YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway

YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway Hong-Chen Yan, Yu Sun, Ming-Yu Zhang, Shu-Er Zhang, Jia-Dong Sun, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-414853/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2021 Read the published version in Histochemistry and Cell Biology → Version 1 posted You are reading this latest preprint version Abstract Background Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of ASCs is a hot topic. Porcine models have close similarities to humans and porcine SDSCs (pSDSCs) offer an ideal in vitro model to investigate human ASCs. To date, studies concerning the role of yes-associated protein (YAP) in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this paper, we explore the link between the transcriptional regulator YAP and the fate of pSDSCs. Results We found that YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Sox2, Oct4. The overexpression of YAP prevented the differentiation of pSDSCs and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/ β -catenin signaling pathway. When an activator of the Wnt/ β -catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939 an inhibitor of Wnt/ β -catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Conclusions our results suggested that, YAP and the Wnt/ β -catenin signaling pathway interact to regulate the fate of pSDSCs. Animal Science Biotechnology and Bioengineering pSDSCs YAP β-catenin Wnt pathway self-renewal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Embryonic stem cells (ESCs) and adult stem cells (ASCs) have the ability of self-renewal and differentiation [ 1 ]. Past studies have shown that ESCs and/or ASCs derived from human, mouse and pig have the capacity to differentiate into germ cells, oocyte-like cells (OLC) and even produced offspring [ 2 – 4 ]. Fetal skin harbors populations of stem cells responsible for development and maintenance into adulthood, making skin an attractive source for ASCs [ 5 ]. Due to ethical considerations human embryos are not accessible for research, resulting in the use of porcine models. Porcine models have close similarities to humans with respect to their genetic relationship and the onset of epigenetic programs, making them ideal animal models for investigating human biology [ 6 ]. Porcine skin-derived stem cells (pSDSCs) offer an ideal in vitro model to investigate human ASCs [ 7 ]. During last 10 years, germ cells differentiated from pSDSCs have been extensively investigated [ 2 , 5 , 7 ]. But the efficiency of pSDSCs differentiation to porcine primordial germ cell-like cells (pPGCLCs) was very low [ 7 , 8 ]. This problem can be solved by improving the differentiation efficiency or generating large amounts of pSDSc through enhancing self-renewal. Recent studies have suggested that YAP is required for the efficient self-renewal of stem cells [ 9 – 11 ]. Hippo signaling pathway has been widely studied in mammalian cells and animal models [ 12 , 13 ]. Hippo signaling regulates diverse cellular processes including cell proliferation and differentiation [ 14 ]. In mammals, The core components of the Hippo pathway were mammalian Ste2-like kinases (MST1/2, Hpo orthologs) and large tumor suppressor kinase 1/2 (Lats1/2, Wts orthologs). Activation of Hippo signaling results in YAP being phosphorylated, sequestered to the cytoplasm, and degraded [ 15 ]. Recently, emerging evidence suggests that YAP is a crucial regulator for maintaining the self-renewal of ESCs, induced pluripotent stem cells (iPSC), and ASCs in vitro and in vivo [ 9 , 10 , 16 – 18 ]. Additionally, YAP also regulates self-renewal of tissue-specific progenitor cells, such as skin epidermal progenitor cells, cardiac progenitor cells, muscle progenitor cells, and neural progenitor cells [ 19 – 22 ]. For instance, Zhang et al found that activation of the transcriptional auxiliary activity of epidermal YAP in mice can inhibit their terminal differentiation leading to the expansion of epidermal progenitor cells in the basement membrane [ 23 ]. Canonical Wnt signaling is a highly conserved pathway [ 24 ] that is dependent on β -catenin whose role is primarily based on whether β -catenin is stable in the cytoplasm or translocated to the nucleus. In the basic state, β -catenin in the cytoplasm is mainly inhibited by GSK3 β , which holds Wnt signaling in an inhibited state [ 24 , 25 ]. It was found that there is a multipoint cross correlation between the Wnt and Hippo signaling pathway [ 26 ]. The interaction between Hippo signaling and Wnt signaling lies in the mutual regulation of their core molecules. The Hippo signaling pathway can affect the expression of Wnt target genes by changing the transcriptional activity of β -catenin. Qin et al reported that YAP overexpressing hESCs display lower levels of active β -catenin and Wnt target genes [ 9 ]. Zhao et al found that YAP can interact with β -catenin and induce the expression of Wnt target genes in mouse heart tissue [ 27 ]. These studies confirmed that Hippo and Wnt signaling indeed regulate each other's activity. Researchers suggested that the Hippo signaling pathway plays different roles in different kinds of stem cells, either being inhibitory or activating, depending on biological contexts [ 10 , 14 , 27 , 28 ]. To date, studies concerning the role of YAP in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this study, we used the pSDSCs to explore the effect of YAP on ASCs self-renewal. Our data suggested that the upregulation of YAP contributes to the maintenance and self-renewal capacity of pSDSCs by inhibiting Wnt signalling. Materials And Methods pSDSCs isolation, culture and differentiation Pigs were housed in Laixi, Shandong province. This research was approved by the Ethics Committee of Qingdao Agriculture University (Agreement No. 2019-011). Porcine back skin tissue was collected from day 40–45 gestation fetus (E40-45). The isolation, culture and differentiation of pSDSCs were described previously [ 2 , 7 ]. Briefly, the porcine skin tissue was mechanically dissociated using a pipette. The pSDSCs were incubated in SDSCs medium [ 7 ]. After 12 days culture, the cells are positive for Sox2, Oct4 and YAP (Fig. 1 ). These cells could differentiate into pPGCLCs under in vitro differentiation conditions [ 7 ]. Briefly, the pSDSCs colonies were disaggregated into single cells and 1×10 5 cells were seeded into 60 mm tissue culture dish within differentiation medium [ 7 ]. Every 2 days the differentiation medium was changed. At ~ 20 days cells with the morphological characteristics of pPGCLCs were present. Indirect immunofluorescence assay (IFA) Nanog, Sox2, Oct4 and YAP expression in pSDSCs were examined by IFA. The pSDSCs were collected and fixed in 4% paraformaldehyde for 30 min, then the samples were transferred to microscope slides treated with poly-L-lysine. The samples were permeabilized with 0.5% Triton-X-100 for 10 min and then blocked with 10% bovine serum albumin overnight at 4°C. The pSDSCs samples were covered with 50 µl primary antibody (Table S1) and incubated at 4°C overnight. After washing three times with PBS, the samples were incubated for 45 min at 37°C in a moisture chamber with secondary antibody (Table S1). The slides were washed three times with PBS and then incubated with Hoechst 33342 for staining the cell nucleus. Finally, the samples were viewed under a fluorescence microscope (Leica, Germany) and photographed. Fluorescence activated cell sorting (FACS) analysis The prepared pSDSCs (1×10 7 cells/ml) were fixed in 80% methanol. The pSDSCs were incubated successively with primary antibodies and secondary antibody (Table S1) at 37°C for 60 min. Then, the cells were centrifuged and were analyzed by Flow cytometry (Becton Dickinson, Mountain View, CA). Data were analyzed using WinMDI 2.9 software. Small interfering RNA (siRNA) transfection Three fluorescein-labeled siRNA targeted to YAP (siYAP-1, siYAP-2, siYAP-3) and fluorescein-labeled non-sense siRNA (siYAP-negative control, siYAP-NC) were purchased form GenePharma Co., Ltd. (Shanghai, China) (Table S2). Each siRNAs were transfected at a concentration of 30 pmol into the cells using lipofectamin 2000 (11,668 − 019; Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. The pSDSCs were collected for further experiment 48h post transfection. All the siRNA experiments were performed three times. RNA extraction and RT-qPCR As previously described [ 7 ], the total RNA was extracted from pSDSCs with the RNA prep pure Micro Kit (Aidlab, RN07, Beijing, China). The cDNA was synthesized by TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AT311-03, Beijing, China). RT-qPCR amplification with specific primers (Table S3) was done using a Light Cycler 480 (Roche, Germany). The relative transcript abundance were calculated by the 2 −ΔΔCt method [ 29 ] and normalized according to the housekeeping gene Gapdh . Western blot analysis Proteins were extracted using RIPA buffer (Beyotime, P0013C, Nantong, China). SDS was mixed with the samples and boiled for 5 min in water to denature proteins, then SDS-PAGE was used to separate proteins. The proteins were transferred onto a poly-vinylidene fluoride (PVDF) membrane (Millipore, ISEQ00010, USA), and blocked in TBST containing 6% BSA for 4 hours. The membranes were incubated with primary antibody (Table S1) at 4°C, and then a horseradish peroxidase (HRP)-conjugated corresponding secondary antibody (Beyotime) was incubated at room temperature for 120 min on the second day. The BeyoECL Plus Kit (Beyotime, P0018) was used for chemiluminescence detection. The relative expression level of the target protein to GAPDH was calculated using AlphaView SA (ProteinSimple, CA, USA) software. Statistical Analysis GraphPad Prism 8 (GraphPad Software, San Diego, CA) was used for data analysis with One way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons; P < 0.05 means statistically significant difference, P 0.05 means