Yan Jin,Yunkun Lu,Lianyu Lin,Chao Liu,Xiaojie Ma,Xi Chen,Ziyu Zhou,Zhensheng Hu,Jiaqi Pu,Guo Chen,Qian Deng,Liling Jiang,Yuhan Li,Yulong Zhao,Hao Wang,Junfen Fu,Wei Li,Saiyong Zhu
Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception
chemical reprogramming,CD3254,RXRα,RNA exosome,transposable elements
Significance Chemistry-alone approach of converting one cell type into another has recently been demonstrated, representing a breakthrough in biology. However, chemical reprogramming is hampered by low efficiency, and the underlying molecular mechanisms remain unclear. Here, we study the key question of how do small molecules work as a driving force for reinstating pluripotency in somatic cells. We show that small molecule CD3254 activates endogenous transcription factor RXRα to significantly promote chemical reprogramming. Mechanistically, CD3254–RXRα axis can directly activate all 11 RNA exosome component genes at transcriptional level. RNA exosome modulates the degradation of transposable element-associated RNAs and then inflammation is reduced, contributing to successful reprogramming. In summary, our study provides conceptual advances for cellular reprogramming and technical improvement for regenerative medicine.
Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception

Chemistry-alone approach of converting one cell type into another has recently been demonstrated, representing a breakthrough in biology. However, chemical reprogramming is hampered by low efficiency, and the underlying molecular mechanisms remain unclear. Here, we study the key question of how do small molecules work as a driving force for reinstating pluripotency in somatic cells. We show that small molecule CD3254 activates endogenous transcription factor RXRα to significantly promote chemical reprogramming. Mechanistically, CD3254–RXRα axis can directly activate all 11 RNA exosome component genes at transcriptional level. RNA exosome modulates the degradation of transposable element-associated RNAs and then inflammation is reduced, contributing to successful reprogramming. In summary, our study provides conceptual advances for cellular reprogramming and technical improvement for regenerative medicine.

Cellular reprogramming has fascinated the scientific community and general public for decades (1). Advances in the field provide essential approaches for regenerative medicine (2). Transcription factors (TFs) are a natural choice for reprogramming, but exogenous TFs have to be delivered into starting cells, which is labor intensive and raises potential safety concerns. Chemical compounds not only are more convenient and nonintegrating for manipulating cell fates, but also can provide a better understanding of cell-fate transitions (3, 4). Unfortunately, chemical reprogramming remains a slow and inefficient process, hindering its broad applications (5–11). Then, more efforts are required to identify new small molecules for rapid and efficient chemical reprogramming. Besides technical innovations and advances, many efforts were also made to elucidate the molecular mechanisms of cellular reprogramming. Several important cellular and molecular mechanisms have been identified as hallmarks of pluripotent reprogramming, including epigenetic regulation, mesenchymal-to-epithelial transition, and metabolic rewiring (12–17). So far, chemical reprogramming is still in its infancy, and the molecular understanding is very limited. There are many interesting questions in this research area: for example, how small molecules silence somatic genes, activate endogenous pluripotent gene regulatory network, and finally enable successful cell-fate transitions? Nuclear receptors are interesting and special groups of transcription factors that can directly translate chemical signals into distinct physiologic effects and play important roles in development and physiology (18). Retinoid X receptor (RXR) can heterodimerize with other nuclear receptors, such as RAR and PPAR, and control multiple intertwined signaling pathways. RXR signaling pathway plays essential roles in development and has been harnessed for blood, brown fat, and pancreatic differentiation (4, 19–21). In addition, RXR signaling has been identified to be involved in tissue repair and regeneration (22). However, the precise molecular mechanisms are not completely understood. With the advent of new technologies, including RNA-Seq and cleavage under targets and tagmentation (CUT&Tag) (23), now we have the power to comprehensively dissect the direct downstream target genes of RXR, providing better understanding of nuclear receptor-mediated cellular functions. Particularly, starting from agonist–nuclear receptor axis can provide us a unique opportunity to dissect the fundamental questions about reinstating pluripotency in somatic cells by only environmental cues. RNA surveillance ensures precise control of RNA homeostasis in a spatiotemporally controlled manner and safeguards normal development (24–28). RNA exosome, a multiprotein complex with 3′ to 5′ ribonuclease activity, including 11 subunits (Exosc1–10 and Dis3) in total, is a key component involved in RNA surveillance (29–32). Cell-fate transitions, with the accompanying rapid degradation and synthesis of a plethora of RNAs, present a challenge for precise quality control, and therefore, necessitate comprehensive RNA surveillance (33–36). Currently, the precise contributions of RNA surveillance to cellular reprogramming remain to be elucidated. In addition, how to harness RNA surveillance for a better control of cell fates? Definitely, it will have broad applications if we can control the expression levels of RNA exosome components at a whole with small molecules rather than genetic tool. Here, we carry out a chemical screening and find that RXRα-specific agonist CD3254 can significantly promote chemical reprogramming of fibroblasts. Further mechanistic studies provide interesting findings that CD3254–RXRα axis can transcriptionally activate RNA exosome with all the 11 components in total, including Exosc1–10 and Dis3. Unexpectedly, RNA exosome mainly regulates the degradation of transposable element (TE)-associated repeat RNAs, particularly MMVL30-int, which functions as a new barrier for cellular reprogramming. In turn, the degradation of MMVL30 mediates the reduction of inflammation, contributing to the promotion of chemical reprogramming. Overall, these studies provide us a better understanding of cell-fate transitions.

