Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling. Summary: A broad repertoire of conventional human ESCs and transgene-independent iPSC lines can be reverted to stable naive states using WNT, MEK/ERK and tankyrase inhibition.

Complex determinants may collectively influence the functional pluripotency of both hiPSCs and hESCs. For example, one critical variable impacting the functional pluripotency of conventional hPSCs is their developmental, molecular and epigenetic commonality with ʻprimed' mouse post-implantation epiblast stem cells (mEpiSCs) (Tesar et al., 2007;Brons et al., 2007;Chou et al., 2008;Kojima et al., 2014;Weinberger et al., 2016), which possess a less primitive pluripotency than inner cell mass (ICM)derived mouse ESC (mESCs). For example, mEpiSCs cannot fully contribute to a blastocyst chimera and are resistant to chemical reversion to ICM-like naïve ʻground state' pluripotency with 'LIF-2i' (MEK/ERK and GSK3β signal inhibition) (Bernemann et al., 2011;Ying et al., 2008;Marks et al., 2012). Conventional human pluripotent stem cells (hPSCs) rely on self-renewal signaling pathways more similar to those of mEpiSCs than ESCs, and these hPSCs might exist in developmentally primed states that display mEpiSC-like lineage skewing following directed differentiation. Although several hPSC naïve reversion approaches were recently described, none was maintained with classical MEK/ERK/WNT 2i signaling inhibition alone (Hanna et al., 2010;Chan et al., 2013;Gafni et al., 2013;Takashima et al., 2014;Theunissen et al., 2014;Ware et al., 2014). Thus, although various methods may achieve pluripotent states reminiscent of the human ICM, the determinants required for stable human rewiring to an mESC-like ground state remain undefined and might represent unknown species-specific differences.
The roles of the derivation method and of lineage priming of conventional hiPSCs in the amenability to naïve reversion have not been fully evaluated. For example, although human hematopoietic progenitors are more efficiently reprogrammed than fibroblast donors via standard methods (Eminli et al., 2009;Park et al., 2012;Guo et al., 2014), both donor types generated hiPSCs with diminished and lineage-skewed differentiation potencies that were attributed to the retention of donor epigenetic memory (Kim et al., 2011;Hu et al., 2011). By contrast, hiPSCs reprogrammed efficiently from cord bloodderived CD33 + CD45 (PTPRC) + myeloid progenitors (MPs) (Park et al., 2012) displayed reduced interline variability or differentiation bias (Burridge et al., 2011;Park et al., 2014). These MP-iPSCs generated vascular progenitors (VPs) with less culture senescence, decreased sensitivity to DNA damage, and greater in vivo engraftment potential than VPs generated from standard fibroblast-derived hiPSCs . MP-iPSCs also generated physiologically functional photoreceptors that elicited action potentials in a three-dimensional retinal differentiation system .
Since murine and human MPs may represent a 'privileged' somatic donor type (Park et al., 2012;Guo et al., 2014), we tested the hypothesis that efficient myeloid reprogramming generates an improved primed functional pluripotency with reduced lineage priming and increased amenability to naïve ground state reversion. Here, we demonstrate that effective reprogramming of human CD33 + CD45 + MP donors generates hiPSCs with an improved multilineage differentiation potency that lacks the lineage-priming differentiation bias characteristic of hiPSCs derived via standard reprogramming methods. Moreover, supplementation of classical LIF-2i with only the tankyrase inhibitor XAV939 (LIF-3i) permitted a large repertoire of hiPSCs to efficiently revert to a stable mESClike naïve state that possessed further improved multilineage functional pluripotency. Interestingly, MP-iPSCs reverted to this stable naïve state more efficiently than hiPSCs derived via less efficient methods.