not significant (ns). The data were expressed as mean ± standard error (S.E.) and all the experiments were repeated at least three times. Results Endogenous YAP expression in pSDSCs pSDSCs were cultured using a previously described method [ 7 ]. Spherical aggregates of cells were clearly visible by day 8 of culture (Fig. 1 A). FACS analysis during the culturing of the pSDSCs showed dynamic marker changes with the percentage positive for Sox2 being 0.09% at 4 days, 0.19% at 8 days, and 11.69% at 12 days; for Oct4, at 4 days 0.51%, 8 days 4.37%, and 12 days 27.87%; for YAP, at 4 days 5.75%, 8 days 8.76%, 12 days 41.37%. Using FACS and western blot analysis revealed that, during the culturing of pSDSCs, the percentage of the pluripotency markers Sox2 and Oct4 [ 30 ] increased dramatically. Concomitant with the increase of stem cell markers, YAP positive cells were also significantly upregulated (Fig. 1 B and 1 C). These results confirmed that YAP expression is increased during the culturing of pSDSCs suggesting it may play a role in self-renewal. LPC activates YAP and promotes pSDSCs pluripotency We explored existing data to identify a small molecule that could increase YAP expression. We found that lysophosphatidylcholine (LPC) could be hydrolyzed into lysophosphatidic acid (LPA) by autotaxin (ATX) [ 31 ] and lead to the activation of YAP in cells [ 32 ]. In order to investigate the potential effects of different concentrations of LPC on regulating YAP in pSDSCs 0, 5, 10 and 25 µM LPC was added to the pSDSCs medium, respectively. As show in Fig. 2 A, after 12 days culture, spherical colonies were observed in all treatments. FACS analysis of the pSDSCs in the various treatments showed that the percentage of Oct4 positive cells increased in the LPC-treated groups in a dose-dependent manner up to 10 µM (44.84% in 5 µM LPC, 53.79% in 10 µM LPC and 41.84% in 25 µM LPC) compared to the control group (28.67 %) (Fig. 2 B). Based on these results, we used 10 µM LPC for the subsequent studies. In agreement with previous findings [ 31 ], exposure to LPC led to an increase in the mRNA and protein levels of YAP in the pSDSCs (Fig. 2 C and 2 D). These observations demonstrated that YAP overexpression can be induced by supplementing the culture medium with 10 µM LPC. To investigate the potential effects of LPC induced YAP expression on regulating the self-renewal of pSDSCs, we examined the protein expression levels of Oct4 and Sox2. As shown in Fig. 2 D, both Oct4 and Sox2 were upregulated, indicating that YAP plays an important role in the self-renewal of pSDSCs. LPC induced YAP counteract pSDSCs transdifferentiated into pPGCLCs To evaluated the importance of YAP in pSDSCs self-renewal, we examined their ability to differentiate to pPGCLCs in the presence of LPC [ 2 , 7 ]. The pSDSCs were plated in differentiation medium in 6 cm dishes, and incubated for 20 days to induce the formation of pPGCLCs. In the untreated control pSDSCs formed numerous pPGCLCs and high expression of the pPGC marker Vasa (Fig. 3 A and 3 B); In contrast, LPC treated pSDSCs exhibited resistant to the differentiation and significantly reduced Vasa protein levels under the same differentiation conditions (Fig. 3 A and 3 B). Supporting this, FACS analysis found the percentage of Vasa-positive cells was 48.73% in the untreated control and 7.93% in the LPC treatment group (Fig. 3 C). YAP shuttles between the cytoplasm (inactive) and the nucleus (active) [ 10 , 14 ]. Therefore YAP localization is essential in maintaining the pluripotent properties of stem cells [ 10 , 14 ]. In our experiments, immunofluorescent staining revealed that YAP was predominantly located in the nucleus of undifferentiated pSDSCs, while significant YAP positive signals was found in the cytoplasm of differentiated cells (Fig. 3 D). Similar results to those found in a previous study using human ESCs [ 33 ]. In conclusion, these results demonstrate that YAP activation maintains pSDSCs pluripotency even under differentiation conditions in vitro . Knockdown of YAP in pSDSCs by siRNA To further confirm the role of YAP in pSDSC self-renewal and differentiation, sequence-specific siRNAs against YAP were introduced. Fluorescein-labeled non-sense siRNA (siRNA-NC) was used to optimize the transfection conditions. The expression of the red fluorescent protein was examined after transfection for 48 h by fluorescence microscope. The results in Fig. 4 A demonstrating a successful transfection. In order to further investigate the transfection effect of siRNA, FACS was employed. The results showed the percentage of CY3 positive cells were similar in the siRNA-NC group and the siYAP-1 groups (Fig. 4 B). In addition, RT-qPCR and western blot demonstrated that siYAP-1 had the strongest downregulation effect on YAP compared to the other three siRNA groups (Fig. 4 C and 4 D). Knockdown of YAP gene expression suppressed pSDSCs pluripotency and promoted pSDSCs differentiation Striking morphological changes were observed in the siRNA transfected group (Fig. 5 A). Knockdown of YAP induced the pSDSCs cells to acquire a fibroblast-like morphology. The pSDSCs colonies lost their suspended culture and attached and spread on the culture surface. To gain an insight into the molecular mechanisms underlying this morphological phenomenon, the expression levels of two pluripotency markers, Oct4 and Sox2, and a defined terminal differentiation marker involucrin (IVL) [ 11 ] were analyzed. As shown in Fig. 5 B, YAP knockdown resulted in the downregulation of Oct4 and Sox2 at the mRNA level and upregulated the expression of IVL and β -catenin. Western blot analysis of protein expression also indicated the same tendencies (Fig. 5 C). In addition, the loss of pluripotency in the siYAP transfected pSDSCs was further confirmed by an increase in the percentage of IVL positive cells using FACS analysis. The results showed the percentage of IVL positive cells in the control group was 13.77%, but in the siYAP treated group, the percentage was increased to 24.19% (Fig. 5 D). YAP promotes pSDSCs self-renewal by suppressing the Wnt/ β -catenin signaling pathway Previous studies demonstrated that YAP promotes human and mouse stem cell self-renewal in response to suppressing the differentiation-inducing effects of the Wnt/ β -catenin pathway [ 9 , 17 , 30 ]. To elucidate the role of YAP in pSDSCs self-renewal and its connection with the Wnt/ β -catenin signaling we investigated whether YAP regulates β -catenin activity. Western blot analysis revealed that the protein expression level of β -catenin was effectively suppressed in LPC treated pSDSCs (Fig. 2 D), while β -catenin was robustly activated after siYAP-1 transfection (Fig. 5 C). These results demonstrate a functional connection between YAP and the Wnt/ β -catenin signaling pathway. To further confirm the functional connection between YAP and the Wnt pathway and the role in maintaining the pluripotency of pSDSCs, we assessed the effect of the GSK3 inhibitor CHIR99021 (CHIR), which activates Wnt/ β -catenin signaling [ 30 ], on the differentiation of pSDSCs in LPC treatment. Three dosages (1 µM, 3 µM, and 6 µM) of CHIR were used. Interestingly, increasing the concentration of CHIR in pSDSCs induced cells to acquire a flattened phenotype, indicating a loss of characteristic pSDSC morphology (Fig. 6 A). FACS analysis indicated that there was a significant upregulation in the percentage of IVL positive cells in the 3 µM and 6 µM CHIR treated groups when compared with the control group (Fig. 6 B). In addition, the protein expression levels of β -catenin and IVL were increased, but both Oct4 and Sox2 were significant decreased and the degrees of reduction of both self-renewal markers were closely correlated with the concentration of CHIR (Fig. 6 C). Based on the above results, we used 6 µM CHIR for subsequent studies. As shown in Fig. 6 E, the Wnt pathway was effectively repressed in the 10 µM LPC-treated pSDSCs (L-pSDSCs). Upon treating L-pSDSCs with 6 µM CHIR, the expression of β -catenin increased but Oct4 and Sox2 decreased (Fig. 6 D and 6 E). These results show that activating Wnt/ β -catenin signaling partially restores the differentiation ability of the L-pSDSC group, as evidenced by the western blot results showing higher expression of IVL (Fig. 6 E). Therefore, the results investigating the addition with CHIR imply that YAP promotes pSDSCs self-renewal by suppressing the Wnt/ β - catenin signaling pathway. YAP prevents pSDSCs differentiation by modulating Wnt/ β -catenin signalling In order to further elucidate the mechanism by which YAP regulates pSDSCs differentiation through Wnt signaling, we investigated the effect of XAV939, a Wnt signaling antagonist, on the self-renewal of pSDSCs following YAP knockdown. Three dosages of XAV939 were tested (1 µM, 5 µM, and 10 µM). With increasing concentration of XAV939 the pSDSCs showed an elevated clonal growth rate, and propagated readily in culture for more than 5 passages (data not show). Western blot analysis indicated that β -catenin protein was effectively suppressed in the 10 µM XAV939 treated groups but Oct4 and Sox2 were markedly increased (Fig. 7 A). Moreover, FACS analysis showed that the percentage of Oct4 positive cells was significantly upregulated in the 10 µM XAV939 treatment group when compared with the control group (Fig. 7 B). Next, using the siRNA system, we evaluated how YAP modulates pSDSCs self-renewal in the 10 µM XAV939 treated cells. The results indicated that siYAP led to increased expression of β -catenin in the pSDSCs but in the siYAP plus XAV939 group, β -catenin returned to the control levels (Fig. 7 C). These results indicated that the Wnt pathway is important for the differentiation of pSDSCs. Moreover, it appears that YAP inhibits pSDSCs differentiation in response to Wnt/ β -catenin signaling. Discussion Self-renewal is a critical aspect of stem cells allowing them to increase in number while maintaining pluripotency [ 19 ]. The molecular