Chemical reprogramming provides a unique assay to investigate the interplay between environmental cues and cell-fate decisions. However, chemical reprogramming is a slow and inefficient process and takes about 40 days in total with multiple stages (Stages 1 to 3). Therefore, we decided to identify small molecules to improve the reprogramming technology, which is still in its infancy. A mouse chemical reprogramming protocol recently refined in our lab was applied as a starting point for screening (10). Briefly, we used OG2 MEFs as starting materials, treated cells with Stage 1 medium from day 0 to day 12, and screened small molecules during Stage 1, using Sall4-positive colony number as readout (Fig. 1A). Interestingly, CD3254 was the top candidate, and the effect could be further confirmed (Fig. 1 B–D). Next, we extended the reprogramming process to Stage 2 and observed the increase of pOct4-GFP-positive colony number (Fig. 1 E and F). The optimal concentration of CD3254 was 0.5 μM, and additionally, CD3254 mainly functioned at Stage 1 (Fig. 1 G and H). Using real-time quantitative PCR (RT-qPCR), we observed that CD3254 induced the expression of early pluripotent genes, including Sall4, Cdh1, Epcam, and Lin28a (Fig. 1I). Other RXR agonists bexarotene and SR11237 could also significantly enhance reprogramming (SI Appendix, Fig. S1 A and B), while RXR antagonist HX531 blocked the effect of CD3254 (SI Appendix, Fig. S1 C and D). In addition, CD3254 enhanced the efficiency of chemical reprogramming, rather than the kinetics (SI Appendix, Fig. S1E). We further applied CD3254 to transcription factor (TF)-mediated reprogramming and observed a limited effect (SI Appendix, Fig. S1F). Overall, through chemical screening, we have identified a unique small molecule CD3254, a specific and potent RXRα agonist, that could significantly enhance the efficiency of chemical reprogramming, overcoming one challenging issue of chemical reprogramming. Based on these strong and replicable effects, we decided to focus our following studies on CD3254 for a better understanding of chemical reprogramming, which remains largely unexplored. After further culturing the day-24 reprogramming intermediates for another 12 to 16 d, we established several pOct4-GFP-positive ciPSC lines and characterized them in detail (Fig. 2A). These pOct4-GFP-positive cells had the typical mouse ESC morphology (Fig. 2B). Immunostaining demonstrated that these ciPSCs expressed typical pluripotent markers, including Oct4, Sox2, and Nanog (Fig. 2C). RT-qPCR showed the robust expression of pluripotency genes, such as Oct4, Sox2, and Nanog (Fig. 2D). RNA-Seq suggested that these ciPSCs are similar to mESCs at transcriptome level (Fig. 2E). Bisulfite sequencing analysis revealed that the Oct4 and Nanog promoters of these ciPSCs were largely demethylated, providing further evidence for the successful epigenetic rewiring (Fig. 2F). Karyotyping results showed that these ciPSCs maintained a normal karyotype (Fig. 2G). By embryoid body differentiation, we could observe all the three germ layer cells, such as Tuj1-positive ectoderm, α-SMA-positive mesoderm, and Foxa2-positive endoderm (Fig. 2H). Teratoma assay showed that these ciPSCs could generate typical teratomas containing derivatives of all the three germ layers, including ectoderm, mesoderm, and endoderm (Fig. 2I). CD3254–ciPSCs also could generate chimeric mice (Fig. 2J), further addressing the pluripotency in vivo. In aggregate, the above comprehensive characterizations demonstrated that these ciPSCs are molecularly and functionally similar to mESCs, proving the successful pluripotent reprogramming by a chemical approach.

