Principles of signaling pathway modulation for enhancing human naive pluripotency induction

•Inhibition of SRC, PKC, and WNT consolidates human naive pluripotency induction•Competitiveness p53 depleted PSCs in cross-species chimeric embryos•Opposing net effect for ACTIVIN on mouse versus pluripotency•2i ERKi independent alternative naive-like PSC conditions Isolating MEK/ERK signaling-independent pluripotent stem cells (PSCs) with characteristics while maintaining differentiation competence (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening defined induce molecular functional features pluripotency. Synergistic inhibition WNT/?-CATENIN, protein kinase C (PKC), SRC signaling induction teratoma-competent PSCs, capacity to differentiate into trophoblast (TSCs) extraembryonic endodermal (nEND) vitro. Divergent transcriptional requirements boosting were found between human. P53 depletion hPSCs increased their contribution mouse-human embryos upon priming differentiation. Finally, can be substituted NOTCH/RBPj, which induces a diminished risk deleterious global DNA hypomethylation. Our findings set framework defining foundations Conventional embryonic (hESCs) retain variety epigenetic properties are consistent possessing primed state. These include an inability tolerate (MEKi/ERKi), predominant utilization proximal enhancer element maintain OCT4 expression, X-chromosome inactivation, high levels methylation (Weinberger et al., 2016Weinberger L. Ayyash M. Novershtern N. Hanna J.H. Dynamic cell states: rodents humans.Nat. Rev. Mol. Cell Biol. 2016; 17: 155-169Crossref PubMed Scopus (331) Google Scholar). Several groups have previously established deriving genetically unmodified (hPSCs). For example, medium (NHSM) do not require use exogenous transgenes or feeder cells, teratoma formation competence, contain following components: 2iLIF, P38i/JNKi, inhibitor (PKCi), ROCKi, ACTIVIN, FGF2 (Gafni 2013Gafni O. Weinberger Mansour A.A. Manor Y.S. Chomsky E. Ben-Yosef D. Kalma Y. Viukov S. Maza I. Zviran A. al.Derivation novel ground state cells.Nature. 2013; 504: 282-286Crossref (710) Under NHSM conditions, exhibit some features, most notably preservation is inhibited, hallmark mammalian (Takashima 2014Takashima Guo G. Loos R. Nichols J. Ficz Krueger F. Oxley Santos Clarke Mansfield W. al.Resetting transcription factor control circuitry toward ground-state human.Cell. 2014; 158: 1254-1269Abstract Full Text PDF (542) Scholar), TFE3 nuclear localization, vitro reconstitution primordial germ (PGCs), mild reduction Scholar; Irie 2015Irie Tang W.W.C. Kobayashi T. Dietmann Surani M.A. SOX17 critical specifier fate.Cell. 2015; 160: 253-268Abstract (458) The latter profoundly weaker than one observed suggesting sub-optimal growth conditions. Alternative generate MEK-independent more compelling milieu markers developed. components (2i, PKCi, FGF2) supplemented BRAF inhibitors fibroblast (MEF)-dependent lines (termed 5iLA) (Theunissen 2014Theunissen T.W. Powell B.E. Wang H. Mitalipova Faddah D.A. Reddy Fan Z.P. Maetzel Ganz K. Shi al.Systematic identification culture maintenance pluripotency.Cell Stem Cell. 15: 471-487Abstract (497) Globally, these generated pronounced downregulation upregulation markers. However, hypomethylation under was accompanied by immediate loss imprinting over all imprinted loci (Bar 2017Bar Schachter Eldar-Geva Benvenisty Large-Scale Analysis Loss Imprinting Human Pluripotent Cells.Cell Rep. 2017; 19: 957-968Abstract (38) Pastor 2016Pastor W.A. Chen Liu Kim Sahakyan Lukianchikov Plath Jacobsen S.E. Clark A.T. Naive Cells Feature Methylation Landscape Devoid Blastocyst Germline Memory.Cell 18: 323-329Abstract (148) Scholar) confounding chromosomal abnormalities nearly within only 10 passages (Liu 2017Liu X. Nefzger C.M. Rossello F.J. Knaupp A.S. Firas Ford Pflueger Paynter J.M. Chy H.S. al.Comprehensive characterization distinct states reprogramming.Nat. Methods. 14: 1055-1062Crossref (69) Derivation hESCs t2iL-Go (or t2iL-GoY), combination small molecules included our previous transgene-free has been reported NANOG-KLF2 induction, yet shown thus far robustly form teratomas vivo after extended 3-week transfer (Guo 2016Guo von Meyenn Reik Bertone P. Smith Derived Directly from Isolated Inner Mass.Stem Reports. 