| 1. |
- Birtele, Marcella, et al.
(författare)
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Dual modulation of neuron-specific microRNAs and the REST complex promotes functional maturation of human adult induced neurons
- 2019
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 593:23, s. 3370-3380
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Tidskriftsartikel (refereegranskat)abstract
- Direct neuronal reprogramming can be achieved using different approaches: by expressing neuronal transcription factors or microRNAs; and by knocking down neuronal repressive elements. However, there still exists a high variability in terms of the quality and maturity of the induced neurons obtained, depending on the reprogramming strategy employed. Here, we evaluate different long-term culture conditions and study the effect of expressing the neuronal-specific microRNAs, miR124 and miR9/9*, while reprogramming with forced expression of the transcription factors Ascl1, Brn2, and knockdown of the neuronal repressor REST. We show that the addition of microRNAs supports neuronal maturation in terms of gene and protein expression, as well as in terms of electrophysiological properties.
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| 2. |
- Drouin-Ouellet, Janelle, et al.
(författare)
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REST suppression mediates neural conversion of adult human fibroblasts via microRNA-dependent and -independent pathways
- 2017
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4684 .- 1757-4676. ; 9:8, s. 1117-1131
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Tidskriftsartikel (refereegranskat)abstract
- Direct conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs), was achieved for the first time 6 years ago. This technology offers a promising shortcut for obtaining patient- and disease-specific neurons for disease modeling, drug screening, and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors, and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient-derived material for large-scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST-controlled neuron-specific microRNAs miR-9 and miR-124, we show that the effect of REST inhibition is only partially mediated via microRNA up-regulation. Transcriptional analysis confirmed that REST knockdown activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes that are not activated via microRNA overexpression. Based on this, we developed an optimized one-step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single-vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson's, Huntington's, as well as Alzheimer's disease.
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| 3. |
- Lau, Shong, et al.
(författare)
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Direct neural conversion from human fibroblasts using self-regulating and nonintegrating viral vectors.
- 2014
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 9:5, s. 1673-1680
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Tidskriftsartikel (refereegranskat)abstract
- Recent findings show that human fibroblasts can be directly programmed into functional neurons without passing via a proliferative stem cell intermediate. These findings open up the possibility of generating subtype-specific neurons of human origin for therapeutic use from fetal cell, from patients themselves, or from matched donors. In this study, we present an improved system for direct neural conversion of human fibroblasts. The neural reprogramming genes are regulated by the neuron-specific microRNA, miR-124, such that each cell turns off expression of the reprogramming genes once the cell has reached a stable neuronal fate. The regulated system can be combined with integrase-deficient vectors, providing a nonintegrative and self-regulated conversion system that rids problems associated with the integration of viral transgenes into the host genome. These modifications make the system suitable for clinical use and therefore represent a major step forward in the development of induced neurons for cell therapy.
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| 4. |
- Pereira, Maria J M, et al.
(författare)
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Highly efficient generation of induced neurons from human fibroblasts that survive transplantation into the adult rat brain.
- 2014
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Induced neurons (iNs) offer a novel source of human neurons that can be explored for applications of disease modelling, diagnostics, drug screening and cell replacement therapy. Here we present a protocol for highly efficient generation of functional iNs from fetal human fibroblasts, and also demonstrate the ability of these converted human iNs (hiNs) to survive transplantation and maintain their phenotype in the adult rat brain. The protocol encompasses a delay in transgene activation after viral transduction that resulted in a significant increase in conversion efficiency. Combining this approach with treatment of small molecules that inhibit SMAD signalling and activate WNT signalling provides a further increase in the conversion efficiency and neuronal purity, resulting in a protocol that provides a highly efficient method for the generation of large numbers of functional and transplantable iNs from human fibroblasts without the use of a selection step. When transplanting the converted neurons from different stages of in vitro culture into the brain of adult rats, we observed robust survival and maintenance of neuronal identity four weeks post-transplantation. Interestingly, the positive effect of small molecule treatment observed in vitro did not result in a higher yield of iNs surviving transplantation.
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| 5. |
- Torper, Olof, et al.
(författare)
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Generation of induced neurons via direct conversion in vivo.
- 2013
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 110:17, s. 7038-7043
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Tidskriftsartikel (refereegranskat)abstract
- Cellular reprogramming is a new and rapidly emerging field in which somatic cells can be turned into pluripotent stem cells or other somatic cell types simply by the expression of specific combinations of genes. By viral expression of neural fate determinants, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons. The resulting cells are nonproliferating and present an alternative to induced pluripotent stem cells for obtaining patient- and disease-specific neurons to be used for disease modeling and for development of cell therapy. In addition, because the cells do not pass a stem cell intermediate, direct neural conversion has the potential to be performed in vivo. In this study, we show that transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, convert into neurons when reprogramming genes are activated after transplantation. Using a transgenic mouse model to specifically direct expression of reprogramming genes to parenchymal astrocytes residing in the striatum, we also show that endogenous mouse astrocytes can be directly converted into neural nuclei (NeuN)-expressing neurons in situ. Taken together, our data provide proof of principle that direct neural conversion can take place in the adult rodent brain when using transplanted human cells or endogenous mouse cells as a starting cell for neural conversion.
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| 6. |
- Torper, Olof, et al.
(författare)
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In Vivo Reprogramming of Striatal NG2 Glia into Functional Neurons that Integrate into Local Host Circuitry.
- 2015
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 12:3, s. 474-481
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Tidskriftsartikel (refereegranskat)abstract
- The possibility of directly converting non-neuronal cells into neurons in situ in the brain would open therapeutic avenues aimed at repairing the brain after injury or degenerative disease. We have developed an adeno-associated virus (AAV)-based reporter system that allows selective GFP labeling of reprogrammed neurons. In this system, GFP is turned on only in reprogrammed neurons where it is stable and maintained for long time periods, allowing for histological and functional characterization of mature neurons. When combined with a modified rabies virus-based trans-synaptic tracing methodology, the system allows mapping of 3D circuitry integration into local and distal brain regions and shows that the newly reprogrammed neurons are integrated into host brain.
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