Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells Zhuojin Xua,b, Aaron M. Robitailleb,c, Jason D. Berndta,b,c, Kathryn C. Davidsond, Karin A. Fischerb,e, Julie Mathieub,e, Jennifer C. Pottera,b, Hannele Ruohola-Bakerb,e, and Randall T. Moona,b,c,1 aHoward Hughes Medical Institute, University of Washington School of Medicine, Seattle, WA 98109; bInstitute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA 98109; cDepartment of Pharmacology, University of Washington School of Medicine, Seattle, WA 98109; dAustralian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia; and eDepartment of Biochemistry, University of Washington School of Medicine, Seattle, WA 98109 Contributed by Randall T. Moon, August 24, 2016 (sent for review January 13, 2016; reviewed by Kouichi Hasegawa and Renee A. Reijo Pera) In both mice and humans, pluripotent stem cells (PSCs) exist in at least primed state (e.g., LIS1, WIN1, and H1-4iLIF lines) (3, 11–13)orby two distinct states of pluripotency, known as the naïve and primed deriving new hESC lines from human six- to eight-cell embryos (e.g., states. Our understanding of the intrinsic and extrinsic factors that ELF1 line) using naïve-state growth conditions (12). The molecular enable PSCs to self-renew and to transition between different plurip- programs used to support naïve pluripotency in hESCs and the de- otent states is important for understanding early development. In gree of conservation of the different states of pluripotency between mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC human and mouse PSCs is an area of active investigation (14). self-renewal and support the naïve state. In human embryonic stem Wnt/β-catenin signaling is a key signaling pathway involved in cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs embryonic development and is tightly regulated in PSCs (15–17). and is reduced or absent in primed-state hESCs. However, the role Wnt ligands are evolutionarily conserved secreted glycoproteins of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. involved in autocrine and paracrine cell signaling. In the absence Here, we demonstrate that inhibition of the secretion of Wnts or of Wnts, a “destruction complex” consisting of the cytosolic pro- β inhibition of the stabilization of -catenin in naïve hESCs reduces cell teins glycogen synthase kinase 3 (GSK3), AXIN, adenomatous proliferation and colony formation. Moreover, we show that addition polyposis coli (APC) protein, and casein kinase 1α phosphorylates of recombinant Wnt3a partially rescues cell proliferation in naïve β-catenin (encoded by the CTNNB1 gene). This pool of phos- hESCs caused by inhibition of Wnt secretion. Notably, inhibition of β β phorylated -catenin is ubiquitylated and thereby targeted to the Wnt/ -catenin signaling in naïve hESCs did not cause differentiation. proteasome for degradation. In the presence of Wnt ligands, Instead, it induced primed hESC-like proteomic and metabolic profiles. binding of Wnts to a heteromeric receptor complex leads to in- Thus, our results suggest that naïve hESCs secrete Wnts that activate hibition of the destruction complex, thus enabling β-catenin to autocrine or paracrine Wnt/β-catenin signaling to promote efficient accumulate in the cytoplasm. β-catenin translocates to the nucleus, self-renewal and inhibit the transition to the primed state. where it acts as a transcriptional coactivator for the T-cell factor Wnt/β-catenin signaling | naïve pluripotency | naïve-to-primed transition | (TCF) and lymphoid enhancing factor (LEF) family of DNA- human embryonic stem cells | self-renewal binding transcription factors (18). Wnt/β-catenin signaling is important in mESCs (19–22). Activation of Wnt/β-catenin signaling by recombinant Wnt3a or by inhibition luripotent stem cells (PSCs) undergo unlimited self-renewal of GSK3 synergizes with activation of JAK/STAT signaling by Pand can differentiate into cells representative of all three recombinant leukemia inhibitory factor (Lif) to promote self-renewal germ layers of the adult body (1). In both mice and humans, PSCs exist in at least two different states of pluripotency in vitro, commonly referred to as naïve and primed states. The different Significance states of pluripotency likely reflect different stages of embryonic development in vivo (2–5). Understanding the molecular un- Pluripotent stem cells (PSCs) can exist in a naïve or primed pluri- derpinnings of PSC behaviors in different pluripotency states and potency state. Naïve PSCs correspond to an earlier developmental identifying the molecular factors that control transitions between state more closely related to cells from the preimplantation em- these states may provide insight into early embryonic develop- bryo and may have more robust developmental potential than ment and contribute to efforts designed to use PSCs and their primed PSCs. However, understanding the molecular mechanisms derivatives for research and