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(1)저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l. 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다.. 다음과 같은 조건을 따라야 합니다:. 저작자표시. 귀하는 원저작자를 표시하여야 합니다.. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다.. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.. l l. 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다.. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer. (2) Master’s Thesis of Pharmacy. Shp2 activity balances Naïve and Primed pluripotency by regulating MAPK and Jak/Stat3 pathway in Mouse Embryonic Stem Cells. 마우스 배아 줄기세포에서 Shp2 활성화에 의한 MAPK, Jak/Stat3 pathway 변화를 통한 Naïve 와 Primed 전분화성 조절 연구. August 2021. Graduate School of Pharmacy Seoul National University Pharmaceutical Biosciences Major. Seong-Min Kim. (3) (4) Abstract For maintaining naïve pluripotency, simultaneous inhibition of Mek1 as well as Gsk3 by chemical inhibitors is critical, suggesting that Mek1-Erk1/2 (Mek/Erk) signaling pathway is activated upon leukemia inhibitory factor (LIF) stimulation. Previously, Ptpn11 (encoding the Shp2 protein, which serves both as a tyrosine phosphatase and adapter) was predicted as one of the key factors that negatively modulates naïve pluripotency through LIF-dependent Jak/Stat3 signaling. In this study, we demonstrated that roles of Shp2 differed between naïve and primed pluripotency by using an isogenic pair of naïve and primed mouse embryonic stem cells (mESCs). Activated Shp2 by LIF modulated the naïve pluripotency. Suppression of Shp2 led to elevated naive pluripotency by promoting Jak/Stat3 signaling and disturbed in vivo differentiation potential. In sharp contrast, Shp2 depletion significantly impeded the self-renewal of ESCs under primed culture conditions, along with a reduction in Mek/Erk signaling. Similarly, during the primed conversion of metastable Pluripotent Stem Cells (PSCs), the treatment with an allosteric Shp2 inhibitor (iShp2) led to eliminated primed pluripotency, which led to predominant naïve pluripotency. Moreover, upon treatment of iShp2, the cells sustained Stat3 phosphorylation and decoupled Mek/Erk signaling, thus replacing the use of iMek1 both for the maintenance of naïve pluripotency and for the establishment of naïve ESCs through reprogramming. Considering the irreversible effect of iMek1 on genomic integrity, iShp2 would be a promising alternative for iMek1.. 1. (5) Taken together, our findings highlight the differential roles of Shp2 in naïve and primed pluripotency and propose the usage of iShp2 instead of iMek1 for the efficient maintenance and establishment of naïve pluripotency.. Keywords: Shp2, Ptpn11, Naïve pluripotency, Primed pluripotency, mESCs, MAPK, Jak/Stat3, LIF. Student Number: 2019-22467. 2. (6) Table of Contents Abstract…………………………………………………………….……………1 Table of Contents…………………………………………………….…………3 List of Figure……………………………………………………..……………..5 List of Table………………………………………………………….………….6 Abbreviations…………………………………………...………………………7 Introduction……………………………………………………………………..8 1. Naïve and Primed pluripotency 2. LIF/Shp2 signaling pathway 3. Dichotomous effect of Shp2 suppression in Naïve and Primed ESCs. Materials and Methods………………………………………………………13 1. Reagents 2. Cell culture 3. Establishment of Shp2KD cell line 4. Live cell imaging 5. Flow cytometric analysis 6. RNA isolation and quantitative RT-PCR analysis 7. Dual Luciferase Reporter assay. 8. RNA-sequencing analysis 9. Teratoma formation assay 10. Gene Set Enrichment Analysis (GSEA) 11. Alkaline phosphatase assay 12. Statistical analysis 3. (7) Results…………………………………………………………………………19 1. Transient LIF-dependent response 2. Shp2 activation by LIF negatively affects naïve pluripotency 3. Role of Shp2 in naïve pluripotency 4. Role of Shp2 in primed pluripotency 5. Defects in the differentiation of naïve ESCs by Shp2 depletion 6. Shp2 chemical inhibitor as an iMek1 replacement 7. Reprogramming of cells to naïve pluripotency using an Shp2 chemical inhibitor. Discussion…………………………………………………………….…….….51 Bibliography…………………………………………………………………..54 국문 초록……………………………………………………………..………..57 Acknowledgements……………………………………….…………………..59. 4. (8) List of Figure. Figure 1. Naïve (Ground) and Primed pluripotency Figure 2. Shp2 mediated signaling pathways in Naïve mouse ESCs Figure 3. Transient LIF dependent response Figure4.Derivation of IL-6/JAK/STAT3 signaling pathway as a main pathwayinnaïve pluripotency Figure 5. Putative negative and positive regulators for naïve pluripotency Figure 6. Shp2 activation by LIF negatively affects naïve pluripotency Figure 7.Naive and primed mESC pair used in the study Figure 8. Enhanced naïve pluripotency after genetic perturbation of Shp2 Figure 9. Decreased iMek dependency after Shp2 genetic perturbation Figure 10.Primed unfavorable effect after Shp2 genetic perturbation Figure 11.Marginal effect on in vitro differentiation of naïve ESCs by Shp2depletion Figure 12.Defective in vivo differentiation of naïve ESCs by Shp2 depletion Figure 13.Naïve favorable effect after Shp2 chemical perturbation Figure 14.Decreased iMek dependency after Shp2 chemical perturbation Figure 15. Primed unfavorable effect of Shp2 chemical perturbation Figure 16. Naïve reprogramming with Shp2 chemical inhibitor Figure 17. Graphical scheme for the study. 5. (9) List of Table. Materials and Methods -Table 1. Primer sequences for RT-qPCR. 6. (10) Abbreviations Shp2 Src homology region 2 domain-containing phosphatase-2 mESC mouse Embryonic Stem Cell LIF. Leukemia Inhibitory Factor. 2i. two inhibitors, Mek1 inhibitor and GSK3β inhibitor. iGsk3 Gsk3 inhibitor, CHIR99021 iMek1. Mek1 inhibitor, PD0325901. iShp2. Shp2 inhibitor, RMC-4550. Sos Son of Sevenless Grb2. Growth factor receptor-bound protein 2. MAPK. Mitogen-activated protein kinase. Jak. Janus Kinase. Stat3. Signal Transducer and Activator of Transcription 3. OG2. Oct4-ΔPE-GFP cell. POG2. Primed- Oct4-ΔPE-GFP cell. OSKM MEF. Oct4, Sox2, Klf4, c-Myc Mouse Embryonic Fibroblast. GSEA Gene set enrichment analysis DMSO. Dimethyl sulfoxide. DMEM. Dulbecco‟s modified eagle medium. FBS. Fetal bovine serum. 7. (11) Introduction Naïve and Primed pluripotency Although mouse embryonic stem cells (mESCs) have been widely used to characterize the early embryonic development of mammals, these cells exhibit clear differences from human ESCs at the cellular (e.g., colony morphology and surface markers). 1. and molecular levels, such as different epigenetic states, X-. chromosome inactivation, chimera formation, major signaling pathways glucose metabolism 4, among others. 5. 2, 3. , and. . Based on recent progress in our. understanding of pluripotency, two discrete pluripotent states, naïve (or ground) and primed, which represent pre- and post- implanted epiblast stem cells (Epi-SCs) respectively 6, account for most of the differences between mouse and human ESCs7 when naive hESCs are established by conversion from primed hESCs8. A constant supply of leukemia inhibitory factor (LIF) along with two chemical inhibitors for Mek1 (iMek1) and Gsk3 (iGsk3) (hereinafter referred to as „LIF+2i‟) is essential9 not only for maintenance but also for the establishment of naïve ESCs, which means the „LIF+2i‟ condition mimics the characteristics of naïve pluripotency of pre-implantation embryos in vitro. LIF-mediated maintenance and establishment of naïve ESCs depend on the Jak/Stat3 pathway 10, which is also conserved in embryo development and implantation. 