mechanisms involved in maintaining the self-renewal of ESCs and ASCs in vitro has been a key issue in stem cell research. Emerging evidence has indicated that YAP may play an important role in maintaining the pluripotency of stem cells [ 9 , 10 , 27 ]. To date, reports concerning the influence of YAP on the pluripotency of ASCs are limited. In this paper, we confirmed that YAP helped maintain pSDSCs ability for self-renewal through suppressing the Wnt/ β -catenin signaling pathway. Our observations demonstrated that concomitantly with the increase of YAP positive cells, the protein levels of Oct4 and Sox2, were also significantly upregulated (Fig. 1 C and 2 D). On the other hand, YAP knockdown leads to a loss of pluripotency in pSDSCs (Fig. 5 ). Interestingly, higher expression of YAP prevented pSDSCs differentiation in vitro even under differentiation inducing conditions (Fig. 3 A, 3 B and 3 C). Furthermore, we found evidence that the Wnt pathway regulates pSDSCs differentiation. Our experiments indicated that the addition of CHIR, an activator of the Wnt/ β -catenin signaling pathway, in pSDSCs medium results in a rapid loss of stem cell properties, including morphological changes and decreased expression of the pluripotency markers, Oct4 and Sox2 (Fig. 6 A and 6 C). Conversely, when the Wnt/ β -catenin signaling pathway is inhibited using XAV939 we confirmed that stem cell markers were positively upregulated (Fig. 7 A and 7 B). We also found evidence supporting a direct relationship between YAP and the Wnt/ β -catenin signaling pathway. YAP overexpression or knockdown could affect the protein expression level of β -catenin, a core component of the Wnt pathway, and thereby inhibit or promote the differentiation of pSDSC. The results in Fig. 6 D and 6 E support that pSDSCs treated with LPC plus CHIR resulted in increased protein levels of β -catenin and IVL and decreased protein levels of Oct4 and Sox2 compared with that of LPC treatment alone (Fig. 6 D and 6 E). In addition, we also confirmed that knockdown of YAP would induce the differentiation of pSDSCs, but treatment with XAV939 after YAP knockdown prevented this differentiation (Fig. 7 C). To summarize, these observations mutually corroborate each other and further establish that YAP plays an important role in maintaining the pluripotency of pSDSCs partly through repressing the Wnt/β-catenin signaling pathway. Recent studies have shown that core transcription factors, such as Oct4, Sox2 and Nanog, play an essential role in establishing and controlling gene expression programs which define the identity of human and mouse ESCs [ 34 ]. In particular, these core transcription factors are important for establishing the ability of ESCs and ASCs to self-renew and differentiate [ 15 , 35 , 36 ]. Several studies have presented data showing that YAP can facilitate self-renewal through the upregulation of core transcription factors, such as Oct4, Sox2 and Nanog, and inhibiting YAP function results in the differentiation of ESCs [ 10 , 16 , 17 ]. In addition, based on transcriptional profiling, Ramalho-Santos et al demonstrated that YAP also has a critical role in the self-renewing ability of ASCs, such as neural stem cells and hematopoietic stem cells [ 18 ]. These results strongly support our work, for instance, in this paper self-renewal of the pSDSCs was identified based on the expression level of protein markers, Oct4, Sox2 and Nanog (Figure S1), and our results showed a striking correlation between the protein expression level of YAP and that of Oct4 and Sox2 (Fig. 1 ). YAP was highly expressed in self-renewing pSDSCs and was downregulated when cells were induced to differentiate. Meanwhile, studies described in this manuscript revealed that overexpression of YAP prevented the differentiation of pSDSCs (Fig. 2 ) and the depletion of YAP by siRNAs suppressed the self-renewal of pSDSCs (Fig. 5 ). These observations strongly confirm a particularly important role for YAP in maintaining the “stemness” of pSDSCs. Lian et al. found that YAP can positively regulate pluripotent genes such as Nanog, Oct4 and Sox2 by genome-wide analysis of mouse ESCs [ 17 ]. Bora-Singhal et al. further found that the structural domain of the YAP protein can interact with the transcription factor Oct4 to induce the transcriptional activity of Sox2 regulated downstream targets and maintain the self-renewal ability of stem cells [ 37 ]. Reciprocally, Seo et al showed YAP expression is regulated by Sox2 and could compensate for the self-renewal defect caused by Sox2 inactivation in mesenchymal stem cells (MSCs) [ 15 ]. Given these facts, the relationship between YAP and Oct4 or Sox2 is rather interesting and complex and requires further investigation. Extensive crosstalk between YAP and β -catenin, a key protein of the Wnt signaling pathway, has been observed [ 9 , 15 , 28 , 38 ]. Qin et al. confirmed that YAP promotes human ESCs naive pluripotency by interfering with β -catenin and retaining it in the cytoplasm to suppress Wnt signaling.[ 9 ]. Seo et at found that in MSCs, YAP binds β -catenin and inhibits canonical Wnt signaling to prevent MSCs differentiation [ 15 ]. Our results are in line with these previous findings, after overexpression of YAP, the expression of β -catenin was decreased (Fig. 2 C and 2 D). Conversely, the expression of β -catenin increased when we knockdown YAP using RNAi (Fig. 5 B and 5 C). Similarly, when we added CHIR to the LPC-treated pSDSCs we found that the differentiation ability of pSDSCs was partially restored through activation of the Wnt pathway (Fig. 6 D and 6 E). This suggests that YAP was sufficient to maintain the self-renewal characteristics of pSDSCs by inhibiting the Wnt pathway. In order to more clearly investigate the mechanism of YAP regulating the self-renewal of pSDSCs through the Wnt/ β -catenin signaling pathway, we added the inhibitor of the Wnt pathway XAV939 to the pSDSCs that had YAP knocked down. With the addition of XAV939, we found that the cell fate of pSDSCs was altered. Compared with pSDSCs with low YAP, the addition of XAV939 resulted in pSDSCs with higher self-renewal ability (Fig. 7 ). Taken together, this supports YAP regulating the fate of pSDSCs though its effects on the Wnt/ β -catenin signaling pathway (Fig. 8 ). Conclusions In conclusion, we have shown that YAP regulates the self-renewal of pSDSCs by inhibiting the Wnt pathway. However, the specific processes and mechanisms of the interaction between the Hippo and Wnt pathways are not fully understood. Further study of the interactions between the members of these two signaling pathways are expected to provide new insights for the molecular basis of many biological processes. Abbreviations SDSCs: Skin-derived stem cells; ASCs: Adult stem cells; pSDSCs: Porcine SDSCs; YAP: Yes-associated protein; siRNAs: Small interfering RNA; ESCs: Embryonic stem cells; OLC: Oocyte-like cells; pPGCLCs: Porcine primordial germ cell-like cells; MST1/2: Mammalian Ste2-like kinases; Lats1/2: Large tumor suppressor kinase 1/2; iPSC: Induced pluripotent stem cells; IFA: Indirect immunofluorescence assay; FACS: Fluorescence activated cell sorting; LPC: Lysophosphatidylcholine; LPA: Lysophosphatidic acid; ATX: autotaxin; IVL: involucrin; CHIR: CHIR99021; L-pSDSCs: LPC-treated pSDSCs; MSCs: mesenchymal stem cells; Declarations Acknowledgments We thank all the members of the institute of Reproductive Sciences, Qingdao Agricultural University who make efforts to these experiments. Authors’ contributions SW and CSF conceived the study; YHC, SY, ZMY and SJD carried out experiments and data analysis; ZSE, Paul WD, Francesca GK and Massimo DF interpreted the data and wrote the manuscript. All authors approved the final version. Funding This work was supported by National Natural Science Foundation of China (32072734, 31672423), Shandong Province Key Research and Development Program (2019GSF107082) and Taishan Scholar Construction Foundation of Shandong Province (ts20190946). Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate Experiments involving live animals were conducted in accordance with the "Regulations for the Administration of Affairs Concerning Experimental Animals" promulgated by the State Science and Technology Commission of Shandong Province. The study was approved by the ethics committee of Qingdao Agricultural University. Consent for publication Not applicable. Competing interests Authors declare that no competing interests. Author details 1 College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109, China. 2 Animal Husbandry General Station of Shandong Province, Jinan 250010, China. 3 Department of Animal Sciences, Auburn University, Auburn, AL 36849, USA. 4 Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome 00133, Italy. References Mo JS, Park HW, Guan KL. The Hippo signaling pathway in stem cell biology and cancer. EMBO reports. 2014; 15(6):642-56. Dyce PW, Wen L, Li J. In vitro germline potential of stem cells derived from fetal porcine skin. Nature cell biology. 2006; 8(4):384-90. Ge W, Ma H-G, Cheng S-F, et al. Differentiation of early germ cells from human skin-derived stem cells without exogenous gene integration. Scientific reports. 2015; 5(1):1-9. Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M. 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Hippo pathway effector Yap promotes cardiac regeneration. Proceedings of the National Academy of Sciences. 2013; 110(34):13839-44. Judson RN, Tremblay AM, Knopp P, et al. The Hippo pathway member Yap plays a key role in influencing fate decisions in muscle satellite cells. J Cell Sci. 2012; 125(24):6009-19. Zhang H, Pasolli HA, Fuchs E. Yes-associated protein (YAP) transcriptional coactivator functions in balancing growth and differentiation in skin. Proceedings of the National Academy of Sciences. 2011; 108(6):2270-5. Murillo-Garzón V, Kypta R. WNT signalling in prostate cancer. Nature Reviews Urology. 