Novel Role of Mitochondrial Manganese Superoxide Dismutase in STAT3 Dependent Pluripotency of Mouse Embryonic Stem Cells

Leukemia Inhibitory Factor (LIF)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway maintains the stemness and pluripotency of mouse embryonic stem cells (mESCs). Detailed knowledge on key intermediates in this pathway as well as any parallel pathways is largely missing. We initiated our study by investigating the effect of small molecule Curcumin on various signalling pathways essential for self-renewal. Curcumin sustained the LIF independent self-renewal of mESCs and induced pluripotent stem cells (miPSCs) in a STAT3 activity dependent manner. Gene expression analysis showed LIF/STAT3 and redox signaling components to be majorly modulated. Amongst ROS genes, expression of Manganese Superoxide Dismutase (MnSOD) specifically relied on STAT3 signaling as evidenced by STAT3 inhibition and reporter assay. The silencing of MnSOD, but not Cu-ZnSOD expression, resulted in the loss of mESC pluripotency in presence of LIF, and the overexpression of MnSOD is sufficient for maintaining the expression of pluripotent genes in the absence of STAT3 signaling. Finally, we demonstrate MnSOD to stabilize the turnover of pluripotent proteins at the post-translational level by modulating proteasomal activity. In conclusion, our findings unravel a novel role of STAT3 mediated MnSOD in the self-renewal of mESCs.

For experimental conditions, mESCs were cultured on both feeder and gelatin coated feeder-free conditions, in presence or absence of LIF conditions at the rate of 10,000 cells /well of six well plates in 2 mL medium. miPSCs were cultured only on feeder cells. After 24 hrs, the culture medium was replaced with mES medium lacking LIF, and curcumin dissolved in DMSO was added at various concentrations, while maintaining control cultures with the vehicle control (DMSO). Media change was given every 48 hrs to the cells and on confluency, the cells were harvested for gene and protein expression analysis.
For clonal analysis, mESCs were plated on gelatin coated 12 well plates with 10 cells/well in 1 mL medium. The cells were cultured in mES medium in presence or absence of LIF, and in presence of 5 µM Curcumin in no LIF condition. A single cell in each condition was identified, marked and traced for a period of 6 days to look for colony formation.
For Embryoid Body culture, the mESCs were cultured in low adherent plates (Corning) in the absence of LIF, with a media change every 3 days. The cells were harvested for gene expression analysis after 14 days of culture.
For Alkaline Phosphatase staining, mESCs cultured in presence and absence of curcumin were washed with PBS, fixed and stained with alkaline phosphatase substrate as per manufacturer's instruction (Millipore). After incubation with substrate for 15 min, colonies were stained purple and the image was captured using a phase contrast microscope (Nikon Eclipse TE2000S, Japan) and the number of alkaline phosphatase positive and negative colonies were enumerated.

80,000 cells were cultured on feeder cells under LIF and No LIF conditions and in the presence of 5µM
Curcumin. On the fourth day, the cells were trypsinized and 80,000 cells were replated on feeder cells while maintaining the said conditions through 8 passages. The cumulative population doubling was calculated and a graph was plotted as the number of cells/mL vs. the number of days in culture.

Gene Expression Analysis
RNA isolation was performed using RNA micro kit (Qiagen) as per manufacturer's instruction. cDNA was prepared using 1 µg of RNA according to the manufacturer's instructions (SuperScript III First Strand cDNA Synthesis kit, Invitrogen). Conventional Polymerase Chain Reaction (PCR) was performed using 2X Master Mix (Fermentas) and 5µM gene specific primers (IDT technologies). Quantitative PCR was carried out using SYBR Green (Kapa Biosystems). The samples were analysed using the 7500 RT-PCR (ABI Biosystems) and were normalized with Gapdh to obtain the relative Δ Ct values among samples.

Immunofluorescence
Cultured ESCs in the presence and absence of LIF and medium containing 5 µM curcumin or MnSOD overexpressed or MnSOD ShRNA cells were fixed in 4 % paraformaldehyde at 4°C for 20 mins and then permeabilized with 0.2 % Triton X-100 in PBS for 30 minutes, and blocked in blocking buffer containing 3 % BSA containing 10 % Donkey serum in 1X PBS Solution and incubated at room temperature (RT) for 30 mins. Antibodies were diluted in blocking buffer and cells were incubated in the indicated antibody solution at 4 °C overnight, followed by incubation for 2 hrs at RT with Secondary antibody. The cells are washed with 0.1 % PBS-Tween 20 and stained with DAPI (Sigma Aldrich) for 1 minute and observed under fluorescent microscope (Nikon Eclipse TE2000U, Japan). Both Purified and conjugated antibodies were used. The primary antibodies and dilutions used are mentioned in the supplementary table 2.

Cloning
The Open Reading Frame (ORF) of MnSOD was PCR amplified from mESC cDNA using gene specific primers as mentioned in supplementary table 1. The PCR product corresponding to the complete size of MnSOD was cloned in between BamHI and XhoI sites of pCDNA3.1 (+) (for transient transfection) and pMIG vector (for retroviral transfection) and transformed into competent DH5α cells. The ampicillin resistant colonies were selected and screened for positive clones and confirmed by sequencing (Amnion Biosciences, Bangalore).
mESC genomic DNA was isolated using genomic DNA isolation kit according to manufacturer's protocol (Genetix Biotech). As reported earlier, 1779 bp upstream region from the start site of MnSOD was identified, PCR amplified and cloned into KpnI and XhoI regions of pGL3 basic vector (Addgene).