11. . Two. inhibitors (2i) are required for maintaining naïve pluripotency, which is likely due to the innate mechanism of Gsk3/ inhibition in the inner cell mass (ICM) of blastocysts. 12. coupled with a lower basal activity of Erk1/2 (hereinafter Erk) in. early blastocysts 13, the latter of which is possibly attributed to the expression of Erk-specific dual-specificity phosphatases (DUSPs) 14, 15. 8. (12) 2.. ed pluripotency Primed to differentiation. Pluripotency maintenance. Differentiation. etic landscape Implantation. d.p.c 3.5 ICM. d.p.c 6.5 EpiSC. Naïve Pluripotency. ent, 2015. Primed Pluripotency bFGF/Activin. LIF / 2i. Mek1 and GSK3β inhibition. Metabolism. Signaling. SK3β for what? Model. Epigenome Transcriptome. 1st question. Biology? nd 2. question. 2i : iMek1 and iGSK3β. Summary/Discussion. Figure 1. Naïve (Ground) and Primed pluripotency Naïve pluripotency is established in ICM(inner cell mass) of pre-implantation epiblast. After implantation, the epiblast become „primed‟ to differentiate. Epiblast stem cells from primed epiblast have primed pluripotency. In vitro conversion between the two pluripotency status can be done.. 9. (13) LIF/Shp2 signaling pathway Src homology region 2 domain-containing phosphatase 2 (Shp2), which is encoded by protein tyrosine phosphatase non-receptor type 11 (Ptpn11), transmits the receptor signaling to Ras-to-Erk16 and negatively modulates Jak/Stat3 signaling 17. Phosphorylation of Shp2 upon cytokine stimulation by interleukin-6 (IL-6) or LIF, leads to intramolecular conformational changes to relieve autoinhibition of tyrosine phosphatase activity. 18. and dephosphorylates the Jak/Stat3. pathway 18, 19. In parallel, the recruitment of phosphorylated Shp2 to gp130 forms a protein complex with Gab1 to transduce signals from the receptor to Ras/Raf/Mek/Erk20. Considering the role of Shp2 as an adaptor in Ras-dependent Erk activation (Ras-to-Erk), as well as in oncogenic Ras-to-Erk21, 22 and target therapy resistance23, small molecules to inhibit the bivalent roles of Shp2 have been developed as novel anti-cancer therapeutic agents24. Shp2 deficiencies enhance self-renewal capacity and suppress differentiation of mESC through increased sensitivity to LIF on Jak/Stat3 and attenuation of Ras-toErk17. Similarly, failure to recruit Shp2 25 and Shp1 2 to the site of Stat3 activation sensitizes mESCs to LIF in the Jak/Stat3 signaling pathway, thus favoring selfrenewal. In contrast, the molecular mechanisms that control the pluripotency and differentiation of hESCs via Shp2 depletion are different from those of mESCs26, which suggests the dichotomous role of Shp2 in naïve and primed ESCs.. 10. (14) Figure 2. Shp2 mediated signaling pathways in Naïve mouse ESCs (A) Protein structure of Shp2.Shp2 has two src homology 2 (SH2) domains and a protein tyrosine phosphatase (PTP) domain. (B) Shp2 acts as both an adaptor and a phosphatase. (C) LIF mediated signaling pathways are critical to maintain pluripotency of mESCs. Mainly, Jak/Stat3 signaling pathway, which is critical to maintain naïve pluripotency becomes activated. Simultaneously, Ras-to-Erk signaling, which leads to differentiation becomes activated. Upon LIF binding to LIF receptor, Shp2 recruits to the receptor and becomes activated. Active Shp2 mediates signal to Ras-to-Erk as a mediator by allowing Sos to activate Ras, while dephosphorylates Jak and Stat3 as a phosphatase.. 11. (15) Dichotomous effect of Shp2 suppression in Naïve and Primed ESCs Here, we showed that transient activation of Shp2 by LIF negatively modulated Jak/Stat3 signaling and transmitted the signal to Ras-to-Erk, which consequently led to an attenuation of naïve pluripotency. Accordingly, genetic depletion of Shp2 or iShp2 (a chemical inhibitor of Shp2) treatment favors naïve pluripotency by sensitizing the Jak/Stat3 signaling pathway to LIF stimulation and decoupling Ras-to-Erk signaling, whereas primed ESCs were intolerant to Shp2 perturbation. The dichotomous effect of Shp2 inhibition thus favored the enrichment of naïve ESCs. Additionally, iShp2 could efficiently substitute iMek1 for the maintenance and establishment of naïve pluripotency.. 12. (16) Materials and Methods 1. Reagents The primary antibodies against α-tubulin (#sc- 8035), -actin (#sc-47778), Stat3 (#sc-8019), Erk2 (#sc-154) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against phospho-Shp2 (Tyr542) (#3151), phospho-Stat3 (#9145s), phospho-Erk (#9101) were purchased from Cell Signaling Technology. Antibody against Shp2 (#VL3159027A) was purchased from Invitrogen. siRNAs targeting Negative Control (#SN-1003) and the others (listed at supplement table) were obtained from Bioneer. Expression vector of 6X Stat3-luciferase was kindly gifted by Prof. HyewonYoun from Seoul National University.. 2. Cell culture Naïve mouse ESCs were cultured on 0.5% porcine gelatin-coated dish in Naïve mESC culture media -DMEM high glucose (Gibco) supplemented with 15% FBS (Gibco), 1% Glutamax (Gibco), 1% MEM-nonessential amino acids (Gibco), 0.1% Gentamycin (Gibco), 0.1 mM -mercaptoethanol (Gibco), 1,000 U/ml mouse leukemia inhibitory factor (mLIF) (Millipore, Merk), 1 M PD0325901 (Peprotech) and 3 M CHIR99021- at 37oC and 5% CO2 incubating condition. Cells were passaged 1:10 ratio every 3 days using 0.25% Trypsin/EDTA (Wellgene) as a dissociation reagent. Primed mouse ESCs were cultured on Matrigel (Corning# 354277)-coated dish in EpiSC culture media -DMEM/F12 (Gibco) supplemented with 15% KnockOut SR (Gibco), 1% GlutaMAX (Gibco), 1% MEM-nonessential amino acids (Gibco), 0.1% Gentamycin (Gibco), 10 ng/ml 13. (17) murine bFgf (Peprotech), 20 ng/ml murine Activin (Peprotech)- at 37oC and 5% CO2 condition. Primed mESCs were passaged every 3-4 days using Dispase (Gibco) as a colony detachment reagent. Detached colony clumps were transferred 1:15-1:20 ratio on Matrigel (Corning# 354277) coated dishes. Culture media was changed every day for all cell lines. Oct4-ΔPE-GFP/ΔDE-RFP ESCs were cultured on mitogen-inactivated C57BL/6 feeder cells on 0.15% porcine gelatin-coated dish in culture media - DMEM Low (Gibco) supplemented with 15% FBS (Gibco), 1% P.S.G (Gibco), 1% MEM-nonessential amino acids (Gibco),0.1 mM β-mercaptoethanol (Gibco) and 1,000 U/ml mouse leukemia inhibitory factor (mLIF) (Millipore, Merk)- at 37oC and 5% CO2 incubating condition.. 3. Establishment of Shp2KD cell line Shp2 stable knocked-down cell line was acquired by introducing shPtpn11 piggybac vector. Transfection was performed using Lipofectamine 3000 reagent (#L3000-001, Invitrogen). G418 selection (600g/ml) was done for 9 days. Single colony selection was performed from heterogenous Shp2KD pool cells.. 4. Live Cell imaging Brightfield images and GFP images of cells on specific area were captured every hour by JuLi Stage (NanoEntek). Acquired images were spliced together to create time-lapse cell growth graphs using JuLi-Edit.. 14. (18) 5. Flow cytometric analysis GFP intensity of OG2 cells were measured by Flow cytometric analysis. Cells were dissociated with Accutase (#561527, BD Bio-sciences) and washed three times with DPBS. Cells were analyzed through FACS Carlibur (BD Bioscience). GFP intensity was determined by measuring FITC channel.. 