For morphological analysis of pluripotency states, HUES8 primed hESCs were grown on Matrigel and dedifferentiated to naïve pluripotency using previously reported conditions in an mTeSR-based medium supplemented with MEK (PD0325901) and GSK3 (CHIR99021) inhibitors, the adenylyl cyclase activator forskolin, human leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and ascorbic acid (Duggal et al., 2015; Qin et al., 2016). We found that this treatment induced colonies to develop a prominent dome-shape by day 6 of dedifferentiation and increase expression of naïve pluripotency markers DNMT3L, DPPA3, KLF2, and KLF4, as determined by rt-PCR (Figure 1A and B), which confirms the transition to a naïve state. Staining fixed cells for actin filaments with phalloidin revealed that naïve but not primed colonies had a ring of bundled actin filaments at the colony periphery (Figure 1C). A similar actin ring also formed around colonies of naïve H9 cells and naïve WTC11 induced PSCs (iPSCs) (Figure 1—figure supplement 1A) as well as HUES8 cells dedifferentiated by alternative medium supplements (Figure 1—figure supplement 1B). Moreover, the actin ring assembled independently of naïve colony size (Figure 1—figure supplement 1C).

An actin filament ring is reported to encircle colonies of clonal human PSCs to provide a mechanosensitive element linked to focal adhesions (Närvä et al., 2017). The actin filament ring we observed around naïve hESC colonies was instead tethered to adherens junctions, as indicated by co-labeling for β-catenin, with separated interdigitated adherens junctions suggesting a contractile force (Figure 1D, crosshairs). The contractile property of the ring was also suggested by the actin ring around naïve hESC colonies being decorated with pMLC as determined by immunolabeling (Figure 1E). In contrast, primed hESC colonies had irregular aggregates of pMLC with limited co-localization with actin filaments. Additionally, immunolabeling for cortactin, a regulator of cortical branched actin filaments, partly overlapped with the actin ring (Figure 1F). The subcellular localization of the actin ring was confirmed with 3D reconstruction (Figure 1—figure supplement 1D). Together, these data indicate a contractile actin ring surrounding naïve but not primed hESC colonies, with the ring likely composed of unbranched actin filaments, which bind pMLC, and branched filaments, which bind cortactin.

The nature of the actin ring enclosing colonies of naïve but not primed hESC colonies suggested a potential difference in colony mechanics, which we determined by using traction force microscopy. Increased cell-matrix traction forces are associated with destabilized adherens junctions in epithelial monolayers (Mertz et al., 2013; Scarpa et al., 2015). Consistent with pMLC localization, primed colonies exhibited elevated cell-substrate tractions that were distributed throughout the colony (Figure 1G, left; Figure 1—figure supplement 1E). In contrast, naïve colonies exhibited overall low magnitude cell-substrate tractions that were localized to the colony periphery and largely absent from the colony interior (Figure 1G, right; Figure 1—figure supplement 1E), suggesting decreased cell-substrate tensional force and a likely shift to more stabilized cell-cell forces. Along with pMLC localization, these low traction forces are consistent with uniform cell-cell adhesion in naïve hESC colonies. Together these data reveal a significant reorganization of the actin cytoskeleton during the transition to a naïve state of pluripotency that includes the assembly of a contractile actin ring surrounding naïve cell colonies, coincident with attenuated cell-substrate traction forces and a transition to enhanced cell-cell junction traction force within the colony unit.

The assembly of an actin ring in naïve but not primed hESC colonies led us to ask whether the actin ring has a functional significance in the transition to naïve pluripotency. New actin filaments are predominantly generated by two distinct nucleators, the Arp2/3 complex, which generates branched filaments, and formins, which generate unbranched filaments (Pollard, 2007). We found that the actin ring assembled when naïve cells are generated in the presence of SMIFH2, a broad-spectrum inhibitor of formin activity (Rizvi et al., 2009; Ganguly et al., 2015) but not CK666, a selective pharmacological inhibitor of Arp2/3 complex activity (Nolen et al., 2009; Yang et al., 2012 ; Figure 2A), despite the ring likely being composed of both unbranched and branched actin filaments as indicated by pMLC and cortactin immunolabeling. Additionally, CK666 blocked increased expression of markers of naïve pluripotency seen in controls, determined by quantitative PCR (qPCR) of PECAM1, ESRRB, KLF4, and DNMT3L (Figure 2B). To eliminate the possibility that CK666 treatment led cells to exit pluripotency and differentiate, we immunolabeled for the general pluripotency markers OCT4 and SOX2 and found that CK666-treated cells remained broadly pluripotent (Figure 2—figure supplement 1A and B). To further confirm that Arp2/3 complex activity is necessary for the actin filament ring, we also treated cells with CK869, another inhibitor of Arp2/3 complex activity (Hetrick et al., 2013) . Consistent with our finding using CK666, CK869 blocked the formation of the actin ring without impairing pluripotency (Figure 2—figure supplement 1A and B).

