Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H 2 S production
2021; Springer Nature; Volume: 40; Issue: 11 Linguagem: Inglês
10.15252/embj.2020106771
ISSN1460-2075
AutoresWeiyun Wang, Shaofang Ren, Yunkun Lu, Xi Chen, Juanjuan Qu, Xiaojie Ma, Qian Deng, Zhensheng Hu, Yan Jin, Ziyu Zhou, Wenyan Ge, Yibing Zhu, Nannan Yang, Qin Li, Jiaqi Pu, Guo Chen, Cunqi Ye, Hao Wang, Xiaoyang Zhao, Zhi‐Qiang Liu, Saiyong Zhu,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle28 April 2021free access Transparent process Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2S production Weiyun Wang Weiyun Wang orcid.org/0000-0001-7152-1565 The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Shaofang Ren Shaofang Ren State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Search for more papers by this author Yunkun Lu Yunkun Lu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Xi Chen Xi Chen The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Juanjuan Qu Juanjuan Qu College of Life Science, Shanxi University, Taiyuan, China Search for more papers by this author Xiaojie Ma Xiaojie Ma The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Qian Deng Qian Deng The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Zhensheng Hu Zhensheng Hu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yan Jin Yan Jin The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Ziyu Zhou Ziyu Zhou The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Wenyan Ge Wenyan Ge The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yibing Zhu Yibing Zhu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Nannan Yang Nannan Yang Prenatal Diagnosis Center, Hangzhou Women's Hospital, Hangzhou, China Search for more papers by this author Qin Li Qin Li The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Jiaqi Pu Jiaqi Pu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Guo Chen Guo Chen The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Cunqi Ye Cunqi Ye The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Hao Wang Hao Wang Prenatal Diagnosis Center, Hangzhou Women's Hospital, Hangzhou, China Department of Cell Biology and Medical Genetics, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Xiaoyang Zhao Xiaoyang Zhao State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Search for more papers by this author Zhiqiang Liu Zhiqiang Liu College of Life Science, Shanxi University, Taiyuan, China Search for more papers by this author Saiyong Zhu Corresponding Author Saiyong Zhu [email protected] orcid.org/0000-0002-2294-8092 The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Weiyun Wang Weiyun Wang orcid.org/0000-0001-7152-1565 The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Shaofang Ren Shaofang Ren State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Search for more papers by this author Yunkun Lu Yunkun Lu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Xi Chen Xi Chen The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Juanjuan Qu Juanjuan Qu College of Life Science, Shanxi University, Taiyuan, China Search for more papers by this author Xiaojie Ma Xiaojie Ma The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Qian Deng Qian Deng The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Zhensheng Hu Zhensheng Hu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yan Jin Yan Jin The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Ziyu Zhou Ziyu Zhou The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Wenyan Ge Wenyan Ge The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yibing Zhu Yibing Zhu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Nannan Yang Nannan Yang Prenatal Diagnosis Center, Hangzhou Women's Hospital, Hangzhou, China Search for more papers by this author Qin Li Qin Li The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Jiaqi Pu Jiaqi Pu The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Guo Chen Guo Chen The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Cunqi Ye Cunqi Ye The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Hao Wang Hao Wang Prenatal Diagnosis Center, Hangzhou Women's Hospital, Hangzhou, China Department of Cell Biology and Medical Genetics, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Xiaoyang Zhao Xiaoyang Zhao State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China Search for more papers by this author Zhiqiang Liu Zhiqiang Liu College of Life Science, Shanxi University, Taiyuan, China Search for more papers