Arp2/3 Complex Activity Enables Nuclear YAP for Naïve Pluripotency of Human Embryonic Stem Cells

Our understanding of transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency that includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, uniform cell mechanics within naïve colonies, nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.


Introduction
Derivation of clonal pluripotent stem cells (PSCs) from embryos yields cells with a spectrum of pluripotent states, dependent on developmental progression of the embryo, species, and culture condition. Clonal mouse embryonic stem cells (mESCs) represent a ground-state of pluripotency and closely recapitulate the naïve blastocyst from which they are isolated 1 . In contrast, clonal human and other primate PSCs, as conventionally isolated and maintained, exist in a primed state of pluripotency and more closely resemble the postimplantation epiblast 2 . To study the naïve state of clonal human PSCs, culture conditions were developed that dedifferentiate primed human embryonic stem cells (hESCs) to a naïve state of pluripotency [3][4][5][6] . Development of culture conditions that convert and sustain a naïve pluripotent state in human PSCs has provided an opportunity to study human development before gastrulation 7 .
These in vitro models of naïve pluripotency have provided insights on the transcriptomic, epigenetic, and proteomic programs that maintain a functional naïve pluripotency state in stem cells 4,8,9 . We have limited understanding, however, of how established morphological changes during the transit from primed to naive states are regulated and whether morphological changes regulate state transitions. It is the cytoskeleton, an intracellular network of proteins, which responds to the external microenvironment and facilitates changes in cell behavior such as cell shape. This network is primarily composed of microtubules, intermediate filaments, and actin filaments. We know that pluripotent stem cell fate is intricately regulated by biophysical cues primarily transmitted through the actin cytoskeleton, which control gene expression, proliferation, and differentiation 10 . The corresponding coordinated changes in cell shape are essential for developmental embryogenesis and are largely mediated by the actin cytoskeleton 11 . Accordingly, mechanoregulation has been studied for roles in exit from the pluripotent state toward targeted cell fates including endodermal 12 , ectodermal 13 , and mesodermal 14 lineages 15 . Directly targeting actin filament dynamics has also been shown to regulate pluripotent stem cell fate [16][17][18] . Other components of the cytoskeleton such as microtubules and intermediate filaments have also been established to modulate stem cell behavior although studies have primarily focused recently on how they impact nucleus morphology and activity [19][20][21] .
Actin-associated proteins, including β-catenin for enabling Wnt pathway activity and ezrin-radixin-moesin (ERM) proteins for enabling tensional forces at the plasma membrane, facilitate maintenance of the naïve pluripotent state in mESCs 22 . During murine preimplantation development, actin filaments generate mechanical forces that contribute to differentiation throughout the blastocyst stage by modulating mechanosensitive signaling pathways such as Hippo signaling 23,24 . These actin structures allow cells within the developing blastocyst to organize based on contractility, coupling mechanosensing and fate specification 25 . Despite evidence that morphological changes and actin filament remodeling determine naïve pluripotency during mouse development, their roles in hESC naïve pluripotency remain unclear.
In asking the role of morphological changes during hESC dedifferentiation to a naïve state of pluripotency, we identified the assembly of a ring of contractile actin filaments encapsulating naïve but not primed colonies that is tethered to adherens junctions and decorated with phosphorylated myosin light chain (pMLC) and moesin. We found that nucleating activity of the Arp2/3 complex but not formins is necessary for the actin ring, naïve cell mechanics, including decreased cell-substrate tensional forces and colony formation, and transition to naïve pluripotency. RNAseq analysis revealed a role for Hippo pathway signaling in Arp2/3 regulated naïve pluripotency, which we confirmed by showing increased nuclear localization of the transcriptional co-activators YAP and TAZ in naïve compared with primed hESCs that is blocked by inhibiting Arp2/3 complex activity. Consistent with these findings, naïve pluripotency that is blocked with inhibiting Arp2/3 complex activity is restored by expressing a nuclear-localized non-phosphorylatable YAP (YAP-S127A) 26 . These data provide new mechanistic insights on how actin filament dynamics regulates the naïve state of hESCs pluripotency and the integration between actin filament remodeling and pluripotency.