To further assess the pluripotent state of cells dedifferentiated in the presence of CK666, we immunolabeled for the primed pluripotent marker SSEA3 (Trusler et al., 2018). SSEA3 expression significantly decreased with dedifferentiation in control conditions, as previously reported (Liu et al., 2017) but remained at levels similar to primed cells in the presence of CK666 (Figure 2C and D). Additionally, the naïve pluripotency marker KLF4 (Takashima et al., 2014) translocated from the cytoplasm to the nucleus with control dedifferentiation but not in the presence of CK666 (Figure 2C and E). Although branched actin filaments generated by Arp2/3 complex promote cell cycle progression and proliferation (Molinie et al., 2019), we used an EdU pulse (1 hour) to show no difference in EdU incorporation in the absence or presence of CK666 or CK869 (Figure 2—figure supplement 1C), indicating that the inhibitors did not change cell proliferation. Consistent with CK666 disrupting the actin filament ring, it also disrupted localization of cortactin around naïve colonies (Figure 2—figure supplement 1D).

We further tested effects of CK666 on a functional naïve pluripotent state by staining for alkaline phosphatase and scoring for colony formation, which indicates the capacity for clonogenic expansion and self-renewal (Rostovskaya et al., 2019). Primed and naïve hESCs were passaged and plated at clonogenic cell numbers and maintained for 5 days without or with CK666. In controls, colony formation was greater in naïve compared with primed hESC, as previously reported (Chen et al., 2022). However, with CK666 but not CK689, an inactive analog of CK666, there was no increase in colony formation in naïve compared with primed cells (Figure 2F). Additionally, traction force microscopy revealed that elevated cell-substrate tractions throughout colonies of primed but not naïve cells (Figure 1G) were retained when hESCs were dedifferentiated in the presence of CK666 (Figure 2G; Figure 2—figure supplement 1E). These data identify an essential role for the Arp2/3 complex in promoting an actin filament ring and uniform naïve colony mechanics as well as acquiring a naïve pluripotent state in hESCs.

To understand how Arp2/3 complex activity affects the transcriptional circuitry required for naïve pluripotency, we performed bulk RNA-sequencing (RNAseq) on primed and naïve hESCs and hESCs dedifferentiated in the presence of CK666 (Figure 3A). We found that primed, naïve, and CK666-treated cells had a total of 12,817 differentially expressed genes (DEGs) with an adjusted pval<0.05 (false discovery rate [FDR]-corrected by Benjamini-Hochberg procedure). Of these DEGs, 182 were unique to control primed cells compared with control naïve cells and were not differentially expressed in CK666-treated cells; CK666-treated cells compared with control primed or control naïve cells had 102 and 502 DEGs, respectively. To determine the transcriptional networks involved in the dedifferentiation from primed to naïve pluripotency, we identified KEGG pathways in control naïve dedifferentiation which revealed Hippo signaling as the top candidate (Figure 3B). Additionally, transcription factor binding motif analysis revealed that one of the top candidates was TEAD2 (Figure 3C), which is a downstream effector of Hippo signaling.

The Hippo effector protein YAP is a known regulator of the human naïve pluripotent state, with overexpression of YAP in PSCs promoting the acquisition of naïve pluripotency (Qin et al., 2016). Although actin filament dynamics is reported to regulate YAP signaling (Hsiao et al., 2016; Furukawa et al., 2017), to our knowledge a role for Arp2/3 complex activity regulating YAP or TAZ activity in human naïve pluripotency has not been reported. For an unbiased global analysis of known YAP target genes, we used two publicly available datasets (Estarás et al., 2017; Pagliari et al., 2021) and found that of the 3744 YAP target genes identified in our RNAseq dataset, 3156 (84%) were not differentially expressed in any condition and 588 (16%) were enriched in one or multiple conditions. Of those 588 enriched YAP target genes, 174 (30%) were significantly enriched in the control naïve dedifferentiation condition versus the control primed condition; 407 (69%) were significantly enriched among CK666-treated dedifferentiation condition versus the control primed condition; and 7 (1%) were significantly enriched in both conditions versus the control primed condition (Figure 3D, adjusted pval>0.05).

