Figure 1.
Characterisation of hiPSC lines derived from normal dermal human fibroblasts.
A) Two hiPSC lines were selected for in-depth analyses, NHDF-1 and NHDF-2. These reprogrammed colonies showed hESC-like morphology, for example, tightly packed colonies with high nucleus to cytoplasm ratio (top row). These colonies expressed pluripotency markers hNanog, hOCT4, SSEA-4 and TRA-1-60. Scale bar: 100 µm. B) Genome integrity analysis was performed using both the Illumina Human CytoSNP-12 beadchip with analysis using Karyostudio and using M-FISH. The top panel shows the Illumina analysis of three derived hiPSC clones (iPS-NHDF-1, -2 and -6), using the parental fibroblasts as reference (NHDF). The two analyses indicated a normal chromosomal complement and no gross structural abnormalities for iPS-NHDF-1 and iPS-NHDF-2, but iPS-NHDF-6 showed a translocation and amplification of part of chromosome 2, t(X:2), and was not used in further experiments. GEO: GSE43904. C) iPS-NHDF-1 and iPS-NHDF-2 cell lines are competent at differentiation to three germ layers in vitro. Left column shows embryoid body (EB) formation 4 days after forced aggregation of hiPSCs using Aggrewells. Second column, directed differentiation to neuroectoderm, showing neurons stained for βIII tubulin, using a TujI antibody. Third column, directed differentiation to mesoderm, showing cells stained with antibody against α-sarcomeric actin (ASA). Fourth column, directed differentiation to endoderm, showing nuclei stained with an antibody against the transcription factor, FoxA2. Nuclei are counterstained with DAPI (blue). Scale bar:100 µm. D) PluriTest analysis of Illumina HT12v4 transcriptome array data shows iPS-NHDF-1 and iPS-NHDF-2 to cluster with pluripotent stem cells (red cloud) and not with partly- or differentiated cells (blue clouds). Each circle represents one iPSC line: 1 = iPS-NHDF-1; 2 = iPS-NHDF-2; C = control iPSC line iPS-DF19.9.11TH. GEO: GSE43904.
Figure 2.
Differentiation of human dopaminergic neurons.
A) hiPS cells were directed to differentiate into dopaminergic neurons. Following midbrain patterning, neural progenitor cells were manually passaged (day 16+). Dopaminergic neurons were matured for a total of 6–10 weeks before experimentation. B) RT-PCR analysis of gene expression in cells at different stages of differentiation (EB: day 0, Rosette: day 16, Neuron: day 35). Pluripotency genes (OCT4 and SOX2) were down-regulated as cells differentiate and concurrently midbrain dopaminergic gene expression was upregulated. Midbrain dopaminergic neuronal markers (PITX3, NURR1, EN1, TH and GIRK2) were strongly expressed in differentiated neuronal populations. C) Immunocytochemical staining for TH (bottom panels) revealed an abundant population of neurons. Large clusters of neurons expressed the floorplate marker FOXA2 (top panels). Scale bar: 70 µm. D) The midbrain transcription factors, PITX3 and NURR1 were expressed in differentiated neurons. Scale bar: 70 µm. E) Efficiency of dopaminergic differentiation was quantified from at least 3 independent differentiations. iPS- NHDF1 and 2 lines differentiated with similar efficiencies into neurons (39.59% ±3.56 and 41.73% ±2.02, expressed as Tuj1/DAPI). The proportion of neurons that were dopaminergic was also similar (NHDF1: 50.35% ±3.4 and NHDF2: 52.54% ±2.76). The total number of cells that were TH+ were 19.21% ±2.05 and 21.5% ±1.53 for NHDF1 and NHDF2, respectively. Data are expressed as the mean ± SEM. F) Co-labelling of differentiated neurons revealed expression of dopaminergic neuronal proteins such as DAT and VMAT2 together with TH. The A9 dopaminergic neuron marker, GIRK2 was also expressed in some TH-positive cells. Scale bar: 70 µm. G) Analysis of MAPT exon splicing indicated that differentiated neurons have a splicing pattern similar to that of the human adult cortex. Exon skipping of exons 2, 3 and 10 was observed in undifferentiated hiPSCs, which is typical of embryonic neural tissues.
Figure 3.
Functional dopamine synthesis and homeostasis in differentiated neuronal cultures.
