Generation of four postmortem dura-derived iPS cell lines from four control individuals with genotypic and brain-region-specific transcriptomic data available through the BrainSEQ consortium

In this study, we established induced pluripotent stem (iPS) cell lines from postmortem dura-derived fibroblasts of four control individuals with low polygenic risk score for psychiatric disorders including schizophrenia and bipolar disorder. The fibroblasts were reprogrammed into iPS cells using episomal vectors carrying OCT3/4, SOX2, KLF4, L-Myc, LIN28 and shRNA-p53. All iPS cell lines showed the same genotype with parental postmortem brain tissues, expressed pluripotency markers, and exhibited the differentiation potency into three embryonic germ layers.


Resource Utility
The iPS cell lines generated from postmortem dura-derived fibroblasts of four nonpsychiatric healthy individuals whose genotypic and transcriptomic data of multiple brain regions are available through BrainSEQ consortium can be differentiated into multiple types of brain cells by 2D and 3D methods and provide control lines to model neuropsychiatric disorders.

Resource Details
Recent progresses in induced pluripotent stem cell technologies have enabled the modelling of human brain development and investigation of molecular and cellular mechanisms underlying neurological/neuropsychiatric disorders in vitro.However, in most cases, it is difficult to access the clinical/pathological information obtained from the donor's brains.BrainSEQ consortium (BrainSeq: A Human Brain Genomics Consortium.Neuron.2015) is a project to characterize the genetic and epigenetic regulation of multiple facets of transcription in multiple brain regions across the human lifespan in samples of major neuropsychiatric disorders and controls using the Lieber Institute for brain development (LIBD)'s brain tissue repository (https://www.libd.org/brain-repository/).These comprehensive database and resources make it possible to model neuropsychiatric disorders with iPS cells derived from postmortem tissues and compare/validate the endophenotypes with the data from postmortem brains with the same donors.
In this study, we established iPS cells from postmortem dura-derived fibroblasts of nonpsychiatric control individuals (Table 1) selected from the LIBD brain repository using episomal vectors carrying OCT3/4, SOX2, KLF4, L-Myc, LIN28 and shRNA-p53 and characterized them (Table 2).Each line exhibited typical human embryonic stem (ES) cell-like morphology (Fig. 1A).The expression of pluripotent stem cell markers, SOX2, OCT4, DNMT3B and NANOG, was confirmed by immunostaining (Fig. 1B) and/or RT-qPCR (Fig. 1C).We also confirmed the absence of genomic integration of episomal vectors utilized for reprogramming by genomic PCR (Fig. 1D).The pluripotency of established iPS cell lines was validated by in vitro spontaneous differentiation assay.RT-qPCR analysis revealed that the expression of three germ layer markers: α-fetoprotein (AFP) and GATA4 (endoderm), RUNX1 and HAND1 (mesoderm) and NCAM and SOX1 (ectoderm) was upregulated upon differentiation compared with undifferentiated iPS cells (Fig 1E).All lines were negative for mycoplasma (Supplementary Table 1).
Genomic integrity analysis was performed by copy-number analysis of genome-wide genotyping array at passage number 5 and each cell line was confirmed to contain 46,XY without any chromosomal abnormalities or copy number variations compared with hg19 reference genome (Supplementary Figure 1).As a note, this method will not detect balanced translocations.To confirm the identity of established iPS cell line, we compared DNA profile between established iPS cell lines and parental brain tissues (dbGaP: phs000979.v1.p1).Comparison of 1,478,103 single nucleotide polymorphisms (SNPs) of iPS cells with the original brains showed a similarity score (hamming distance) of at least >0.9 (Fig 1F ) demonstrating that each iPS cell line is genetically identical to the donor's postmortem brain tissue.

Case selection
Four Caucasian males without any psychiatric symptoms carrying low polygenic risk scores of psychiatric disorders (Wray et al., Genome Res. 2007; Schizophrenia Working Group of the Psychiatric Genomics Consortium.Nature.2014) were selected from the LIBD brain repository.