Of the genes significantly enriched in the control naïve condition compared with the control primed condition, known naïve pluripotency markers such as OTX2, DLG2, and CRY1 were significantly upregulated, and these naïve markers were not significantly increased in CK666-treated cells (Figure 3E, left). As expected, genes significantly enriched among both DEG lists included known YAP and Hippo targets such as ANKRD1, SLIT2, and CHD10 (Figure 3E, right). Genes significantly enriched among the CK666-treated condition include the negative Hippo regulator AMOT (Zhao et al., 2011), and lineage-commitment genes such as SOX6 and SPEF2 (Figure 3E, middle). To verify this prediction, we immunolabeled cells to determine YAP localization and found increased nuclear to cytoplasmic ratios of YAP (Figure 3F and G) and TAZ (Figure 3—figure supplement 1A and B) with control dedifferentiation that was blocked by CK666. Immunoblotting total cell lysates for total YAP abundance indicated no difference between primed and naïve hESCs and a small but significant increase in hESCs treated with CK666 (Figure 3—figure supplement 1C and D). These data indicate that a Hippo signaling pathway program, driven by mediators such as YAP, is active during dedifferentiation to naïve pluripotency but is disrupted by inhibiting Arp2/3 complex activity.

Consistent with our findings, actin filaments and associated proteins generate mechanical forces during preimplantation development that contribute to differentiation throughout the blastocyst stage by modulating mechanosensitive pathways such as Hippo (Hirate et al., 2015; Zenker et al., 2018). These actin structures allow cells within the developing blastocyst to organize based on contractility, coupling mechanosensing, and fate specification (Maître et al., 2016). Therefore, we hypothesized that Arp2/3 complex activity facilitated naïve dedifferentiation through increasing YAP nuclear localization. To test this prediction, we asked whether primed hESCs stably expressing a constitutively active, nuclear-localized YAP (YAP-S127A) could restore naïve dedifferentiation in the presence of CK666. Accordingly, we found that that expression of YAP-S127A in the presence of CK666 restored two markers of the naïve state, increased nuclear localization of KLF4 (Figure 4A and B) and decreased SSEA3 (Figure 4C and D). In contrast, acquisition of a naïve pluripotency state remained blocked with CK666 treatment in cells overexpressing wildtype YAP (YAP-WT) (Figure 4A–D). Colony formation, a functional form of naïve pluripotency, was also restored by heterologous expression of YAP-S127A but not YAP-WT in the presence of CK666 (Figure 4E), indicating that Arp2/3 complex activity is necessary for active nuclear-localized YAP to induce a naïve pluripotency state. In addition, we found that expressing YAP-S127A but not YAP-WT in the presence of CK666 partially restored a contractile actin ring enclosing naïve cell colonies (Figure 4F), indicating bidirectional signaling between actin filament remodeling and active nuclear-localized YAP for assembly of the actin filament ring around naïve hESC colonies. Thus, we conclude that both the transition from primed to naïve hESC pluripotency includes an Arp2/3 complex-dependent actin filament remodeling that enables active nuclear-localized YAP, and that nuclear-localized YAP enables actin filament remodeling for naïve pluripotency.

Primed hESC lines HUES8 (RRID: CVCL_B207), H9 (RRID: CVCL_1240), and WTC11 (RRID: CVCL_Y803) were maintained on Matrigel (Corning Life Science #354277) in feeder-free mTeSR-1 medium (STEMCELL Technologies # 85850) at 37°C with 5% CO₂ with daily medium changes. Cells were passaged approximately every 3 days, by dissociating with Accutase (STEMCELL Technologies #07920) and including the Rho-associated coiled-coil kinase (ROCKi) inhibitor Y-276932 (10 µM; Selleckchem #S1049) in the plating medium to facilitate survival. All cell lines were routinely confirmed to be negative for mycoplasma by testing with a MycoAlert Mycoplasma Detection Kit (Lonza # LT07-701). The HUES8 cell line was authenticated using STR Profiling (Human Cell STR Profiling Service; ATCC).