To evaluate the quality of sa-MP reprogramming, we generated a library of over 40 unique MP-iPSC lines derived with and without sa from PB-, CB-and FL-derived CD33 + MPs (Table S2, supplementary Materials and Methods). To delineate the effects of reprogramming-associated donor-specific genetic variability (Kyttälä et al., 2016), independent MP-iPSC lines from unique as well as identical MP donors were generated. This repertoire of MP-iPSCs was complemented with hiPSCs generated via standard methods: 7F-E mononuclear CB cell-derived hiPSCs (Hu et al., 2011), 7F-E and 4F viral (4F-V) fetal (f )/adult (Ad) fibroblastderived iPSCs (fibro-iPSCs: fF-iPSCs, AdF-iPSCs) and 7F-E adult skin keratinocyte-derived iPSCs (Ker-iPSCs) (Park et al., 2012;Byrne et al., 2009). We compared whole-genome transcriptomes of this MP-iPSC repertoire with comparable passage standard hiPSC and hESC lines (Fig. S2A). In contrast to standard fibro-iPSCs, which incompletely resemble hESCs in their gene signatures (Chin et al., 2009), CB-derived sa-MP-iPSCs attained global expression profiles that were indistinguishable [Pearson coefficient (R 2 )=0.99] from standard hESCs, and in a manner that was irrespective of donor genome origin (Fig. S2A). Whole-genome CpG DNA methylation analysis further revealed that sa-MP-iPSCs (from both unique and the same donors) clustered as a function of sa-MP reprogramming into an epigenetically distinct group relative to hESCs and standard fibro-iPSCs (Fig. S2B).
To determine the effects of sa-MP reprogramming on genetic variability, we evaluated MP-iPSCs from independent, unique MP donors generated with and without stromal STAT3 activation, and compared their whole-genome transcriptomes with standard hiPSCs and hESCs. These bioinformatics analyses revealed that sa-MP reprogramming significantly reduced hiPSC gene variability relative to hESC controls, and in a manner that was independent of individual donor source (Fig. 1D). Gene ontology (GO) and gene set enrichment analysis (GSEA) revealed that, relative to standard reprogrammed hiPSCs, sa-MP reprogramming generated hiPSCs with significantly reduced expression of genes associated with lineage priming (e.g. Polycomb complex targets), and distinct changes in the expression of genes involved in cell cycle regulation and metabolism (Fig. 1E, Fig. S5).
We next assessed the quality of sa-MP-iPSC pluripotency circuits by performing principal component analysis (PCA) of microarray data for the expression of pluripotency-associated ESC and core module circuits (Table S1) (Kim et al., 2010a;Boyer et al., 2005) for each hiPSC class. These analyses revealed that, in comparison to standard fibro-iPSC lines, low-passage sa-MP-iPSCs had already attained high-fidelity transcription of these pluripotency circuits that was indistinguishable from hESCs (hESC to CB-derived sa-MP-iPSC ESC module, R 2 =0.99; core module, R 2 =0.98; Fig. 1F). Collectively, these multilineage differentiation and bioinformatics studies revealed that sa-MP-iPSCs, as a class, possessed high molecular and functional pluripotency and lacked the lineage-specific differentiation skewing and increased lineage-primed gene expression variability typically observed in hiPSCs derived via standard methods.
A broad repertoire of conventional hPSC lines stably reverts to naïve morphologies in LIF-3i We evaluated 23 independent non-integrated, conventional, primed hPSC lines for their capacity to tolerate stable, clonogenic self-renewal of SSEA4 + TRA-1-81 + cells for at least ten passages in LIF-3i. (Fig. S8A,B, Table S3B). Long-term stability of colonies with undifferentiated dome-shaped morphologies for >10-20 passages via direct LIF-3i reversion alone was most reproducible for sa-MP-iPSCs and select hESC lines (e.g. H9). However, brief adaptation (one passage) in LIF-3i plus two additional molecules, namely forskolin and purmorphamine (LIF-5i), increased the initial survival of enzyme-digested hPSC single cells, and facilitated a broader repertoire of hPSCs to tolerate subsequent stable clonal selfrenewal in LIF-3i alone (Fig. S7A-C and Fig. S8A,B, Table S3B). This initial LIF-5i modification permitted a wide repertoire of ∼16 conventional hPSC lines to revert with long-term stability in LIF-3i alone.
