Generation of Human Neurons by microRNA-Mediated Direct Conversion of Dermal Fibroblasts
MicroRNAs (miRNAs), miR-9/9*, and miR-124 (miR-9/9*-124) display fate-reprogramming activities when ectopically expressed in human fibroblasts by erasing the fibroblast identity and evoking a pan-neuronal state. In contrast to induced pluripotent stem cel
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Introduction During mammalian neural development, microRNAs-9/9* and -124 (miR-9/9*-124) are upregulated at the onset of neurogenesis and function as a molecular switch to promote neuronal differentiation. Some of these switching mechanisms include the specification of neuron-specific subunit composition of BAF chromatin remodeling complexes, turning off anti-neurogenic transcription factors, and switching to neuron-specific alternative splicing [1–4]. Interestingly, many of the direct targets of miR-9/ 9*-124 are genes typically expressed in most non-neural somatic cell types. Further, when ectopically expressed in non-neural cell
Victoria A. Church, Kitra Cates, Lucia Capano, Shivani Aryal and Woo Kyung Kim contributed equally to this work. Kejin Hu (ed.), Nuclear Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 2239, https://doi.org/10.1007/978-1-0716-1084-8_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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types, these miRNAs exhibit neurogenic activities such as the direct fate conversion of skin fibroblasts to neurons [5, 6]. Importantly, miR-9/9*-124 can reprogram human fibroblasts from donors across the life spectrum while retaining the epigenetic age information stored in fibroblasts [7], thereby allowing generation of human neurons that reflect all ages [8]. Studies from human adult fibroblast conversion identified that miR-9/9*-124 drive fate conversion via reconstruction of the chromatin landscape [9] in part through repression of the neuronal RE1-silencing transcription factor (REST) [10] and induction of neuronal BAF subunit switching [2]. As such, the switch to the neuronal BAF complex drives chromatin remodeling that activates neuronal genes, leaving cells in the miRNA-induced neuronal state, poised to respond to transcription factors that promote neuronal maturity and subtype specificity [9]. The concordant expression of neuronal transcription factors with miR-9/9*-124 generates subtype-specific neuronal programs and increases neuronal maturity. Currently, the miRNA-based reprogramming protocols allow the generation of the following neuronal subtypes: cortical neurons (CNs), striatal medium spiny neurons (MSNs), and spinal motor neurons (MNs). These neuronal subtypes are afflicted in neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Amyotrophic lateral sclerosis, and thus generation of neurons with miR-9/9*-124 in combination with neuron-specification transcription factors opens the door to the investigation of subtype-specific disease pathology. Aging is the greatest risk factor in most forms of neurodegenerative diseases and thus age needs to be recapitulated in reprogrammed, patient-derived cells for modeling late-onset disorders. This requirement poses an experimental challenge for human neurons differentiated from induced pluripotent stem cells since the pluripotency induction reverts the reprogramming cells to an embryonic state, which produces neurons that mimic fetal stages
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