Dedifferentiation was completed using previously published methods (Duggal et al., 2015; Qin et al., 2016). In brief, cells were plated at a density of 10,000 cells per cm (Nakamura et al., 2016) in the presence of ROCKi (10 µM). After 24 hr, cells were washed three times with PBS and incubated in naïve dedifferentiation medium of mTeSR-1 supplemented with 12 ng/mL bFGF (Peprotech #AF-100-18B), 1 µM PD0325901 (MEKi, Selleckchem #S1036), 3 µM CHIR99021 (GSK3βi, Selleckchem #S2924), 10 µM forskolin (STEMCELL Technologies #72112), 50 ng/mL ascorbic acid (Sigma # A92902), and 1000 U recombinant human LIF (STEMCELL Technologies #78055). Medium was replaced daily, and cells were passaged every 3 days with Accutase. Where indicated, the naïve dedifferentiation medium 2iFL was used, which consisted of mTeSR-1 supplemented with 0.5 μM PD0325901, 3 μM CHIR9902, 10 μM forskolin, and 1000 U recombinant human LIF. For actin nucleator experiments, naïve dedifferentiation media was supplemented with either 80 μM CK666 (EMD Millipore #182515), 80 μM CK689 (EMD Millipore #182517), or 50 μM SMIFH2 (Sigma #S4826) throughout the entire dedifferentiation process. When passaging, media was supplemented with both ROCKi (10 µM) and the appropriate inhibitor.

Total RNA was isolated using RNAeasy Mini Plus (QIAGEN #74134) kits and cDNA was generated using iScript cDNA Synthesis kits (Bio-Rad #1708890) as per the manufacturer’s specifications. Quantitative PCR was performed using iQ SYBR Green Supermix (Bio-Rad #1708882) and analyzed on a QuantStudio six Flex Real-Time PCR System (Applied Biosystems).

For microscopy, cells were plated on Matrigel-coated glass coverslips prepared using an ultrasonic cleaning bath (Branson). In brief, coverslips were sonicated for 20 min in the presence of diluted Versa-Clear (Fisher Scientific #18-200-700) in double distilled H₂O (ddH₂O), washed three times using ddH₂O, sonicated for 20 min in ddH₂O, washed three times using ddH₂O, and sterilized and stored in 70% ethanol until use. Cells were maintained for indicated times, typically 3 days, washed briefly with PBS, fixed with 4% PFA for 12 min at room temperature (RT), permeabilized with 0.1% Triton X-100 in PBS for 5 min, and incubated with blocking buffer consisting of 0.1% Triton X-100 in PBS and 1% BSA for 1 hr. Cells were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C, washed with PBS three times, and incubated for 1 hr at RT with secondary antibodies, followed by a final PBS 3× wash, with the second wash containing Hoechst 33342 (1:10,000; Molecular Probes #H-3570) to stain nuclei. To stain for actin filaments, either rhodamine phalloidin (1:400, Invitrogen #R415) or Phalloidin-iFluor 647 (1:1000, Abcam #ab176759) was added to the secondary antibody incubation. Secondary antibodies used were Alexa Fluor 488 or 594 for the appropriate species primaries (1:500, Invitrogen #A-11037). To assess proliferation, EdU staining was performed according to the manufacturer’s guidelines with a 1 hr EdU pulse (Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dye, Fisher Scientific, #C10340).

Cells were imaged using an inverted microscope system (Nikon Eclipse TE2000 Perfect Focus System; Nikon Instruments) equipped with a spinning-disk confocal scanner unit (CSU10; Yokogawa), a 488 nm solid-state laser (LMM5; Spectral Applied Research), and a multipoint stage (MS-2000; Applied Scientific Instruments). A CoolSnap HQ2 cooled charge-coupled camera (Photometrics) was used to take images with a camera triggered electronic shutter controlled by NIS Elements Imaging Software (Nikon) and a 60× Plan Apochromat TIRF 1.45 NA oil immersion objective equipped with a Borealis (Andor) to normalize illumination. High-resolution and super-resolution images were acquired using a Yokogawa CSU-W1/SoRa spinning disk confocal system (Yokogawa) and an ORCA Fusion BT sCMOS camera (Hamamatsu) using 2×2 camera binning. Nuclear-to-cytoplasmic ratios of immunolabeled proteins and number of puncta per cell were quantified using NIS Elements Imaging Software (Nikon). Briefly, the fluorescence in the nucleus (detected by Hoescht) and in the cytoplasm were manually sampled by selection of regions-of-interest. Three regions-of-interest outside of any cell were used to calculate background fluorescence and was subtracted from both nuclear and cytoplasmic fluorescence values. The ratio of fluorescence was then determined by diving the nuclear fluorescence intensity with that of the cytoplasm for a given cell. Quantification of puncta for SSEA3 was done by creating a 3D projection of full-cell z-stacks by using NIS Elements Imaging Software. Surfaces were created using the 3D thresholding tool normalized across all images and the total number of puncta was recorded. The total number of cells was then counted, as determined by the number of Hoescht positive nuclei, and the number of puncta per cell was calculated by dividing the total number of puncta by the number of cells in each field of view. Percentage of positive cells for Oct4/Sox2 staining and EdU incorporation was determined by manually counting the total number of nuclei in each field of view. Oct4, Sox2, or EdU were manually counted and the total number of positive cells was divided by the total number of cells to determine the percent positive. We used IMARIS software (Oxford Instruments) to generate 3D renderings.