A) Western blot analysis of proteins involved in dopamine synthesis and homeostasis in cells at different stages of maturation (EB: day 0, Rosette: day 16, Neuron: day 35). hiPSCs do not express TH, DAT or AADC but these were expressed as the cells differentiated in culture. DAT is expressed only in differentiated neurons. B) Immunostaining for dopamine in differentiated neurons. This labelling co-localised with TH expression. Scale bar: 70 µm. C) Dopamine content of differentiated neurons was measured by HPLC. Differentiated neurons (35 days total in culture) produced dopamine (48.98 pmol/mg ±0.389) and were responsive to L-DOPA (933.14 pmol/mg ±220.71), indicating high AADC activity. Data shown are from 4–6 wells each of 2 independent experiments and is expressed as mean ± SEM. D) Functional dopamine transporter activity was assessed by 3HDA uptake in differentiated neurons. DAT-specific uptake was demonstrated by inhibition by mazindol. Data are expressed as mean ± SEM.
Figure 4.
Functional characterisation of the maturation of human midbrain neuronal cultures.
(A) Representative current-clamp recordings (400 ms current pulses and inj 40–50 pA) from hiPSC-derived neurons at specified time points (from left panel: 6, 7, 8, 9, and 10 weeks in culture). Neurons show changes in resting membrane potentials reflecting maturation (6 weeks: ∼ −35–40 mV, 10 week: ∼ −65–70 mV). These were recorded in neurons that were not showing pace-making activity. (B) Representative pace-making firing traces in whole-cell patch current clamp configuration and (C) representative traces of the spontaneous EPSCs recorded from 10 week old neuronal cultures (n >6 neurons showed spontaneous pace-making activity). Inset shows immunostaining on fixed cells of co-expression of TH and synaptic vesicle protein, SV2 in differentiated neurons. Similar recordings were obtained from NHDF-1 and -2 lines.
Figure 5.
Dynamic changes in Ca2+ signals during neuronal maturation.
Traces show recordings of spontaneous changes in Ca2+ signals after induction of neuronal differentiation from hiPS cells into dopaminergic neurons. Progressive changes in their kinetics were observed, with increase rhythmicity apparent from 6 weeks and onwards in differentiation. By 8 weeks in culture, oscillatory activity typical of dopaminergic neurons was observed in both soma and in the neuronal processes (n >6, for each timepoint). Recordings were obtained from NHDF-1 and -2 lines and gave similar results.
Figure 6.
Spontaneous intracellular calcium signals in human hiPS cell-derived neurons.
(A) Immunostaining in fixed TH-positive neurons (red) expressed inositol tri-phosphate receptors (IP3Rs) in both the soma and in dendritic processes (green). Live cell imaging showed that IP3-BM-evoked calcium signals were detected differentiated neurons, both in the processes (B, upper montages) and also from the soma (B lower panels). Yellow puncta in the upper montages represent a time-dependent fluorescence change which indicated propagation of the calcium wave through neuronal processes. The fluorescence profile in the lower panel shows the fluorescence change obtained from the soma (1. before and 2. after elevation of calcium concentration). (C) Left panel indicates the typical distribution of LysoTracker-sensitive acidic organelles in differentiated neurons. Right-hand images illustrate that the propagation of calcium waves upon application of NAADP/AM originated from the neuronal soma toward a dendritic process. Recordings were obtained from 2 different lines (n >3).
Figure 7.
Measurements of mitochondrial membrane potentials and their sensitivity to MPP+.
(A) Differentiated human neuronal cultures were loaded with mitochondrial membrane indicator TMRM. Changes in fluorescence were measured before (left upper) and after (left lower) application of the membrane potential uncoupler, CCCP. Right panels show quantification of fluorescence changes obtained from the 3 regions of interest marked in the left panels. Induced and spontaneous fluctuations in membrane potential were observed in some cells and changes could be detected at the level of a single mitochondrion (ROI 3). (B) Upper graph represents mean mitochondrial resting potential measured from cell bodies and the effect of exposure to the dopaminergic neuron toxin MPP+ for 24 h. Mean resting fluorescence intensity was significantly lower after MPP+ treatment (control, F.U. 68.8±13.4×103 and 40 µM MPP+, F.U. 43.6±2.9×103, t <0.05, n >40). Lower panels indicate mitochondrial morphological changes following MPP+ treatment.