To characterize the epigenetic status of naïve-reverted hPSCs, we performed Infinium CpG DNA 450K methylation array analysis of these 12 LIF-3i-reverted lines and their isogenic conventional counterparts. This methodology interrogates more than 485,000 methylation sites at single nucleotide resolution, and covers 96% of CpG islands of 99% of RefSeq genes, with an average of 17 CpG sites per gene region distributed across the promoter, 5′UTR, first exon, gene body, and 3′UTR. These studies revealed that all LIF-3ireverted hPSCs possess similar and significantly decreased (P<0.001) epigenome-wide CpG DNA methylation at differentially methylated regions (DMRs) compared with their primed hPSC sources (Fig. 4B,  top panel). Additionally, quantification of dot blot immunoassays of global CpG 5-methylcytosine isoforms (5hMC versus 5MC) revealed reduced global 5MC activities and increased ratios of 5hMC/5MC CpG DNA methylation, which was most evident for sa-MP-iPSC E5C3 (Fig. 4B, bottom panel). Taken together, these data were consistent with a more epigenetically open configuration, and a potential role for TET-mediated CpG DNA demethylation activities in sustaining naïve pluripotency in LIF-3i-reverted hPSCs (Leitch et al., 2013). Interestingly, in contrast to previous reports that naïve reversion results in loss of CpG methylation at known imprinted genomic sites (Pastor et al., 2016), allele-specific analysis of over 1400 known imprinted CpG sites in these 12 independent isogenic hPSC lines (before and after LIF-3i reversion) revealed stability of methylation imprints established in conventional hPSCs, with no systematic loss of imprinted methylation patterns resulting from LIF-3i culture (Table S4A).
To query for naïve-specific epigenetic functionality, we assayed for the activation of the proximal (PE) and distal (DE) enhancers of the OCT4 (POU5F1) promoter in LIF-3i-reverted versus primed hPSCs. Using both transient luciferase reporter assays and stable transgenic genomic DE/PE sequence mutant reporter hPSC lines , we demonstrated that LIF-3i reversion potentiated naïve ESClike activation of the DE of the OCT4 promoter, whereas primed hPSCs displayed preferential mEpiSC-like PE OCT4 activity (Fig. 4C,D).
Finally, to probe the status of pluripotency circuits in naïvereverted hPSCs, we conducted modular GSEA and bioinformatics  Table S1). Z, this study and includes the n=12 independent hPSC lines in analysis of expression and methylation arrays for key pluripotencyassociated stem cell circuits (e.g. the ESC module and SOX2-NANOG-OCT4-regulated core module; Table S1) in these 12 lines, before and after isogenic LIF-3i-reversion. These studies revealed that LIF-3i reversion significantly rewired both core and ESC module genes in naïve hPSCs, with (∼50%) decreases in gene promoter CpG DNA methylation and corresponding increases in gene expression of these pluripotency circuits (Fig. 4E,F).
We next compared the whole-genome transcriptional signatures of LIF-3i-reverted hPSCs with both human ICM-derived epiblasts and mESCs by PCA normalized to published human blastocyst/morula, and with mESC/mESC-LIF-2i data sets as benchmark controls (Fig. 5C,D) (Bao et al., 2009;Vassena et al., 2011). We employed modular bioinformatics using the most differentially expressed genes in E4-E5 human epiblast cells (Petropoulos et al., 2016), and revealed that the patterns of gene expression in LIF-3i-reverted hPSCs cluster closely with human morula/blastocyst epiblast cells. Additional PCA demonstrated that LIF-3i-reverted hPSCs were not only transcriptionally similar to mESCs, but also to naïve-reverted hPSCs derived by others with alternate methods.