2017; 14(11):683. Huang S-MA, Mishina YM, Liu S, et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature. 2009; 461(7264):614-20. Piccolo S, Dupont S, Cordenonsi M. The biology of YAP/TAZ: hippo signaling and beyond. Physiological reviews. 2014; 94(4):1287-312. Zhao B, Tumaneng K, Guan K-L. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal. Nature cell biology. 2011; 13(8):877-83. Imajo M, Miyatake K, Iimura A, Miyamoto A, Nishida E. A molecular mechanism that links Hippo signalling to the inhibition of Wnt/β‐catenin signalling. The EMBO journal. 2012; 31(5):1109-22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4):402-8. doi:10.1006/meth.2001.1262 Estaras C, Hsu HT, Huang L, Jones KA. YAP repression of the WNT3 gene controls hESC differentiation along the cardiac mesoderm lineage. Genes Dev. 2017; 31(22):2250-63. doi:10.1101/gad.307512.117 Bao L, Qi J, Wang YW, et al. The atherogenic actions of LPC on vascular smooth muscle cells and its LPA receptor mediated mechanism. Biochemical and biophysical research communications. 2018; 503(3):1911-8. doi:10.1016/j.bbrc.2018.07.135 Yu FX, Zhao B, Panupinthu N, et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell. 2012; 150(4):780-91. doi:10.1016/j.cell.2012.06.037 Lian I, Kim J, Okazawa H, et al. The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes & development. 2010; 24(11):1106-18. doi:10.1101/gad.1903310 Boyer LA, Lee TI, Cole MF, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005; 122(6):947-56. doi:10.1016/j.cell.2005.08.020 Kim J, Chu J, Shen X, Wang J, Orkin SH. An extended transcriptional network for pluripotency of embryonic stem cells. Cell. 2008; 132(6):1049-61. doi:10.1016/j.cell.2008.02.039 Chambers I, Smith A. Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene. 2004; 23(43):7150-60. doi:10.1038/sj.onc.1207930 Bora‐Singhal N, Nguyen J, Schaal C, et al. YAP 1 Regulates OCT 4 Activity and SOX 2 Expression to Facilitate Self‐Renewal and Vascular Mimicry of Stem‐Like Cells. Stem cells. 2015; 33(6):1705-18. Pan J-X, Xiong L, Zhao K, et al. YAP promotes osteogenesis and suppresses adipogenic differentiation by regulating β-catenin signaling. Bone research. 2018; 6(1):1-12. Supplementary Files OnlineFigs.1.png The immunofluorescence with Sox2, Oct4 and Nanog antibodies (red) and Hoechst33342 (blue) of pSDSCs. TableS1202147.docx TableS2202147.docx TableS3202147.docx Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2021 Read the published version in Histochemistry and Cell Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-414853","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":20935663,"identity":"7b7cc935-db6e-4c72-96c4-fe27eeebdbbf","order_by":0,"name":"Hong-Chen Yan","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hong-Chen","middleName":"","lastName":"Yan","suffix":""},{"id":20935664,"identity":"3a875b8d-f309-4adc-b0cc-a914b89ca522","order_by":1,"name":"Yu Sun","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Sun","suffix":""},{"id":20935665,"identity":"774db581-ccbc-405c-8f94-facec4aa7d46","order_by":2,"name":"Ming-Yu Zhang","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming-Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":20935666,"identity":"b4fb2d01-f942-4b64-9706-d431dcc235d9","order_by":3,"name":"Shu-Er Zhang","email":"","orcid":"","institution":"Animal Husbandry General Station of shandong Province","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shu-Er","middleName":"","lastName":"Zhang","suffix":""},{"id":20935667,"identity":"26665b33-654e-4b2a-8ebf-ce92cbcf62a7","order_by":4,"name":"Jia-Dong Sun","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia-Dong","middleName":"","lastName":"Sun","suffix":""},{"id":20935668,"identity":"b1f62959-2e3b-4dd9-9c35-8d394b8b13ad","order_by":5,"name":"Paul W. Dyce","email":"","orcid":"","institution":"Auburn University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paul","middleName":"W.","lastName":"Dyce","suffix":""},{"id":20935669,"identity":"b6c3161b-9880-43f9-9792-84e4cec1386f","order_by":6,"name":"Francesca Gioia Klinger","email":"","orcid":"","institution":"University of Rome Tor Vergata: Universita degli Studi di Roma Tor Vergata","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"Gioia","lastName":"Klinger","suffix":""},{"id":20935670,"identity":"50da7f76-a3e5-41d1-836b-40f1056fcce2","order_by":7,"name":"Massimo De Felici","email":"","orcid":"","institution":"University of Rome Tor Vergata: Universita degli Studi di Roma Tor Vergata","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Massimo","middleName":"","lastName":"De Felici","suffix":""},{"id":20935671,"identity":"c5ff934a-222b-45b5-9487-285b59e485c6","order_by":8,"name":"Wei Shen","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Shen","suffix":""},{"id":20935672,"identity":"4343cc7a-63f3-4a9a-9f3f-416cfdb65ae8","order_by":9,"name":"Shun-Feng Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDAC5gNsIEoOwmMjRgtbAliZMelaEhuI1iLfxvzswccdtel9x88YMHwoO8zAP7sBvxaDY2zmhjPPHM+deSbHgHHGucMMEncOENAi38Mmzdt2LHfDgRwDZt62wwwGEgmEHMbDJv237Vi6wfk3Bsx/idHCcAyohbGtJsHgBtAWRmK0AP1iJtnbdsBw5o1nBQd7zqXzSNwg6DDmZxI/2+rk+c4nb3zwo8xajn8GIYdBwGEGhgNgxMBDlHogqIOoHwWjYBSMglGADQAAzFNDjxHgQLAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2836-2762","institution":"Qingdao Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shun-Feng","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2021-04-12 14:51:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-414853/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-414853/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00418-021-02034-4","type":"published","date":"2021-09-29T12:10:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":8024996,"identity":"0c91e4aa-cc65-45e0-abbb-3c7a81aa617f","added_by":"auto","created_at":"2021-04-14 23:05:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":386530,"visible":true,"origin":"","legend":"The culture and characterization of porcine skin derived stem cells (pSDSCs). (A) The morphology of pSDSCs at different culture stages. Separation and culturing of pSDSCs and the process of passaging. Scale bar=100 μm. (B) Flow cytometry analysis of Sox2, Oct4, and YAP in pSDSCs. (C) Representative WB and relative densitometric analysis of Sox2, Oct4 and YAP expression in pSDSCs at different stages. The results are presented as mean ± SD. *P \u003c 0.05; **P \u003c 0.01.","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/a116eb820fb4f5d4187dd26f.png"},{"id":8025423,"identity":"0630b216-1002-4370-8e91-bce89c0ba04e","added_by":"auto","created_at":"2021-04-14 23:11:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":473782,"visible":true,"origin":"","legend":"LPC increased the expression of YAP and promoted the pluripotency of pSDSCs. (A) Morphology of pSDSCs cultured with LPC at different concentrations. Scale bar=100 μm. (B) The percentage of cells positive for Oct4 was detected by FACs of pSDSCs cultured with different concentrations of LPC. (C) Relative mRNA expression levels of ATX, YAP, Sox2, Oct4 and β-catenin in control and 10 μM treated LPC groups. (D) Representative WB and relative densitometric analysis of ATX, YAP, Sox2, Oct4 and β-catenin in control and 10 μM LPC treated groups. The results are presented as mean ± SD. *P \u003c 0.05; **P \u003c 0.01.","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/36cc1b59683b5c1866ed5d95.png"},{"id":8024999,"identity":"16df59ea-4df1-4d17-bdb7-d2309e55ab82","added_by":"auto","created_at":"2021-04-14 23:05:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301177,"visible":true,"origin":"","legend":"LPC inhibited the differentiation of pSDSCs into pPGCLCs. (A) Morphology of pPGCLCs cultured in control and with 10 μM LPC following differentiation. (B) Representative WB and relative densitometric analysis of Vasa in control and 10 μM LPC groups following differentiation. The results are presented as mean ± SD. *P \u003c 0.05; **P \u003c 0.01. (C) The positive rate of Vasa was detected by FACs of pPGCLCs cultured with and without 10 μM LPC following differentiation. (D) The immunofluorescence with YAP antibody (red) and Hoechst33342 (blue) of pSDSCs and pPGCLCs. ","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/a0994fdd5133cd47b6376d6a.png"},{"id":8025151,"identity":"4ee9a560-abeb-4394-b220-b0469bc8c915","added_by":"auto","created_at":"2021-04-14 23:08:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":439702,"visible":true,"origin":"","legend":"siYAP decreased the expression of YAP. (A) Representative image of pPGCLCs cultured with negative control (NC) and siYAP groups. (B) The positive rate of CY3 was detected by FACs of pSDSCs in control and siYAP-1 groups. (C) Relative mRNA expression level of YAP in control, NC and three YAP siRNAs groups. (D) Representative WB and relative densitometric analysis of YAP in control, NC and three YAP siRNAs groups. The results are presented as mean ± SD. *P \u003c 0.05; **P \u003c 0.01. ","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/63d50a8feaae0f08df79c3bb.png"},{"id":8025424,"identity":"61c2eeaf-4646-4149-ad95-d5afc25ed45f","added_by":"auto","created_at":"2021-04-14 23:11:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":372394,"visible":true,"origin":"","legend":"YAP knockdown leads to a loss of pluripotency in pSDSCs. (A) Morphology of pSDSCs with and without the knockdown of YAP. Scale bar=100 μm. (B) Relative mRNA expression level of IVL, Sox2, Oct4 and β-catenin in control and siYAP groups. (C) Representative WB and relative densitometric analysis of IVL, Sox2, Oct4 and β-catenin in control and siYAP groups. (D) The positive rate of IVL was detected by FACs of pPGCLCs in the control, NC and siYAP groups. The results are presented as mean ± SD. *P \u003c 0.05; **P \u003c 0.01.","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/c8be5bba452aac92c8db5af5.png"},{"id":8025152,"identity":"846d204d-23fe-4ea2-a713-3eacab5edc93","added_by":"auto","created_at":"2021-04-14 23:08:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":740522,"visible":true,"origin":"","legend":"Analysis