6. RNA isolation and quantitative RT-PCR analysis Total RNA was isolated from cells using Easy-BlueTM total RNA isolation kit (iNtRON Biotech) following the manufacturer‟s instructions. 5x PrimeScript TM RT mix (TaKaRa) was used during reverse transcription to acquire cDNA. Quantitative real-time PCR was performed using TB-Green premix (TaKaRa) by LightCycler-480® II (Roche). Rn18s gene was used as internal loading control for normalizing gene expression data.. 7. Dual Luciferase Reporter assay Cells were transfected with 6x Stat3 reporter vector and pRL vector using Lipofectamine 2000 reagent (#11668019, Invitrogen). After 3 hours of incubation with 1x passive lysis buffer followed by centrifugation, supernatant from the cell lysate was acquired. The supernatant was used for the reaction with LARII and Stop & Glo reagent. Stat3 luciferase assay was performed using Dual Luciferase Reporter Assay System Kit (#E1980, Promega), and detected by SpectraMax® i3x Multi-Mode Microplate Reader.. 15. (19) 8. RNA-sequencing analysis Total RNA was isolated from cells by using Easy-BLUETM RNA isolation kit (iNtRON Biotechnology). 1mg of total RNA was processed for preparing mRNA sequencing library using MGI Easy RNA Directional Library Prep Kit (MGI) following manufacturer‟s instruction. The mRNA is fragmented into small pieces under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved in the RT directional buffer and second strand cDNA synthesis was done. Single „A‟ base was added to the cDNA fragments, followed by ligation of the adapter. The products are purified and enriched by PCR procedure to acquire the final cDNA library. The cDNA library is quantified using QuantiFluor ONE dsDNA System (Promega). Using DNA nanoball (DNB) enzyme, the library is incubated at 30°C for 25min to make DNB. Finally, sequencing of the prepared DNB was performed using MGIseq system (MGI) with 100bp paired-end reads.. 9. Teratoma formation assay 5x105 cells of each OG2 and OG2Shp2KD mESCs were injected into the testes of 5-weeks-old male BALB/C nude mice (n=3), and subcutaneously injected in the neck (n=5) and abdomen (n=5) of 5-weeks-old male BALB/C nude mice. Mice were euthanized 6 weeks after injection. These animal experiments were conducted under the permission of Seoul National University Institutional Animal Care and Use Committee (Permission number: SNU-190716-5-2).. 16. (20) 10. Gene Set Enrichment Analysis (GSEA) FPKM values of RNA sequencing data were formatted and loaded to run Gene Set Enrichment Analysis. GSEA software and the Gene Sets (Hallmark _ IL6 _ JAK _ STAT3 signaling, KEGG _ JAK _ STAT3 signaling, ABBUD _ LIF signaling_up, Hallmark _ KRAS _ signaling _ DN, Hallmark _ KRAS _ signaling _. UP). were. downloaded. from. Molecular. Signatures. Database. v7.4. (https://www.gsea-msigdb.org).. 11. Alkaline phosphatase assay After reprogramming of iOSKM MEF, Alkaline phosphatase staining assaywas performed by using AP staining kit (Cat# 86R-1KT; Sigma-Aldrich) to visualize the reprogrammed cells. Overall procedure was followed by the manufacturer‟s instructions.. 12. Statistical analysis The quantitative data are presented as the mean values ± standard deviation (SD). Unpaired two-tailed t-tests or one-way ANOVA following Dunnett multiple comparison, was performed to analyze the statistical significance of each response variable. p-values less than 0.05 were considered statistically significant (* < 0.05, ** <0.001, ***<0.0001, ****<0.0001 and n.s. for not significant).. 17. (21) Table1. Primer sequences for RT-qPCR Primer sequences (5‟-3‟). Primer pairs Ptpn11. Dppa3. Rex1. NrOb1. Klf4. Esrrb. Klf2. Fgf5. Cer1. Pou5f1. Sox2. Nanog. c-Myc. Forward. AGTCCAAAGTGACCCACGTC. Reverse. CCATCATGCAGAACGACCCT. Forward. CGTACCTGTGGAGAACAAGAGTG. Reverse. CATTCTCAGAGGGATCCCATCTTTG. Forward. CTTCGAAAGCTTGGAGGAAGTGGAG. Reverse. GGACACTCCAGCATCGATAAGACAC. Forward. ACAGAGCAGCCACAGATGGTGTC. Reverse. GATGTGCTCAGTAAGGATCTGCTG. Forward. GAACAGCCACCCACACTTGTGAC. Reverse. CTGTCACACTTCTGGCACTGAAAG. Forward. GATTCTCATCTTGGGCATCGTGTAC. Reverse. CTGACTCAGCTCATAGTCCTGCAG. Forward. CACACATACTTGCAGCTACACCAAC. Reverse. CAAGTGGCACTGAAAGGGTCTGTG. Forward. CATCGGTTTCCATCTGCAGATCTAC. Reverse. GTTCTGTGGATCGCGGACGCATAG. Forward. GTGGAAAGCGATCATGTCTCATCG. Reverse. GCAAAGGTTGTTCTGGACAACGAC. Forward. GAGAAAGCGAACTAGCATTGAGAAC. Reverse. TGTAGCCTCATACTCTTCTCGTTG. Forward. ATGGGCTCTGTGGTCAAGTC. Reverse. CCCTCCCAATTCCCTTGTAT. Forward. GTGCACTCAAGGACAGGTTTCAG. Reverse. CTGCAATGGATGCTGGGATACTC. Forward. ACCACCAGCAGCGACTCTGA. Reverse. TGCCTCTTCTCCACAGACACC. 18. (22) Results Transient LIF-dependent response Constant iMek1 and LIF supplementation would be necessary to inhibit Erk activation on Klf4 and Nanog destabilization. 27, 28. . We also observed Erk. activation along with Stat3 phosphorylation upon LIF stimulation (Fig. 1A). To determine the occurrence of naïve pluripotency, we took advantage of OG2 mESCs, which express naïve specific green fluorescent protein (GFP) under the control of the Oct4 promoter due to the lack of a proximal enhancer 29. Consistent with previous reports 6, a lack of 2i resulted in the loss of the dome shape that characterizes naïve specific colony morphology (Fig. 1B) and significantly attenuated GFP signals even under LIF stimulation (Fig. 1C). Due to negative feedback mechanism(s) toward Stat330, triggered just after LIF stimulation, Stat3 phosphorylation (Fig. 1A) and its transcriptional activity (Fig. 1D) were attenuated along with Erk activation through a series of protein interactions. 31. .. Therefore, the putative negative feedback mechanism(s) and Erk activation after LIF stimulation would be responsible for the constant supply of LIF+2i for maintaining naïve pluripotency.. 19. (23) Figure 3. Transient LIF dependent response (A) Immunoblotting at indicative time after LIF stimulation, Erk2 andβ-actin were used as loading controls. (B) Fluorescent microscopic images at indicated time under LIF+2i, LIF only and LIF deprivation (LIF-) media respectively(scale bars : 500μm). (C) Flow cytometry of GFP after 60hr stimulated by LIF+2i, LIF only, LIF- media respectively (left panel), Graphical presentation of GFP positive population (right panel). (D) Luciferase reporter activity of Stat3 in OG2 cells at indicated time after LIF stimulation (*, p < 0.05, **, p < 0.001, ***, p < 0.0001, n.s. for not significant).. 20. (24) Shp2 activation by LIF negatively affects naïve pluripotency A previous study identified a set of negative and positive regulators of naïve pluripotency. 32. through genome-wide CRISPR knockout (KO) screening. A total. of 156 genes, whose perturbation significantly affected naïve pluripotency. 