To identify the molecular mechanisms of CD3254 in chemical reprogramming, the reprogramming intermediates treated with and without CD3254 were harvested to perform RNA-Seq. The datasets were replicable (SI Appendix, Fig. S2A). Next, we conducted differential gene expression analysis between the two treatment conditions. In total, 1,330 genes were up-regulated and 1,330 genes were down-regulated after CD3254 treatment (fold change >1.5 or <0.67, adjusted P-value < 0.05) (Figs. 3A and SI Appendix, Fig. S2B). Next, we did Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The KEGG terms among up-regulated genes included DNA replication, homologous recombination, spliceosome, and RNA degradation (Fig. 3B). The down-regulated KEGG terms included steroid biosynthesis, MAPK signaling pathway, focal adhesion, and TGF-beta signaling (Fig. 3C). In particular, early pluripotency genes (e.g., Sall4, Lin28a, Esrrb, Klf4, cMyc) and epithelial genes (e.g., Cdh1, Cldn4, Tjp3) were up-regulated, while mesenchymal genes (e.g., Zeb1, Twist1, Snail1) were down-regulated (Fig. 3 D–F). We also observed the downregulation of key genes involved in TGF-beta signaling pathway (e.g., Tgfb1, Grem1, Smad3) (Fig. 3G). In addition, gene ontology (GO) analysis showed up-regulated terms, including stem cell population maintenance, chromatin organization, RNA metabolic process, and DNA replication. Down-regulated GO terms included exocellular matrix, epithelial-to-mesenchymal transition, TGF-beta, and MAPK signaling pathway (Fig. 3H). Gene set enrichment analysis (GSEA) results further suggested the upregulation of ribosome, spliceosome, DNA repair, pyrimidine metabolism, and downregulation of lysosome, cytokine–cytokine receptor interaction, and Hedgehog signaling pathway (SI Appendix, Fig. S2C). Overall, CD3254 treatment promotes the rewiring of gene regulatory network, accelerating MET and pluripotency induction.

To further dissect whether CD3254 promotes mouse chemical reprogramming through its cognate target RXRα, we tested the functions of Rxra knockdown and overexpression (Fig. 4 A and B). Rxra knockdown by shRNAs inhibited Sall4-positive colony number and the expression of early pluripotency marker genes, such as Sall4, Cdh1, Epcam, and Lin28a (Fig. 4 C and D). Unexpectedly, the effect of Rxra overexpression was even more significant, and could increase the number of Sall4-postive colonies by about 10-fold, further suggesting that CD3254–RXRα axis plays critical roles in chemical reprogramming (Fig. 4 E–G). Again, using RNA-Seq, we evaluated the gene expression changes by Rxra overexpression. In total, 1,953 genes were up-regulated and 2,147 genes were down-regulated after Rxra overexpression (fold change > 1.5 or < 0.67, adjusted P-value < 0.05) (Fig. 5A, and SI Appendix, Fig. S3 A and B). The up-regulated categories included stem cell population maintenance, chromatin remodeling, DNA replication, RNA degradation, and mRNA surveillance pathway (Fig. 5B and SI Appendix, Fig. S3D). On the contrary, the down-regulated genes belonged to the categories of lysosome, focal adhesion, MAPK signaling pathway, TGF-beta signaling pathway, autophagy, and response to hypoxia (SI Appendix, Fig. S3 C and D). Similar to CD3254 treatment, we found that early pluripotency genes and epithelial genes were up-regulated, while mesenchymal genes and key genes involved in TGF-beta signaling pathway were down-regulated (SI Appendix, Fig. S3F). GSEA further suggested the upregulation of homologous recombination, and downregulation of cytokine–cytokine receptor interaction, lysosome, and chemokine signaling pathway (SI Appendix, Fig. S3E). Overall, Rxra overexpression also enhances (even more efficiently than CD3254) the rewiring of gene regulatory network from somatic to pluripotent state.