DISCUSSION
Stable reversion to a naïve epiblast-like ground pluripotent state may improve the functional utility of conventional hPSCs. Here, we comprehensively evaluated how the variables of derivation method, lineage priming, and baseline-primed functional pluripotency influence the stability of subsequent reversion to a human naïve epiblast-like pluripotent state. Our studies revealed that stable, long-term reversion to an mESC-like state could be achieved from a wide spectrum of EpiSC-like lineage-primed hPSC states via supplementation of classical LIF-2i with only a tankyrase inhibitor (LIF-3i). LIF-3i-reverted hiPSCs were highly proliferative, generated well-differentiated tri-lineage teratomas, possessed normal karyotypes and stable genomic CpG methylation imprints within a globally more hypomethylated genome that was highly transcriptional, and could be stably passaged as undifferentiated, clonal SSEA4 + TRA-1-81 + dome-shaped colonies for at least 30 passages.
Although human chimera generation and germ line contribution is the most stringent measure of naïve pluripotency and could not be tested here, LIF-3i-reverted hPSCs possessed most of the accepted characteristics of mESCs that we tested. These characteristics included high clonal proliferation rates, MEK/ERK independence, bFGF signaling unresponsiveness, STAT3 phosphorylation and signaling, JAK/STAT3 and BMP4 signal dependence, increased naïve-specific transcript expression (e.g. STELLA, NR5A2), upregulation of core pluripotency networks with concomitant decrease in lineage-primed gene circuits, whole-genome transcriptomic clustering with both human preimplantation epiblasts and mESCs, dominant distal OCT4 enhancer usage, global DNA CpG hypomethylation with increased 5hMC/5MC ratios, X-chromosome activation, decreased class I MHC, increased E-cadherin expression, and augmented expression of cytoplasmic and nuclear activated β-catenin. Importantly, LIF-3i-reverted hPSCs had significantly reduced lineage-primed gene expression and improved multilineage differentiation potency relative to their primed states. The derivation of naïve hPSC lines with improved functional pluripotency has broad impact for optimizing future hiPSC-based cellular therapies.
Recently, hPSC naïve reversion approaches have variably required the imposition of transgenic core factor overexpression, complex anti-apoptosis cocktails (e.g. ROCK, BRAF, SRC or JNK inhibition) to sustain survival/proliferation, HDAC inhibition to reset global epigenetic barriers, and/or EpiSC-specific growth factor reinforcement (e.g. bFGF, activin, TGFβ or BMP inhibition) (Table S5). These studies suggest that hPSCs might be generally 'non-permissive' to classical mESC 2i WNT and MEK/ERK pathway reversion, or that human and murine naïve states might be fundamentally non-equivalent. Although our comparative bioinformatics meta-analyses suggested a common pathway between other reversion methods and ours, we demonstrated that a stable human naive epiblast-like state could be maintained in conventional hPSCs via LIF-2i and only a tankyrase inhibitor.
XAV939, an inhibitor of the poly-ADP-ribosylating enzymes tankyrase 1 and 2 (TNKS and TNKS2; also known as PARP5A/B, ARTDF5/6), was originally identified for its capacity to stimulate β-catenin degradation via stabilizing axin. The sole use of XAV939 in cancer cells inhibited WNT signaling (Huang et al., 2009) by reinforcing the β-catenin destruction complex. However, dual combination of tankyrase inhibition (XAV939) and GSK3β inhibition (CHIR99021) in primed rodent mEpiSCs and primed conventional hESCs paradoxically increased WNT signaling by increasing axin expression, stabilizing the axin-catenin complex, and increasing cytoplasmic retention of β-catenin (Kim et al., 2013;Schmitz et al., 2013). Our hPSC studies herein demonstrated that XAV939 similarly synergizes with CHIR99021 in naïve conditions (i.e. in the absence of MEK/ERK signal; PD0325901) to paradoxically stabilize and augment the expression of activated β-catenin in both nuclear and cytoplasmic compartments.