32. ,. were examined via gene ontology (GO) and KEGG pathway analysis. As expected based on the significance of LIF-mediated signaling on naïve pluripotency described in other studies, these 156 genes were highly enriched in several associated pathways including „Jak/Stat3 signaling‟ (Fig 4A), „Interleukin6 signaling‟ (Fig 4B and C),‟ and „PluriNetWork‟ (Fig. 4B). Particularly, a few genes among a set of 27 positive regulators (shown in red) and 128 negative regulators (shown in blue) were associated with the „pluripotency signaling‟ (Fig. 5A), „JAK/STAT signaling‟ (Fig. 5B), and „Ras signaling‟ (Fig. 5C) pathways. Among 27 putative negative regulators, three genes-Grb2, Ptpn2, and Ptpn11belonged to the “IL-6/JAK/STAT signaling” pathway according to MSigDB Hallmark 2020 (https://maayanlab.cloud/Enrichr/)33 (Fig. 5D). We next focused on Ptpn11, which encodes the Shp2 protein that dephosphorylates Stat3, thus serving as an important negative regulator of IL-6 stimulation 34, and transduces signals toward Ras-to-Erk possibly through Grb2 35 (Fig. 5C).. 21. (25) Figure 4.Derivation of IL-6/JAK/STAT3 signaling pathway as a main pathway in naïve pluripotency (A, B, C) Top enriched categories of GO from BioPlanet 2019 (A), Wiki Pathways 2019 Mouse (B) and MSigDB Hallmark 2020 (C) from DEG, ranked by -log (p-value).. 22. (26) Figure 5. Putative negative and positive regulators for naïve pluripotency (A, B and C) KEGG pathways for Signaling Pathways regulating Pluripotency of Stem Cells (A), Jak/Stat signaling pathway (B), and Ras signaling pathway (C) in Mus musculus. 27 and 128 putative negative and positive regulators for naïve pluripotency were shown in red and blue respectively.(D) List of 27 genes that were predicted as putative negative regulators of naïve pluripotency (top), gene list involved in „IL-6/JAK/STAT3 Signaling Pathway‟ (bottom).. 23. (27) Considering the close similarity between LIF and IL-636, we presumed that the activation of Shp2 by LIF would likely act as a negative regulator on naïve pluripotency by Stat3 dephosphorylation and Erk1/2 activation. As predicted, active phosphorylation of Shp2 (Y542) occurred promptly after LIF stimulation in parallel with signal transduction to Ras-to-Erk, thus relieving its auto-inhibitory regulation on phosphatase activity18 (Fig. 6A). In parallel with Shp2 active phosphorylation (Fig. 6A), the phosphatase activity of Shp2 [inhibited by Shp2 inhibitor (iShp2) treatment], was also clearly induced by LIF stimulation (Fig. 6B). These data suggest that Shp2 activation by LIF simultaneously controls both Jak/Stat3 and Ras-to-Erk signaling to affect naïve pluripotency in a bivalent manner (Fig. 6C).. 24. (28) A Mock 10. LIF Shock. Phosphatase activity. 30 (min) pShp2 Shp2. pSrc pMek1. 3. Fold change. pStat3. C. B. **. **. 2 1 0. Mock. LIF LIF+iShp2. pErk1/2 α-tubulin. Figure 6. Shp2 activation by LIF negatively affects naïve pluripotency (A) Immunoblotting for indicative proteins at 10 and 30 min after LIF stimulation, α-tubulin was used as a loading control. (B) Graphical presentation of phosphatase activity of OG2 ESCS at 10 min after LIF stimulation with or without iShp2 (10µM) for 1 hour (**, p <0.001, n=4). (C) Graphical illustration of LIF-Shp2 mediated signaling pathways of naïve ESCs.. 25. (29) Role of Shp2 in naïve pluripotency Although the roles of Shp2 were well-characterized in mESCs with knockdown (KD) or knockout (KO) models17, we noticed that the effect of Shp2 in human ESCs (hESCs) was less evident than that of mESCs26. Given that hESCs share the molecular and cellular characteristics of primed ESCs7, we hypothesized that the effect of Shp2 in naïve pluripotency would differ from that of primed pluripotency. To evaluate this hypothesis, we utilized primed ESCs (P-OG2) converted from naïve ESCs (OG2). As previously described 37, naïve ESCs with a „colonial dome shape‟ exhibit GFP signals unlike primed ESCs, which exhibit a „flat disc shape‟ (Fig. 7A), expressing typical marker genes of the naïve and primed status (Figs. 7B and C).. 26. (30) OG2. A. Bright field. POG2. GFP+. GFP-. Ptpn11. C. Naïve2.0 pluripotency markers. ****. ***. 0.5. 1. 0.0 Rex1. Dppa3. 100. 1.0. OG2 POG2. 10. 0.5. 1. 0.0. 2. 2 EsrrbOGO G Klf2 P. O POG2 G 2 O POG2 G 2. 0.1. ****. NrOb1. 2 2 2 2 2 0.1 OG OG Fgf5 OG. G. ****. ****. 1000 1.5. G. ****. 1.0. Ptpn11. Primed pluripotency markers 2.0. PO. 10. 1.5. Fold change. 100. ****. Fold change. 1000. OG2 POG2. PO. B. Fold change. Flat disc shape. G. Colonial dome shape. PO. LIF+2i. GFP. OG2. Fold change. POG2. bFgf/Activin A. 2. Cer1. Figure 7.Naive and primed mESC pair used in the study (A) Graphical illustration of isogenic pair of naïve (OG2) and primed (POG2) ESCs (left panel), brightfield and fluorescent microscopic images of OG2 and POG2 ESCs (scale bars: 500μm) (right panel). (B) Relative mRNA expressions of naïve pluripotency markers in OG2 and POG2 mESCs(***, p < 0.0001 and ****, p < 0.00001, n=3). (C) Relative mRNA expressions of primed pluripotency markers in OG2 and POG2 mESCs (****, p < 0.00001, n=3).. 27. (31) Stable knockdown of Ptpn11 was performedusing a pair of naïve and primed ESCs. As expected based on previous studies performed in mESCs, naïve ESCs with clear Ptpn11 knockdown (hereinafter referred to as Shp2 KD or KD naïve ESCs) (Fig. 8A) exhibited a clear „colonial dome shape‟ with an increased GFP signal (Fig. 8D). Consistently, naïve cell-specific marker genes were also significantly enhanced in KD Naïve ESCs (Fig. 8B), whereas core pluripotency genes only exhibited marginal changes (Fig. 8C). Interestingly, KD Naïve ESCs maintained a stable GFP signal even under LIF treatment without 2i supplement (LIF only), which significantly affected the aforementioned „colonial dome shape‟ morphology (Fig. 8F) as well as theGFP signal of wild-type (WT) cells (Fig. 8E).Based on the clear increase in active phosphorylation (Fig. 6A) and phosphatase activity (Fig. 6B) of Shp2 by LIF, the prolonged naïve characteristics of KD naïve ESCs under the LIF-only condition resulted from sustained Stat3 phosphorylation as observed via stable (Fig. 8H) and transient (Fig. 8I) Shp2 depletion. These results were also supported by the gene set enrichment analysis (GSEA)38of FPKM values from the RNA-sequencing transcriptomes of WT and KD. Consistent from the former results, the gene set for „Hallmark IL6 JAK STAT3 signaling‟, „KEGG JAK STAT3 signaling pathway,‟ and „LIF signaling 1 UP‟ were significantly enriched in the KD cells compared with the WT counterparts (Fig. 8G). However, we still could not fully explain the marginal effect of 2i withdrawal on GFP signals (Fig. 8E), as well as the morphological changes (Fig. 8F).. 28. (32) *. 0.5 0.5 0.0 0.0. WT. WT WT. 15. ***. KD. 1.0. 10. 5. 0. 10. *** ** ***. ***. 0. Rex1. NrOb1. Dppa3. Rex1. Klf4. NrOb1. Sox2. 40h. WT. LIF+2i. + LIF Mock 0 10 30 60 (min) pStat3. 60h. I. ABBUD_LIF _SIGNALING_1_UP. NES: 1.508. KD. WT. NES: 1.694. KD. Stat3 α-tub. Erk2 +siNC. KD. WT. + LIF + LIF 0 10 30 60 0 10 30 60 (min) pStat3. Stat3. WT. Nanog. KD. Hallmark_IL6_JAK_STAT3 KEGG_JAK_STAT3 _SIGNALING_PATHWAY _SIGNALING. KD. + LIF. Pou5f1. 20h. NES: 1.858. Mock 0 10 30 60. 0.5. WT. LIF only. G. LIF+2i. KD. ns. KD 1.0. 0.0. Klf4. 0h. WT 1.5. KD. Bright field GFP WT. **. WT KD. ns. WT. KD. LIF only. H. **. E WT. KD. KD. **. D. F. WT. Core pluripotency markers Core pluripotency ns. WT. 5. Dppa3. KD KD. C. Naïve pluripotency markers. Fold mRNA expression. 