Neurogenin 1; Mitf- Microphthalmia-associated transcription factor; N-cad, N-cadherin; Nurr- nuclear receptor-related factor; CHAT- choline acetyltransferase; SLC- solute carrier; GAD- glutamate decarboxylase; GFAP- glial fibrillary acidic protein.
Mentions: Default differentiation of mouse ESC to neuroectoderm occurs when culturing cells in serum-free medium and in the absence of any exogenous GFs [37,42]. Previous reports have also shown a vital role of endogenous signals in inducing mesoderm and endoderm lineages when differentiated without exogenous GFs [43,44]. Therefore, we sought to investigate if the effects of N-cadherin substrate on neurite growth and neuronal conversion could be replicated without exogenously added soluble GFs and inhibitors. Compared to CK/SB-free differentiation, a significant enhancement in neurite outgrowth was observed when differentiations were performed under the guidance of CK/SB (Fig 2A). Surprisingly, the expression level of well described markers for NPCs including nestin, neurogenin 1 (Ngn1), and βIII-tubulin (Tuj) was similar at day 7 in both conditions, as analyzed by qRT-PCR (Fig 2B).

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