MicroRNA Profiling of Neurons Generated Using Induced Pluripotent Stem Cells Derived from Patients with Schizophrenia and Schizoaffective Disorder, and 22q11.2 Del.

Zhao D, Lin M, Chen J, Pedrosa E, Hrabovsky A, Fourcade HM, Zheng D, Lachman HM.
Source: PLoS ONE
Publication Date: (2015)
Issue: 10(7): 1-24
Research Area:
Stem Cells
Cells used in publication:
Fibroblast, dermal(NHDF-Ad), human adult
Species: human
Tissue Origin: dermal
4D-Nucleofector® X-Unit

iPSC lines were generated from fibroblasts obtained from skin biopsies performed by boardcertified physicians. The procedure for growing fibroblasts in preparation for reprogramming into iPSCs is detailed in S1 Text. Briefly, iPSC reprogramming was carried out by nucleofection. One vial of cells was thawed out and placed in a T75 flask in DMEM/F12 supplemented with 10% FBS and fed every 2 days. Cells were grown to ~50% confluence (~4–5 days), after which they were trypsinized and subjected to nucleofection (~6 x105 cells). Reprogramming was carried out using an Amaxa 4D-Nucleofector (P2 Primary Cell Kit from Lonza catalog# V4XP-2012, Program FF-135) with non-integrating plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, and a p53 shRNA vector (Addgene Cat. # 27077, 27078, 27080), according to Okita et al., with some modifications [63,64,69]. iPSCs were maintained on Matrigel plates in mTeSR1 medium (Stem Cell Technologies) with daily feeding in 37°C/5% CO2/85% humidity.


We are using induced pluripotent stem cell (iPSC) technology to study neuropsychiatric disorders associated with 22q11.2 microdeletions (del), the most common known schizophrenia (SZ)-associated genetic factor. Several genes in the region have been implicated; a promising candidate is DGCR8, which codes for a protein involved in microRNA (miRNA) biogenesis. We carried out miRNA expression profiling (miRNA-seq) on neurons generated from iPSCs derived from controls and SZ patients with 22q11.2 del. Using thresholds of p<0.01 for nominal significance and 1.5-fold differences in expression, 45 differentially expressed miRNAs were detected (13 lower in SZ and 32 higher). Of these, 6 were significantly down-regulated in patients after correcting for genome wide significance (FDR<0.05), including 4 miRNAs that map to the 22q11.2 del region. In addition, a nominally significant increase in the expression of several miRNAs was found in the 22q11.2 neurons that were previously found to be differentially expressed in autopsy samples and peripheral blood in SZ and autism spectrum disorders (e.g., miR-34, miR-4449, miR-146b-3p, and miR-23a-5p). Pathway and function analysis of predicted mRNA targets of the differentially expressed miRNAs showed enrichment for genes involved in neurological disease and psychological disorders for both up and down regulated miRNAs. Our findings suggest that: i. neurons with 22q11.2 del recapitulate the miRNA expression patterns expected of 22q11.2 haploinsufficiency, ii. differentially expressed miRNAs previously identified using autopsy samples and peripheral cells, both of which have significant methodological problems, are indeed disrupted in neuropsychiatric disorders and likely have an underlying genetic basis.