1.5 1.0. Bïve pluripotency markers Na 15. Fold mRNA expression. FoldmRNA mRNAexpression expression. Ptpn11 Ptpn11. Fold mRNA expression. A. +siPtpn11. Figure 8. Enhanced naïve pluripotency after genetic perturbation of Shp2 (A) Fold mRNA expressions of Ptpn11 in WT (blue) and KD (red) ESCs (*, p < 0.05). (B) Fold mRNA expression of typical naïve pluripotency marker genes in WT and KD ESCs (**, p < 0.001 and ***, p < 0.0001, n=3). (C) Fold mRNA 29. (33) expressions of core pluripotency markers in WT and KD ESCs (n.s., nonsignificant). (D) Representative light microscopic images of WT and KD ESCs (scale bars : 200μm) (top panel) and fluorescent microscopic images of WT and KD ESCs (scale bars : 500μm) (bottom panel). (E) Fluorescent microscopic images of WT and KD ESCs at indicated time under LIF+2i and LIF only culture condition (scale bars : 500μm).(F) Light microscopic images of WT and KD ESCs under indicated media conditions (scale bars: 500μm).(G) The normalized enrichment score by gene set enrichment analysis (GSEA) of WT and KD ESCs for Hallmark_IL6_JAK_STAT3 signaling (left panel), KEGG_JAK_STAT3 signaling (middle panel), and ABBUD_LIF signaling up (right panel).(H) Immunoblotting for pStat3, Stat3 and Erk2 in WT and KD ESCs at indicated time after LIF stimulation, Erk2 was used as a loading control. (I) Immunoblotting of OG2 ESCs with control (siNC) or Ptpn11 siRNA (siPtpn11) at indicated time after LIF stimulation, Erk2 was used as a loading control.. 30. (34) Therefore, the RNA-sequencing transcriptomes of WT and KD naïve ESCs after depletion of iMek1 or iGsk3 were analyzed next. Compared to the transcriptome of LIF+2i, the WT transcriptome lacking 2i (WT LIF only) exhibited the largest number of DEGs, thus manifesting the most severe alterations. Therefore, to compare the effect of depletion of each inhibitor, „WT LIF only‟ DEGs were designated as a gene pool named „gene sets for 2i‟ for comparison. Within the „gene sets for 2i‟, KD naïve ESCs lacking iMek1 [KD (-) iMek1] were altered the least (Fig. 9A) compared to the other cells, suggesting that KD naïve ESCs would be more tolerant to iMek1 depletion. As predicted, unlike WT cells, the „colonial dome shape‟ morphology of KD naïve ESCs remained unaltered without iMek1 supplementation but was quickly lost after iGsk3 withdrawal (Fig. 9B). Given that Shp2 has another role as a signal transducer to Ras-to-Erk in addition to tyrosine phosphatase, a lack of Shp2 may decouple the signal transduction to Erk upon LIF stimulation. Consistent with the results in Figure 8G and Figure 9A, phosphorylated Mek1 and Erk (Fig. 9C) was attenuated in KD naïve ESCs, whereas no clear alteration in GSK3 phosphorylation was observed (Fig. 9D). These data were also supported by GSEA, demonstrating that the gene signatures of “Hallmark KRAS signaling DN and UP” were more enriched in KD naïve ESCs when iMek1 was depleted (Fig. 9E).. 31. (35) Figure 9. Decreased iMek dependency after Shp2 genetic perturbation (A)DEGs of WT LIF+2i vs WT (-)2i were assigned as „gene sets for 2i‟. The number of Up-DEGs and Down-DEGs within the „gene sets for 2i‟ group are presented. (B) Light microscopic images of WT and KD 48 hours after indicated culture condition (scale bars: 200µm). (C) Immunoblotting analysis for Shp2, pMek1/2, Erk2 and α-tubulin in WT and KD ESCs. (D) Immunoblotting analysis for pGsk3β, pErk1/2, pStat3 and Vinculin of WT and KD ESCs at indicated time after LIF stimulation. Vinculin was used as a loading control. (E) The normalized enrichment score by gene set enrichment analysis (GSEA) of WT and KD ESCs for HALLMARK _ KRAS _ SIGNALING _ DN (left) and HALLMARK _ KRAS _ SIGNALING _ UP (right).. 32. (36) Role of Shp2 in primed pluripotency Unlike naïve ESCs, primed ESCs were likely intolerant to the absence of Shp2. Despite multiple trials, the establishment of primed ESCs with stable Shp2 depletion was unsuccessful due to the severe growth retardation of primed ESCS after Shp2 knockdown (Fig. 10A). Alternatively, KD naïve ESCs were subjected to primed culture conditions (with bFGF/Activin) to establish KD primed-like ESCs. Although KD naïve ESCs propagated well in naïve culture conditions, they did not successfully grow in primed culture conditions (Fig. 10B and C). This dichotomous effect of Shp2 depletion on primed ESCs compared to naïve ESCs was further highlighted by clonogenic assays (Fig. 10D and E), which account for prompt culling of KD ESCs (labeled with GFP only) from the WT ESCs (labeled with RF/GFP) mixture under primed conditions unlike naïve conditions (Fig. 10F). As similar as that of naïve ESCs, signaling to Erk upon bFGF/Activin stimulation was markedly attenuated in KD ESCs (Fig. 10G). These results indicate that the self-renewal of primed ESCs depends on Shp2-dependent signaling unlike that of naïve ESCs.. 33. (37) F RF+/GFP+. G. RF+/GFP+. bFGF/Activin WT KD RF+/GFP+. Shp2 pErk1/2. LIF+2i GFP. Erk2. bFGF/Activin RF GFP. RF. β-actin. 34. (38) Figure 10. Primed unfavorable effect after Shp2 genetic perturbation (A)Light microscopic images of POG2-WT and KD cells (scale bars: 500μm).(B) Light microscopic images of WT and KD ESCs at indicated times under LIF+2i or bFGF/Activin culture condition(scale bars: 500μm). (C) Graphical presentation of cell number at indicated time after seeding of 1x104 ESCs. (D) Images of clonogenic assay of WT and KD ESCs at indicated time after culture in LIF+2i or bFGF/Activin. (E) Graphical presentation of area ratio (A.U.) of clones at day 2 and day 4 determined by ImageJ. (F) Graphical illustration of competition assay with WT or KD ESCs, labeled with red fluorescence (RF)/GFP using Far-red dye or GFP only respectively (top), Fluorescence microscopic images of WT or KD ESCs at 2 days after indicated culture condition, arrowheads indicate KD ESCs (labeled with GFP only) (scale bars: 500μm). (G) Immunoblotting analysis of WT and KD ESCs grown under bFGF/Activin for Shp2, pStat3, pErk1/2, Erk2 and βactin. β-actin was used as a loading control.. 35. (39) Defects in the differentiation of naïve ESCs by Shp2 depletion Shp2 null (Shp2 -/-) mice failed to develop as a result of peri-implantation lethality, which is caused by failure of trophoblast formation39. Similar to the high bFGF signaling dependence described above, trophoblast stem cells (TS) and epiblast stem cells (EpiSCs), which share a close similarity to primed ESCs, rely on bFGF signaling40. As previouslyproposed. 39. , embryo development failure in. Shp2 null mice may result from defects in the transition from naïve to primed ESCs in accordance with the TSimpediment. Naïve ESCs after Shp2 depletion tended not only to maintain naïve pluripotency (Fig. 8) but also to hinder the progression to primed ESCs (Fig 10), and therefore the differentiation potential of KD naïve ESCs would be disturbed. Thus, WT and KD naïve ESCs were subjected to spontaneous differentiation via LIF/2i withdrawal from embryoid body (EB) formation (Fig. 11A and B). During spontaneous differentiation, typical marker genes of naïve pluripotency were clearly upregulated in the embryoid bodies of KD naïve ESCs (Fig. 11A), remaining marginally high one day after differentiation induction and becoming sharply suppressed when core pluripotency marker genes were similarly repressed (Fig. 11C). Thus, spontaneous differentiation was enforced with the serum-induced exit from naïve pluripotency regardless of Shp2.. 