Bipolar disorder-iPSC derived neural progenitor cells exhibit dysregulation of store-operated Ca2+ entry and accelerated differentiation

While most of the efforts to uncover mechanisms contributing to bipolar disorder (BD) focused on phenotypes at the mature neuron stage, little research has considered events that may occur during earlier timepoints of neurodevelopment. Further, although aberrant calcium (Ca2+) signaling has been implicated in the etiology of this condition, the possible contribution of store-operated Ca2+ entry (SOCE) is not well understood. Here, we report Ca2+ and developmental dysregulations related to SOCE in BD patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells (BD-NPCs) and cortical-like glutamatergic neurons. First, using a Ca2+ re-addition assay we found that BD-NPCs and neurons had attenuated SOCE. Intrigued by this finding, we then performed RNA-sequencing and uncovered a unique transcriptome profile in BD-NPCs suggesting accelerated neurodifferentiation. Consistent with these results, we measured a slower rate of proliferation, increased neurite outgrowth, and decreased size in neurosphere formations with BD-NPCs. Also, we observed decreased subventricular areas in developing BD cerebral organoids. Finally, BD NPCs demonstrated high expression of the let-7 family while BD neurons had increased miR-34a, both being microRNAs previously implicated in neurodevelopmental deviations and BD etiology. In summary, we present evidence supporting an accelerated transition towards the neuronal stage in BD-NPCs that may be indicative of early pathophysiological features of the disorder.

imaging solution with 2 µM Tg.For TRPC4/5 and NMDA inhibition, the imaging solutions with 10 µM ML 204 (Tocris), 10 µM AC 1903 (Tocris), and 50 µM APV (Tocris), sequentially.5 minutes of imaging was conducted after each treatment.All experiments were performed at room temperature.Image processing and analysis was done using NIS Elements (Nikon, Minato City, TYO) and Microsoft Excel.

Fura-2 Ca 2+ imaging
For ratiometric Ca 2+ imaging, iPSC/NPCs were incubated in imaging solution with 2 µM of the Fura-2 AM dye (Thermo Fisher Scientific) similar to the Fluo-4 protocol but without addition of Tg after the last wash.Intracellular Ca 2+ concentration ([Ca 2+ ]i) was estimated according to the following standard equation ( 1): In this equation, R is the F340/F380 fluorescence ratio while an in situ Fura-2 calibration session was done to estimate the values of Rmin and Rmax.Specifically, Rmin and Rmax were calculated from the fluorescence ratios with imaging solutions containing 1 mM EGTA + 2 µM ionomycin and 20 mM Ca 2+ + 2 µM ionomycin, respectively.For its part, Q was determined from the Fmin/Fmax ratio at 380 nm and Kd represents the apparent dissociation constant of Fura-2 binding to Ca 2+ (135 nM).

Puncta formation assay
iPSC/NPCs (5.0 x 10 5 cells) were seeded onto Geltrex-coated 35 mm glass-bottom dishes (MatTek) 1 day prior to transfection with Lipofectamine 2000 (Thermo Fisher Scientific) as per the manufacturer's protocol.48 hours after transfection the cells were incubated with 2 µM Tg for 10 minutes prior to imaging using a Nikon Eclipse Ti2 microscope.Image analysis, including total puncta count and cell area measurements, were conducted in ImageJ (NIH, Rockville, MD).