by this author Saiyong Zhu Corresponding Author Saiyong Zhu [email protected] orcid.org/0000-0002-2294-8092 The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Author Information Weiyun Wang1, Shaofang Ren2, Yunkun Lu1, Xi Chen1, Juanjuan Qu3, Xiaojie Ma1, Qian Deng1, Zhensheng Hu1, Yan Jin1, Ziyu Zhou1, Wenyan Ge1, Yibing Zhu1, Nannan Yang4, Qin Li1, Jiaqi Pu1, Guo Chen1, Cunqi Ye1, Hao Wang4,5, Xiaoyang Zhao2, Zhiqiang Liu3 and Saiyong Zhu *,1,6 1The MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China 2State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China 3College of Life Science, Shanxi University, Taiyuan, China 4Prenatal Diagnosis Center, Hangzhou Women's Hospital, Hangzhou, China 5Department of Cell Biology and Medical Genetics, School of Medicine, Zhejiang University, Hangzhou, China 6Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China *Corresponding author. Tel: +86 571 88981797; E-mail: [email protected] The EMBO Journal (2021)40:e106771https://doi.org/10.15252/embj.2020106771 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Chemical compounds have recently been introduced as alternative and non-integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling-mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H2S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk-Cn-NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates. Synopsis Chemically-induced pluripotent stem cells (ciPSCs) hold great potential for regenerative medicine, but their induction efficiency remains low and the underlying molecular mechanisms unclear. Here, suppression of spleen tyrosine kinase (Syk) is shown to enhance murine ciPSC formation via modulation of amino acid metabolism and redox homeostasis, suggesting novel approaches for controlling cell fates. Syk inhibitor R406 promotes chemical reprogramming of mouse embryonic fibroblasts. Syk inhibition alleviates calcineurin/NFAT-mediated suppression of glycine, serine and threonine metabolic gene expression and impacted metabolites. Syk inhibition enhances cysteine biosynthesis and cellular hydrogen sulfide (H2S) production. Increased H2S levels reduce oxidative phosphorylation and ROS levels, facilitating ciPSC induction. Introduction Somatic cells can be reprogrammed to pluripotent stem cells (PSCs) by somatic cell nuclear transfer, cell fusion, and transcription factors (Gurdon, 1962; Tada et al, 2001; Takahashi & Yamanaka, 2006). Chemical compounds not only are more convenient and non-integrating for manipulating cell fates, but also can provide a better understanding of cell-fate transitions (Li et al, 2014; Theunissen & Jaenisch, 2014; Ma et al, 2017). Now, it is possible to generate chemical-induced PSCs (ciPSCs) from mouse somatic cells (Hou et al, 2013; Long et al, 2015; Zhao et al, 2015; Cao et al, 2018; Fu et al, 2018; Yang et al, 2020). However, the process is still time consuming and labor intensive; and therefore, more efforts are required to identify additional small molecules for rapid and efficient chemical reprogramming. Besides technical innovations and advances, many efforts were also made to elucidate the molecular mechanisms of pluripotent reprogramming (Hochedlinger & Jaenisch, 2015). Important cellular and molecular mechanisms have been identified as hallmarks of pluripotent reprogramming, including epigenetic modulations, mesenchymal-to-epithelial transition, and metabolic regulations (Theunissen & Jaenisch, 2014; Hochedlinger & Jaenisch, 2015). Particularly, the interplays between cellular metabolism and cell-fate transition are being more appreciated in recent years. As early as 2010, we have identified the Warburg effect as a classical hallmark of pluripotent reprogramming, and that chemical compounds modulating the Warburg effect plus small molecules targeting epigenetics enabled single factor OCT4-mediated pluripotent reprogramming (Zhu et al, 2010). Later, many studies supported our initial discovery, and further expanded the knowledge about metabolic regulations of pluripotency induction, including the Warburg effect, mitochondrial dynamics, autophagy, and lipid metabolism (Folmes et al, 2012; Zhang et al, 2012; Ma et al, 2015; Wu et al, 2015; Wu et al, 2016; Wang et al, 2017; Ying et al, 2018; Wu et al, 2019; Cheng et al, 2020; Zhu et al, 2020). Even with all these efforts, currently, the upstream signaling pathways regulating metabolism during reprogramming and how metabolic pathways regulate reprogramming