36. (40) Figure 11. Marginal effect on in vitro differentiation of naïve ESCs by Shp2 depletion (A) Light microscopic images of embryoid body (EB) cultured for 3 days of WT or KD ESCs (scale bars: 500μm) (left), Graphical presentation of relative mRNA expression of typical naïve pluripotency and core pluripotency markers of WT and KD (right). (B) Light microscopic images of WT and KD before [LIF/2i] or 48 hours after differentiation [(-)LIF/2i]. (C) Relative mRNA expressions of naïve pluripotency (left) and core pluripotency (right) markers at indicative time after spontaneous differentiation of WT and KD.. 37. (41) However, unlike in vitro differentiation, the formation of teratoma from KD naïve ESCs was significantly impaired in multiple injection sites compared to those from WT (Fig. 12A, B and C). One teratoma-like mass which was formed out of a total of 13 injections of KD naïve ESCs (Fig. 12C and D) only exhibited a few ectoderm and endoderm tissue structures without clear mesoderm tissue formation, unlike the well-developed teratoma from WT (Fig. 12E). It is also worth noting that. Shp2 is. required. for. gastrulation and. developmentduring mouse 41 and Xenopus 42embryo development.. 38. mesoderm. (42) Figure 12. Defective in vivo differentiation of naïve ESCs by Shp2 depletion (A) Representative images of teratomas formed in mouse testes at 6 weeks after injection of WT (top) and KD (bottom) ESCs. (B) Representative images of teratomas formed in mouse subcutaneous area at 6 weeks after injection of WT. (C) Summary of teratoma formation of WT and KD in three different areas. (D) Graphical presentation of teratoma volume [V = a × b × c × π / 6 (a = length, b = width, and c = depth)] of total 13 teratoma from WT or KD ESCs. Volumes of normal testis after teratoma injection were considered as 0. (*, p < 0.05). (E) Hematoxylin & eosin (H&E) (for ectoderm and endoderm) and Masson‟s trichrome (for mesoderm) staining of teratoma section from WT and KD, uncharacterized tissue from teratoma of KD were shown in the box.. 39. (43) Shp2 chemical inhibitor as an iMek1 replacement There is emerging evidence that Shp2 contributes to chemoresistance and cancer development. 21, 22, 43. . Thus,Shp2 allosteric inhibitors that interfere in both. phosphatase activity and signal transduction by leading the whole conformation change have been developed as novel anti-cancer therapeutic agents. 21. . We first. examined whether an allosteric Shp2 inhibitor (Fig. 13A, RMC-4550: iShp2), which was initially developed to decouple the oncogenic Ras-to-Erk signaling in human cancers. 21. , also inhibits LIF-dependent Erk activation. At an iShp2. concentration known to decrease the phosphorylation of Erk (Fig. 13B), Stat3 phosphorylation was significantly sustained after LIF stimulation in naïve ESCs (Fig. 13C), which was associated with higher Stat3 reporter activity in the presence of iShp2 8 hours after the first LIF stimulus (Fig. 13D). Additionally, iShp2 treatment also preserved Stat3 phosphorylation even at a 1/100-fold LIF concentration (Fig. 13E) and clearly rescued naïve ESCs from cell death at a low LIF concentration (Fig. 13F).. 40. (44) Figure13. Naïve favorable effect after Shp2 chemical perturbation (A). Chemical. structure. of. Shp2. inhibitor. (iShp2:. RMC-4550).. (B). Immunoblotting analysis of WT ESCs at 30 min after indicative dose of iShp2 treatment, α-tubulin was used as a loading control. (C) Immunoblotting analysis of WT ESCs at indicated time after 1000 units of LIF stimulation, pretreated with either DMSO [Mock] or 5M of iShp2 [+iShp2] for 1 hour (L/2i: LIF+2i control). (D) Graphical presentation of luciferase reporter activity of Stat3 at 8 hours after LIF deprivation with either DMSO [Mock] or 5M of iShp2 [+iShp2] for 1 hour compared to prior to LIF deprivation as a control [LIF](***, p <0.0001).(E) 41. (45) Immunoblotting analysis for pStat3 after 30 min of LIF stimulation. (F) Light microscopic images after indicative dose of LIF stimulation in WT ESCs for 48 hours supplemented with either DMSO [Mock] or 5μM of iShp2 [+iShp2](scale bars: 500μm).. 42. (46) Similar to our observations in KD naïve ESCs (Fig. 9A and B), iShp2 treatment could likely replace the effects of iMek1 (but not iGSK3) on the „colonial dome shape‟ morphology of naïve ESCs (Fig. 14A), which may result from the clear decoupling of LIF mediated Ras-to-Erk signaling by iShp2 treatment (Fig. 13C). Similarly, typical naïve marker expressions (Fig. 14B) and the GFP intensity (Fig. 14C and D) indicated that iShp2 treatment compensated for the loss of iMek1 in naïve ESCs.. 43. (47) A. ( - ) iGSK3!. ( - ) iMek1. ( - ) 2i. +iShp2. Mock. LIF+2i. mRNA expression. *. Rex1 Rex1. 0.0010. *. ns. 0.0005. 0.0000. LIF+2i. 0.0005. Mock +iShp2. (-)iMek (-)iGsk3β. mRNA expression. B. ***. D. C. LIF+2i. Mock +iShp2 Mock. % of GFP positive cells. (-)iMek. +iShp2. *. 0.0003. *. 0.0002 0.0001. Mock +iShp2. GFP high. Mock +iShp2. *. 0.0004. 0.0000. (-)2i. Esrrb Esrrb. *. LIF+2i. (-)iMek (-)iGsk3β. (-)2i. ****. 100. 50. 0. LIF+2i. ( - ) iMek1. Figure 14. Decreased iMek dependency after Shp2 chemical perturbation (A) Light microscopic images of WT ESCs under control [LIF+2i], deprivation of iMek1 [(-) iMek1], iGsk3 [(-)iGsk3] or iMek1/iGsk3[(-2i)] with DMSO [Mock] or 5μM of iShp2 [+iShp2] treatment for 2 days (scale bars: 200μm). (B) Relative gene expression of Rex1 (left) and Essrb (right) at indicated culture condition with DMSO (Mock: blue) or 5µM of iShp2 (+iShp2: red) for 2 days (*, p < 0.05, **, p <0.001, ***, p <0.0001, n.s. for not significant). (C) Flow cytometry of GFP intensity of WT ESCs with either LIF+2i control [LIF+2i] or iMek1 depletion [(-)iMek1] for 2 days, pretreated with DMSO [Mock] or 5M of iShp2 [+iShp2](D) Quantification of „GFP high‟ in (C) (****, p < 0.00001, n = 6).. 44. (48) Next, in order to validate the dichotomous effect of iShp2 in naïve and primed ESCs, we took advantage of mESCs expressing GFP and/or RFP due to distinct enhancer activity of Pou5f1 (encoding Oct4) in naïve (or ICM) [under control of distal enhancer (DE)] and primed (or epiblast) [under control of proximal enhancer (PE)] ESCs. 29. (Fig. 15A). As illustrated in Figure 6J, while ESCs of. intermediate status expressing both GFP and RFP proliferate under LIF only condition (Fig. 15B), naïve (e.g., GFP+ only) and primed (e.g., RFP+ only) ESCs would exclusively expand under LIF+2i and bFGF/Activin culture condition respectively (Fig. 15B). As expected, both GFP and RFP signal from intermediate ESCs was gradually increased under „LIF only‟ condition (Fig. 15E). To contrast, GFP but not RFP signal became readily dominant under LIF+2i while RFP signal was only marginally affected by bFGF/Activin culture (Figs. 15F and G). As conversion from the intermediate status to primed ESCs (expressing only RFP+) requires multiple passaging as described previously 29, RFP as well as GFP signal just barely maintained by bFGF/Activin. Of note, iShp2 treatment was likely to interfere in the increase of RFP rather than GFP signal under LIF+2i (vs LIF+2i‟) and bFGF/Activin (vs F.A+iShp2) (Fig.15C and D), implying that Shp2 inhibition would be unfavorable for ESCs that are under control PE of Pou5f1.. 