6: 437-446Abstract (206) Takashima striking difference rodent initiate autonomous needed formative capacitating signals (Ying 2008Ying Q.-L. Wray Batlle-Morera Doble B. Woodgett Cohen self-renewal.Nature. 2008; 453: 519-523Crossref (2406) Collectively, prior studies suggest none devised so optimal reduce gap characteristics. In this study, out devise enhanced enable stabilization stringent preserving cell-autonomous embryonic, extra-embryonic lineages. This complemented efforts decipher regulatory such PSCs. Simultaneously, aimed establish robust obligatory transgenes, broad histone deacytelase Recent ability 2i/LIF knocking repressors like Mettl3 Dnmt1 (m6A mRNA methyltransferase enzymes, respectively), epiblast (EpiSCs) undergo apoptosis complete ablation same factors (Geula 2015Geula Moshitch-Moshkovitz Dominissini A.A.F. Kol Salmon-Divon Hershkovitz V. Peer Mor al.m6A facilitates resolution naïve differentiation.Science. 347: 1002-1006Crossref (844) Liao 2015Liao Karnik Gu Ziller M.J. Clement Tsankov A.M. Akopian Gifford C.A. Donaghey Galonska C. al.Targeted disruption DNMT1, DNMT3A DNMT3B cells.Nat. Genet. 47: 469-478Crossref (274) noted differences led us hypothesize they might utilized screen define endow feature (i.e., absence repressors). We engineered WIBR3 hESC inducible METTL3 expression (Figures 1A 1B ). Primed Tet-OFF-METTL3 expanded could sustained presence DOX (Figure 1C). MEFs, poorly maintained described but other 1C, S1A, S1B). also support when ablated. test candidate may enrich DOX. tested whether supplementing various implicated induced (iPSC) derivation efficiency achieve goal 1D). Remarkably, adding molecule IWR1 enabled expansion 1E). (WNTi) stabilizes AXIN cytoplasm inhibiting Tankyrase enzyme (TNKi). Adding specific TNKi, XAV939, yielded similar 1F). Notably, number recent publications TNKi among ingredients generating naive, potential, (Smith 2012Smith Z.D. Chan M.M. Mikkelsen T.S. Gnirke Regev Meissner A unique phase early embryo.Nature. 2012; 484: 339-344Crossref (685) Yang 2017aYang Ryan D.J. Tsang J.C.H. Lan Masaki Gao Antunes Yu Zhu Z. al.Establishment potential 550: 393-397Crossref (123) Scholar, 2017bYang Xu Wu Dong Lai Vivo Embryonic Extraembryonic Potency.Cell. 169: 243-257.e25Abstract (224) Zimmerlin 2016Zimmerlin Park Huo J.S. Verma Pather S.R. Talbot Jr., C.C. Agarwal Steppan Zhang Y.W. Considine al.Tankyrase promotes stable improved functionality.Development. 143: 4368-4380Crossref (49) OCT4+ DNMT1 S1A S1B), likely due key synergistically used ?PE-OCT4-GFP S1C) supplementation increase GFP 1G S1C). Consistent conducted mice (Kim 2013Kim Ye Tai C.-I. Zhou Yan Li Pera Ying Modulation ?-catenin function maintains self-renewal.Nat. Commun. 4: 2403Crossref (107) rendered dispensable 1G). Further, as inhibits signaling, validated including GSK3 (GSK3i) and, fact, compromises intensity signal S1D). Although dispensable, JNK/P38 boosted marker viability therefore, media herein optional secondary booster S1E). When above modifications, uniformly differentiated transforming (TGF) receptor (TGFR) introduced 1H, 1I, S1F). employed second targeted strategy effort identify whose addition generates without need supplementation. To this, added lacking independently (on DOX) 2A). While fraction rapidly deteriorated, (SRCi = CGP77675) stability dome-like 2B). assemble FGF/TGF/ACTIVIN/MEF-independent serum-free condition termed (HENSM) 2C–2E). Supplementation SRCi Geltrex ACTIVIN-A-containing media, although essential, supported consistency morphology S1G S1H) HENSM-ACT). expressed KLF17 HENSM S2A). mature passage them first injection 2F). extend repressor machineries, DGCR8 null retained S2B). enables expanding TET-OFF 2G) (Liao transferred NHSM, 5i/LA. t2iL-GO, TESR/3iL-MEF 2H 2I). Strikingly, HENSM-ACT allowed unlimited ESCs results demonstrate closely mimic generation ablated repressors. As 5i-LA, (Sperber 2015Sperber Mathieu Ferreccio Hesson Fischer K.A. Devi Detraux al.The metabolome regulates landscape during naive-to-primed transition.Nat. 1523-1535Crossref (223) oxidative phosphorylation (OXPHOS) activity S2C). newly identified metabolic endogenously synthesize glutamine at sufficient adequate alpha-ketogluterate (?KG) (Carey 2015Carey B.W. Finley L.W.S. Cross J.R. Allis C.D. Thompson C.B. Intracellular ?