Correction to: Neuroendocrine characteristics of induced pluripotent stem cells from polycystic ovary syndrome women

In the original publication the Fig. 2 and the Supplementary Material 1 was incorrect. The correct version of Fig. 2 and the Supplementary Material are provided in this correction article. NESTIN should be corrected to PAX6 in Fig. 2C legend and at page 528 and Supplementary Material 1. NANOG should be corrected to PAX6 in Fig. 2C picture. Fig. 2. Differentiation and identification of NSCs from PCOS-derived iPSCs. (A) Schematic procedure of NSCs differentiation from iPSCs. NSC: Neural stem cell; EB: embryoid body. (B) The phenotype of specific differentiated NSCs. Scale bars = 100 µm. (C) Immunofluorescence images of the NSC markers SOX2 and PAX6. Scale bars = 50 µm. ZOOM, scale bars = 25 μm. (D) The mitochondrial respiration function of PCOS- and non-PCOS-derived iPSCs and NSCs. (E) Quantitative analysis of basal oxygen consumption, ATP production, maximal respiration, and proton leak. (F) Proposed neuroendocrine state in normal and PCOS patients. In normal patients, the GnRH pulsatile frequency is critical for steroidogenesis and follicular development. Low frequency pulses prefer FSH, and high frequency pulses favour LH. In PCOS, the increased GnRH release led to a high level of LH pulsatility, impairing the preferential release of FSH and follicular maturation, thus leading to polycystic ovaries. Red: increased; Blue: decreased. Solid arrow: up regulated; Dotted arrow: down regulated.


Generation and culture of PCOS-derived iPSCs
Human fibroblasts were derived from skin cells of patients. The iPSC clones were reprogrammed as described previously. Briefly, epithelial cells were transduced with OCT4-, SOX2-, KLF4-and C-MYC-expressing lentiviral vectors in MEF (mitomycin-C-treated mouse embryonic fibroblasts) medium without serum. On day 5, the medium was replaced with iPSC medium [DMEM/F12 supplemented with 20% (v/v) knock out serum replacer (Knockout SR), 2 mM L-glutamine, 2 mM non-essential amino acids, and 0.1 mM β-mercaptoethanol (Invitrogen) with no additional bFGF]. Putative PCOS-derived iPSC colonies were emerged within 21 days after transduction. Generated colonies were mechanically dissociated for passage.

Neural stem cell differentiation from iPSCs
NSC differentiation was performed as described previously. iPSC were picked from the MEF feeder and suspended cultured for 4 days in iPSC medium without bFGF, to induce embryoid bodies (EB). For NSC differentiation, RA (Retinoic acid) was added at final concentration of 1-2 µM after EB formation. After 4 days as a floating culture, EBs were collected and plated onto matrigel coated dishes cultured in EB medium without RA.

Microarray analysis
Microarray hybridization was carried out at CapitalBio (Beijing, China). Total RNA (100 ng) was used to prepare twice-amplified and labelled RNA for hybridization with HG-UI33 plus 2.0 arrays (n=3).

Quantitative real-time PCR
According to the manufacturer's protocol, total RNA was extracted using Trizol reagent, and cDNA was synthesized by a ReverAid First Strand cDNA Synthesis Kit (Invitrogen). The PCR products were amplified using the SYBR Green mix kit by an QuantStudio3 system (Applied Biosystems). Fold-change by RT-PCR was measured and calculated by normalizing to the housekeeping gene (β-actin) and calculating the

Immunofluorescence staining
Cells were fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 20 min and then blocked with PBS containing Triton X-100 (Sigma-Aldrich) for 30 min at room temperature. After blocking, the cells were incubated with primary antibodies overnight at 4 °C, then incubated with secondary antibodies 1hr at room temperature. The primary antibodies (all at a 1:250 dilution, Abcam) were used to detect OCT4, SOX2, and NANOG expression of iPSC. NSC was stained by primary antibody of SOX2 and PAX6 (proteintech). We visualized antigen localization using goat anti-mouse/rabbit Alexa Fluor 488, 555 and 594. The nuclei were stained with Hoechst (Invitrogen).

ELISA
The culture medium of iPSC were collected and centrifuged with 12,000 rpm in 4 ℃ before assays. The testosterone and estrodiol levels were detected using ELISA kit (R&D system).

Mitochondrial oxygen consumption detection
The mitochondrial respiration was detected using the XF24 extracellular flux analyzer from Seahorse Bioscience (Billerica). A classical Mito stress test was performed based on the following procedure: (1) the basal respiration was measured before adding chemicals; (2) oligomycin (2.0 μM) was added to inhibit ATP production; (3) The maximal respiration was measured by adding the uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP); and (4) rotenone and antimycin A (0.5 μM) were applied in combination to block respiration. The final results were normalized to cell number (10 5 cells per well).

Statistical analysis
The microarray data were analysed to identify statistically significantly different expression. The list of identified genes (fold change, FC>2; FDR<0.05) was submitted to AmiGO2 and DAVID database to identify the Gene Ontology (GO) terms associated with biological functions and pathways. Data are presented as the mean±SD and were analysed using GraphPad Prism 5 program (GraphPad Software) and R language package.