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OCT6 inhibits differentiation of porcine-induced pluripotent stem cells through MAPK and PI3K signaling regulation

As a transcription factor of the Pit-Oct-Unc (POU) domain family, octamer-binding transcription factor 6 (OCT6) participates in various aspects of stem cell development and differentiation. At present, however, its role in porcine-induced pluripotent stem cells (piPSCs) remains unclear. Here, we explored the function of OCT6 in piPSCs. We found that piPSCs overexpressing OCT6 maintained colony morphology and pluripotency under differentiation conditions, with a similar gene expression pattern to that of non-differentiated piPSCs. Functional analysis revealed that OCT6 attenuated the adverse effects of extracellular signal-regulated kinase (ERK) signaling pathway inhibition on piPSC pluripotency by activating phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling activity. Our research sheds new light on the mechanism by which OCT6 promotes PSC maintenance.


INTRODUCTION
Octamer-binding factor 6 (Oct-6, Pou3f1, SCIP, Tst-1) is a transcription factor of the Pit-Oct-Unc (POU) family . The DNA-binding (POU) domain of these proteins is highly conserved, consisting of a POU-specific domain and a POU homeodomain (Sock et al., 1996). POU transcription factors recognize the common DNA octamer motif (ATGCAAAT) (Jauch et al., 2011). They play distinct roles in stem cell development and function and exhibit spatiotemporal expression patterns . OCT6 is not widely expressed in all cells but has been detected in embryonic stem cells (ESCs) (Meijer et al., 1990), Schwann cells (Friedrich et al., 2005), glial cells (Kuhlbrodt et al., 1998), and neonatal testicular cells . To date, most studies have explored the function of OCT6 in Schwann cells, while little is known regarding their functions in other cell types. For induced pluripotent stem cells (iPSCs), OCT6 partially replaces OCT4 to initiate reprogramming (Jerabek et al., 2017). OCT6 is also implicated in the maintenance of iPSC pluripotency and plays a decisive role in neural differentiation (Malik et al., 2019). However, few studies have investigated the potential functions and mechanisms of OCT6 in porcine iPSCs (piPSCs).
We previously reported that porcine PSCs can be induced in medium containing leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and other cytokines, with the addition of signal transduction inhibitors (Chir099021, SB431542) and feeder cells to maintain colony morphology and pluripotency (Ma et al., 2018). Here, as OCT6 is not expressed in piPSCs, we explored the function of OCT6 in piPSCs by its overexpression.
Notably, piPSCs overexpressing OCT6 showed smoother colony edges in the presence of feeder cells (feeders) and maintained normal colony morphology and pluripotency after feeder removal. RNA sequencing (RNA-seq) analysis revealed that OCT6 expression promoted the up-regulation of extracellular matrix (ECM)-related genes and inhibited the differentiation of piPSCs. In addition, OCT6 activated PI3K and inhibited ERK in piPSCs, consistent with its ability to maintain piPSC colony morphology and pluripotency upon feeder removal.

Overall experimental scheme
In this study, we used lentivirus packaging to transfect PCDH-Teton-3×Flag-OCT6-Puro and PCDH-Teton-3×Flag-Puro into piPSCs. The piPSCs transfected with the OCT6 gene vector (OE-OCT6 piPSCs) were used as the experimental group, and the piPSCs transfected with empty vector (OE-NC piPSCs) were used as the control group. OCT6 expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR), cellular immunofluorescence staining, and western blot analysis. We performed total RNA-seq of piPSCs and detected the expression levels of related genes using qRT-PCR, western blotting, and immunofluorescence staining. Small molecular compounds were used to activate or inhibit the ERK and PI3K-AKT signaling pathways, respectively, to compensate for the maintenance of pluripotency caused by overexpression of OCT6.