-ketoglutarate 518: 413-416Crossref (521) benefit L-glutamine supplementation, it essential stability, it. EpiSCs cannot period then exogenously 2J). failed naivety 2J), obtained even S2D). five new blastocysts (LIS36, LIS42, LIS46 LIS41 LIS49 HENSM). dermal blood reprogrammed iPSCs hiPSC positive (OCT4, NANOG, SSEA4, TRA1-60, TRA1-81, STELLA, DNMT3L, TFCP2L1, KLF17) S2E); formed S2F); differentiated, priming, directly bona fide S2G) primitive endoderm (PrE nEND cells) S2H S2I) (Linneberg-Agerholm 2019Linneberg-Agerholm Wong Y.F. Herrera J.A.R. Monteiro R.S. Anderson K.G.V. Brickman Naïve respond Wnt, Nodal LIF signalling produce expandable endoderm.Development. 2019; 146: dev180620Crossref (47) Okae 2018Okae Toh Sato Hiura Takahashi Shirane Kabayama Suyama Sasaki Arima Trophoblast 2018; 22: 50-63.e6Abstract (245) hPSC passaged TryplE every 4–5 days S2J). normal karyotype passaging S2K). indicates resetting does cause select pre-existing abnormal variants, seen 5iLA-MEF Unbiased clustering genome-wide profiles demonstrated hiPSCs possess gene pattern cluster separately 3A 3B Transcripts associated significantly upregulated KLF17, KLF4, STELLA (DPPA3), DPPA5, KHDC1L, ARGFX 3C S3A; Tables S1, S2, S3). ESRRB, purported regulator detectable herein, its lack ICM (Petropoulos 2016Petropoulos Edsgärd Reinius Deng Q. Panula S.P. Codeluppi Plaza Reyes Linnarsson Sandberg Lanner Single-Cell RNA-Seq Reveals Lineage X Chromosome Dynamics Preimplantation Embryos.Cell. 165: 1012-1026Abstract (401) had downregulated lineage commitment genes T, OTX2, ZIC2, VIM1 S3B; Table S1). published datasets 5iLA t2iL-Go, note clustered samples 3B). Similar qualities detected Xeno-free (HENSM-XF conditions) BioLaminin511-coated plates 3A). SUSD2 surface tested; however, meaningfully 50% S3D), provides cautionary regarding claims exclusive (Bredenkamp 2019bBredenkamp Stirparo G.G. Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment Cells.Stem 12: 1212-1222Abstract (35) Fluorescence-activated sorting (FACS) analysis confirmed Gb3/CD77 IL6ST/CD130 CD24 S3E) (Collier 2017Collier A.J. Schell J.P. Chovanec Petropoulos Corcoran A.E. Walker Douagi Rugg-Gunn P.J. Comprehensive Surface Protein Profiling Identifies Specific Markers States.Cell 20: 874-890.e7Abstract (91) By comparing transcriptome pre-implantation development, showed enrichment late morula-early blastocyst-specific signature 3D). importance 5i/LA (Pastor 2018Pastor Ho Hunt T.J. Polo TFAP2C opening enhancers.Nat. 553-564Crossref (59) knockout (KO) S3C). explored change chromatin accessibility each ATAC-seq compared different stages. how many show decrease hESCs. Like t2iL-Go-MEF, corresponds earlier stages 3E). endowment transposon (Table S4) profile next pre-X-chromosome configuration. WIBR2 carrying knockin MECP2-tdTomato MECP2-GFP alleles 4A) 2016Theunissen Friedli He Planet O’Neil R.C. Markoulaki Pontis Iouranova Imbeault al.Molecular Criteria Defining State.Cell 502-515Abstract (264) Upon transferring >88% both fluorescent markers, reactivation chromosomes. Transferring back inactivation chromosome, evident obtaining GFP-/tdTomato+ >95% reprimed non-random manner 4A). report emphasizing FGFR random (An 2020An Feng Cao Lu Overcoming Autocrine FGF Signaling-Induced Heterogeneity Enables Modeling Random Inactivation.Cell 2020; 27: 482-497.e4Abstract (12) FGFRi increasing concentration capable S3F S3G). sampled status whole-genome bisulfite sequencing (WGBS). Lines displayed profound 4B 4C), ?81% ?55% ?66% hPSCs. It rapid level partial UHRF1 (von 2016von Iurlaro Habibi N.Q. Salehzadeh-Yazdi Petrini Milagre Xie al.Impairment Maintenance Is Main Cause Global Demethylation Cells.Mol. 62: 848-861Abstract (116) HENSM, maintained, partially 4D), underlie Supplementing 5i/LAF leads dramatic well 2H) explain partial, did observe S3H), contrary what Theunissen Plur