Integrating the RNA-Seq datasets of CD3254 treatment and Rxra overexpression, we found that RNA exosome component genes were notably induced. RNA exosome is a complex of 11 subunits in total and involved in RNA degradation (28, 29) (Fig. 5 B and C). The roles of RNA exosome in cellular reprogramming have not been described and remain largely unknown. As shown in Fig. 5D, both CD3254 treatment and Rxra overexpression increased the expression of all the 11 subunits of RNA exosome, including Exosc1–10 and Dis3. We used RT-qPCR to further confirm the upregulation of Exosc3, Exosc7, Exosc8, and Dis3 after CD3254 treatment and Rxra overexpression (Fig. 5 E and F). In addition, both CD3254 treatment and Rxra overexpression could up-regulate the protein levels of Exosc3 and Dis3 (SI Appendix, Fig. S3G). We conducted transcription factor (TF) regulatory potential index analysis of RNA exosome component genes using Cistrome database, and RXRα was identified as a predictive upstream regulatory TF of RNA exosome component genes (SI Appendix, Fig. S4 A and B). To determine whether RNA exosome subunit genes are the direct downstream targets of RXRα, we performed CUT&Tag assay (23). Indeed, we observed that RXRα could directly bind to the genomic regions of all the 11 RNA exosome component genes (Fig. 5 G and H and SI Appendix, Fig. S4C). Next, the direct binding of RXRα on the genomic regions of RNA exosome component genes Exosc3 and Dis3 was further confirmed by CUT&Tag-qPCR assay (Fig. 5 I and J). In sum, we have identified RXRα as a direct upstream regulator of all component genes of RNA exosome.

Next, we tested the function of RNA exosome in chemical reprogramming. We used shRNAs to knock down Exosc3, a core component of RNA exosome (Fig. 6 A and B). We found that Exosc3 knockdown decreased the number of Sall4-positive colonies and the expression of early pluripotency maker genes Sall4, Cdh1, Epcam, and Lin28a (Fig. 6 C and D). Additionally, knockdown of Dis3, another key component of RNA exosome, demonstrated a similar phenotype (SI Appendix, Fig. S5 A–D). Using RNA-Seq, we evaluated the gene expression changes after Exosc3 knockdown. In total, 314 genes were up-regulated and 1,093 genes were down-regulated after Exosc3 knockdown (fold change > 1.5 or < 0.67, adjusted P-value < 0.05) (Fig. 6 E and F). The up-regulated genes belong to terms of ECM–receptor interaction, response to wounding, and cytokine–cytokine receptor interaction (Fig. 6 G and I). On the contrary, the down-regulated terms included DNA replication, chromatin organization, glycine, serine and threonine metabolism, and signaling pathways regulating pluripotency of stem cells (Fig. 6 H and I). Taken together, we have identified that RNA exosome is a positive regulator for chemical reprogramming.