Acquisition and production of piPSCs
The piPSCs were obtained from previous research. We established doxycycline (Dox)-induced porcine PSCs, in which the expression of transgenic genes can be switched on/off by Dox and lentiviral particles of TetO-FUW-OSKM (OCT4, SOX2, KLF4, and c-MYC) and FUW-M2rtTA can be infected into porcine embryonic fibroblasts (PEFs). Here, the somatic cells were reprogrammed into porcine iPSCs induced by Dox (DOX-iPSCs), which displayed a normal karyotype of 38 chromosomes, formed embryonic bodies in vitro, and spontaneously differentiated into three germ layers (Ma et al., 2018).

Vector construction and cloning
To explore the function of OCT6 in piPSCs, total RNA was extracted from porcine testes using TRIzol Reagent (Takara, Japan), then reverse transcribed (Thermo Fisher Scientific, USA) into cDNA. The porcine OCT6 gene was PCR-amplified from cDNA, and subcloned into an in-house PCDH-Teton-3×Flag-MCS-Puro vector using a NovoRec ® plus One step PCR Cloning Kit (Novoprotein, China). All primers used in this study are provided in Supplementary Table S1, and the core coding sequence of the recombinant vector was verified by sequencing.

Lentiviral packaging
After HEK293T cells were grown to 80%-90% density in 6well plates, lentiviral plasmids (VSV-G, PAX2) for packaging were transfected into the cells using polyethyleneimine (PEI). In total, 1 μg of VSV-G, 1 μg of PAX2, and 2 μg of lentiviral plasmids were mixed with 12 μL of PEI (1 mg/mL), rested for 15 min, followed by the addition of 200 μL of Opti-MEM to the HEK293T cells. After 12 h, the culture medium was changed to DMEM. After 48-72 h, the lentiviral particles were collected, and cell fragments were filtered with a 0.45 μmol/L filter. Viruses were used after 12 h of rest at 4 ℃.

Lentiviral particle transduction
The passaged piPSCs were inoculated into a 12-well plate covered with feeders. The viral supernatant containing lentiviral particles was mixed with the piPSC culture medium (1:1) and 4 μg/mL polystyrene. The piPSCs in mixed medium were cultured at 37 ℃ for 8-12 h, after which the medium was replaced with new piPSC medium. Puromycin was used to select purine-positive cells after 7 days of culture.

RNA extraction, reverse transcription, and qRT-PCR detection
RNAiso Plus Reagent (Takara, Japan) was selected to extract total RNA. Quality of the extracted RNA was detected using a Nanodrop microspectrophotometer (Thermo Fisher Scientific, USA) and agarose gel electrophoresis. After removing the feeders, the cells were passaged once to remove feeder interference, followed by total RNA extraction. The RNA (2 μg) was then reverse transcribed using a RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) to obtain cDNA. Finally, qRT-PCR was performed through a three-step process using SuperReal PreMix Plus (SYBR Green) (Tiangen, China) . The primers for qRT-PCR are shown in Supplementary Table S1.

Immunofluorescence staining
The piPSCs were washed twice with phosphate-buffered saline (PBS) and fixed at room temperature for 15 min with 4% paraformaldehyde. The cell membrane was perforated with 0.1% Triton-100 for 10 min, then sealed at room temperature for 1 h with 10% FBS. The cells were incubated with primary antibodies (Flag; 1:1 000; Sigma-Aldrich, USA) at 4 ℃ for 12 h, then washed with PBS three times and incubated with goat anti-mouse IgG (H+L) secondary antibody AlexaFluor488 (1:500; ZSGB-BIO, China) conjugated at room temperature for 1 h. Cells were counterstained at room temperature with Hoechst33342 (1:1 000) for 5 min. Finally, images were collected using an EVOS fluorescence microscope (Thermo Fisher Scientific, USA).
The PVDF membranes were washed in TBST buffer for 30 min, and new TBST buffer was replaced every 10 min. The membranes were incubated with goat anti-mouse/rabbit IgG (H+L) (Cell Signaling Technology, USA) at 37 ℃ for 1 h, then washed with TBS-T buffer at room temperature for 15 min. Finally, the Tanon-5200 automatic chemiluminescence image analysis system (Tanon, China) was used to detect horseradish peroxidase (HRP) signals. ImageJ (v1.53c) was used to analyze the relative grayscale of western blots.

