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. 2020 Feb 25;9(3):532.
doi: 10.3390/cells9030532.

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

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Free PMC article

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

Mitsuru Ishikawa et al. Cells. .
Free PMC article

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.

Keywords: Alzheimer’s disease; excitatory neuron; human pluripotent stem cell; microRNA-124; microRNA-9/9*; neurogenin2; presenilin1; presenilin2.

Conflict of interest statement

H.O. is a scientific consultant for SanBio, Co. Ltd., and K Pharma Inc. The other authors declare neither financial nor non-financial competing interests.

Figures

Figure 1
Figure 1
Bcl-xL genes (BmiRs) expression alone promotes neural differentiation from human pluripotent stem cells (hPSCs). (A) Overview of the protocol for differentiation into neurons by Tet-On-inducible Bcl-xL gene miR9/9*-124 overexpression. (B) Design of the lentiviral vector for Bcl-xL-miR-9/9*-124-mediated conversion of hESCs to neuronal cells. mKO1 and rtTA2M2 are abbreviations for monomeric Kusabira Orange, a fluorescent protein, and reverse tetracycline transactivator, respectively. (C) Representative bright field images illustrating the neuronally converted KhES1 cells at day 14. Scale bar: 100 µm. (D) Immunofluorescent (Tubb3) images of KhES1 in three different culture conditions: neuronal medium without Dox; hPSC medium or neuronal medium with 2 µg/ml Dox at day 20. Arrowheads indicate Tubb3+ neurite morphology. Scale bar: 500 µm. (E) Relative mRNA expression levels of Pou5f1, Sox2, Sox1, Tubb3, Map2, and Gfap in KhES1. (PSM: medium for pluripotent stem cells; NM: medium for neurons). Each data was standardized by the control cells cultured in PSM. (n = 4 independent experiments; mean ± SEM).
Figure 2
Figure 2
The effect of the combination of Ngn2 and BmiRs expression on neural differentiation. (A) The list of control hPSC line used in this study. (B) Overview of the protocol for piggybac vector transfection into hPSCs with the piggybac vectors. (C) Overview of the protocol for lentiviral infection into hPSCs and neuronal induction using Dox. (D,E) Design of the transposase expression vectors: Piggybac vectors for Ngn2-mediated neuronal conversion and lentiviral vector for Bcl-xL-miR-9/9*-124 -mediated neuronal conversion. Puro.R, Hygro.R, Blast.R, and rtTA3G indicates puromycin, hygromycin, blastcidin resistant sequences, and 3rd generation reverse tetracycline transactivator, respectively. (F) Representative low-power field (upper) and high-power field (lower) of the neurons (khES1) 20 days after transient expression of both Ngn2 and BmiRs. (G) Immunocytochemical analysis of neuronal markers (Tubb3, MAP2 and NeuN). The photograph on the right is an enlarged view of the part surrounded by yellow on the left. Scale Bar: 100 µm. (H) Ratio of positive cells for each marker: Tubb3+ cells/all cells (Tubb3+/Ho); MAP2+ cells/ neurons (MAP2+/Tubb3+); NeuN+ cells / neurons (NeuN+/Tubb3+) (n = 4 independent experiments; mean ± SD; * p < 0.05; ** p < 0.01, Dunnett’s test).
Figure 2
Figure 2
The effect of the combination of Ngn2 and BmiRs expression on neural differentiation. (A) The list of control hPSC line used in this study. (B) Overview of the protocol for piggybac vector transfection into hPSCs with the piggybac vectors. (C) Overview of the protocol for lentiviral infection into hPSCs and neuronal induction using Dox. (D,E) Design of the transposase expression vectors: Piggybac vectors for Ngn2-mediated neuronal conversion and lentiviral vector for Bcl-xL-miR-9/9*-124 -mediated neuronal conversion. Puro.R, Hygro.R, Blast.R, and rtTA3G indicates puromycin, hygromycin, blastcidin resistant sequences, and 3rd generation reverse tetracycline transactivator, respectively. (F) Representative low-power field (upper) and high-power field (lower) of the neurons (khES1) 20 days after transient expression of both Ngn2 and BmiRs. (G) Immunocytochemical analysis of neuronal markers (Tubb3, MAP2 and NeuN). The photograph on the right is an enlarged view of the part surrounded by yellow on the left. Scale Bar: 100 µm. (H) Ratio of positive cells for each marker: Tubb3+ cells/all cells (Tubb3+/Ho); MAP2+ cells/ neurons (MAP2+/Tubb3+); NeuN+ cells / neurons (NeuN+/Tubb3+) (n = 4 independent experiments; mean ± SD; * p < 0.05; ** p < 0.01, Dunnett’s test).
Figure 3
Figure 3
Gene expression profiling of the differentiated neurons. Using the KhES1, 201B7, 414C2, and 1210B2 lines, cells in each indicated differentiated state (PSCs, NSCs (day 20), NSCs-derived neurons (day 40) and Ngn2-induced neurons with or without BmiRs expression (day 20 and 40)) were harvested and subjected to Real-time RT-PCR. Analyzed data were summarized asa heatmap and corresponds to the -ΔΔCt value. The numerical value in each cell is the averaged -ΔΔCt value in each target, which is standardized by the data of Ngn2 without BmiRs-induced neurons (Day 20).
Figure 4
Figure 4
