Research subjects: Subjects included six HC and six patients with SZs, including schizoaffective disorder (together: SZ), recruited from the clinical services at McLean Hospital. Participants were assessed using the Structured Clinical Interview for the DSM-V (SCID)23 and did not have significant acute or substantial chronic medical illness or conditions. Subjects were studied for brain myelin content in a previous protocol2. Detailed inclusion and exclusion criteria are given in that reference. There are no previous data on the degree of oligodendroglia production abnormalities in SZ, but sample size was chosen on the basis of the myelin and WM abnormalities previously reported2. Our prior expectation was that we would be able to see differences as great as those observed on brain imaging. Subject characteristics are described in Table 1.

Table 1 Subject characteristics Full size table

All protocols were approved by the Partners Healthcare Institutional Review Board, and subjects provided written informed consents before participation in any studies.

Derivation and expansion of fibroblasts

Fibroblasts were grown from 3 mm skin biopsies, cut into small pieces, placed in plate wells with 2 ml minimal essential media plus 15% fetal bovine serum and 1% Penn/Strep under coverslips for 7 days in an incubator at 37 °C, 5% CO2. Coverslips were removed when cells were clearly seen migrating out of tissue pieces. Cells were passaged when 80–95% confluent, first to one 100 mm dish, then to five 150 mm dishes, then frozen, and stored. Fibroblast stocks were tested for mycoplasma.

Conversion to induced pluripotent stem cells

Fibroblasts were sent to either the New York Stem Cell Foundation Research Institute (NYSCF) or Cellular Reprogramming, Inc. (San Diego, CA) for RNA reprogramming. Four SZ and five HC lines were derived at NYSCF and two SZ and one HC lines (converted as part of another study) were from Cellular Reprogramming. NYSCF reprogramming is based on mRNA/miRNA transfection as detailed in Paull et al.24. Fibroblasts sent to Cellular Reprogramming were similarly converted and the resulting iPS cell colonies were stabilized and expanded. Each fibroblast line was plated to six-well plates without feeders at three different plating densities and subjected to messenger RNA reprogramming. Colonies were bulk-passaged from the most productive well to establish passage 1 iPS cell cultures on rLaminin-521 (BioLamina) in Nutristem XF media (Biological Industries) and expanded in the same culture system until at least passage 3 before being characterized by DAPI/OCT4/TRA-1-60 immunostaining and frozen down. Cells from both sources were made with the same technology and had similar properties.

Characterization of iPSC

After conversion, iPSC lines from NYSCF were characterized as in Paull et al.24. Briefly, Nanostring analysis was performed using a 25 gene probe set for markers of pluripotency. Differentiation potential into the three germ layers was assessed using an expression panel of 100 gene probes, after spontaneous differentiation of iPSCs into embryoid bodies. Additionally, cells were karyotyped with the Nanostring nCounter Plex2 Assay Kit. iPSCs reprogrammed at Cellular Reprogramming showed human embryonic stem cell-like morphology and expressed human pluripotent stem cell markers. Characterized iPSCs from both sources were returned to McLean for subsequent studies. iPSCs were mycoplasma tested prior to plating for differentiation.

OL differentiation

The 12 subject lines of iPSCs were differentiated three times to OLs as in Douvaras and Fossati25 following the fast version of the differentiation protocol. A Scepter 2.0 automated cell counter (Millipore Sigma) was used for all cell counts.

Excitatory neuron differentiation

iPSCs were differentiated into excitatory neurons (hpiNs, human patterned induced neurons) and harvested after 28 days in culture as previously described26.

Single cell RNA sequence characterization of OLs at D65

Plated neurosphere cultures were enzymatically harvested at D65 by Accutase treatment for 1 h at 37 °C; DNAse 1 was added for 5 min to digest genomic DNA released from ruptured cells. The cells were then passed through a 70 μm filter to obtain a single cell suspension. After pelleting, cells were re-suspended in 100 μl of their growth medium containing the appropriate amount of O4-488 conjugated antibody. Stained cells were then pelleted and washed with phosphate-buffered saline and sorted into a 96-well plate using a BD Biosciences FACSAria III, 100μm ceramic nozzle, on the slow setting. SmartSeq2 libraries were prepared according to the SmartSeq2 protocol27,28.