Alkaline phosphatase (AP) staining
Suitable piPSCs were fixed with 4% paraformaldehyde (pH 7.4) for 15 min, then washed 2-3 times with PBS. The cells were stained with AST Fast Red TR and α-naphthol AS MX phosphate (Sigma-Aldrich, USA) according to the manufacturer's instructions. In brief, 1.0 mg/mL AST Fast Red TR, 0.4 mg/mL α-naphthol AS MX phosphate, and 0.1 mmol/L Tris-HCL 8.8 buffers were used to prepare the AP staining solution. The cells were incubated for 20 min with AP stain, which was then replaced with PBS. The AP-positive piPSC clones showed red and images were captured using a phase contrast microscope (Nikon, Japan).

RNA-seq analysis
To explore the molecular mechanism of OCT6 in piPSCs, we performed RNA-seq of OE-OCT6 and OE-NC piPSCs after feeder removal . We extracted total RNA using RNAisoPlus Reagent (Takara, Japan) and guanidine isothiocyanate phenol-chloroform extraction. Total RNA quality was detected using a Nanodrop spectrophotometer, and total RNA integrity was detected by agarose gel electrophoresis. Total RNA was digested using DNase Ⅰ, mRNA was enriched using Oligo (dT) magnetic beads, and cDNA was synthesized by adding random primers after mRNA was interrupted (second-strand cDNA was synthesized with dUTP instead of dTTP). Next, 3'adenylated and adaptor ligation was performed at the ends of the synthesized double-stranded DNA. Joint connection and PCR amplification of the connection products were carried out. A DNA library was constructed and qualitychecked, with the resulting product then cyclized . The DNBSEQ platform was used for computer sequencing, and quality control (QC) of the obtained raw reads was carried out. The filtered clean reads were compared to the porcine genome Ssc11.1, and expression level was normalized to reads per kilobase per million mapped reads (RPKM) using gene annotation files. EdgeR was used to analyze differences in samples. Heat-maps were drawn using pheatmap (v1.0.12) to show specific gene expression levels. ClusterProfiler (v4.2.2) was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Adjusted P<0.05 or Q<0.05 was used to define the working threshold of statistical significance.

Statistical analysis
Student t-test was used to determine significant differences between two groups, and univariate or multivariate analysis of variance (ANOVA) was used to determine significant differences between multiple groups. All data were expressed as mean±standard error (SE) and differences were considered significant at P<0.05.

Cloning and overexpression of porcine OCT6 gene in piPSCs
To determine whether overexpression of OCT6 can improve the anti-differentiation ability of piPSCs, we introduced the OCT6 gene cloned from porcine testicular tissue into the lentiviral vector. The OCT6 protein sequence was highly conserved, with only three amino acid differences among Homo sapiens, Mus musculus, and Sus scrofa ( Figure 1A).
To further investigate the role of porcine OCT6 in piPSCs, we generated piPSC lines stably expressing porcine OCT6 using the TetOn lentiviral vector ( Figure 1B). Quantitative analysis showed that the OCT6 gene was up-regulated (1.25×10 4 times) compared with the negative control (NC, TetOn-3×Flag empty vector). After three consecutive passages, AP staining was performed. Results showed that overexpression of OCT6 did not affect piPSC pluripotency, and the edge of the piPSC clones was smoother than that of the NC ( Figure 1D). Both protein and immunofluorescence analyses showed that the OCT6 protein expression was significantly up-regulated in OCT6 piPSCs but could not be detected by Flag or OCT6 antibodies in the NC group ( Figures  1E, F). These results suggest that OCT6 helps maintain colony morphology of piPSCs.