Evaluation of neuronal functionality using GCaMP and quantification of synapse-related proteins. (A) Design of the lentiviral vector for Synapsin1: jGCaMP7s-T2A-NLS -mCherry. (B) Ratio of positive cells for Synapsin1: mCherry+ cells in live cells. The number of total cells was defined as the total number of Ho signal. (n = 4 independent experiments; mean ± SEM; * p < 0.05, Dunnett’s test). (C) Representative GCaMP7s and mCherry images. The circled ROIs are depicted in (D) as time-dependent changes in GFP fluorescence intensity. Scale bar: 50 µm. (D) Representative images of GCaMP spikes and display of parameters (ΔFmax). (E) Comparison of ΔFmax values. The average value of the control group (BmiRs) was used as the reference value. (n = 4 independent experiments; mean ± SD; *** p < 0.001, Dunnett’s test) (F) Ratio of calcium spike+ cells. The number of cells having a ΔFmax value of more than 0.01 in (D) was quantified. (n = 4 independent experiments; mean ± SEM; *** p < 0.001, Dunnett’s test) (G) Representative confocal microscope images of immunocytochemical analyses of neuronal markers and synaptic markers using confocal microscope (Scale bar: 10 µm). The enlargement of the area enclosed by the yellow rectangle in the merge photograph are shown on the right. (H,I) Comparison of SynapsinI+ (H) and PSD95+ (I) area colocalized with Tubb3+ area per Tubb3+ neurite length. The average value of the control group (BmiRs-) was used as the reference value. (n = 4 independent experiments; mean ± SEM; * p < 0.05, Dunnett’s test).
Figure 5
Figure 5
Sequential evaluation of neuronal activity in the neuronal culture using microelectrode array (MEA). The electrophysiological analyses were continuously performed using MEA from day 7 to 35 after neuronal induction. The number of active electrodes (A), mean firing rate (B), the number of burst (C), the number of bursting electrodes (D), and network burst frequency (E) were calculated using Neural Metric Tool (Axion Biosystems). (n = 4 independent experiments; mean ± SEM) (F) A typical 3-minute raster plot drawn with KhES1-derived neurons induced by the overexpression of Ngn2 with BmiRs on day 35. The figure below is an enlargement of the first 15 seconds. The black lines and the blue lines indicate single spikes and spikes defined in a burst, and the time surrounded by magenta indicates the moment when a network burst occurs. See the method section for detailed description of the analysis.
Figure 6
Figure 6
Early phenotype expression of the PS1, PS2 mutant iPS cell-derived neurons by the introduction of miRs. (A) Overview of the neuronal culture protocol and phenotype assay. (B) Design of the lentiviral vector for Bcl-xL-free miR-9-124-mediated neuronal conversion. The sequences for pri-miR-9-3 and pri-miR-124-2 were included in order to induce the expressions of miR-9 and miR-124, respectively. (C) Secreted Aβ40 and Aβ42 levels in the neuronal cell culture derived from the healthy control induced-pluripotent stem cells (iPSCs) and the iPSCs with PSEN1 or 2 mutation at day 20 and day 40. (n = 5 independent experiments; mean ± SEM, ## p < 0.01 by Turkey’s test versus CT (miRs-, Day20); * p < 0.05, Turkey’s test versus the healthy donor group) (D) The ratio of Aβ42/40 (n = 5 independent experiments; mean ± SEM, * p < 0.05, Turkey’s test versus the healthy donor group) (E,F) Functional analysis of mitochondrial respiration in the neurons at day12. Basal oxygen consumption rate (OCR), maximal respiration and spare respiration capacity in healthy control and Alzheimer’s disease (AD) neurons induced by overexpression of Ngn2 with or without miRs in the same neuronal medium used in the experiments above (E). Total OCR profile along the assay time course is described in Figure S6B. Spare respiration capacity in a neuronal medium without antioxidant factors are shown in (F). (n = 5 independent experiments; mean ± SEM; # p < 0.05; ## p < 0.01, Tukey’s test vs. Ngn2 without miRs, * p < 0.05, ** p < 0.01, *** p < 0.001, Turkey’s test vs. healthy control group) (G) Representative images of immunocytochemistry for neurons using CellROX, a marker for reactive oxygen species. Arrowheads indicate CellROX signals. (H) CellROX+ cell population in neurons. (n = 4 independent experiments; mean ± SEM; * p < 0.05, Turkey’s test vs. healthy control group).
Figure 7
Figure 7
Accelerated pathological analysis and drug discovery screening resulting from early neural differentiation and maturation from pluripotent stem cells by combining the expression of Neurogenin2 and microRNA-9/9*-124. In contrast to the conventional induction method using dual SMAD inhibitors, transient expression of Ngn2 gene enables a purified population of neurons to be obtained. On the other hand, the Ngn2 method is insufficient in terms of neuronal maturation. Thus, by adding BmiRs expression used in this study, neuronal function can be rapidly enhanced. This enables phenotypic evaluation, which originally required long-term culture, to be performed within short-term culture.

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