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Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons

The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients’ cells play an imp...

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Autores principales: Kraskovskaya, Nina, Bolshakova, Anastasia, Khotin, Mikhail, Bezprozvanny, Ilya, Mikhailova, Natalia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095147/
https://www.ncbi.nlm.nih.gov/pubmed/37047770
http://dx.doi.org/10.3390/ijms24076799
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author Kraskovskaya, Nina
Bolshakova, Anastasia
Khotin, Mikhail
Bezprozvanny, Ilya
Mikhailova, Natalia
author_facet Kraskovskaya, Nina
Bolshakova, Anastasia
Khotin, Mikhail
Bezprozvanny, Ilya
Mikhailova, Natalia
author_sort Kraskovskaya, Nina
collection PubMed
description The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients’ cells play an important role in such studies. There are two ways to obtain induced neuronal cells. One is based on induced pluripotent stem cells. The other is based on direct reprogramming, which allows us to obtain mature neuronal cells from adult somatic cells, such as dermal fibroblasts. Moreover, the latter method makes it possible to better preserve the age-related aspects of neuropathology, which is valuable for diseases that occur with age. However, direct methods of reprogramming have a significant drawback associated with low cell viability during procedures. Furthermore, the number of reprogrammable neurons available for morphological and functional studies is limited by the initial number of somatic cells. In this article, we propose modifications of a previously developed direct reprogramming method, based on the combination of microRNA and transcription factors, which allowed us to obtain a population of functionally active induced striatal neurons (iSNs) with a high efficiency. We also overcame the problem of the presence of multinucleated neurons associated with the cellular division of starting fibroblasts. Synchronization cells in the G1 phase increased the homogeneity of the fibroblast population, increased the survival rate of induced neurons, and eliminated the presence of multinucleated cells at the end of the reprogramming procedure. We have demonstrated that iSNs are functionally active and able to form synaptic connections in co-cultures with mouse cortical neurons. The proposed modifications can also be used to obtain a population of other induced neuronal types, such as motor and dopaminergic ones, by selecting transcription factors that determine differentiation into a region-specific neuron.
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spelling pubmed-100951472023-04-13 Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons Kraskovskaya, Nina Bolshakova, Anastasia Khotin, Mikhail Bezprozvanny, Ilya Mikhailova, Natalia Int J Mol Sci Article The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients’ cells play an important role in such studies. There are two ways to obtain induced neuronal cells. One is based on induced pluripotent stem cells. The other is based on direct reprogramming, which allows us to obtain mature neuronal cells from adult somatic cells, such as dermal fibroblasts. Moreover, the latter method makes it possible to better preserve the age-related aspects of neuropathology, which is valuable for diseases that occur with age. However, direct methods of reprogramming have a significant drawback associated with low cell viability during procedures. Furthermore, the number of reprogrammable neurons available for morphological and functional studies is limited by the initial number of somatic cells. In this article, we propose modifications of a previously developed direct reprogramming method, based on the combination of microRNA and transcription factors, which allowed us to obtain a population of functionally active induced striatal neurons (iSNs) with a high efficiency. We also overcame the problem of the presence of multinucleated neurons associated with the cellular division of starting fibroblasts. Synchronization cells in the G1 phase increased the homogeneity of the fibroblast population, increased the survival rate of induced neurons, and eliminated the presence of multinucleated cells at the end of the reprogramming procedure. We have demonstrated that iSNs are functionally active and able to form synaptic connections in co-cultures with mouse cortical neurons. The proposed modifications can also be used to obtain a population of other induced neuronal types, such as motor and dopaminergic ones, by selecting transcription factors that determine differentiation into a region-specific neuron. MDPI 2023-04-05 /pmc/articles/PMC10095147/ /pubmed/37047770 http://dx.doi.org/10.3390/ijms24076799 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kraskovskaya, Nina
Bolshakova, Anastasia
Khotin, Mikhail
Bezprozvanny, Ilya
Mikhailova, Natalia
Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons
title Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons
title_full Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons
title_fullStr Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons
title_full_unstemmed Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons
title_short Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons
title_sort protocol optimization for direct reprogramming of primary human fibroblast into induced striatal neurons
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095147/
https://www.ncbi.nlm.nih.gov/pubmed/37047770
http://dx.doi.org/10.3390/ijms24076799
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