remain incompletely understood. Spleen tyrosine kinase (Syk) has a crucial role in immunity, such as B-cell response and autoimmune diseases (Turner et al, 1995; Mocsai et al, 2010), and other biological processes, such as endothelial cell development and brown adipocyte differentiation (Yanagi et al, 2001; Knoll et al, 2017). Syk activation subsequently activates cellular calcium (Ca2+) signaling, and one downstream signaling axis of Syk-Ca2+ is Cn-NFAT (Crabtree & Olson, 2002). In the nucleus, NFAT can transcriptionally active or repress downstream genes (Baksh et al, 2002; Nguyen et al, 2009; Goodyer et al, 2012; Moreno et al, 2015; Yao et al, 2016). Cn-NFAT modulates diverse physiological processes, including T-cell and B-cell activation, embryonic and adult stem cell self-renewal and differentiation, aging, and tissue regeneration (Horsley et al, 2008; Kao et al, 2009; Li et al, 2011; Kujawski et al, 2014). Whether and how the Syk-Cn-NFAT signaling axis regulates somatic cell reprogramming remains largely unknown. In this study, we conducted a small molecule screen and identified Syk inhibitor R406 that can significantly promote mouse chemical reprogramming, and then studied the Syk-Cn-NFAT signaling axis in chemical reprogramming. After further investigating the molecular mechanisms of R406, we identified that glycine, serine, and threonine metabolic genes were notably induced by R406. We found that endogenous H2S, as one downstream metabolite of glycine, serine, and threonine metabolism, transsulfuration cysteine biosynthesis pathway, and cysteine metabolism, was significantly upregulated after R406 treatment, and consequently modulated redox homeostasis and promoted chemical reprogramming. Results Identification of Syk inhibitor R406 that can significantly promote mouse chemical reprogramming Mouse chemical reprogramming remains a slow and labor-intensive process and takes about 40–60 days in total (Hou et al, 2013; Zhao et al, 2015; Cao et al, 2018), which prompted us to identify new small molecules that can improve the reprogramming process. As a starting point, we carried out a chemical screen using a chemical library mainly targeting kinases. Based on our recent work on successfully identifying small molecules that can promote CRISPR-Cpf1-based genome editing (Ma et al, 2018), we hypothesized that we can further explore this potent chemical library in the mouse chemical reprogramming system. We used MEFs with pOct4-GFP reporter as starting materials, treated cells with Stage 1 medium from day 0 to day 12, and screened small molecules during Stage 1. Then, we cultured the reprogramming intermediates for another 12 days with Stage 2 medium and counted GFP+ colony number on day 24 (Fig 1A). Interestingly, the top three small molecules that we identified were R406 and R788, two Syk inhibitors, and Lenvatinib, a VEGFR inhibitor (Fig 1B). From independent replication experiments, we further confirmed that these three small molecules could significantly increase GFP+ colony number (Fig 1C). For further comparison, we tested previously reported small molecules that could promote chemical reprogramming at different stages and found that R406 had the highest effect on promoting chemical reprogramming when added at Stage 1 (Fig EV1A). Crotonic acid and BrdU, which were identified from the late stage (Stage 2), could not promote chemical reprogramming when they were added at Stage 1 (Fig EV1A) (Long et al, 2015; Fu et al, 2018). This result indicated that our screening helped us identify new small molecules that specifically worked at Stage 1, which so far has not been extensively explored. Then, we focused our studies on the roles of R406 in mouse chemical reprogramming. Both GFP+ and alkaline phosphatase-positive (AP+) colony number were significantly increased after R406 treatment (Figs 1D, and EV1B and C). The optimal concentration of R406 was 1 μM (Figs 1E and F, and EV1D). Next, we confirmed that R406 functioned at the early stage of chemical reprogramming and could increase typical early pluripotent marker Sall4 (Fig 1G and H). Interestingly, the effect of R406 could be observed even when we treated cells during d0-d4 (Fig EV1E). Based on these observations, we could conclude that the effect of R406 was stage-specific, and the optimal stage of treatment was Stage 1. Using real-time qualitative PCR (RT–qPCR), we observed that R406 and R788 promoted the induction of early pluripotent genes, including