Actin Filament Remodeling as hESCs Transition to a Naïve State
For morphological analysis of pluripotency states, HUES8 primed human embryonic stem cells (hESCs) were grown on Matrigel and dedifferentiated to naïve pluripotency using previously reported conditions in an mTeSR-based medium supplemented with ERK (PD0325901) and GSK3 (CHIR99021) inhibitors, the adenylyl cyclase activator forskolin, human leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and ascorbic acid 4,27 . We confirmed transition to a naïve state by showing that colonies have prominent doming by day 6 of dedifferentiation and increased expression of pluripotency markers DNMT3L, DPPA3, KLF2, and KLF4, as determined by rt-PCR ( Fig. 1A-B). 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 (Fig. 1C). These supracellular actin rings also formed around colonies of naïve H9 cells and naïve WTC11 iPSCs (Supp. Fig. 1A) as well as HUES8 cells dedifferentiated by alternative medium supplements (Supp. Fig. 1B). Moreover, the actin ring assembled independently of naïve colony size (Supp. Fig. 1C).
A similar actin ring is reported to encircle colonies of clonal human pluripotent stem cells to provide a mechanosensitive element linked to focal adhesions 28 . 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 (Fig. 1D, crosshairs). The contractile property of the ring was also suggested by the actin ring being decorated with phosphorylated myosin light chain (pMLC) as determined by immunolabeling (Fig. 1E). In contrast, primed hESC colonies had irregular aggregates of pMLC with limited co-localization with actin filaments. Immunolabeling with pan-ERM antibodies showed co-localization with the actin filament ring in naïve cells, and ERM-specific antibodies revealed co-localization of moesin but not ezrin or radixin (Fig. 1F, Supp. Fig. 1D). Together, these data indicate a contractile actin ring surrounding naïve but not primed hESC colonies.
The supracellular nature of the actin ring and differential pMLC labeling between 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 29,30 . Consistent with pMLC localization, primed colonies exhibited elevated cell-substrate tractions that were distributed throughout the colony (Fig. 1G, left; Supp. Fig. 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 (Fig. 1G, right; Supp. Fig. 1E), suggesting decreased cell-substrate tensional force and a likely shift to more stabilized cell-cell forces.
Along with pMLC localization, these low tractions are consistent with uniform cell-cell adhesion in naïve hESC colonies. Together these data identify a significant reorganization of the actin cytoskeleton during 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.

Arp2/3 Complex Activity is Necessary for Transition of hESC to Naïve Pluripotency
The assembly of an actin ring in naïve but not primed human embryonic stem cell (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 31 . We found that the actin ring assembled when naïve cells are generated in the presence of SMIFH2, a broad-spectrum inhibitor of formin activity 32,33 but not CK666, a selective pharmacological inhibitor of Arp2/3 complex activity 34,35 (Fig. 2A). Additionally, CK666 blocked increased expression of markers of naïve pluripotency seen in controls, determined by qPCR of PECAM1, ESRRB, KLF4, and DNMT3L (Fig. 2B). To eliminate the possibility that CK666 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 (Supp. Fig. 2). To further assess the pluripotent state of cells dedifferentiated in the presence of CK666, we immunolabeled for the primed pluripotent marker SSEA3 36 . In controls, SSEA3 expression significantly decreased with dedifferentiation, as previously reported 37 but not with CK666 ( Fig. 2C, D). Additionally, the naïve pluripotency marker KLF4 38 translocated from the cytoplasm to the nucleus with control dedifferentiation but not in the presence of CK666 (Fig. 2C, E).
We further tested for a functional naïve pluripotent state by staining for alkaline phosphatase (AKP) and scoring for colony formation, which indicates the capacity for clonogenic expansion and self-renewal 39 . Primed and naïve hESCs were passaged and plated at clonogenic cell numbers and maintained for five days without or with CK666. In controls, colony formation was greater in naïve compared with primed hESC, as previously reported 40 .
However, with CK666, but not CK689, an inactive analog of CK666, there was no increase in colony formation in naïve compared with primed cells (Fig. 2F). Additionally, traction force microscopy revealed that elevated cell-substrate tractions throughout colonies of primed but not naïve cells (Fig. 1G), were retained when hESCs were dedifferentiated in the presence of CK666 ( Fig. 2G; Supp. Fig. 2B). 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.

Arp2/3 Complex Activity Enables Active YAP for Naïve Pluripotency
To understand how Arp2/3 complex activity affects the transcriptional circuitry required for naïve pluripotency, we performed bulk RNA-sequencing (RNAseq) on primed cells, naïve cells, and primed human embryonic stem cells (hESCs) dedifferentiated in the presence of CK666 (Fig. 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. 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 that revealed Hippo signaling as the top candidate ( Fig. 3B). Additionally, transcription factor binding motif analysis identified TEAD2 as a top candidate, which is a downstream effector of Hippo signaling (Fig. 3C).
The Hippo effector protein YAP is a known regulator of the human naïve pluripotent state, with overexpression of YAP in pluripotent stem cells promoting the acquisition of naïve pluripotency 27 . Although actin filament dynamics, including a contractile ring of actin, regulate YAP signaling 41,42 , 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 43,44  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; these naïve markers failed to significantly increase in the CK666treated condition (Fig 3E, left). As expected, genes significantly enriched among both DEG lists include known YAP and Hippo targets such as ANKRD1, SLIT2, and CHD10. (Fig. 3E, right).
Genes significantly enriched among the CK666-treated condition include the negative Hippo regulator AMOT 45 , and lineage-commitment genes such as SOX6 and SPEF2 (Fig. 3E, middle).
These data suggested a Hippo signaling pathway program, driven by mediators such as YAP, occurs during dedifferentiation to naïve pluripotency but is disrupted by inhibiting Arp2/3complex activity. To verify this prediction, we immunolabeled cells to determine YAP localization and found increased nuclear to cytoplasmic ratios of YAP ( Fig. 3F-G) and TAZ (Supp. Fig. 3) with control dedifferentiation that was blocked by CK666.
Consistent with these data, during preimplantation development, actin filaments and associated proteins generate mechanical forces that contribute to differentiation throughout the blastocyst stage through modulation of mechanosensitive pathways such as Hippo signaling 23,24 . These actin structures allow cells within the developing blastocyst to organize based on contractility, coupling mechanosensing and fate specification 25 . 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 form of YAP (YAP-S127A) could restore naïve dedifferentiation in the presence of CK666. Accordingly, we observed that two immunofluorescence-based markers of the naïve state, increased nuclear localization of KLF4 ( Fig. 4A-B) and decreased SSEA3 (Fig. 4C-D), were rescued by overexpression YAP-S127A in the presence of CK666. In contrast, acquisition of a naïve state of pluripotency remained blocked with CK666 treatment in cells overexpressing wildtype YAP (WT YAP) (Fig. 4A-D).