The overall much higher number of down-regulated mRNAs (314 up versus 1,093 down) after Exosc3 knockdown is counterintuitive, as RNA exosome is an RNA degrader. Such observation is also distinct from previous observations in other biological systems (31, 35, 37). Besides mRNAs, noncoding RNAs (ncRNAs) including long noncoding RNAs (lncRNAs), enhancer RNAs (eRNAs), promoter-associated RNAs (paRNAs), and transposable element-associated RNAs (repeat RNAs) have also been shown as the substrates of RNA exosome (31, 35, 37). To investigate the effects of RNA exosome on ncRNAs, we quantified the expressional changes of lncRNAs, eRNAs, and paRNAs upon Exosc3 knockdown. Overall, the expression levels of these ncRNAs showed downward trends in shExosc3, similar with mRNAs (SI Appendix, Fig. S6 A, E, and F). Moreover, we observed that the expression patterns of some lncRNAs are in correlation with that of the nearby genes, indicating the close expressional regulation of the lncRNAs on their nearby genes. For example, after Exosc3 knockdown, the expression of Gm14261, which is upstream of Sall4, was decreased, and the expression of Gm10638 located at downstream of Siah1a was increased (SI Appendix, Fig. S6 B–D). Unexpectedly, the expression of repeat RNAs was significantly increased upon Exosc3 knockdown, suggesting that RNA exosome mainly targets repeat RNAs for degradation during reprogramming (Fig. 7A). We discovered that majority of the differentially expressed repeat RNAs were up-regulated (n = 3,403), while only a few were down-regulated (n = 35) (Fig. 7B). The increased repeat RNAs included the majority of TE classes, such as long terminal repeat (LTR)-containing endogenous retroviruses (ERVs) and non-LTR elements, including long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) (Fig. 7C). Interestingly, through precisely investigating the expressional changes of repeat RNAs, we found that eight out of the top 10 up-regulated repeat RNAs (ranked by log2 fold change and P-values) are MMVL30-int, which belongs to VL30 ERV1 family (38) (Fig. 7 B and C). In addition, the proportion of the up-regulated MMVL30-int duplicates was significantly higher than that of the all expressed TEs, implying that MMVL30-int was the major target of RNA exosome (Fig. 7D). To further validate this hypothesis, we investigated the expressional changes of repeat RNAs after CD3254 treatment and Rxra overexpression. Again, MMVL30-int was significantly enriched in the high-ranking down-regulated TEs (SI Appendix, Fig. S6 G and H). Integrated analysis of these RNA-Seq datasets showed that 13 MMVL30-int duplicates were regulated by Exosc3 knockdown, CD3254 treatment, and Rxra overexpression (Fig. 7E). The expression patterns of these duplicates showed distinct manners in CD3254 treatment, Rxra overexpression, and Exosc3 knockdown, implying that RNA exosome is indeed responsible for degradation of MMVL30-int transcripts (Fig. 7 F and G). RT-qPCR confirmed the downregulation of MMVL30 expression by CD3254 treatment and Rxra overexpression and the upregulation of MMVL30 expression by Exosc3 knockdown (Fig. 7H). Moreover, we performed Exosc3 RNA immunoprecipitation with RT-qPCR (RIP-qPCR) and validated that Exosc3 could directly bind MMVL30 (Fig. 7I). Next, we analyzed the RNA-Seq data of reprogramming intermediates treated with transcription inhibitor actinomycin D (Act D) and collected at 0 h, 4 h, and 8 h. We found that the stability of MMVL30-int was significantly decreased in CD3254 treatment cells (Fig. 7J). Functionally, we found that MMVL30 knockdown could increase Sall4-positive colonies (Fig. 7K and SI Appendix, Fig. S7 A–C) and promote the induction of early pluripotent genes, including Sall4, Cdh1, Epcam, and Lin28a (SI Appendix, Fig. S7D). Therefore, MMVL30 functions as a new barrier for chemical reprogramming. Collectively, by further data exploration, we identified that CD3254–RXRα–RNA exosome axis promotes the degradation of transposable element (mainly MMVL30) during cell-fate transitions.

