MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons
Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors, microRNAs or by culturing in the presence of small molecules. This was first achieved by expressing Brn2, Ascl1 and Myt1L...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-10-01
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| Series: | Stem Cell Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1873506120302853 |
| _version_ | 1818199000023564288 |
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| author | Mette Habekost Arne Lund Jørgensen Per Qvist Mark Denham |
| author_facet | Mette Habekost Arne Lund Jørgensen Per Qvist Mark Denham |
| author_sort | Mette Habekost |
| collection | DOAJ |
| description | Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors, microRNAs or by culturing in the presence of small molecules. This was first achieved by expressing Brn2, Ascl1 and Myt1L in mouse fibroblasts, and was later achieved in human cells by the inclusion of additional factors such as NeuroD1. Here, we present the first protocol for directly converting porcine fibroblasts into induced neurons. We used lentivirus-mediated delivery of previously identified neuron-specifying transcription factors and microRNAs and evaluated morphology and neuron marker expression after ten days of conversion. We found that Ascl1 and microRNAs, miR-9/9* and miR-124 together generated more neuronal cells than other conditions tested. The porcine induced neurons expressed common mature markers such as MAP2 and Synaptophysin after four weeks of conversion. Transcriptomic analysis revealed that fibroblast-specific signatures were silenced early in the conversion process, while the neuron-specific genes became more abundant during conversion. We generated a heterogeneous population of glutamatergic and GABAergic neurons. |
| first_indexed | 2024-12-12T02:14:48Z |
| format | Article |
| id | doaj.art-95ea23caf1254e61865b2935584e05e3 |
| institution | Directory Open Access Journal |
| issn | 1873-5061 |
| language | English |
| last_indexed | 2024-12-12T02:14:48Z |
| publishDate | 2020-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Stem Cell Research |
| spelling | doaj.art-95ea23caf1254e61865b2935584e05e32022-12-22T00:41:50ZengElsevierStem Cell Research1873-50612020-10-0148101984MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neuronsMette Habekost0Arne Lund Jørgensen1Per Qvist2Mark Denham3Department of Biomedicine, Aarhus University, 8000C Aarhus, Denmark; Danish Research Institute of Translational Neuroscience, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, 8000C Aarhus, DenmarkDepartment of Biomedicine, Aarhus University, 8000C Aarhus, DenmarkDepartment of Biomedicine, Aarhus University, 8000C Aarhus, Denmark; Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, 8000C Aarhus, Denmark; Centre for Integrative Sequencing, iSEQ, Aarhus University, 8000C Aarhus, Denmark; Centre for Genomics and Personalized Medicine, CGPM, Aarhus University, 8000C Aarhus, DenmarkDepartment of Biomedicine, Aarhus University, 8000C Aarhus, Denmark; Danish Research Institute of Translational Neuroscience, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, 8000C Aarhus, Denmark; Corresponding author.Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors, microRNAs or by culturing in the presence of small molecules. This was first achieved by expressing Brn2, Ascl1 and Myt1L in mouse fibroblasts, and was later achieved in human cells by the inclusion of additional factors such as NeuroD1. Here, we present the first protocol for directly converting porcine fibroblasts into induced neurons. We used lentivirus-mediated delivery of previously identified neuron-specifying transcription factors and microRNAs and evaluated morphology and neuron marker expression after ten days of conversion. We found that Ascl1 and microRNAs, miR-9/9* and miR-124 together generated more neuronal cells than other conditions tested. The porcine induced neurons expressed common mature markers such as MAP2 and Synaptophysin after four weeks of conversion. Transcriptomic analysis revealed that fibroblast-specific signatures were silenced early in the conversion process, while the neuron-specific genes became more abundant during conversion. We generated a heterogeneous population of glutamatergic and GABAergic neurons.http://www.sciencedirect.com/science/article/pii/S1873506120302853Disease modellingPorcine induced neuronsDirect reprogrammingNeuronal conversionRegenerative medicineMicroRNA |
| spellingShingle | Mette Habekost Arne Lund Jørgensen Per Qvist Mark Denham MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons Stem Cell Research Disease modelling Porcine induced neurons Direct reprogramming Neuronal conversion Regenerative medicine MicroRNA |
| title | MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons |
| title_full | MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons |
| title_fullStr | MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons |
| title_full_unstemmed | MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons |
| title_short | MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons |
| title_sort | micrornas and ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons |
| topic | Disease modelling Porcine induced neurons Direct reprogramming Neuronal conversion Regenerative medicine MicroRNA |
| url | http://www.sciencedirect.com/science/article/pii/S1873506120302853 |
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