miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro

Abstract

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer’s disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.

MiR-124 synergism with ELAVL3 enhances target gene expression to promote neuronal maturity

“…By chemically inhibiting LGE in rat neurons, mimicking the transcriptomic phenotype observed in HNs with reduce miR-124 and ELAVL3 activity, we observed a variety of altered electrical properties. This finding also indicates that miR-9/9*-124 is highly neurogenic as these miRNAs not only allow for the conversion of HAFs into neurons (Abernathy et al 2017; Yoo et al 2011), but also enhanced the expression of neuronal markers, such as MAP2 , when overexpressed during neuronal differentiation of human pluripotent stem cell-derived neurons (Ishikawa et al, 2020; Sun et al, 2016). Although further tests are required to see if all the identified transcripts targeted by both miR-124 and nELAVLs share the same mechanism as PTBP2 , our loss-of-function results from in both reprogramming and primary human neuron systems, and its effect on other neuronal genes lend support to the general role of miR-124 as a positive regulator of select target genes in neurons.…”

How to reprogram human fibroblasts to neurons

“…Notably, the resulting neurons showed increased calcium activity and synaptic formation. In addition, the microelectrode array analyses showed that the electrical network activity was very high [36]. Moreover, a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/ Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor, a medium with bFGF and N2B27 supplements and the human ESC medium mTeSR1 was used to successfully established a feeder-free reprogramming condition, greatly improved the episomal reprogramming efficiency, and succeeded in generating iPSCs, then the iPSCs were differentiated into motor neurons based on dual SMAD inhibition [37,38].…”

Stem Cell-Based Disease Modeling and Cell Therapy

“…Moreover, when applying this method to iPSCs from Alzheimer’s disease (AD) patients, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42 was observed in a shorter culture period than those previously reported. Thus, the induction method combining Ngn2 and miR-9/9*-124 might enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery [ 31 ].…”

“…This Special Issue includes both research [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ] and reviews articles [ 10 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ] which cover wide ranges of stem cell research: adult stem cells, cancer stem cells, pluripotent stem cells, and complex 3D organoid/cell aggregate models [ 26 , 27 , 33 ], with the focuses on stem cell biology/technology [ 10 , 23 , 24 , 25 , 26 , 31 , 32 , 34 ], and stem cell-based disease modeling [ 10 , 27 , 29 , 31 , 33 , 38 , 43 ] and cell therapy [ 24 , 28 , 30 , 32 , 35 , 36 , 37 , 39 , 40 , 41 ].…”