of pSDSCs treated with CHIR99021. (A) Morphology of pSDSCs cultured with different concentrations of CHIR. Scale bar=100 μm. (B)The positive rate of IVL was detected by FACs of pSDSCs cultured with different concentrations of CHIR. (C) Representative WB and relative densitometric analysis of β-catenin, IVL, Sox2 and Oct4 of pSDSCs cultured with different concentrations of CHIR. (D) Relative mRNA expression level of β-catenin, IVL, Sox2 and Oct4 in control, LPC and LPC+CHIR groups. E Representative WB and relative densitometric analysis of β-catenin, IVL, Sox2 and Oct4 in control, LPC and LPC+CHIR groups. The results are presented as mean ± SD. *P \u003c 0.05; **P \u003c 0.01.","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/89aeadfc123e252d388de026.png"},{"id":8025154,"identity":"fed167a0-bc6c-4794-8109-c9b0781c02aa","added_by":"auto","created_at":"2021-04-14 23:08:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":349486,"visible":true,"origin":"","legend":"Analysis of pSDSCs treated with XAV939. (A) Representative WB and relative densitometric analysis of β-catenin, Sox2 and Oct4 in pSDSCs cultured with different concentrations of XAV939. (B) The positive rate of Oct4 was detected by FACs of pPGCLCs cultured with different concentrations of XAV939. (C) Representative WB and relative densitometric analysis of β-catenin, Sox2 and Oct4 of pSDSCs in control, SiYAP and SiYAP+XAV groups. The results are presented as mean ± SD. *P \u003c 0.05; **P \u003c 0.01.","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/7cf2eed98e9d333c40f6e23c.png"},{"id":8025150,"identity":"a55235c9-fbcd-437e-9468-216eb8f9b70d","added_by":"auto","created_at":"2021-04-14 23:08:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":100213,"visible":true,"origin":"","legend":"Schematic model for the mechanism of YAP regulating the fate of pSDSCs is related to the Wnt/β-catenin pathway. Overexpress YAP promotes pSDSCs self-renewal by suppressing the Wnt/β-catenin signaling pathway. Knockdown of YAP induces the differentiation of pSDSCs through activating the Wnt/β-catenin signaling pathway. ","description":"","filename":"OnlineFig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/d62c33782567b80fd88a2f95.png"},{"id":17200760,"identity":"f572e5a5-4346-46fb-bb51-b913cd01f563","added_by":"auto","created_at":"2022-01-11 12:10:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":960427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/778fc81a-6a09-4dfd-bc0b-be8d66455d3b.pdf"},{"id":8025148,"identity":"6c6333a2-82d8-43c2-af00-38e07fa42d44","added_by":"auto","created_at":"2021-04-14 23:08:58","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":126434,"visible":true,"origin":"","legend":"The immunofluorescence with Sox2, Oct4 and Nanog antibodies (red) and Hoechst33342 (blue) of pSDSCs.","description":"","filename":"OnlineFigs.1.png","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/75b6365ad1bb36588e070b3c.png"},{"id":8024994,"identity":"1e324b4a-5d4b-430c-ad13-fcb482a610f5","added_by":"auto","created_at":"2021-04-14 23:05:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15670,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1202147.docx","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/3958967bb7cf775c1d57c51a.docx"},{"id":8024991,"identity":"748462e2-3568-4c0a-8da2-99c86f4d7b51","added_by":"auto","created_at":"2021-04-14 23:05:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14574,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2202147.docx","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/455cccd99af013a70440f09c.docx"},{"id":8024998,"identity":"05e852dc-d012-47b7-bb73-79e8744546de","added_by":"auto","created_at":"2021-04-14 23:05:58","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15173,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3202147.docx","url":"https://assets-eu.researchsquare.com/files/rs-414853/v1/b1615ec379ef36b9a6f52d0e.docx"}],"financialInterests":"","formattedTitle":"YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway","fulltext":[{"header":"Introduction","content":" \u003cp\u003eEmbryonic stem cells (ESCs) and adult stem cells (ASCs) have the ability of self-renewal and differentiation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Past studies have shown that ESCs and/or ASCs derived from human, mouse and pig have the capacity to differentiate into germ cells, oocyte-like cells (OLC) and even produced offspring [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Fetal skin harbors populations of stem cells responsible for development and maintenance into adulthood, making skin an attractive source for ASCs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to ethical considerations human embryos are not accessible for research, resulting in the use of porcine models. Porcine models have close similarities to humans with respect to their genetic relationship and the onset of epigenetic programs, making them ideal animal models for investigating human biology [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Porcine skin-derived stem cells (pSDSCs) offer an ideal \u003cem\u003ein vitro\u003c/em\u003e model to investigate human ASCs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. During last 10 years, germ cells differentiated from pSDSCs have been extensively investigated [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. But the efficiency of pSDSCs differentiation to porcine primordial germ cell-like cells (pPGCLCs) was very low [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This problem can be solved by improving the differentiation efficiency or generating large amounts of pSDSc through enhancing self-renewal. Recent studies have suggested that YAP is required for the efficient self-renewal of stem cells [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHippo signaling pathway has been widely studied in mammalian cells and animal models [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Hippo signaling regulates diverse cellular processes including cell proliferation and differentiation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In mammals, The core components of the Hippo pathway were mammalian Ste2-like kinases (MST1/2, Hpo orthologs) and large tumor suppressor kinase 1/2 (Lats1/2, Wts orthologs). Activation of Hippo signaling results in YAP being phosphorylated, sequestered to the cytoplasm, and degraded [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recently, emerging evidence suggests that YAP is a crucial regulator for maintaining the self-renewal of ESCs, induced pluripotent stem cells (iPSC), and ASCs \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, YAP also regulates self-renewal of tissue-specific progenitor cells, such as skin epidermal progenitor cells, cardiac progenitor cells, muscle progenitor cells, and neural progenitor cells [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For instance, Zhang et al found that activation of the transcriptional auxiliary activity of epidermal YAP in mice can inhibit their terminal differentiation leading to the expansion of epidermal progenitor cells in the basement membrane [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCanonical Wnt signaling is a highly conserved pathway [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] that is dependent on \u003cem\u003eβ\u003c/em\u003e-catenin whose role is primarily based on whether \u003cem\u003eβ\u003c/em\u003e-catenin is stable in the cytoplasm or translocated to the nucleus. In the basic state, \u003cem\u003eβ\u003c/em\u003e-catenin in the cytoplasm is mainly inhibited by GSK3\u003cem\u003eβ\u003c/em\u003e, which holds Wnt signaling in an inhibited state [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It was found that there is a multipoint cross correlation between the Wnt and Hippo signaling pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The interaction between Hippo signaling and Wnt signaling lies in the mutual regulation of their core molecules. The Hippo signaling pathway can affect the expression of Wnt target genes by changing the transcriptional activity of \u003cem\u003eβ\u003c/em\u003e-catenin. Qin et al reported that YAP overexpressing hESCs display lower levels of active \u003cem\u003eβ\u003c/em\u003e-catenin and Wnt target genes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Zhao et al found that YAP can interact with \u003cem\u003eβ\u003c/em\u003e-catenin and induce the expression of Wnt target genes in mouse heart tissue [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These studies confirmed that Hippo and Wnt signaling indeed regulate each other's activity.\u003c/p\u003e \u003cp\u003eResearchers suggested that the Hippo signaling pathway plays different roles in different kinds of stem cells, either being inhibitory or activating, depending on biological contexts [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To date, studies concerning the role of YAP in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this study, we used the pSDSCs to explore the effect of YAP on ASCs self-renewal. Our data suggested that the upregulation of YAP contributes to the maintenance and self-renewal capacity of pSDSCs by inhibiting Wnt signalling.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003epSDSCs isolation, culture and differentiation\u003c/h2\u003e \u003cp\u003ePigs were housed in Laixi, Shandong province. This research was approved by the Ethics Committee of Qingdao Agriculture University (Agreement No. 2019-011).