TY - JOUR
T1 - Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons
AU - Kraskovskaya, Nina
AU - Bolshakova, Anastasia
AU - Khotin, Mikhail
AU - Bezprozvanny, Ilya
AU - Mikhailova, Natalia
N1 - Funding Information:
This research was funded by the Ministry of Science and Higher Education of the Russian Federation, Agreement No. 075-15-2021-1063 (N.M), by Russian Science Foundation (RSF) grant No. 22-75-00106 (N.K.) and by RSF grant 22-15-00049 (I.B.). The financial support was divided in the following way: the experiments and results depicted in Figure 1 were funded from Agreement No. 075-15-2021-1063, in Figure 2 by RSF grant 22-15-00049 (I.B.), and in Figure 3 and Figure 4 by RSF grant 22-75-00106 (N.K.). I.B. holds the Carl J. and Hortense M. Thomsen Chair in Alzheimer’s Disease Research.
Funding Information:
We are thankful to Oleg Shuvalov for his assistance with the flow cytometry experiments. The DF1 and DF2 cell lines were obtained from the shared research facility “Vertebrate cell culture collection” supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 075-15-2021-683).
Publisher Copyright:
© 2023 by the authors.
PY - 2023/4
Y1 - 2023/4
N2 - 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.
AB - 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.
KW - aging
KW - cell synchronization
KW - dendritic spines
KW - direct reprogramming
KW - lentiviruses
KW - medium spiny neurons
KW - microRNA
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U2 - 10.3390/ijms24076799
DO - 10.3390/ijms24076799
M3 - Article
C2 - 37047770
AN - SCOPUS:85152345540
SN - 1661-6596
VL - 24
JO - International journal of molecular sciences
JF - International journal of molecular sciences
IS - 7
M1 - 6799
ER -