We speculate that additional, potentially complex activities of tankyrase beyond WNT signaling may further support stabilization of a human naïve ground state (Fig. 7B). These mechanisms might include the promotion of genomic integrity via telomere recombination/elongation and stability of the non- Smith, 1998), pericentric heterochromatin regulation (Karantzali et al., 2011), centrosome and mitotic spindle regulation Chang et al., 2009) and promotion of homologous recombination (Vidi et al., 2014). Thus, future studies will focus not only on whether differentiated derivatives of naïve hPSCs provide functional advantages over conventional primed hPSCs for cellular transplantation, but also on elucidating the mechanisms of naïve promotion by tankyrase inhibition.
Initial testing was performed using conventional hPSCs (hESC-H9, sa-MP-iPSCs 6.2, E5C3); for ethics relating to the use of hESC lines and details of conventional hPSC culture see the supplementary Materials and Methods. Conventional hPSCs were expanded 5-6 days on MEFs. Single-cell passaging was performed using Accutase (Sigma), with initial cell plating densities of 50,000-100,000 cells/cm 2 followed by 5000-20,000 cells/cm 2 for subsequent passages. Exposure (24-48 h) to small molecules prior to initial passage greatly enhanced survival of conventional hPSCs. Naive-reverted hPSC cultures were passaged every 3-4 days on fresh irradiated MEFs. Morphological changes were photomicrographed using a Nikon Eclipse TE-2000 inverted microscope, DS-Fi1 camera and NIS-Elements software.
Cultures were assayed by flow cytometry using anti-SSEA1 (BD Biosciences), SSEA4 (R&D Systems) and TRA-1-60/TRA-1-81 (BD Biosciences) antibodies. For details of flow cytometry and associated western blotting see the supplementary Materials and Methods. Conditions that supported stable expansion of hPSC lines for >3-5 passages were further evaluated by qRT-PCR using TaqMan assays on a ViAA7 Real-Time PCR System (Life Technologies) for expression of OCT4, NANOG, ZFP42, KLF2, NR5A2 and STELLA. For details of qRT-PCR and primers see the supplementary Materials and Methods. Karyotype analysis was performed by the JHU Cytogenetics Core Facility. RNA-FISH for XIST is described in the supplementary Materials and Methods.
Extensive small-molecule screening (supplementary Materials and Methods, Table S3) identified XAV939 for permitting long-term survival of hPSCs in classical 2i (CHIR99021 and PD0325901). A combination of CHIR99021, PD0325901 and XAV939 was sufficient for directly reverting a series of hPSC lines to stable, clonal cultures. Other hPSC lines required additional pre-treatment for one passage with forskolin and purmorphamine along with LIF-3i (LIF-5i) to enhance initial clonal viability. Cultures were subsequently passaged with LIF-3i alone, and routinely evaluated for expression of SSEA1/SSEA4, TRA-1-60/TRA-1-81 by flow cytometry every two to five passages thereafter.

Characterization of naïve hPSCs
A description of the generation of a repertoire of episomal hiPSC lines for the functional and molecular characterization of the reprogrammed state is provided in Table S2 and in the supplementary Materials and Methods, along with a description of how hESCs and hiPSCs were differentiated into the various mesodermal, ectodermal and endodermal lineages.

Genomic CpG methylation
Assays of global 5-methylcytosine versus 5-hydroxymethylcytosine were carried out on genomic DNA of hPSC lines by dot blot as described in the supplementary Materials and Methods.

OCT4 enhancer usage
The use of the proximal versus distal enhancer of OCT4 was assessed by luciferase assays and using enhancer mutant reporter hPSC lines as described in the supplementary Materials and Methods.

Bioinformatics analysis
Details of bioinformatics analyses, including gene expression and CpG DNA methylation microarrays and transcriptome analysis, are provided in the supplementary Materials and Methods.