\u003c/p\u003e \u003cp\u003ePorcine back skin tissue was collected from day 40\u0026ndash;45 gestation fetus (E40-45). The isolation, culture and differentiation of pSDSCs were described previously [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, the porcine skin tissue was mechanically dissociated using a pipette. The pSDSCs were incubated in SDSCs medium [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. After 12 days culture, the cells are positive for Sox2, Oct4 and YAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These cells could differentiate into pPGCLCs under \u003cem\u003ein vitro\u003c/em\u003e differentiation conditions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, the pSDSCs colonies were disaggregated into single cells and 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded into 60 mm tissue culture dish within differentiation medium [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Every 2 days the differentiation medium was changed. At ~\u0026thinsp;20 days cells with the morphological characteristics of pPGCLCs were present.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIndirect immunofluorescence assay (IFA)\u003c/h2\u003e \u003cp\u003eNanog, Sox2, Oct4 and YAP expression in pSDSCs were examined by IFA. The pSDSCs were collected and fixed in 4% paraformaldehyde for 30 min, then the samples were transferred to microscope slides treated with poly-L-lysine. The samples were permeabilized with 0.5% Triton-X-100 for 10 min and then blocked with 10% bovine serum albumin overnight at 4\u0026deg;C. The pSDSCs samples were covered with 50 \u0026micro;l primary antibody (Table S1) and incubated at 4\u0026deg;C overnight. After washing three times with PBS, the samples were incubated for 45 min at 37\u0026deg;C in a moisture chamber with secondary antibody (Table S1). The slides were washed three times with PBS and then incubated with Hoechst 33342 for staining the cell nucleus. Finally, the samples were viewed under a fluorescence microscope (Leica, Germany) and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence activated cell sorting (FACS) analysis\u003c/h2\u003e \u003cp\u003eThe prepared pSDSCs (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/ml) were fixed in 80% methanol. The pSDSCs were incubated successively with primary antibodies and secondary antibody (Table S1) at 37\u0026deg;C for 60 min. Then, the cells were centrifuged and were analyzed by Flow cytometry (Becton Dickinson, Mountain View, CA). Data were analyzed using WinMDI 2.9 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSmall interfering RNA (siRNA) transfection\u003c/h2\u003e \u003cp\u003eThree fluorescein-labeled siRNA targeted to YAP (siYAP-1, siYAP-2, siYAP-3) and fluorescein-labeled non-sense siRNA (siYAP-negative control, siYAP-NC) were purchased form GenePharma Co., Ltd. (Shanghai, China) (Table S2). Each siRNAs were transfected at a concentration of 30 pmol into the cells using lipofectamin 2000 (11,668\u0026thinsp;\u0026minus;\u0026thinsp;019; Invitrogen, Carlsbad, CA) following the manufacturer\u0026rsquo;s protocol. The pSDSCs were collected for further experiment 48h post transfection. All the siRNA experiments were performed three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and RT-qPCR\u003c/h2\u003e \u003cp\u003eAs previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], the total RNA was extracted from pSDSCs with the RNA prep pure Micro Kit (Aidlab, RN07, Beijing, China). The cDNA was synthesized by TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AT311-03, Beijing, China). RT-qPCR amplification with specific primers (Table S3) was done using a Light Cycler 480 (Roche, Germany). The relative transcript abundance were calculated by the 2 \u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and normalized according to the housekeeping gene \u003cem\u003eGapdh\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted using RIPA buffer (Beyotime, P0013C, Nantong, China). SDS was mixed with the samples and boiled for 5 min in water to denature proteins, then SDS-PAGE was used to separate proteins. The proteins were transferred onto a poly-vinylidene fluoride (PVDF) membrane (Millipore, ISEQ00010, USA), and blocked in TBST containing 6% BSA for 4 hours. The membranes were incubated with primary antibody (Table S1) at 4\u0026deg;C, and then a horseradish peroxidase (HRP)-conjugated corresponding secondary antibody (Beyotime) was incubated at room temperature for 120 min on the second day. The BeyoECL Plus Kit (Beyotime, P0018) was used for chemiluminescence detection. The relative expression level of the target protein to GAPDH was calculated using AlphaView SA (ProteinSimple, CA, USA) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8 (GraphPad Software, San Diego, CA) was used for data analysis with One way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s test for multiple comparisons; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 means statistically significant difference, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 means extremely significant difference, and P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 means not significant (ns). The data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (S.E.) and all the experiments were repeated at least three times.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEndogenous YAP expression in pSDSCs\u003c/h2\u003e \u003cp\u003epSDSCs were cultured using a previously described method [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Spherical aggregates of cells were clearly visible by day 8 of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). FACS analysis during the culturing of the pSDSCs showed dynamic marker changes with the percentage positive for Sox2 being 0.09% at 4 days, 0.19% at 8 days, and 11.69% at 12 days; for Oct4, at 4 days 0.51%, 8 days 4.37%, and 12 days 27.87%; for YAP, at 4 days 5.75%, 8 days 8.76%, 12 days 41.37%. Using FACS and western blot analysis revealed that, during the culturing of pSDSCs, the percentage of the pluripotency markers Sox2 and Oct4 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] increased dramatically. Concomitant with the increase of stem cell markers, YAP positive cells were also significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results confirmed that YAP expression is increased during the culturing of pSDSCs suggesting it may play a role in self-renewal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLPC activates YAP and promotes pSDSCs pluripotency\u003c/h2\u003e \u003cp\u003eWe explored existing data to identify a small molecule that could increase YAP expression. We found that lysophosphatidylcholine (LPC) could be hydrolyzed into lysophosphatidic acid (LPA) by autotaxin (ATX) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and lead to the activation of YAP in cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In order to investigate the potential effects of different concentrations of LPC on regulating YAP in pSDSCs 0, 5, 10 and 25 \u0026micro;M LPC was added to the pSDSCs medium, respectively. As show in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, after 12 days culture, spherical colonies were observed in all treatments. FACS analysis of the pSDSCs in the various treatments showed that the percentage of Oct4 positive cells increased in the LPC-treated groups in a dose-dependent manner up to 10 \u0026micro;M (44.84% in 5 \u0026micro;M LPC, 53.79% in 10 \u0026micro;M LPC and 41.84% in 25 \u0026micro;M LPC) compared to the control group (28.67 %) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Based on these results, we used 10 \u0026micro;M LPC for the subsequent studies. In agreement with previous findings [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], exposure to LPC led to an increase in the mRNA and protein levels of YAP in the pSDSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These observations demonstrated that YAP overexpression can be induced by supplementing the culture medium with 10 \u0026micro;M LPC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the potential effects of LPC induced YAP expression on regulating the self-renewal of pSDSCs, we examined the protein expression levels of Oct4 and Sox2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, both Oct4 and Sox2 were upregulated, indicating that YAP plays an important role in the self-renewal of pSDSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLPC induced YAP counteract pSDSCs transdifferentiated into pPGCLCs\u003c/h2\u003e \u003cp\u003eTo evaluated the importance of YAP in pSDSCs self-renewal, we examined their ability to differentiate to pPGCLCs in the presence of LPC [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The pSDSCs were plated in differentiation medium in 6 cm dishes, and incubated for 20 days to induce the formation of pPGCLCs. In the untreated control pSDSCs formed numerous pPGCLCs and high expression of the pPGC marker Vasa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); In contrast, LPC treated pSDSCs exhibited resistant to the differentiation and significantly reduced Vasa protein levels under the same differentiation conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Supporting this, FACS analysis found the percentage of Vasa-positive cells was 48.73% in the untreated control and 7.93% in the LPC treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). YAP shuttles between the cytoplasm (inactive) and the nucleus (active) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore YAP localization is essential in maintaining the pluripotent properties of stem cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In our experiments, immunofluorescent staining revealed that YAP was predominantly located in the nucleus of undifferentiated pSDSCs, while significant YAP positive signals was found in the cytoplasm of differentiated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Similar results to those found in a previous study using human ESCs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In conclusion, these results demonstrate that YAP activation maintains pSDSCs pluripotency even under differentiation conditions \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of YAP in pSDSCs by siRNA\u003c/h2\u003e \u003cp\u003eTo further confirm the role of YAP in pSDSC self-renewal and differentiation, sequence-specific siRNAs against YAP were introduced. Fluorescein-labeled non-sense siRNA (siRNA-NC) was used to optimize the transfection conditions. The expression of the red fluorescent protein was examined after transfection for 48 h by fluorescence microscope. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA demonstrating a successful transfection. In order to further investigate the transfection effect of siRNA, FACS was employed. The results showed the percentage of CY3 positive cells were similar in the siRNA-NC group and the siYAP-1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, RT-qPCR and western blot demonstrated that siYAP-1 had the strongest downregulation effect on YAP compared to the other three siRNA groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of YAP gene expression suppressed pSDSCs pluripotency and promoted pSDSCs differentiation\u003c/h2\u003e \u003cp\u003eStriking morphological changes were observed in the siRNA transfected group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Knockdown of YAP induced the pSDSCs cells to acquire a fibroblast-like morphology. The pSDSCs colonies lost their suspended culture and attached and spread on the culture surface. To gain an insight into the molecular mechanisms underlying this morphological phenomenon, the expression levels of two pluripotency markers, Oct4 and Sox2, and a defined terminal differentiation marker involucrin (IVL) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] were analyzed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, YAP knockdown resulted in the downregulation of Oct4 and Sox2 at the mRNA level and upregulated the expression of IVL and \u003cem\u003eβ\u003c/em\u003e-catenin. Western blot analysis of protein expression also indicated the same tendencies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In addition, the loss of pluripotency in the siYAP transfected pSDSCs was further confirmed by an increase in the percentage of IVL positive cells using FACS analysis. The results showed the percentage of IVL positive cells in the control group was 13.77%, but in the siYAP treated group, the percentage was increased to 24.19% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eYAP promotes pSDSCs self-renewal by suppressing the Wnt/\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eβ\u003c/span\u003e \u003cb\u003e-catenin signaling pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies demonstrated that YAP promotes human and mouse stem cell self-renewal in response to suppressing the differentiation-inducing effects of the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin pathway [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To elucidate the role of YAP in pSDSCs self-renewal and its connection with the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling we investigated whether YAP regulates \u003cem\u003eβ\u003c/em\u003e-catenin activity. Western blot analysis revealed that the protein expression level of \u003cem\u003eβ\u003c/em\u003e-catenin was effectively suppressed in LPC treated pSDSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), while \u003cem\u003eβ\u003c/em\u003e-catenin was robustly activated after siYAP-1 transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results demonstrate a functional connection between YAP and the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway.\u003c/p\u003e \u003cp\u003eTo further confirm the functional connection between YAP and the Wnt pathway and the role in maintaining the pluripotency of pSDSCs, we assessed the effect of the GSK3 inhibitor CHIR99021 (CHIR), which activates Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], on the differentiation of pSDSCs in LPC treatment. Three dosages (1 \u0026micro;M, 3 \u0026micro;M, and 6 \u0026micro;M) of CHIR were used. Interestingly, increasing the concentration of CHIR in pSDSCs induced cells to acquire a flattened phenotype, indicating a loss of characteristic pSDSC morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). FACS analysis indicated that there was a significant upregulation in the percentage of IVL positive cells in the 3 \u0026micro;M and 6 \u0026micro;M CHIR treated groups when compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, the protein expression levels of \u003cem\u003eβ\u003c/em\u003e-catenin and IVL were increased, but both Oct4 and Sox2 were significant decreased and the degrees of reduction of both self-renewal markers were closely correlated with the concentration of CHIR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Based on the above results, we used 6 \u0026micro;M CHIR for subsequent studies. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, the Wnt pathway was effectively repressed in the 10 \u0026micro;M LPC-treated pSDSCs (L-pSDSCs). Upon treating L-pSDSCs with 6 \u0026micro;M CHIR, the expression of \u003cem\u003eβ\u003c/em\u003e-catenin increased but Oct4 and Sox2 decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These results show that activating Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling partially restores the differentiation ability of the L-pSDSC group, as evidenced by the western blot results showing higher expression of IVL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Therefore, the results investigating the addition with CHIR imply that YAP promotes pSDSCs self-renewal by suppressing the Wnt/\u003cem\u003eβ\u003c/em\u003e- catenin signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eYAP prevents pSDSCs differentiation by modulating Wnt/\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eβ\u003c/span\u003e \u003cb\u003e-catenin signalling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to further elucidate the mechanism by which YAP regulates pSDSCs differentiation through Wnt signaling, we investigated the effect of XAV939, a Wnt signaling antagonist, on the self-renewal of pSDSCs following YAP knockdown. Three dosages of XAV939 were tested (1 \u0026micro;M, 5 \u0026micro;M, and 10 \u0026micro;M). With increasing concentration of XAV939 the pSDSCs showed an elevated clonal growth rate, and propagated readily in culture for more than 5 passages (data not show). Western blot analysis indicated that \u003cem\u003eβ\u003c/em\u003e-catenin protein was effectively suppressed in the 10 \u0026micro;M XAV939 treated groups but Oct4 and Sox2 were markedly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Moreover, FACS analysis showed that the percentage of Oct4 positive cells was significantly upregulated in the 10 \u0026micro;M XAV939 treatment group when compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Next, using the siRNA system, we evaluated how YAP modulates pSDSCs self-renewal in the 10 \u0026micro;M XAV939 treated cells. The results indicated that siYAP led to increased expression of \u003cem\u003eβ\u003c/em\u003e-catenin in the pSDSCs but in the siYAP plus XAV939 group, \u003cem\u003eβ\u003c/em\u003e-catenin returned to the control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These results indicated that the Wnt pathway is important for the differentiation of pSDSCs. Moreover, it appears that YAP inhibits pSDSCs differentiation in response to Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eSelf-renewal is a critical aspect of stem cells allowing them to increase in number while maintaining pluripotency [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The molecular mechanisms involved in maintaining the self-renewal of ESCs and ASCs \u003cem\u003ein vitro\u003c/em\u003e has been a key issue in stem cell research. Emerging evidence has indicated that YAP may play an important role in maintaining the pluripotency of stem cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To date, reports concerning the influence of YAP on the pluripotency of ASCs are limited. In this paper, we confirmed that YAP helped maintain pSDSCs ability for self-renewal through suppressing the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway. Our observations demonstrated that concomitantly with the increase of YAP positive cells, the protein levels of Oct4 and Sox2, were also significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). On the other hand, YAP knockdown leads to a loss of pluripotency in pSDSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interestingly, higher expression of YAP prevented pSDSCs differentiation \u003cem\u003ein vitro\u003c/em\u003e even under differentiation inducing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, we found evidence that the Wnt pathway regulates pSDSCs differentiation. Our experiments indicated that the addition of CHIR, an activator of the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway, in pSDSCs medium results in a rapid loss of stem cell properties, including morphological changes and decreased expression of the pluripotency markers, Oct4 and Sox2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Conversely, when the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway is inhibited using XAV939 we confirmed that stem cell markers were positively upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). We also found evidence supporting a direct relationship between YAP and the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway. YAP overexpression or knockdown could affect the protein expression level of \u003cem\u003eβ\u003c/em\u003e-catenin, a core component of the Wnt pathway, and thereby inhibit or promote the differentiation of pSDSC. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE support that pSDSCs treated with LPC plus CHIR resulted in increased protein levels of \u003cem\u003eβ\u003c/em\u003e-catenin and IVL and decreased protein levels of Oct4 and Sox2 compared with that of LPC treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In addition, we also confirmed that knockdown of YAP would induce the differentiation of pSDSCs, but treatment with XAV939 after YAP knockdown prevented this differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). To summarize, these observations mutually corroborate each other and further establish that YAP plays an important role in maintaining the pluripotency of pSDSCs partly through repressing the Wnt/β-catenin signaling pathway.