Polyacrylamide gels of 7.9 kPa stiffness were made by adjusting acrylamide and bisacrylamide stock solution (Bio-Rad Laboratories, Hercules, CA, USA) concentrations. A solution of 40% acrylamide, 2% bisacrylamide, and 1× PBS was polymerized by adding tetramethylethylene diamine (Fisher BioReagents) and 1% ammonium persulfate. A droplet of the gel solution supplemented with 0.2 μm fluorescent beads solution (Molecular Probe, Fisher Scientific) was deposited on a quartz slide (Fisher Scientific) and covered with a 25 mm glass (Fisher) coverslip pretreated with 3-aminopropyltrimethoxysilane (Sigma-Aldrich) and glutaraldehyde (Sigma-Aldrich). After polymerization, the gel surface attached to the quartz slide was functionalized with Matrigel via polydopamine. The gel was sterilized and stored in 1× PBS before cell seeding. The traction forces exerted by colonies on the polyacrylamide gel substrates were computed by measuring the displacement of fluorescent beads embedded within the gel. Briefly, images of bead motion near the substrate surface, distributed in and around the contact region of a single cell (before and after cell detachment with 10% sodium dodecyl sulfate), were acquired with Yokogawa CSU-21/Zeiss Axiovert 200 M inverted spinning disk microscope with a Zeiss LD C-Apochromat 40×, 1.1 NA water-immersion objective and an Evolve EMCCD camera (Photometrics). The traction stress vector fields were generated using an open-source package of FIJI plugins (https://sites.google.com/site/qingzongtseng/tfm).

To determine clonogenic potential, cells were dissociated with Accutase and plated on Matrigel-coated six-well dishes at a density of 1000 cells per cm (Nakamura et al., 2016) in the presence of ROCKi (10 µM). Five days after plating, cells were stained for alkaline phosphatase as per the manufacturer’s protocol (StemAb Alkaline Phosphatase Staining Kit II, ReproCell #00-0055) and imaged using a Leica DFC 7000t microscope. To quantify the number of alkaline phosphatase positive colonies, images were analyzed using Fiji (Schindelin et al., 2012).

RNA was extracted using RNeasy Mini kits (QIAGEN) according to the manufacturer’s instructions and concentrations were determined by NanoDrop. Library preparation and RNAseq were performed by Novogene Co. Ltd (USA). Briefly, RNA purity was measured using a NanoPhotometer spectrophotometer (IMPLEN). RNA integrity and quantity were determined using a Bioanalyzer 2100 system (Agilent Technologies). Three paired biological replicate libraries were prepared for each condition, with each library generated with 1 µg of RNA per sample. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA). Then 3 µL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Raw data (raw reads) were processed through fastp to remove adapters, poly-N sequences, and reads with low quality. Q20, Q30, and GC content of the clean data were calculated and found to be within the normal range. All the downstream analyses were based on the clean data with high quality. Reference genome (ID: 51) and gene model annotation files were downloaded from genome website browser (NCBI) directly. Paired-end clean reads were aligned to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software. FeatureCounts was used to count the read numbers mapped of each gene. And then RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis was performed using DESeq2 R package. The resulting p values were adjusted using the Benjamini and Hochberg’s approach for controlling the FDR. Genes with a padj<0.05 found by DESeq2 were assigned as differentially expressed. The R package clusterProfiler was used to test the statistical enrichment of differential expression genes in KEGG pathways. KEGG terms with padj<0.05 were considered significant enrichment. Transcription factor binding motif analysis was performed using Enrichr (Chen et al., 2013; Kuleshov et al., 2016). To investigate YAP target gene expression, supplementary tables generated as previously described (see ‘Supplemental Methods’ in Pagliari et al., 2021 and Estarás et al., 2017) were used to generate YAP target gene lists (Estarás et al., 2017; Pagliari et al., 2021).