Sall4, Cdh1, Epcam, and Esrrb (Figs 1I and EV1F). We further tested the effects of R406 and R788 on mouse reprogramming induced by transcription factors (TFs). TF-induced reprogramming by the lentiviral approach took about 8 days. When we counted iPSC colony number at day 24 for chemical reprogramming and at day 8 for TF-induced reprogramming, we observed that the efficiencies were relatively comparable (Fig EV1G and H). We observed that R406 and R788 could also promote TF-induced mouse reprogramming (Fig EV1I). Next, we tested the effects of R406 and R788 in human cells and found that R406 and R788 inhibited human somatic cell reprogramming (Fig EV1J). These results suggested that there are differences between mouse and human somatic cell reprogramming. Such observation is not unexpected, considering the difference between mouse and human PSCs, and the underlying molecular mechanisms can be explored in the future. In addition, genetically knocking down or knocking out Syk gene by shRNA or sgRNAs could also promote chemical reprogramming, indicating that R406 works through its target protein kinase Syk (Figs 1J–L, and EV1K and L). Figure 1. Syk inhibitor R406 can significantly promote mouse chemical reprogramming A. Schematic diagram depicting the procedure of compound screening during mouse chemical reprogramming. B. The chemical screening result evaluated by GFP+ colony number on d24. C. GFP+ colony number of samples treated with R406, R788, and Lenvatinib. n = 3. D. Fluorescence image of colonies with pOct4-GFP expression after DMSO and R406 treatments. Scale bar, 100 μm. E–G. Concentration (F) and stage (G) test of R406 (E) during reprogramming. n = 3. H. Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406-treated cells on d12. n = 5. Scale bar, 100 μm. I. RT–qPCR analysis of Sall4, Cdh1, Epcam, and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3. J. Diagram showing the procedure of shSyk virus infection at the early stage of reprogramming. K. RT–qPCR analysis of Syk expression in MEFs infected with shNC and shSyk viruses. n = 3. L. Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm. M. Schematic diagram of the procedure of establishing ciPSC lines. N. ciPSCs express key pluripotent markers Oct4, Sox2, and Nanog. Scale bar, 100 μm. O. ciPSCs have developmental potentials to form teratomas containing tissues from all three germ layers. Scale bar, 100 μm. P. Chimeric mice were generated from ciPSCs. Data information: All data are presented as mean ± SD. Statistical significance was assessed by the two-tailed Student's t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. See also Fig EV1. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Tests of R406 and other candidate small molecules on different reprogramming contexts and characterization of ciPSCs A. Sall4+ colony number on d12 after treatments with candidate small molecules. n = 5. B. Bright field (bf) of colonies on d24 after DMSO and R406 treatments. Scale bar, 500 μm. C. AP staining of colonies on d24 after DMSO and R406 treatments. Scale bar, 500 μm. AP staining was done after the data acquisition of bright field (B) from the same sample. D. The effects of R406 with different concentrations. Scale bar, 500 μm. E. The effects of R406 in different time windows. n = 3. F. RT–qPCR analysis of early pluripotent genes expression in reprogramming intermediates treated with DMSO and R788. n = 3. G, H. GFP+ colony number on d24 of chemical reprogramming (C-rep) and d8 of TF-induced reprogramming (TF-rep). n = 5. I. The effects of R406 and R788 in TF-induced reprogramming. n = 5. J. The effects of R406 and R788 in human reprogramming. n = 4. K. RT–qPCR analysis of pluripotent genes expression in reprogramming intermediates treated with shNC and shSyk. n = 3. L. Immunofluorescence of Sall4 in reprogramming intermediates infected with sgNC and Syk sgRNA viruses. n = 4. Scale bar, 100 μm. M–O. Characterization of ciPSCs: AP staining and Sall4 staining (M), karyotyping analysis of ciPSCs at passage 7 and passage 15 (N), and Tuj1, α-SMA, and Foxa2 immunostaining after EB differentiation (O). Scale bar, 100 μm. Data information: All data are presented as mean ± SD. Statistical significance was assessed by the two-tailed Student's t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint To generate ciPSCs after R406 treatment, we cultured the day-24 cells for another 12–16 days and then established pOct4-GFP+ ciPSC lines (Fig 1M). These ciPSCs were