\u003c/p\u003e \u003cp\u003eRecent studies have shown that core transcription factors, such as Oct4, Sox2 and Nanog, play an essential role in establishing and controlling gene expression programs which define the identity of human and mouse ESCs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In particular, these core transcription factors are important for establishing the ability of ESCs and ASCs to self-renew and differentiate [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Several studies have presented data showing that YAP can facilitate self-renewal through the upregulation of core transcription factors, such as Oct4, Sox2 and Nanog, and inhibiting YAP function results in the differentiation of ESCs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, based on transcriptional profiling, Ramalho-Santos et al demonstrated that YAP also has a critical role in the self-renewing ability of ASCs, such as neural stem cells and hematopoietic stem cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These results strongly support our work, for instance, in this paper self-renewal of the pSDSCs was identified based on the expression level of protein markers, Oct4, Sox2 and Nanog (Figure S1), and our results showed a striking correlation between the protein expression level of YAP and that of Oct4 and Sox2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). YAP was highly expressed in self-renewing pSDSCs and was downregulated when cells were induced to differentiate. Meanwhile, studies described in this manuscript revealed that overexpression of YAP prevented the differentiation of pSDSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and the depletion of YAP by siRNAs suppressed the self-renewal of pSDSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These observations strongly confirm a particularly important role for YAP in maintaining the \u0026ldquo;stemness\u0026rdquo; of pSDSCs.\u003c/p\u003e \u003cp\u003eLian et al. found that YAP can positively regulate pluripotent genes such as Nanog, Oct4 and Sox2 by genome-wide analysis of mouse ESCs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Bora-Singhal et al. further found that the structural domain of the YAP protein can interact with the transcription factor Oct4 to induce the transcriptional activity of Sox2 regulated downstream targets and maintain the self-renewal ability of stem cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Reciprocally, Seo et al showed YAP expression is regulated by Sox2 and could compensate for the self-renewal defect caused by Sox2 inactivation in mesenchymal stem cells (MSCs) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Given these facts, the relationship between YAP and Oct4 or Sox2 is rather interesting and complex and requires further investigation.\u003c/p\u003e \u003cp\u003eExtensive crosstalk between YAP and \u003cem\u003eβ\u003c/em\u003e-catenin, a key protein of the Wnt signaling pathway, has been observed [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Qin et al. confirmed that YAP promotes human ESCs naive pluripotency by interfering with \u003cem\u003eβ\u003c/em\u003e-catenin and retaining it in the cytoplasm to suppress Wnt signaling.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Seo et at found that in MSCs, YAP binds \u003cem\u003eβ\u003c/em\u003e-catenin and inhibits canonical Wnt signaling to prevent MSCs differentiation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Our results are in line with these previous findings, after overexpression of YAP, the expression of \u003cem\u003eβ\u003c/em\u003e-catenin was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Conversely, the expression of \u003cem\u003eβ\u003c/em\u003e-catenin increased when we knockdown YAP using RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Similarly, when we added CHIR to the LPC-treated pSDSCs we found that the differentiation ability of pSDSCs was partially restored through activation of the Wnt pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). This suggests that YAP was sufficient to maintain the self-renewal characteristics of pSDSCs by inhibiting the Wnt pathway. In order to more clearly investigate the mechanism of YAP regulating the self-renewal of pSDSCs through the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway, we added the inhibitor of the Wnt pathway XAV939 to the pSDSCs that had YAP knocked down. With the addition of XAV939, we found that the cell fate of pSDSCs was altered. Compared with pSDSCs with low YAP, the addition of XAV939 resulted in pSDSCs with higher self-renewal ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Taken together, this supports YAP regulating the fate of pSDSCs though its effects on the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, we have shown that YAP regulates the self-renewal of pSDSCs by inhibiting the Wnt pathway. However, the specific processes and mechanisms of the interaction between the Hippo and Wnt pathways are not fully understood. Further study of the interactions between the members of these two signaling pathways are expected to provide new insights for the molecular basis of many biological processes.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eSDSCs: Skin-derived stem cells; ASCs: Adult stem cells; pSDSCs: Porcine SDSCs; YAP: Yes-associated protein; siRNAs: Small interfering RNA; ESCs: Embryonic stem cells; OLC: Oocyte-like cells; pPGCLCs: Porcine primordial germ cell-like cells; MST1/2: Mammalian Ste2-like kinases; Lats1/2: Large tumor suppressor kinase 1/2; iPSC: Induced pluripotent stem cells; IFA: Indirect immunofluorescence assay; FACS: Fluorescence activated cell sorting; LPC: Lysophosphatidylcholine; LPA: Lysophosphatidic acid; ATX: autotaxin; IVL: involucrin; CHIR: CHIR99021; L-pSDSCs: LPC-treated pSDSCs; MSCs: mesenchymal stem cells;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the members of the institute of Reproductive Sciences, Qingdao Agricultural University who make efforts to these experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSW and CSF conceived the study; YHC, SY, ZMY and SJD carried out experiments and data analysis; ZSE, Paul WD, Francesca GK and Massimo DF interpreted the data\u003c/p\u003e\n\u003cp\u003eand wrote the manuscript. All authors approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (32072734, 31672423), Shandong Province Key Research and Development Program (2019GSF107082) and Taishan Scholar Construction Foundation of Shandong Province (ts20190946).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments involving live animals were conducted in accordance with the \"Regulations for the Administration of Affairs Concerning Experimental Animals\" promulgated by the State Science and Technology Commission of Shandong Province. The study was approved by the ethics committee of Qingdao Agricultural University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2 \u003c/sup\u003eAnimal Husbandry General Station of Shandong Province, Jinan 250010, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Department of Animal Sciences, Auburn University, Auburn, AL 36849, USA.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003e Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome 00133, Italy.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMo JS, Park HW, Guan KL. 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Bone research. 2018; 6(1):1-12.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"pSDSCs, YAP, β-catenin, Wnt pathway, self-renewal ","lastPublishedDoi":"10.21203/rs.3.rs-414853/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-414853/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSkin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of ASCs is a hot topic. Porcine models have close similarities to humans and porcine SDSCs (pSDSCs) offer an ideal \u003cem\u003ein vitro\u003c/em\u003e model to investigate human ASCs. To date, studies concerning the role of yes-associated protein (YAP) in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this paper, we explore the link between the transcriptional regulator YAP and the fate of pSDSCs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Sox2, Oct4. The overexpression of YAP prevented the differentiation of pSDSCs and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway. When an activator of the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939 an inhibitor of Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eour results suggested that, YAP and the Wnt/\u003cem\u003eβ\u003c/em\u003e-catenin signaling pathway interact to regulate the fate of pSDSCs.\u003c/p\u003e","manuscriptTitle":"YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-14 23:05:56","doi":"10.21203/rs.3.rs-414853/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d90bded-838c-4ef7-b7a6-06e3f0f5b75c","owner":[],"postedDate":"April 14th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":3625421,"name":"Animal Science"},{"id":3625422,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2022-01-11T12:10:23+00:00","versionOfRecord":{"articleIdentity":"rs-414853","link":"https://doi.org/10.1007/s00418-021-02034-4","journal":{"identity":"histochemistry-and-cell-biology","isVorOnly":false,"title":"Histochemistry and Cell Biology"},"publishedOn":"2021-09-29 12:10:23","publishedOnDateReadable":"September 29th, 2021"},"versionCreatedAt":"2021-04-14 23:05:56","video":"","vorDoi":"10.1007/s00418-021-02034-4","vorDoiUrl":"https://doi.org/10.1007/s00418-021-02034-4","workflowStages":[]},"version":"v1","identity":"rs-414853","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-414853","identity":"rs-414853","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}