Seahorse mitochondrial stress test
To evaluate mitochondrial function in HC-iPSC/NPCs compared to BD-iPSC/NPCs, an XF Mito Stress Test was performed using the Agilent Seahorse XF Analyzer (Santa Clara, CA) as per the manufacturer's protocol.Media was made fresh using the Seahorse XF Base DMEM with added 2 mM L-glutamine, 10 mM glucose and 1 mM sodium pyruvate (bicarbonate-free). 1 day before 1.5 x 10 5 cells/well for each NPC line were seeded onto 20 wells of a Geltrex-coated 24-well XF cell culture microplate (Seahorse Bioscience).The calibration plate was hydrated in a non-CO2 37ºC incubator overnight.Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were recorded 4 times in 20 mins, after which 3 different compounds were added sequentially (2 µM oligomycin, 2 µM FCCP and 0.5 µM antimycin A/rotenone) to measure the 4 fundamental parameters of mitochondrial function and their derivatives: basal respiration, maximal respiration, ATP production, spare respiratory capacity and coupling efficiency.At the end of the experiment run, cell densities from each well were quantified by using crystal violet staining and used for normalization of the results.
Images were captured on a Nikon Eclipse Ti2 microscope using the NIS Elements software.
Analysis was conducted using ImageJ (NIH) as follows.After a threshold fluorescence was set the image was made binary and watershed was used to separate clumped cells.Particles between 150 and 5,500 pixels were analyzed for a final count of total and EdU positive cells.Rate of proliferation was measured using EdU positive cells over total cell count.

TUNEL assay
The Roche In Situ Cell Death Detection Kit (Millipore Sigma) was used as per the manufacturer's protocol to assay cell viability between HC-and BD-NPCs under normoxia (ambient O2, 5% CO2, 6 and 37ºC) and hypoxia (1% O2, 5% CO2, and 37ºC).Briefly, after treatment, cells were fixed using 4% PFA for 20 minutes.After permeabilization with 0.1% Triton-X for 2 mins, the cells were incubated in TUNEL reaction solution for 30 mins in the dark at 37ºC with humidity.After a PBS rinse, cells were incubated in DAPI staining solution before being mounted onto slides for visualization as above.

Neurosphere assay
iPSC/NPCs were seeded at 1.0 x 10 4 in ultra-low adhesion U-bottom 96 well plates (Corning) and were agitated at 75 rpm in NEM for 72 hours.Neurospheres were then transferred to Geltrexcoated 24-well plates and incubated in differentiation media for 72 hours.Phase contrast images were collected on a Nikon Eclipse Ti2 microscope before fixing the neurospheres in 4% paraformaldehyde (PFA) for 20 minutes.

Immunocytochemistry
Immunocytochemistry was carried out for neurospheres and 2D NPC cultures grown on Geltrexcoated coverslips in 24-well plates as previously described (2,3).Imaging was done with a Nikon Eclipse Ti2 microscope and processing was conducted using ImageJ (NIH).

Immunoblotting
Cell protein lysates and immunoblotting were performed as previously described (3).For quantification of immunoblot data, the intensity of each band of interest was divided by the intensity of its respective loading control (GAPDH) to provide the normalized expression value used for statistical analysis.