positive for AP and typical pluripotent makers Oct4, Sox2, Nanog, and Sall4 (Figs 1N and EV1M). Karyotyping results showed that these ciPSCs maintained a normal karyotype during expansion process (Fig EV1N). Using embryoid body differentiation, we could detect all three germ layer cells, including Tuj1+ ectoderm, α-SMA+ mesoderm, and Foxa2+ endoderm (Fig EV1O). In addition, we did teratoma assay and found that these ciPSCs could generate typical teratomas containing derivatives of all three germ layers, suggesting that these ciPSCs are pluripotent (Fig 1O). Chimeric mice could also be generated from ciPSCs (Fig 1P), further supporting their in vivo developmental potentials. Collectively, above characterizations demonstrated that these ciPSCs are morphologically, cellularly, and functionally similar to mESCs. Overall, through chemical screening, we have identified a novel small molecule R406, a specific Syk inhibitor, that could significantly promote the early stage of chemical reprogramming, overcoming one challenging issue of the mouse chemical reprogramming protocol. Furthermore, we confirmed that the mouse chemical reprogramming system was effective for deriving ciPSCs. Based on our observations, we decided to focus our studies on how R406 and its target Syk regulated mouse chemical reprogramming. R406 works through the Syk-Cn-NFAT axis during chemical reprogramming One main downstream signaling axis of Syk is Cn-NFAT (Li et al, 2011; Moreno et al, 2015), and therefore, we tested whether Cn and NFAT could also regulate chemical reprogramming (Fig 2A). Firstly, we examined the functional effects of FK506, a specific Cn inhibitor, and found that FK506 treatment indeed could increase the number of Sall4+ colonies at day 12 and AP+ colonies at day 24 (Figs 2B and EV2A–C). The optimal concentration of FK506 was 0.5 μM (Fig 2C). Consistent with R406, FK506 also worked specifically at Stage 1 (Fig 2D). Using RT–qPCR, we observed that FK506 notably promoted the induction of early pluripotent genes Sall4, Cdh1, Epcam, and Esrrb (Fig 2E). We have also noticed the literatures indicating that calcineurin-NFAT is activated and required for reprogramming (Sun et al, 2016; Khodeer & Era, 2017), so we detected NFAT activity during chemical reprogramming by immunostaining. We found that R406 treatment indeed inhibited NFATc1 and the fluorescence signal of NFATc1 could not be detected in colonies on d4 of reprogramming (Fig EV2D–F). Next, we genetically knocked down Ppp3ca, one of the genes that encode Cn proteins, and found that knocking down Ppp3ca by shRNA could increase Sall4+ colony number (Fig 2F–H) and promote the induction of early pluripotent genes Sall4, Cdh1, Epcam, and Esrrb (Fig 2I). In addition, we found that knocking down Nfatc1 could also enhance chemical reprogramming (Fig 2J–M). Conclusively, these results suggested that R406 works through the Syk-Cn-NFAT signaling cascade in chemical reprogramming. Figure 2. Inhibition of Syk-Cn-NFAT signaling pathway promotes chemical reprogramming A. Schematic diagram showing Syk-Cn-NFAT pathway axis. B. Immunofluorescence of Sall4 in reprogramming intermediates treated with DMSO and FK506. n = 5. Scale bar, 100 μm. C, D. Concentration (C) and stage (D) test of FK506 during reprogramming. n = 3. E. RT–qPCR analysis of Sall4, Cdh1, Epcam, and Esrrb gene expression in reprogramming intermediates treated with DMSO and FK506. n = 3. F. Diagram showing the procedure of Ppp3ca knockdown at the early stage of reprogramming. G. RT–qPCR analysis of Ppp3ca expression in MEFs infected with shNC and shPpp3ca viruses. n = 3. H. Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm. I. RT–qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3. J. Diagram showing the procedure of shNfatc1 virus infection at the early stage of reprogramming. K. RT–qPCR analysis of Nfatc1 expression in MEFs infected with shNC and shNfatc1 viruses. n = 3. L. Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm. M. RT–qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3. Data information: All data are presented as mean ± SD. Statistical significance was assessed by the two-tailed Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See also Fig EV2. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. FK506 can promote chemical reprogramming and the regulation of NFAT by R406 A. Sall4+ colony number on d12 after FK506 and R406 treatments. n = 5. Statisti
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