Transfection
For transient transfection, 5 µg of plasmid [MnSOD and vector control pCDNA 3.1(+)] was transfected into confluent mESCs using Xtremegene HP transfection reagent according to manufacturer's instructions (Roche diagnostics). 24 hrs post transfection, the transfected cells were subjected to different conditions (With LIF, No LIF) and cultured till confluency. The cells were then passaged. On confluency, the cells were analysed for their gene and protein expression pattern.
pMIG-MnSOD and pMIG-GFP were transduced into mESCs using retroviral particles. Briefly, pMIG-MnSOD and pMIG-GFP plasmids were packaged into retroviral packaging vectors pVSVG and pGAGPOL in 293T cells. Retroviral transduction was performed on 80% confluent mESCs twice in 24 hr intervals. 48 hrs post-transduction, the mESCs were trypsinised using 0.25% trypsin and plated on inactivated MEF and cultured in the presence and absence of LIF. Media change was given every 48 hrs to the cells and on confluency, the cells were passaged and at P10, the cells were harvested for gene expression analysis.
MnSOD and Cu-Zn SOD ShRNA were transduced into mESCs using lentiviral particles. Briefly, pLKO-MnSODshRNA and pLKO-Cu-ZnSODshRNA (Sigma Aldrich, Germany) along with the scrambled control, were each packaged into lentiviral packaging plasmids -psPAS2 and pMD.2G, along with the scrambled control. Lentiviral transduction was performed on 80 % mESCs twice in 24 hr intervals. 24 hrs post 2 nd transduction, the transduced mESCs were trypsinised using 0.25 % trypsin and plated on gelatine coated 6 well plate. The cells were then selected with 1µg of puromycin, with media change every 48 hrs. On reaching 80 % confluency in the second passage, the cells were harvested for gene and protein expression analysis.

Western Blotting:
Cells were harvested and were lysed with ice-cold RIPA cell lysis buffer with freshly added protease inhibitors and phosphatase inhibitor. Protein concentration was determined using BCA kit (Novagen). 20-50 µg of protein was used for western blotting experiments. The protein were run on 8-12 % SDS-PAGE gels and transferred onto charged PVDF membrane (Millipore). Post transfer, the membrane was blocked using 3 % Skimmed milk/BSA in 0.1% TBST solution and probed with primary antibody overnight at 4 ºC and subsequently with horseradish-peroxidase (HRP)-conjugated secondary antibody for 1 hr at RT. The protein specific bands were detected by chemiluminiscence analyser (GE ImageQuant LAS 4000) or using a TMB substrate (Sigma Aldrich). Details of the antibodies used are mentioned in the supplementary table 2.

Teratoma Assay
To evaluate the pluripotency of curcumin treated cells, approximately 1-3 *10 6 ES cells cultured in absence of LIF and in presence of curcumin were harvested and injected subcutaneously (s.c.) into the dorsal flank of 4-5 week old female athymic nude mice. Following 4 weeks, mice were sacrificed and the tissues were removed, fixed with formalin and embedded with paraffin. Further sectioning and H&E staining was performed by Anand Diagnostics, Bangalore. The tissue sections were then observed under the microscope for the identification of different lineage cell types using a phase contrast microscope (Nikon Eclipse TE2000S, Japan).

Flow cytometric analysis
Oct4-GFP iPSCs were cultured for 4-5 days in absence of LIF and in presence or absence of Curcumin and were trypsinized and subjected to flow cytometric analysis to enumerate the number of GFP expressing cells using a flow cytometer (BD FACS Calibur, U.S.A).
For Propidium iodide staining, confluent mESCs treated in the presence and absence of LIF and in the presence of curcumin were stained with propidium iodide (Sigma Aldrich) for cell cycle analysis by flow cytometry.
MnSOD overexpressed cells were analysed for SSEA1 expressing population by Flow cytometry. Briefly, MnSOD overexpressed cells were trypsinised and washed with 1X PBS. The cells were then stained with Anti-SSEA1 antibody, for 1 hr on ice. The cells were then washed in PBS and stained with PE conjugated anti-mouse IgG antibody for an hour on ice. The cells were then fixed with 4% PFA for 1 hr on ice and then analysed flow cytometrically. The number of positive SSEA1 cells were then selected by gating out the secondary alone and cells alone controls and plotted.

BrDU based Cell Proliferation Assay
700-1000 cells were plated in each well of a 96 well plate. 24 hrs post plating, 0.2 µg/mL of BrDU was added to the proliferating cells and incubated for 12-16 hrs at 37 ºC, 5 % CO 2 . Cells were then fixed with ice cold 70 % ethanol for 10 mins at RT. The cells were permeabilised with 0.1 % Triton X-100 in PBS for 1 hr at RT and the BrDU labelled nuclei were detected using anti-BrDU antibody (1:1000 dilution; Sigma Aldrich, B8434) and secondary anti-mouse HRP conjugated antibody (1:2000 dilution; GE, Catalogue number NA931V) for 1 hr at RT and developed using TMB solution. The reaction was stopped using 3M H 2 SO 4 and the absorbance was read at 450 nm.