pGAMA-YAP was a gift from Miguel Ramalho-Santos (Addgene plasmid #74942). Site-directed mutagenesis was performed on the pGAMA-YAP construct to create p-GAMA-YAP-S127A using the QuikChange Lightning kit (Agilent Technologies #210513). Forward primers used for the Ser-to-Ala substitution were as follows: 5′-GTTCGAGCTCATGCCTCTCCAGC-3′ and 5’-GCTGGAGAGGCATGAGCTCGAAC-3’. The pGAMA-YAP-S127A plasmid was confirmed via DNA sequencing. To prepare lentivirus, HEK293-FT (Invitrogen #R70007) cells were grown in Dulbecco’s Modified Eagle Medium (Thermo Fisher #11965118) supplemented with 10% fetal bovine serum (Peak Serum #PS-FB4), non-essential amino acids (UCSF CCF #CCFGA001), pen/strep (UCSF CCF #CCFGK003), and sodium pyruvate (UCSF CCF #CCFGE001) and maintained at 37°C with 5% CO₂. Lentivirus was generated according to the manufacturer’s specifications by co-transfecting HEK293-FTs with a mixture of packaging plasmids (ViraPower Lentivirus Expression System; Thermo Fisher #K497500). Briefly, 5×10⁶ HEK293-FTs were seeded onto a 10 cm dish containing 10 mL of complete medium without antibiotics. After 24 hr, cells were transfected with a mixture of 3 µg of the lentiviral plasmid containing the gene of interest and 9 µg of the ViraPower Packaging Mix using Lipofectamine 2000 (Thermo Fisher #11668030). At 72 hr post-transfection, supernatant was collected, filtered, and concentrated using Lenti-X Concentrator (Takarabio #631231). Concentrated viral supernatant was aliquoted and stored at –80°C.

To generate hESC lines stably expressing YAP-WT and YAP-S127A, primed HUES8 cells were grown to approximately 60% confluency in one well of a six-well plate. Medium was aspirated, washed once with PBS, and cells were then fed with 1 mL fresh media containing 2 µg of polybrene (Millipore Sigma #TR-1003-G), and incubated for 15 min at 37°C. Concentrated virus supernatant (100 µL) was added and after 6–8 hr 1 mL of fresh medium was added. After 36 hr, viral particles were removed by replacing medium. Three days after virus infection, hESCs were passaged and expanded to three wells in a six-well plate. After wells reached ~75% confluency, hESCs were sorted for high mCherry expression by using a BD FACS Aria3u. Sorted cells were maintained as described above with the addition of penicillin and streptomycin for 3 days, after which cells were then maintained in standard antibiotic-free mTeSR-1 media.

Total lysates were obtained from primed cells, naïve cells, and cells dedifferentiated in the presence of 80 µM CK666 at day 6 which were plated on Matrigel-coated six-well plates. Cells were washed 3× in cold PBS on ice, lysed in 100 µL/well RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate, 0.1% SDS supplemented with protease inhibitors PEFA) while rotating at 4°C for 10 min. Cells were then scrapped into microfuge tubes, and a post-nuclear supernatant was collected after centrifugation at 12,000 rpm for 5 min at 4°C. Proteins in lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline (TBS) containing 0.1% Tween and 5% non-fat dry milk (TBST) for 60 min at RT and incubated overnight at 4°C with antibodies in TBST containing 5% non-fat dry milk. After washing, membranes were incubated with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) in TBST for 1 hr at RT, and bound antibodies were developed by enhanced chemiluminescence using SuperSignal West Femto (Thermo Fisher Scientific) and imaged using an Alpha Innotech FluorChem Q (Alpha Innotech). For quantification, the average intensity of each YAP band was normalized to that of the GAPDH band in each sample.

All statistical tests and sample sizes are included in the Figure Legends and text. All data shown are mean ± SD. In all cases, p values are represented as follows: ****p<0.0001, ***p<0.001, **p<0.001, and *p<0.05. All quantifications were statistically analyzed using ANOVA with Tukey post hoc tests. Statistical analysis was performed using GraphPad Prism version 10.1.2.