Sequencing was carried out as paired-end 2 × 25 bp with an additional eight cycles for each index. Data were separated by barcode and aligned using Tophat version 2.0.10 (ref. 29) with default settings. Transcripts were quantified using the Cufflinks suite version 2.2.1 (ref. 30). Data were normalized for gene length and library size using Cuffnorm.

Immunofluorescent characterization of the O4-expressing OLs

Primary antibody anti-O4 or anti-O1 diluted (1:100) and 5% goat serum was added to live cultures and incubated for 45 min at 37 °C. After one wash with pre-warmed media, secondary goat anti-mouse Alexa Fluor-488 or Alexa Fluor-568 was added for 30 min at 37 °C. Cultures were washed and fixed with 4% paraformaldehyde in 100 mM phosphate buffer pH 7.4. All other immunocytochemistry was done post fixation. Primary antibodies were used at the following dilutions: rabbit anti-OLIG2 (1:500), goat anti-SOX10 (1:100), and rat anti-MBP (1:200). Secondary antibodies, donkey or goat anti- rabbit, mouse, goat or rat Alexa Fluor-488, 555, or 568 were used at 1:500. DAPI staining was used to visualize cell nuclei in ICC protocols. Antibody manufacturer, catalog number, and dilution information are listed in Table 2.

Table 2 List of antibodies used Full size table

Flow cytometry analysis of O4-expressing cells

Plated neurosphere cultures were enzymatically harvested as detailed above at designated developmental time points. Sorting was done on a BD Biosciences Accuri C6 Cell Sorter using a 100 μm ceramic nozzle, on the slow setting. Fibroblasts that did not express O4 were used as a biological control for gating. Propidium iodine (PI) was used to identify and exclude dead cells. Data obtained were analyzed using BD C6 analysis™ software.

Flow cytometry analysis of live and dead cells

Analysis of the percentage of live cells in mixed neurosphere cultures was done by FACS analysis according to the manufacturer’s protocols using the LIVE/DEAD Viability Cytotoxicity Kit, for mammalian cells.

Magnetization transfer ratio measurements

All subjects participating in the study had MR imaging done and MTR data collection was as described in previous publications2,31. Data were collected from a 1 × 3 × 3 cm voxel (Fig. 4a) within the WM of the right prefrontal cortex at 4 Tesla (Varian/UnityInova; Varian Inc., Palo Alto, CA).

Brain WM quantification

At 4 T high-contrast T1-weighted axial and sagittal images were also collected (TE/repetition time = 6.2/11.4 ms, field of view = 22 × 22 × 16 cm3, readout duration = 4 ms, receive bandwidth = ± 32 kHz, matrix size = 256 × 256 × 64, in-plane resolution = 0.94 × 0.94, slice thickness = 2.5 mm, flip angle = 11°). Whole-brain images were segmented into gray matter, WM, and CSF using FSL’s FS-FAST32 routine and percent WM was calculated.

Data analysis

Laboratory staff who isolated, reprogrammed, grew, and analyzed cells in vitro were blind to subject diagnosis and to the in vivo brain imaging results. The statistician (CR) was given access to all these data for the purpose of analysis.

Repeated measures linear regression tests were performed to detect associations of diagnosis, MTR, and percentage WM with percentage OLs in culture. Covariates were diagnosis (SZ patient or HC) and time in culture (in categories: days 65, 75, or 85 of differentiation). O4 percentages for the 3-day 85 replicates were averaged to obtain a single day 85 O4 percentage. We first assessed for statistical evidence of a difference in diagnosis-associated reduction in percentage OLs between time points (a test for interaction between diagnosis and time). In the absence of statistical evidence of that interaction, we tested for evidence of the primary hypothesis, a patient–control difference in percentage OLs in culture in a model assuming the same or similar difference across time points. To test the secondary hypothesis, whether there was an association between MTR or percentage WM (as quantified by FSL-FMRIB Software Laboratory v5.0) in vivo with percentage OLs in vitro, we replaced diagnosis with either brain myelin content (MTR) or percentage brain WM as a covariate in the model. We allowed unstructured covariance between repeated measurements, and used Kenward–Roger degrees of freedom for hypothesis testing. A sensitivity analysis tested for an association of diagnosis with percentage OLs, controlling for age. Finally, Pearson correlations were used to quantify associations between continuous measures.

Analysis of CSPG4 variants

Exome sequencing was done as part of a larger effort at the Stanley Center at the Broad Institute. Variants of CSPG4 were analyzed using the Integrative Genomics Viewer33,34.