Discussion
Our new findings support a model in which naïve pluripotency is characterized by an Arp2/3 complex-dependent remodeling of the actin cytoskeleton that includes formation of a contractile supracellular actin ring enclosing naïve colonies and establishment of uniformity in colony mechanics likely enabled by the actin ring being physically associated with β-catenin and moesin, which are known to play roles in pluripotency 46,47 (Fig. 4F). Moreover, Arp2/3 activity facilitates dedifferentiation to a naïve state of pluripotency through promoting nuclear translocation of YAP and regulating Hippo target gene expression. Consistent with these findings, naïve pluripotency that is blocked with inhibited Arp2/3 complex activity is restored by expressing a constitutively active, nuclear-localized YAP-S127A.
Our findings are distinct from those on a contractile actin filament ring that assembles around colonies of primed pluripotent stem cells 28 and Xenopus neural crest cells 48 , which functions to enhance cell-substrate adhesion and migratory capacity, respectively. Our findings also highlight distinct differences between murine and human embryonic stem cells. Cells within the ICM of mouse blastocysts exclude YAP from the nucleus whereas cells within the ICM of human blastocysts maintain nuclear YAP 27,49 . This difference in YAP localization is retained in vitro, with murine naïve pluripotent stem cells (PSCs) having predominantly cytosolic YAP 50 , and human naïve PSCs having predominantly nuclear YAP (Fig. 3F-G). How this difference in YAP localization occurs between mouse and human is unknown, although a number of cytoskeletal factors regulate YAP localization, including stability of the actin cytoskeleton, contractility, and mechanical regulators such as ERM proteins 42 . The role of the actin cytoskeleton in the exit from the pluripotent state has also highlighted how actin dynamics may facilitate cell fate decisions. For example, cells located at the colony edge of primed human embryonic stem cells (hESCs) have distinct cytoskeletal dynamics and are uniquely poised to exit pluripotency and differentiate 51,52 . Positional differences in differentiation potential such as these have been proposed as a mechanism executed in early embryo symmetry breaking with rearrangement of the actin cytoskeleton being required for the first cell fate decision in the blastocyst 53,54 . Thus, it may be possible that the contractile actin ring we observe at the edge of naïve colonies (Fig 1. C) functions as a hub for regulating cell fate dynamics through similar mechanisms as first cell fate decision including modulation of mechanosensitive signaling such as YAP and through pathways such as Hippo.
Further highlighting differences between human and mouse embryonic stem cells, we recently reported that Arp2/3 complex activity is necessary for the differentiation of clonal mouse naïve PSCs to the primed epiblast state, which is in part mediated by translocation of myocardin-related transcription factor MRTF from the cytosol to the nucleus 55 . Additionally, a recent report suggests that Arp2/3 complex activity may form a positive feedback loop with YAP-TEAD1 transcriptional activity controlling cytoskeletal reorganization 56 ; thus Arp2/3 complex activity may regulate naïve pluripotency at multiple stages including initially to reorganize the actin cytoskeleton, but also during maintenance of naïve pluripotency through regulating YAP localization and hence activity.
Our findings increase our understanding of actin dynamics and cell mechanics as regulators of cell fate transitions. A role for contractile actin filaments as a mechanoresponsive element for pluripotency states is well established 57 , and our work identifies cytoskeletal dynamics essential for uniform colony mechanics and the naïve pluripotent state, the role of Arp2/3 complex activity, and YAP/TAZ activity as a promising target for reprogramming of hESCs and for regenerative medicine.

Cell Culture
Primed human embryonic stem cell lines HUES8, H9, and WTC11 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).

Generation of Naïve hESCs
Dedifferentiation was completed using previously published methods 4,27 . In brief, cells were

Colony Formation Assay
To determine clonogenic potential, cells were dissociated with Accutase and plated on Matrigelcoated 6-well dishes at a density of 1,000 cells per cm 2 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 58 .

Library Preparation and RNA Sequencing
RNA was extracted using RNeasy Mini kits (Qiagen) according to the manufacturer's instructions and concentrations were determined by NanoDrop. Library preparation and RNA sequencing 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 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, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated 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.

RNA Sequencing Analysis
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 False Discovery Rate (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 59,60 . To investigate YAP target gene expression, supplementary tables