Reduced expression of Paternally Expressed Gene-3 enhances somatic cell reprogramming through mitochondrial activity perturbation

Imprinted genes control several cellular and metabolic processes in embryonic and adult tissues. In particular, paternally expressed gene-3 (Peg3) is active in the adult stem cell population and during muscle and neuronal lineage development. Here we have investigated the role of Peg3 in mouse embryonic stem cells (ESCs) and during the process of somatic cell reprogramming towards pluripotency. Our data show that Peg3 knockdown increases expression of pluripotency genes in ESCs and enhances reprogramming efficiency of both mouse embryonic fibroblasts and neural stem cells. Interestingly, we observed that altered activity of Peg3 correlates with major perturbations of mitochondrial gene expression and mitochondrial function, which drive metabolic changes during somatic cell reprogramming. Overall, our study shows that Peg3 is a regulator of pluripotent stem cells and somatic cell reprogramming.

For embryoid body (EB) induction, the differentiation medium consisted of ESC culture medium without LIF. The cells were harvested by trypsinisation, counted, and propagated in hanging drops (400 single ESCs/ 30 µl initial drop) for 2 days, before being transferred to 10 cm 2 bacterial dishes. On day 5, embryoid bodies were transferred onto gelatin-coated 10 cm 2 tissue culture dishes always in differentiation medium.

Flow cytometry
4 For analysis and/or sorting of GFP +/or mCHERRY +/cell populations, cells were trypsinized, washed once in PBS, and resuspended in PBS with 5% FBS plus DAPI (SIGMA 09542). Untreated cells were used as negative staining control and DAPI staining was performed to exclude dead cells from the analysis. Before FACS analysis the samples were filtered with 35µm mesh size filters (Corning Life Sciences 352235) to avoid aggregates.
For apoptosis detection, phosphatidylserine exposure was detected by flow cytometry using APC Annexin V (BD pharmingen 550474) following the manufacturer's instructions, quantified using BD LSR Fortessa and analyzed by Flowjo software.
Unstained cells were used as negative control.
For E-cadherin staining, cells were collected and incubated with Fc receptor blocking reagent (anti-Mouse CD16/CD32, eBioscience 14-0161-82) in PBS with 5% FBS for 10 min at 4°C. The cells were washed once in PBS plus 5% and incubated for 20 min at 4°C with E-cadherin antibody (0,5 µg/10 6 cells, Biolegend 147308) in PBS with 5% FBS and DAPI. E-cadherin expression was quantified by BD LSR Fortessa flow cytometer and analyzed by Flowjo software. Unstained cells were used as negative control and DAPI (SIGMA 09542) staining was performed to exclude dead cells from the analysis. Before FACS analysis the samples were filtered with 35µm mesh size filters (Corning Life Sciences 352235) to avoid aggregates.

Mitochondria live-cell imaging and morphological features quantification
Living cells labeled with MitoTraker Green were cultured onto Thermo Scientific Nunc Lab-Tek chambered coverglass (155411) and fluorescence images were 5 obtained using Leica TCS SP5 CFS confocal microscope. Nuclear staining was performed with Hoechst 33342 (ThermoFisher H1399).
Images processing was performed using Fiji software 4 and mitochondria morphological features were identified and characterized using MiNA macros as previously described 5 . MiNA software is freely available at https://github.com/ScienceToolkit/MiNA.
To silence Oct4 we used the oligonucleotides published by Ang and colleagues 6 . The oligonucleotides used to generate the short hairpins are given in the Supplementary

Immunofluorescence staining
For immunocytochemistry, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and then washed twice with PBS. These fixed cells were then incubated in blocking solution containing 10% goat serum (Sigma) and 0.1% Triton X-100 (Sigma) for 1h at room temperature. The cells were then left overnight at 4°C in blocking solution containing the primary antibody. The next day, the cells were washed three times with PBS and then incubated with the secondary antibody for 1h at room temperature. The primary antibody against rabbit NANOG (Calbiochem #SC1000), rabbit SOX2 (ab97959), mouse OCT3/4 (sc-5279), mouse E-cadherin (BD610182) were used, and the goat anti-rabbit, goat anti-mouse IgG, (1:1000, Life Technologies) conjugated to Alexa Fluor-488 and Alexa Fluor-568 were used as secondary antibody. Nuclear staining was performed with DAPI (SIGMA 09542).

Alkaline phosphatase staining
Alkaline phosphatase is an enzyme expressed by ESCs and is used as a marker of pluripotency. To evaluate the alkaline phosphatase expression, the cells were fixed in 10% Neutral Formalin Buffer for 15 min at 4°C, and washed three times with distilled water. These fixed cells were then incubated for 45 min at room temperature in 2ml of the staining solution prepared as follows: 0,005g Naphthol AS MX-PO4 (Sigma,   (2015).