OCT6 inhibits piPSC differentiation under differentiation conditions
We then applied differentiation conditions to verify the role of OCT6 in maintaining piPSC morphology and pluripotency (Kim et al., , 2021Wu & Yao, 2005). Under differentiation conditions, the piPSCs overexpressing OCT6 maintained basic colony morphology for three consecutive passages without feeders (Figure 2A). AP staining was used to determine the effects of OCT6 on piPSC pluripotency under differentiation conditions. Results showed that piPSCs overexpressing OCT6 maintained pluripotency, even in the culture system without feeders ( Figure 2B). Detection of core pluripotent genes showed a slight but significant increase in exogenous OSKM and endogenous OCT4 expression (1.31 and 1.86 times, respectively) and an obvious up-regulation in endogenous c-Myc expression (2.92 times) in the OE-OCT6 piPSCs ( Figure 2C). Previous studies have shown that c-Myc is the downstream target of the Wingless/Integrated (WNT) signaling pathway, and an increase in c-Myc gene expression may be related to activation of WNT Zhang et al., 2021). We therefore removed the GSK3 inhibitor Chir99021 from the culture medium. However, results showed that OE-OCT6 still maintained colony morphology and pluripotency after Chir99021 removal ( Figure 2D).
To further explore the results caused by overexpression of OCT6, we performed RNA-seq of the two piPSC lines after 5 days of culture. Results showed that transcriptional changes occurred in both directions after overexpression of OCT6 (339 up-regulated genes and 639 down-regulated genes) ( Figure 2E). We detected pluripotency-related genes by qRT-PCR and found that compared with the OE-NC group, the OE-OCT6 group showed elevated c-Myc and OCT4 expression but inhibited LIN28A expression, which may be related to the inhibition of differentiation ( Figure 2C). The most significant differentially expressed genes (DEGs) from RNA-seq were verified by qRT-PCR. Results showed that these hypervariable genes were involved in the inhibition of differentiation and maintenance of stem cell pluripotency. Among them, PAX5, CLU, and COL5A1 affect differentiation (Baghdadi et al., 2018;Mansouri et al., 1996;Oh et al., 2020) and SOX3, SIX6, and L1CAM are involved in the regulation of stem cell pluripotency (Corsinotti et al., 2017;Son et al., 2011;Wolfrum et al., 2010) as well as decreased expression of imprinted gene NNAT (Teichroeb et al., 2011), with epigenetic erasure required for resetting the cell identity to a ground state, which may explain why OCT6 overexpression is beneficial for the cloning and maintenance of pluripotency ( Figure 2F).
We further carried out KEGG and GO analyses of the RNAseq data. KEGG analysis showed that the DEGs were mainly enriched in the MAPK and PI3K-AKT signaling pathways ( Figure 2G). GO analysis indicated that the DEGs were primarily related to the ECM, extracellular space, and whole composition of plasma membrane ( Figure 2H). These findings suggest that OCT6 not only affects the canonical pathways but may also inhibit piPSC differentiation by regulating the components of the ECM and cell membrane.

OCT6 maintains morphology and reduces pluripotency of piPSC colonies by inhibiting MAPK-ERK pathway
KEGG analysis was performed to explore the possible pathways regulated by OCT6. Results indicated that the main enriched pathways were the MAPK, PI3K-ATK, and ECM pathways. As MAPK and PI3K-AKT play important roles in piPSCs, we compared the OE-OCT6 and OE-NC groups with piPSCs cultured with (MOCK group) or without feeders (Fgroup). The heat-map of MAPK-related genes showed that the expression patterns in the OE-NC and F-groups and the OE-OCT6 and MOCK groups were similar ( Figure 3A). The qRT-PCR results further confirmed that the gene expression pattern in the OE-OCT6 group was basically the same as that in the MOCK group, indicating that the OE-OCT6 group maintained the characteristics of piPSCs after feeder removal, and OE-OCT6 may inhibit the ERK pathway via activation of DUSP10 and inhibition of PTPRR, RRAS, and FGFR3 ( Figure 3B, C).
Under feeder removal conditions, the ERK and phosphorylated ERK (p-ERK) protein expression levels in the OE-OCT6 piPSCs relative to the OE-NC piPSCs significantly increased and decreased, respectively ( Figure 3D). We verified the changes in the MAPK-ERK pathway in piPSCs with and without feeders, which indicated that the ERK pathway was inhibited by the removal of feeders ( Figure 3E).
To verify this, we tested PD0325901 (ERK inhibitor) and 12-Otetradecanoyl phorbol-13-acetate (PMA, ERK activator) in the piPSCs. When 1 μmol/L PD0325901 was added to the OE-OCT group, the colonies became smaller; in contrast, piPSCs showed obvious differentiation after the addition of PMA ( Figure 3F). However, the piPSCs maintained basic colony morphology after the addition of PD0325901 ( Figure 3G). These findings confirm that inhibition of ERK may be of significance for the maintenance of piPSCs. After the concentration gradient test, 1 μmol/L PD0325901 and 1 μmol/L PMA were selected as the optimum concentrations for ERK inhibition and activation, respectively (Supplementary Figure S1A, B).

