Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes
Astrocyte-to-neuron reprogramming is a promising therapeutic approach for treatment of neurodegenerative diseases. The use of small molecules as an alternative to the virus-mediated ectopic expression of lineage-specific transcription factors negates the tumorigenic risk associated with viral geneti...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-04-01
|
| Series: | Biomedicines |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2227-9059/10/4/928 |
| _version_ | 1827621596978741248 |
|---|---|
| author | Gary Stanley Fernandes Rishabh Deo Singh Kyeong Kyu Kim |
| author_facet | Gary Stanley Fernandes Rishabh Deo Singh Kyeong Kyu Kim |
| author_sort | Gary Stanley Fernandes |
| collection | DOAJ |
| description | Astrocyte-to-neuron reprogramming is a promising therapeutic approach for treatment of neurodegenerative diseases. The use of small molecules as an alternative to the virus-mediated ectopic expression of lineage-specific transcription factors negates the tumorigenic risk associated with viral genetic manipulation and uncontrolled differentiation of stem cells. However, because previously developed methods for small-molecule reprogramming of astrocytes to neurons are multistep, complex, and lengthy, their applications in biomedicine, including clinical treatment, are limited. Therefore, our objective in this study was to develop a novel chemical-based approach to the cellular reprogramming of astrocytes into neurons with high efficiency and low complexity. To accomplish that, we used C8-D1a, a mouse astrocyte cell line, to assess the role of small molecules in reprogramming protocols that otherwise suffer from inconsistencies caused by variations in donor of the primary cell. We developed a new protocol by which a chemical mixture formulated with Y26732, DAPT, RepSox, CHIR99021, ruxolitinib, and SAG rapidly and efficiently induced the neural reprogramming of astrocytes in four days, with a conversion efficiency of 82 ± 6%. Upon exposure to the maturation medium, those reprogrammed cells acquired a glutaminergic phenotype over the next eleven days. We also demonstrated the neuronal functionality of the induced cells by confirming KCL-induced calcium flux. |
| first_indexed | 2024-03-09T11:06:37Z |
| format | Article |
| id | doaj.art-ed0e713caab14264b227889975b0b71e |
| institution | Directory Open Access Journal |
| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-09T11:06:37Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomedicines |
| spelling | doaj.art-ed0e713caab14264b227889975b0b71e2023-12-01T00:56:42ZengMDPI AGBiomedicines2227-90592022-04-0110492810.3390/biomedicines10040928Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse AstrocytesGary Stanley Fernandes0Rishabh Deo Singh1Kyeong Kyu Kim2Department of Precision Medicine, Graduate School of Basic Medical Science (GSBMS), Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon 16419, KoreaDepartment of Precision Medicine, Graduate School of Basic Medical Science (GSBMS), Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon 16419, KoreaDepartment of Precision Medicine, Graduate School of Basic Medical Science (GSBMS), Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon 16419, KoreaAstrocyte-to-neuron reprogramming is a promising therapeutic approach for treatment of neurodegenerative diseases. The use of small molecules as an alternative to the virus-mediated ectopic expression of lineage-specific transcription factors negates the tumorigenic risk associated with viral genetic manipulation and uncontrolled differentiation of stem cells. However, because previously developed methods for small-molecule reprogramming of astrocytes to neurons are multistep, complex, and lengthy, their applications in biomedicine, including clinical treatment, are limited. Therefore, our objective in this study was to develop a novel chemical-based approach to the cellular reprogramming of astrocytes into neurons with high efficiency and low complexity. To accomplish that, we used C8-D1a, a mouse astrocyte cell line, to assess the role of small molecules in reprogramming protocols that otherwise suffer from inconsistencies caused by variations in donor of the primary cell. We developed a new protocol by which a chemical mixture formulated with Y26732, DAPT, RepSox, CHIR99021, ruxolitinib, and SAG rapidly and efficiently induced the neural reprogramming of astrocytes in four days, with a conversion efficiency of 82 ± 6%. Upon exposure to the maturation medium, those reprogrammed cells acquired a glutaminergic phenotype over the next eleven days. We also demonstrated the neuronal functionality of the induced cells by confirming KCL-induced calcium flux.https://www.mdpi.com/2227-9059/10/4/928neuronal reprogrammingsmall moleculeastrocyteneurons |
| spellingShingle | Gary Stanley Fernandes Rishabh Deo Singh Kyeong Kyu Kim Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes Biomedicines neuronal reprogramming small molecule astrocyte neurons |
| title | Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes |
| title_full | Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes |
| title_fullStr | Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes |
| title_full_unstemmed | Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes |
| title_short | Generation of a Pure Culture of Neuron-like Cells with a Glutamatergic Phenotype from Mouse Astrocytes |
| title_sort | generation of a pure culture of neuron like cells with a glutamatergic phenotype from mouse astrocytes |
| topic | neuronal reprogramming small molecule astrocyte neurons |
| url | https://www.mdpi.com/2227-9059/10/4/928 |
| work_keys_str_mv | AT garystanleyfernandes generationofapurecultureofneuronlikecellswithaglutamatergicphenotypefrommouseastrocytes AT rishabhdeosingh generationofapurecultureofneuronlikecellswithaglutamatergicphenotypefrommouseastrocytes AT kyeongkyukim generationofapurecultureofneuronlikecellswithaglutamatergicphenotypefrommouseastrocytes |