45. (49) A. B. LIF only. LIF+2i. Naïve, ICM. bFGF/Activin. Primed, EpiSCs. C. 1. 0. 10. 20. 30. 2. 1. 0. 1.0 0.5 0.0. 0. (hrs). 10. 20. 30. F.A F.A+iShp2. 1.5. 10. 20. 30. F.A F.A+iShp2. 1.5 1.0 0.5 0.0. 0. RFP. 2.0. 2.0. LIF+2i LIF+2i'. Cell growth ratio. Cell growth ratio. Cell growth ratio. 3. 2. 0. GFP. RFP. LIF+2i LIF+2i'. Cell growth ratio. GFP 3. 0. 10. 20 (hrs). (hrs). (hrs). D LIF only GFP RFP. Slope. 0.03777. LIF+2i 0.02869. LIF+2i' 0.02403. F.A -0.003536. F.A+iShp2 -0.003722. P value. <0.0001. <0.0001. 0.0002. 0.0361. 0.1707. Slope. 0.03211. 0.009804. 0.003503. -0.002779. -0.01609. P value. <0.0001. 0.0001. 0.12. 0.1528. <0.0001. 46. 30. (50) Figure 15. Primed unfavorable effect of Shp2 chemical perturbation (A) Graphical illustration of endogenous Oct4 with distal enhancer (DE) and proximal enhancer (PE)(top), Oct4-PE-GFP (middle) and Oct4-DE-RFP (bottom), activation of DE and PE at naïve and inner cell mass (ICM) and at primed and epiblast stem cells (EpiSCs) respectively. (B) ESCs with GFP+/RFP(Green), GFP+/RFP+ (Yellow) and GFP-/RFP+ (red) at naïve, intermediate and primed status respectively (top), expected growth of GFP+ or RFP+ ESCs under each indicative condition was shown (bottom). (C) Cell growth ratio at indicated time of GFP+ or RFP+ ESCs under LIF+2i / LIF+2i‟ (iShp2 instead of iMek1) and bFGF/Activin with DMSO (F.A) or iShp2 (F.A+iShp2). (D) Table of slope and p value of linear regression of GFP+ or RFP+ at indicated culture condition, effect of iShp2 on slope was highlighted in red. (E) Cell growth ratio of GFP+ and RFP+ ESCs under LIF only culture condition media (left) and fluorescence microscopic images of GFP+ and RFP+ at indicated time (scale bars: 500μm) (right). (F) Fluorescence microscopic images of GFP under control of distal enhancer (DE-GFP) of Oct4-PE-GFP/DE-RFP ESCs under indicated media conditions (scale bars: 500μm). (G) Fluorescence microscopic images of RFP under control of proximal enhancer (PE-RFP) of Oct4-PE-GFP/DE-RFP ESCs under indicated media conditions (scale bars: 500μm).. 47. (51) Reprogramming of cells to naïve pluripotency using an Shp2 chemical inhibitor In order to reprogram cells to naïve pluripotency, LIF+2i supplementation is required with the simultaneous induction of the four Yamanaka factors (4F: Oct4, Sox2, Klf4, and c-Myc, hereinafter referred to as OSKM) with LIF 44. To examine whether iShp2 could replace iMek1 during naïve reprogramming, we used mouse embryonic fibroblasts obtained from inducible-OSKM (iOSKM-MEFs) mice. 45. ,. which readily achieves OSKM induction via doxycycline (Dox)treatment. These iOSKM-MEFs were subjected to naïve reprogramming with each different condition (Fig. 16A). As expected, multiple colonies with „colonial dome shape‟ that were positive to alkaline phosphatase (AP) activity were obtained via LIF+2i supplementation but not the absence of 2i nor iMek1 (Figs. 16B, C and D). In this condition, iShp2 treatment was likely to reverse the effect of iMek1 depletion for naïve reprogramming (Figs. 16B, C and D). Notably, the marker expression of naïve (Fig. 16E) and core pluripotency (Fig. 16F) indicated that a new combination of LIF with iGSK3 and iShp2 (instead of iMek1) could enrich the reprogrammed iPSCs more efficiently than the conventional 2i supplementation.. 48. (52) A LIF+2i. ( - ) 2i. ( - ) iMek+iShp2. ( - ) iMek1. Reprogramming. iOSKM MEF. B. (-)iMek1 ( - ) 2i. +iShp2. Mock. AP. Bright Field. LIF+2i. D. (-)2i. 30. 20. 20. +iShp2. LIF+2i LIF+2i ((- -)2i )2i Mock Mock +iShp2. +iShp2. 10. 10 0. Mock. **. 30. % Area (A.U.). % Area (A.U.). LIF+2i. (-)iMek1. C. 0. LIF+2i. LIF+2i. (-)2i. (-)2i. Mock +iShp2. (-)iMek1 Mock +iShp2. Figure 16. Naïve reprogramming with Shp2 chemical inhibitor (A) Graphical illustration for naïve reprogramming of mouse embryonic fibroblast introducing doxycycline inducible reprogramming factors (iOSKM MEF) under indicated culture condition. (B) Light microscopic images (Bright Field) and alkaline phosphatase staining (AP) of iOSKM MEF reprogrammed for 49. (53) 15 days underindicated culture condition. (C) Representative images of AP staining of iOSKM MEF under indicated culture condition (scale bars: 3 mm). (D)Graphical quantification of AP positive area of (C)under indicated culture condition (right)(arbitrary unit: A.U)(**, p<0.001, n=3). (E) Relative mRNA expressions of naïve pluripotency markers of reprogrammed iOSKM MEF under indicated condition (****, p < 0.00001, n = 3). (F)Relative mRNA expressions of core pluripotency markers of reprogrammed iOSKM MEF under indicated condition (*, p < 0.05 and ****, p < 0.00001, n = 3).. 50. (54) Discussion The disrupted activity of Shp1 2 and 2 25 in mESCs due to a lack of Zap70, a protein that acts as a non-receptor tyrosine kinase upon LIF stimulation, increases Jak/Stat3 signaling and self-renewal. However, although we observed that the effect of Zap70 depletion on hESCs was only marginal unlike mESCs (data not shown), we simply assumed that these discrepancies were largely attributable to species-specific factors and other inherent differences 1. However, recent advances in the characterization of naïve and primed pluripotency have now revealed that such dissimilarities between mouse and human ESCs result from the unique characteristics of naïve and primed pluripotent cells. Unlike in primed ESCs, the simultaneous chemical inhibition of Mek1 and Gsk3 is critical for the maintenance of naïve pluripotency 9, thus highlighting the unique cellular signaling in the ICM of blastocysts12, 46. Particularly, the normal development of Erk2 null embryos until pre-implantation. 47. and low basal Erk. activity in ICM13 suggest that Erk activity is dispensable during the preimplantation state, which is consistent with the characteristics of naïve pluripotency. Similarly, Shp2 null embryo lethality also resulted from trophoblast failure 39 and possible epiblast cell death with primed characteristics. The requirement of DUSPs, Erk1/2 specific phosphatases, in mESCs also demonstrates that Erk activity needs to be maintained at a lower level to hold naïve pluripotency. 14, 15. . Nevertheless, the constant requirement of the Mek1. inhibitor during in vitro culture of naïve ESCs implies that constant Erk activation occurs due to LIF stimulation, which eventually impedes naïve pluripotency via Erk-dependent phosphorylation 27, 28. 51. (55) Meta-analysis of previous datasets from a genome-wide study. 