PI3K-AKT pathway compensates for adverse effects caused by MAPK-ERK suppression
We next asked how OCT6 maintains piPSC pluripotency after feeder removal. Heat-map and qRT-PCR analysis showed that the expression patterns of PI3K-AKT pathway-related genes in the OE-OCT6 group were similar to those in the MOCK group ( Figure 4A). Notably, expression of the MAGI family, LPAR1, and IGTB4 genes increased, while PPP2R3A expression decreased. These genes are associated with cell adhesion and improvement in piPSC colony formation (Gong et al., 2019;Li et al., 2017bLi et al., , 2019 (Figure 4B, C). In the absence of feeders, AKT and phosphorylated AKT were significantly decreased and increased, respectively, in the OE-OCT6 group relative to the OE-NC group ( Figure 4D). We also compared the protein level of AKT in the OE-NC group with or without feeders and found that AKT and phosphorylated AKT were significantly decreased and increased, respectively, in the presence of feeders ( Figure 4E). This change in AKT level in the OE-OCT6 group disappeared after the addition of the  PI3K inhibitor LY294002, consistent with the addition of PD0325901 in the OE-NC group, resulting in colony reduction ( Figure 4F). These results suggest that elevated AKT signaling maintains the pluripotency of piPSCs under differentiation conditions. After adding the PI3K activator 740Y-P to the OE-NC group, pluripotency also recovered under differentiation conditions ( Figure 4G). Therefore, the PI3K/AKT signaling pathway is beneficial for maintaining piPSC pluripotency under differentiation. After the concentration gradient test, 5 μmol/L LY294002 and 5 μmol/L 740Y-P were selected as the optimum concentrations for PI3K inhibition and activation, respectively (Supplementary Figure  S1C, D).