RNA-sequencing
RNA was collected from each of the 6 cell lines at the NPC stage as follows.Approximately 1.0-1.5 x 10 6 NPCs were harvested by manual scraping and pellets collected by centrifugation.Total RNA was extracted from cell pellets using the Qiagen miRNeasy mini kit with a 30 µL RNase free water elution.Agilent Bioanalyzer QC was performed on a 1:100 dilution of the stock RNA using the Pico RNA chip.RNA samples with RIN scores greater than 7 were diluted to 40 ng/µL using concentrations measured with the Qubit HS RNA assay (Invitrogen).Sequencing libraries were prepared with the KAPA Stranded RNA-Seq Kit with RiboErase and sequenced on Illumina Hiseq4000 to achieve ~80M paired end, 100 base pair reads for each sample.
Quality control of the paired-end reads was done with FastQC (v0.11.9) and adaptor trimming using BBduk (v39.00).To account for bias in using a single publicly available RNA-seq data analysis package, we analyzed our results sequentially using multiple packages.First, reads were mapped onto the human genome (GRCh38) using Hisat2 v2.0.5 and counted using FeatureCounts v1.6.2 default parameters.Differential expression analysis was performed using the DESeq2 v1.6.3 package.Reads were then mapped onto the hg38.p13human genome (GENCODE release 41) using STAR aligner (v2.7.10a with the 'alpha_220207' patch).Here, differential expression analysis was performed using the DESeq2 (v1.34.0) and edgeR (v3.36.0) packages.Genes were labelled as significantly altered if adjusted p-value ≤ 0.05 and log2 fold change ≥ 2 or ≤ -2.The differentially expressed transcripts were then grouped by biological process using the GeneOntology online resource.Specific statistics can be found in the figure caption and in Supplementary File 1.
On the day 5, Induction Medium was added and EBs were transferred to a 24-well plate and incubated for 2 days at 37ºC.On the day 7, EBs were transferred into Matrigel droplets and incubated in Expansion Medium at 37ºC for 3 days.On the 10 th day, media was changed to Maturation Medium and the organoids were allowed to mature for 40-50 days at 37ºC on an orbital shaker at 65 rpm.On day 20, 3 µM YM58483 was added to select organoids for a 20 days chronic treatment.Once the organoids had reached maturity (day 40/50), they were frozen in a gelatin/sucrose solution for slicing.Organoids were sliced using a Leica CM1860 cryostat at a thickness of 20 µm.Sections were then stained according to the immunocytochemistry procedure described above and imaged using a Nikon Eclipse Ti2 microscope.

NanoString miRNA profiling
The nCounter Human miRNA Expression Assay Kit v3 (Nanostring Technologies, Seattle, WA, USA) was used for miRNA profiling.This expression panel contains 799 probes that represent >95% of all human miRBase reads.RNA was collected as above from the six cell lines in addition to two others, GM08330 and GM05440, described previously (4).Briefly, 100 ng of total RNA was annealed to the nCounter miRNA Tag reagent, hybridized to the Reporter CodeSet, and run on an nCounter Prep Station.The nSolver analysis software (NanoString Technologies) was used for normalization and analysis.Normalization was done by using the geometric mean of all miRNAs and controlling for negative control signals.

Real-time quantitative PCR for miR-34a
Expression levels of miR-34a and housekeeping miRNAs (RNU6B and SNORD_95) were determined with predesigned TaqMan assays (Life Technologies, Foster City, CA).High-Capacity cDNA synthesis Kit with RNase inhibitor (Thermo Fisher Scientific) was used to synthesize cDNA from 500 ng of total RNA.RT-qPCR experiments were performed on a LightCycler ® 480 Instrument (Roche, Germany) according to the manufacturer's instructions.miR-34a expression level in each sample was normalized to the geometric mean of RNU6B and SNORD_95 expressions.The 2 −ΔΔ CT method (5) was used to determine relative miRNA expression levels.

Statistics
All statistical figures are presented as mean ± standard error of mean (SEM) unless otherwise stated.After testing for normality with Levene's test and equal variance with a Shapiro-Wilk test, the appropriate analysis was carried out.Comparison between 2 groups used two-tailed Student's t-test with Welch's correction when variance was unequal.Multiple comparisons were analyzed using an ANOVA when assumptions of normality and equal variance were met.The Mann-Whitney U test or Kruskal-Wallis test was used when these assumptions were violated.Any pvalue of less than 0.05 was considered to be significant.All analysis was conducted using R. Specific statistical methods are described in the figure legends.

Figure S1 .
Figure S1.HC-NPC and neuronal validation after iPSC induction and differentiation.(A)

Figure S4 .
Figure S4.Example image showing distribution of STIM1 puncta before and after Tg

Figure S5 .
Figure S5.Mitochondria function normally in BD-NPCs.(A) A Seahorse mitochondrial stress

Figure S6 .
Figure S6.TUNEL assay demonstrates no difference in rates of cell death between HC-and

Figure S7 .
Figure S7.HC organoids grown under SOCE inhibition do not have developmental

Figure
Figure S8.qPCR quantifying miR-34a levels in HC-and BD-NPCs and neurons over