32. revealed. that Shp2 was predicted to serve as a negative regulator for naïve pluripotency through its involvement in both LIF-Jak/Stat3 and Ras-to-Erk signaling (Fig. 2). Notably, Shp2, whose catalytic activity was induced by LIF along with tyrosyl phosphorylation18(Fig. 2), serves not only as a negative regulator for Stat3 (Fig. 3) but also as a positive regulator for Ras-to-Erk (Fig. 3). This is why the decoupling of „Ras-to-Erk‟ signaling occurred through Shp2 inhibition, thus liberating the demand of iMek1 for naïve pluripotency (Fig. 6) as well as enhancing naïve pluripotency (Fig. 3). In sharp contrast, Shp2 appeared to be indispensable for primed ESCs, whose pluripotency relies on bFGF/Activin stimuli. Perturbation of Shp2 significantly delayed the self-renewal of ESCs in primed conditions (Figs. 4 and 6J) with concurrent decoupling of Erk signaling (Fig. 4D). Additionally, Shp2 suppression can successfully substitute the usage of iMek1 not only for maintenance of naïve ESCs (Fig. 6) but also during reprogramming (Fig. 7), suggesting that iShp2 can be used as an alternative to improve iPSC formation. Therefore, the interfering dual roles of Shp2 with the allosteric inhibitor of Shp2 21. used in this study prolonged Stat3 and attenuated Erk phosphorylation, which. mimicked the effect of Shp2 depletion on naïve pluripotency (Fig. 6) and naïve reprogramming (Fig. 7). In conclusion,Shp2 serves as a negative regulator for naïve pluripotency due to its dual roles in Jak/Stat3 and Ras-to-Erk signaling, whereas the pluripotency of primed cells depended on bFGF/Activin, which could also be modulated by Shp2dependent signaling. Allosteric inhibition of Shp2 with an inhibitor to disrupt these dual functions can therefore be used to improve naïve pluripotency and 52. (56) replace the use of iMek1. Considering the complex protocol for establishing human naïve pluripotent stem cells. 48. , iShp2 treatment would be a promising. strategy for the efficient establishment of human naïve PSCs, which will be further examined in the future studies.. A. Naïve ESCs. Primed ESCs. LIF. Phosphatase. Jak/Stat3. Phosphatase. Adaptor. Mek1/Erk1/2. Jak/Stat3. iMEK1. Adaptor. Mek/Erk1/2. Primed Pluripotency. Naïve Pluripotency. Primed Pluripotency. Naïve Pluripotency. iShp2. Genetic perturbation Primed pluripotency. Naïve pluripotency. Favorable for Naïve maintenance & Establishment. Figure 17. Graphical scheme for the study (A) Genetic and Chemical suppression of Shp2 led to naïve favorable and primed unfavorable effect. Shp2 chemical inhibitor could be a promising substitute of iMek for maintaining and establishing naïve pluripotency.. 53. (57) Bibliography 1 Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004; 269:360-380. 2 Cha Y, Moon BH, Lee MO et al. Zap70 functions to maintain stemness of mouse embryonic stem cells by negatively regulating Jak1/Stat3/c-Myc signaling. Stem Cells 2010; 28:1476-1486. 3 Jeong HC, Park SJ, Choi JJ et al. 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Structural determinants of SHP-2 function and specificity in Xenopus mesoderm induction. Mol Cell Biol 1998; 18:161-177. 43 Ahmed TA, Adamopoulos C, Karoulia Z et al. SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERKDependent Tumors. Cell Rep 2019; 26:65-78 e65. 44 Zhang J, Ratanasirintrawoot S, Chandrasekaran S et al. LIN28 Regulates Stem Cell Metabolism and Conversion to Primed Pluripotency. Cell Stem Cell 2016; 19:66-80. 45 Stadtfeld M, Maherali N, Borkent M, Hochedlinger K. A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nat Methods 2010; 7:5355. 46 Azami T, Bassalert C, Allegre N et al. Regulation of the ERK signalling pathway in the developing mouse blastocyst. Development 2019; 146. 47 Saba-El-Leil MK, Vella FD, Vernay B et al. An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development. EMBO Rep 2003; 4:964-968. 48 Theunissen TW, Friedli M, He Y et al. Molecular Criteria for Defining the Naive Human Pluripotent State. Cell Stem Cell 2016; 19:502-515.. 56. (60) 국문초록 줄기세포의 나이브전분화성(Naïve pluripotency)을 유지하기 위해서는 LIF (Leukemia Inhibitory Factor)의 첨가 외에 추가적으로Gsk3와 Mek1 의 지속 적 저해가 필수적이고 (LIF+2i), 이것은 MAPK 신호전달과 Jak/Stat3 신호전달 이 LIF에 의해서 함께 활성화된다는 것을 나타낸다. LIF 자극 직후에 Jak/Stat3 의 타이로신 인산가수분해 효소(phosphatase)와 Ras-Erk signaling 의 연결자 단백질(adaptor protein)의 두 역할을 하는 Ptpn11 (Shp2 를 암호화한다)은, Genome wide screening 을 통해 나이브전분화성의 음성 억제제로서 추정되었 던 유전자이다. 이 연구에서 본인은 Shp2 의 역할이 나이브전분화성과 프라임 전분화성 (primed pluripotency) 에서 다르다는 것을 밝혔다. Shp2의 감소는 나이브 배아 줄기세포에서 나이브전분화성을 증가시켰고 분화를 억제시킨 반면, 프라임 배아 줄기세포에서 세포사멸을 유도하고,나이브 배아줄기세포를 프라임화 (primed conversion) 시킬 때 성장억제를 유도했다. 비슷한 결과로, metastable 상태의 전분화성 줄기 세포를 프라임화 (primed conversion) 시킬 때, Shp2 의 두 가지 역할을 모두 억제하는 Shp2 알로스테릭 저해제(iShp2) 처리는 프라임 전분화성 (primed pluripotency)을 감소시켰고, 결과적으로 나이브 전분화성(naive pluripotency) 이 우세해지게 했다. Shp2 감소 후 나이브 배아줄기세포에서 지 속된 Stat3 인산화와 함께 감소된 Erk signaling 을 야기하면서,나이브전분화성 에 있어서 Mek억제제(iMek) 의 필요성을 감소시켰다.또한, Shp2 알로스테릭저 해제(iShp2) 처리는 나이브전분화성 의 자기 재생(self-renewal)을 가능하게 했고, 나이브전분화성의 유지와 확립에 있어서 iMek을 대체했다.iMek이 유전자 온전성에 미치는 비가역적인 효과를 고려했을 때, iShp2 는 iMek의 유망한 대체 57. (61) 제가 될 수 있을 것이다. 결론적으로,이 연구는 Shp2 가 나이브와 프라임 전분화성에 양분(兩分)된 다른 역할을 하는 것을 밝혀냈으며, 나이브전분화성의 유지와 확립에 있어서 iMek 의 대체제로써 iShp2 의 사용 가능성을 제안한다.. 주요어 : Shp2, Ptpn11, 마우스 배아줄기세포 (mESCs), 나이브 전분화성 (Naïve pluripotency), 프라임 전분화성 (Primed pluripotency), MAPK 신호 전달, Jak/Stat3 신호전달, LIF 학번 : 2019-22467. 58. (62) Acknowledgements 2019년 초, 처음 연구실 문을 두드린 후로 서울대학교 약학대학에서 6개월의 연구생과 2년의 학위과정을 마치기까지 짧고도 긴 시간이 흘렀습니다. 무사히 석 사 학위 과정을 마칠 수 있도록 도움을 주신 모든 분들께 진심으로 감사의 인사를 드립니다. 무엇보다도, 부족한 저를 제자로 받아주시고 지도해주신 차혁진 교수님께 감사 를 전합니다. 교수님께서는 제가 스스로 연구의 즐거움을 느낄 수 있도록 격려해 주시고 길을 제시해주셨습니다. 그 덕분에 학위 과정이 보람찬 기억으로 남게 되 었습니다. 실험과 연구가 서툴렀던 저는 때로 한계점에 봉착한 듯한 기분이 들기 도 했지만, 스스로 제 능력을 확장할 수 있게 지도해주시고 연구의 즐거움을 느끼 게 해주신 덕에 더욱 발전할 수 있었습니다. 앞으로도 교수님의 지도 아래 더욱 성 장하는 제자가 되겠습니다. 또한, 바쁘신 와중에도 저의 학위 논문을 심사해주신 이정원 교수님, 도정태 교 수님께 깊은 감사를 드립니다. 교수님들께서 주신 값진 피드백을 기반으로 더욱 발전하는 연구자가 되도록 노력하겠습니다. 다음으로, 학위과정동안 가족보다 많은 시간을 함께 보낸 실험실 멤버들께 감사 를 전합니다. 연구생으로 첫 출근을 시작했을 때 실험실 시니어로서, 그리고 친근 한 선배님으로서 많은 조언을 주신 옥선 언니, 덕분에 마음을 굳게 먹고 학위과정 에 임할 수 있게 되었습니다. 언니께서 꾸준히 열심히 연구하시는 모습을 보고 많 이 느끼고 배울 수 있었습니다. 항상 늦은 시간까지 연구실에서 공부하시고 낮에 는 실험실의 분위기를 리드해주시는 석우오빠, 누구보다 노력하시면서 즐거움도 잃지 않으시려는 오빠를 보며 많은 것을 느꼈고 더욱 본받고 싶습니다. Naïve primed 팀의 사수로서 정말 많은 도움을 주신 근태 오빠, 처음에 실험이 어설펐 59. (63) 던 저를 잘 지도해 주셔서 정말 감사드립니다. 또한 연구 이외의 일에도 사수로서 먼저 신경 써주시고 도움 주셔서 든든하게 학위과정을 마칠 수 있었습니다. 곧 박 사 졸업을 앞두신 영현 오빠, 실험에 관한 질문을 드리면 상세히 알려주시고 도와 주시는 오빠 덕분에 값진 실험 노하우를 전수받았고, 연구의 즐거움을 느낄 수 있 었습니다. 곧 박사님이 되시고도 모든 일이 잘 풀리길 기원합니다. 항상 성실하고 멋있는 모습 보여주는 주미 언니, 외유내강인 언니를 보며 긴 연구 생활에 임해야 하는 마음가짐을 배우게 되었습니다. 놀라운 아이디어와 추진력을 가진 주찬오빠, 프로젝트에 대해 고민하고 해결해나가는 모습을 보면서 연구자로서 많은 긍정적 인 영향을 받게 됩니다. 제가 실험실 생활을 시작하고 가장 먼저 졸업하신 현준오 빠, 매사 차분하고 현명하신 오빠를 보며 많이 배웠고, 오빠께서 해주시는 조언을 바탕으로 더욱 성장할 수 있었습니다. 작년 이맘때쯤 첫 서울대학교 소속으로 졸 업한 정윤언니, 초반에 실험이 서툴렀을 때 친절하게 가르쳐주고 도와줘서 든든했 고 덕분에 잘 적응할 수 있었어. 함께한 지 1년이 되어가는 민정언니, 정과 웃음이 많으셔서 연구실이 활기차진 것 같아요. 덕분에 즐겁게 실험실 생활 하고 있습니 다. 박사과정 동안에도 잘 부탁드립니다. 항상 바른 자세로 성실히 노력하는 윤정 언니, 언니가 최선을 다해 일을 해내는 모습이 멋지다고 생각하고, 덕분에 나도 긍 정적인 영향을 받고 있어. 분위기 메이커인 승연언니, 실험에 임할 때는 진지하면 서도 연구실 분위기를 항상 즐겁게 만들어줘서 고마워. 프로젝트 잘 진행되어서 남은 학위과정도 잘 마무리하길 바랄게. 또 다른 분위기 메이커이자 우리 연구실 의 바이오 인포매틱스 분석을 맡아주고 있는 은지언니, 덕분에 Shp2 프로젝트를 하면서 도움을 많이 받았어. 연구 이외에도 든든하고 재미있는 친구처럼 대해줘서 고마워. Naïve Primed 관련 연구를 하는 지영언니, 항상 즐겁고 열심히 연구하는 언니로부터 많이 배우고 있어. 부족한 나인데도 항상 좋게 말해줘서 고맙고, 1년 후 석사졸업도 잘 마무리하길 바랄게. 막내 범기, 나보다 후배고 동생이지만 연구 60. (64) 에 대한 열정과 냉철함을 많이 배우게 되는 것 같아. 앞으로도 지금처럼 즐겁게 학 위과정 보내길 바랄게. 가족과도 같은 우리 실험실 멤버들 덕분에 즐겁게 연구에 임할 수 있었습니다. 약학대학 생화학실의 다른 연구실 입학동기, 선후배분들께도 큰 감사를 드립니다. 힘들 수도 있는 시간이었으나, 비슷한 시기에 연구를 시작한 동료들이 있었기에 웃으며 이겨낼 수 있었습니다. 학위과정동안 시간이 많지 않은 저의 상황을 이해 해주고 뒤에서 끊임없는 응원 보내주는 친구들, 지인 분들께 감사의 말을 전합니 다. 같은 연구자의 길을 걷고 있는 대학원생 친구들에게도 감사의 말을 전합니다. 서로의 상황을 잘 알고 공감할 수 있는 대학원생 친구들은 큰 힘이 되었습니다. 감사한 분이 많아서 글이 길어졌습니다. 마지막으로, 항상 저를 믿고 응원해주시 는 엄마, 아빠, 국방의 의무를 다하고 있는 동생, 그리고 할머니 할아버지를 비롯 한 가족 구성원들께 가장 큰 감사의 뜻을 전하며 글을 마칩니다. 주변에 감사드리 며 더욱 올곧게 발전하는 사람이 되어 보답하겠습니다.. 2021년 7월 김 성 민 올림. 61. (65)