DISCUSSION
PiPSCs can be effectively obtained by transferring OCT4, SOX, KLF4, and c-Myc into porcine fetal fibroblasts using lentiviral vectors (Ezashi et al., 2009). However, existing culture systems cannot be maintained long-term without feeders (Montserrat et al., 2012). In the absence of feeders, existing piPSCs show obvious differentiation, indicating the existence of several defects in piPSCs compared with humans and mice (Esteban et al., 2009). Here, we attempted to modify gene expression patterns to create a piPSC line that can maintain pluripotency and colony morphology without feeders. We found that introduction of the OCT6 gene enabled piPSCs to achieve maintenance with no xenogeneic cells.
OCT6 and OCT4 are both members of the POU family. To date, however, most studies have focused on their functions in neurons and neural progenitor cells (Kawasaki et al., 2003). Although the structures of OCT6 and OCT4 are similar, the reprogramming response is weaker in OCT6 (Jerabek et al., 2017). OCT6 can cause extensive chromatin opening (Malik et al., 2019) and is reported to be expressed in early embryos (Meijer et al., 1990). As a pluripotency inducer, OCT6 may play a potential role in the maintenance of pluripotency. Interestingly, in the present study, after OCT6 overexpression, piPSCs maintained their pluripotency without feeders, and their expression pattern was essentially the same as regular piPSCs cultured on feeders. Differentiation-related gene expression (e.g., L1CAM (Bao et al., 2008)) and cell adhesion and ECM-related gene expression (e.g., ITGB4 (Zhang et al., 2020b), LPAR1 (Li et al., 2017a)) were decreased and enhanced, respectively, which was beneficial for the maintenance of piPSC colony morphology. The expression of c-Myc also increased, which may provide additional maintenance of self-renewal and pluripotency of piPSCs.
Our study showed that OCT6 regulated the MAPK-ERK and PI3K-AKT pathways to help maintain piPSC pluripotency. Inhibition of MAPK signaling can induce PSCs to transform into the immature state (Hackett & Surani, 2014). There are many conflicting reports regarding the role of the MAPK signal transduction pathway in human embryonic stem cells (hESCs), including maintenance of pluripotency (Armstrong et al., 2006) and promotion of differentiation (Ding et al., 2010). In pigs, inhibition of MAPK can lead to the rapid loss of pluripotency (Gao et al., 2014). Furthermore, when OCT6 is overexpressed, the expression of DUSP10 (Hiratsuka et al., 2020) increases significantly, whereas the expression PTPRR (Su et al., 2013), RRAS (Michael et al., 2016), and FGFR3 (Blick et al., 2013) decreases significantly, potentially inhibiting ERK. Surprisingly, under feeder withdrawal-stimulated differentiation, we found that low-dose PD0325901 led to colony formation, although proliferation ability was significantly decreased. Furthermore, the addition of a MAPK activator abolished the effects of OCT6, resulting in complete differentiation of piPSCs.
Activation of PI3K caused by OCT6 overexpression can overcome the damage caused by inhibition of MAPK (Singh et al., 2012). As an important pathway for controlling ESC function, PI3K-AKT signaling plays a crucial role in cell selfrenewal and pluripotency maintenance in pigs (Welham et al., 2011). Here, we found that overexpression of OCT6 significantly activated the PI3K-AKT signaling pathway, with an increase in MAGI1/2 (Zhang et al., 2020a), LPAR1 (Cui et al., 2019), and ITGB4 (Leng et al., 2016) gene expression, and decrease in PPP2R3A gene expression (Chen et al., 2019). These genes can promote PI3K-AKT signaling pathway activation. When PI3K-AKT signaling pathway inhibitors were added to the OE-OCT6 group, pluripotency was lost, and clones became smaller. These results indicate that inhibition of the PI3K-AKT signaling pathway damages piPSC pluripotency, suggesting that OCT6 maintains piPSC pluripotency in the differentiated state via the PI3K-AKT signaling pathway.
Taken together, our results showed that overexpression of OCT6 enhanced piPSC colony formation in the differentiated state and enhanced piPSC pluripotency, primarily by inhibition of the MAPK-ERK signaling pathway and activation of the PI3K-AKT signaling pathway.
It is worth highlighting that the genetically engineered (OE-OCT6) iPSCs, whose capability to differentiate in vitro was considerably attenuated by transgenically increasing the relative abundance of OCT6 transcripts and extrinsic OCT6 proteins, may provide an excellent source of nuclear donor cells (NDCs) suitable for somatic cell nuclear transfer (SCNT)based assisted reproductive technologies in pigs and other mammalian species (Gorczyca et al., 2021;Olivera et al., 2016;Samiec & Skrzyszowska, 2010;Secher et al., 2017;Zhang et al., 2014). Highly reprogrammable NDCs may exhibit an enhanced capacity to sustainably perpetuate stemnessand pluripotency-related molecular attributes. The latter, in turn, could enhance the epigenetic dedifferentiation capacity of OE-OCT6 iPSCs in SCNT-derived embryos, conceptuses, and progenies generated for transgenic and biomedical research, not only for pig-to-human xenotransplantation and tissue engineering, but also for regenerative and reconstructive surgical treatments in humans and other mammalian species (Berthelsen et al., 2021;Podstawski et al., 2022;Song et al., 2016;Wiater et al., 2021aWiater et al., , 2021bXu et al., 2022;Yu et al., 2021;Zhao et al., 2020).

SUPPLEMENTARY DATA
Supplementary data to this article can be found online.