Fibroblast culture
Human postmortem dural tissues were collected at autopsy and fibroblasts were generated in a previous study (Bliss et al., PLoS One. 2012).Dura-derived fibroblasts were grown in fibroblast medium consisting of DMEM + GlutaMAX (Gibco), 10% fetal bovine serum (Gibco), and 1 x Antibiotic-Antimycotic (Gibco) in the incubator (at 5% CO2 and 37°C).Cells were passaged by trypsinization with 0.25 x Trypsin-EDTA solution (Sigma) for 2 to 3 min at room temperature.

Immunocytochemistry
The feeder-free iPS cells were fixed with 4% PFA (Sigma) at room temperature for 10 min and permeabilized and blocked with 0.3% Triton X-100 (Sigma), 10% normal horse serum (Thermo Fisher) in DPBS (Gibco) at room temperature for 30 min.Cells were then stained with antibodies against SOX2 and NANOG (Table 3) diluted in 5% normal horse serum/ 0.01% Tween-20/D-PBS at 4°C overnight and incubated with fluorescent dye-conjugated secondary antibodies (Table 3) diluted in 0.01% Tween-20/D-PBS at room temperature for 2 h.Cell nuclei were labeled by Hoechst33342 (1:10,000, Invitrogen) for 10 min.Stained cells were images with a laser confocal microscopy (LSM700, Zeiss).

RT-qPCR
Total RNA was extracted with Direct-zol Mini prep kit (Zymo Research).For reverse transcription, we used SuperScript IV VILO Master Mix (ThermoFisher Scientific).The expression of pluripotent stem cell markers (Table 3) was analyzed via qPCR with QuantiTect SYBR Green PCR kit (Qiagen) on QuantStudio 3 (Applied Biosystems).Total RNA from a human embryonic stem cell line (HESC H9) (ScienCell) was used as a positive control and dural fibroblasts (FB) from each individual used as negative controls.

Genome integration analysis
DNA was extracted from iPS cells with DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's instruction.PCR was performed with the OriP primers (Table 3), Platinum Taq DNA Polymerase (Invitrogen) and the following program: initial denaturation for 2 min at 94°C and 25 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min on T100 Thermal Cycler (Bio-Rad).

in vitro spontaneous differentiation
iPS cells on feeder were harvested using CTK solution (Okita et al., Nat. Methods. 2011).The cell clumps were transferred to Ultra Low Attachment plates (Corning) in 20% KSR medium without bFGF.The medium was changed every other day.Twenty-day-old embryoid bodies (EBs) were harvested.The expression of three germ layer markers (Table 3) was analyzed via RT-qPCR.

Genome-wide genotyping array analysis
Genomic DNA was extracted from feeder-free iPS cells with DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's instruction.Genotyping array analysis was performed by Macrogen with an Infinium Omni 2.5-8 kit (illumina).

Copy number variation analysis
Genome Studio (v2.0) was used to calculated log-2 represent probe intensity ratio (Log R) representing a ratio of observed intensity to reference intensity.Segmentation and plotting were generated using aspcf (kmin = 500) and plotGenome utilities from the Bioconductor package copynumber (Nilsen et al., R package version 1.26.0. 2013;Nilsen et al., BMC Genomics. 2012).

Genotype comparison between iPS cells and parental brain tissues
Genotyping with Illumina BeadChips was carried out using DNA extracted from cerebellar tissues and iPS cells.Low quality and rare variants were removed with PLINK.For parental tissues, genotypes were prephased and imputed using genome build hg19.Genotypes were matched between cells and brain tissues with hamming distance used to compare genomic similarity.

Mycoplasma detection
Mycoplasma test was carried out with MycoAlert mycoplasma detection kit (Lonza) according to the manufacturer's instruction.The ratio of Reading B to Reading A is used to determine whether a cell culture is contaminated by mycoplasma with the following cutoff: ratio < 0.9 for negative and > 1.2 for positive.

Summary of lines
Figure 1.