treatment of human diseases. that regulate naïve PSC behaviors such as self-renewal, differen- Much of our knowledge about the differences between naïve tiation, or preservation of the naïve state is incomplete. Here, we and primed states comes from research on mouse PSCs. Naïve report that Wnt/β-catenin signaling promotes the self-renewal of mouse embryonic stem cells (mESCs) are derived from embryonic naïve human embryonic stem cells (hESCs). When grown in con- cells of the preimplantation blastocyst and have a broad and ro- ditions that inhibit Wnt/β-catenin signaling, naïve hESCs remain bust developmental capacity (6, 7). In contrast, primed PSCs, undifferentiated but have a more primed-like protein expression β exemplified by mouse epiblast stem cells (mEpiSCs), display bia- profile. Our results suggest that Wnt/ -catenin signaling plays a ses toward lineage-specific differentiation (8). For example, al- critical role in regulating human naïve pluripotency. though mESCs can be used to generate a whole animal through Author contributions: Z.X. and J.D.B. designed research; Z.X., A.M.R., K.A.F., J.M., and J.C.P. tetraploid complementation when injected into a preimplantation performed research; J.C.P. assisted in cell culture; Z.X., A.M.R., J.D.B., K.C.D., H.R.-B., and R.T.M. blastocyst (9), mEpiSCs exhibit a poor capacity to contribute analyzed data; and Z.X., A.M.R., J.D.B., K.C.D., H.R.-B., and R.T.M. wrote the paper. to such chimeras. Conventional human embryonic stem cells Reviewers: K.H., Kyoto University; and R.A.R.P., Montana State University. (hESCs) exist in the primed state and exhibit marked similarities The authors declare no conflict of interest. in morphological, molecular, and epigenetic characteristics com- 1To whom correspondence should be addressed. Email: [email protected]. pared with mEpiSCs (10). More recently, naïve-state hESCs have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. been generated by toggling conventional hESCs back from the 1073/pnas.1613849113/-/DCSupplemental. E6382–E6390 | PNAS | Published online October 3, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1613849113 Downloaded by guest on September 30, 2021 and inhibit spontaneous differentiation (19, 20). Dual inhibition of using the small molecules XAV939, which promotes degradation PNAS PLUS kinases GSK3 and MEK drives self-renewal and inhibits differ- of β-catenin (25), or IWP2, which blocks the secretion of Wnt li- entiation of naïve mESCs (2). Moreover, paracrine and autocrine gands (26), or by siRNA-mediated knockdown of CTNNB1 (13). Wnt signaling prevent naïve mESCs from converting into a primed We also found that the expression of genes involved in Wnt sig- mEpiSC-like state (22). naling pathways changes early during the naïve-to-primed transi- Whether Wnt/β-catenin signaling plays a similar role in naïve tion in hESCs (13). Nevertheless, we did not establish a causal link hESCs has not been fully investigated. Notably, naïve hESC lines between Wnt/β-catenin signaling and naïve hESC behaviors, such generated without transgenes (3, 12, 23, 24), including ELF1 and as self-renewal, differentiation, or preservation of the naïve state. H1-4iLIF hESCs, were derived and subsequently maintained in the Here, we show that Wnt/β-catenin signaling promotes self- presence of GSK3 inhibitors—a condition that may promote Wnt/ renewal of naïve hESCs but is dispensable for the maintenance β-catenin signaling. Recently, we reported that activation of a of pluripotency marker expression. Moreover, inhibition of Wnt/ β-catenin–activated reporter (BAR) is increased when ELF1 β-catenin signaling in naïve hESCs induces a more primed-like hESCs are grown in naïve conditions compared with primed con- global protein expression profile. Taken together, our results ditions (13). Moreover, the activity of BAR in naïve-state ELF1 implicate Wnt/β-catenin signaling as a positive regulator of hu- hESCs is suppressed by inhibition of Wnt/β-catenin signaling (13), man naïve pluripotency. A B AXIN2 TROY with CHIR w/o CHIR 1.2 naïve 0.8 primed 0.4 normalized Venus to naïve cells * 0.0 * 1.2 DMSO C w/o CHIR 0.8 XAV with CHIR 5 5 ** 10 86.7±5.3% 10 DMSO 0.4 * IWP2 90.3±4.0%0% 4 * 10 4 normalized to 10 0.0 CD9 Fold change 3 3 10 10 3.0 Ctrl si 2 2 * 10 10 0 CTNNB1 2 0 2.0 si -10 2 3 4 5 -10 3 4 5 1.0 AXIN1/2 0 10 10 10 0 10 10 10 Ctrl siRNA si normalized to * 0.0 * * Tra1-60 D d1-d3: DMSO XAV XAV IWP2 IWP2 d4-d6: DMSO XAV DMSO IWP2 DMSO Venus 250 120 120 50 150 200 90 40 33.4% 15.1% 24.9% 90 8.37% 20.2% 150 100 with CHIR 30 60 60 100 20 50 30 30 count 50 10 0 0 0 0 0 3 3 4 5 3 3 4 5 3 3 4 5 3 3 4 5 -103 0 103 104 105 0-10 10 10 10 -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 Venus 20 150 200 100 60 15 80 37.4% 150 16.6% 100 28.2% 6.64% 17.9% 10 40 60 100 w/o CHIR 40 50 count 5.0 20 50 20 0 0 0 0 0 3 3 4 5 3 3 4 5 3 3 4 5 0-10 10 10 10 0-10 10 10 10 -103 0 103 104 105 -103 0 103 104 105 -10 0 10 10 10 Fig.