Recent studies have suggested that accumulation of MMVL30 transcripts can stimulate cytoplasmic nucleotide sensing pathways, and subsequently, interferon response, which function in tumorigenesis, development, and tissue repair (39–41). However, the roles of TEs in cellular reprogramming remain elusive. Therefore, we decided to further explore the consequences after the dysregulation of MMVL30 during chemical reprogramming. Overall, we found that a number of ERVs were induced in the forms of both sense and antisense transcripts upon Exosc3 knockdown (Fig. 8A). These up-regulated ERVs, including MMVL30-int, were expressed in both forward and reverse orientations and could form double-stranded RNAs (dsRNAs) (Fig. 8B). Indeed, fluorescence-activated cell sorting (FACS) analysis identified that CD3254 treatment could decrease the total dsRNA level (Fig. 8C). We also observed the reduction of dsRNAs formed by MMVL30 (Fig. 8D). The accumulation of dsRNAs can trigger inflammation and immune response (39, 40, 42). Indeed, we observed that several inflammation-related genes, including Ifngr1, Tnfrsf1a, and Tnfrsf10b, were down-regulated in the CD3254-treated, Rxra overexpression, and MMVL30 knockdown samples, but, on the contrary, up-regulated in the Exosc3 knockdown samples (Fig. 8E). To further test the roles of IFN-γ and TNF-α pathways, we added IFN-γ and TNF-α during reprogramming and found that they indeed decreased the number of Sall4-positive colonies (Fig. 8F). IFN-γ and TNF-α treatment down-regulated early pluripotent gene Sall4 and epithelial gene Cdh1, while up-regulated mesenchymal gene Zeb2 (Fig. 8G). On the contrary, JAK inhibitor upadacitinib and IKK inhibitor BMS-345541 could promote reprogramming (Fig. 8 H and I). The relationships between inflammation and cell fates are context dependent. Initial studies suggested that activation of TLR3- or IL6-mediated innate immunity is required for efficient pluripotent reprogramming (43–45). Later, several reports have suggested that IFN-γ- or TRAIL-mediated inflammation is a barrier for TF-induced pluripotency (46, 47). Here, our study indicated IFN-γ and TNF-α pathways as a roadblock of chemical reprogramming. In summary, through unbiased chemical screening, we found that RXRα-specific agonist CD3254 can significantly promote chemical reprogramming. Further mechanistic studies provided interesting findings that CD3254–RXRα axis can transcriptionally activate RNA exosome with all the 11 components (Exosc1–10 and Dis3) in total. After dissecting the exotome, we observed that RNA exosome mainly regulates the degradation of TEs (mainly MMVL30-int duplicates) and reduction of inflammation (e.g., IFN-γ and TNF-α pathways). Notably, these studies provided us insights into cell-fate transitions and better strategies to control cell fates (Fig. 8J).

Compared to introducing exogenous transcription factors to abruptly forcing cells to change their identity, chemical approach is much less artificial by triggering innate cellular mechanisms. Here, through chemical screening, we identified CD3254 as a small molecule that can significantly promote mouse chemical reprogramming. Currently, chemical reprogramming is still in its infancy and has not been extensively studied. Particularly, unbiased chemical screening can provide us many unexpected findings and provide insights, as exemplified here and in our recent study of Syk inhibitor R406 in chemical reprogramming (10). Notably, our work is a good example for harnessing endogenous transcription factors directly by small molecules for efficient chemical reprogramming. These efforts will help us to develop efficient chemical reprogramming and to better understand the underlying molecular mechanisms. Mechanistically, we demonstrated that CD3254–RXRα axis can transcriptionally activate RNA exosome with all the 11 components in total. RNA exosome plays important roles in RNA surveillance and ensures precise control of RNA homeostasis in a spatiotemporally controlled manner and safeguards normal development (26, 28). Induction of RNA exosome at a whole by small molecule rather than genetic approach is very attractive. We foresee that it will be interesting to test whether activation of RNA exosome by CD3254 can be applied to other biological systems and further developed as therapeutics for various diseases, including injury and cancers. Previous studies mainly focused on mRNAs as the major targets of RNA exosome, and here, we found that RNA exosome mainly regulates the degradation of transposable elements (TEs), particularly MMVL30, which functions as a new barrier for cellular reprogramming. In addition, MMVL30-mediated inflammation (IFN-γ and TNF-α pathways) is reduced, contributing to the promotion of chemical reprogramming. Previously, there are studies about the relationship between inflammation and cell fates. For example, initial studies suggested that activation of TLR3-mediated innate immunity is required for efficient pluripotent reprogramming (43). Later, injury-induced inflammation and senescence also modulate in vivo reprogramming (44, 45). Distinct to these studies, here we found that inflammation is a barrier for small molecule–induced mouse reprogramming. Therefore, the roles of inflammation in reprogramming are context dependent. Undoubtedly, these studies and further explorations will provide us better understanding of cell-fate transitions. In summary, our studies not only have identified small molecules for improving the chemical reprogramming technique, but also identified unique molecular mechanisms, which opens areas for further exploration. In fact, the pluripotent reprogramming itself represents a valuable approach to study general mechanisms of cell-fate transitions. Deciphering the underlying molecular mechanisms of chemical reprogramming will generate fundamental insights into cell-fate decisions and in turn will provide innovative strategies to make the reprogramming process rapid and efficient, which can be applied for generating functional cells. Notably, these efforts will not only stimulate innovations for producing functional cells for regenerative medicine, but also provide unique approaches to pharmaceutically stimulate rejuvenation and regeneration in vivo.

OG2 mice and 129 mice were mated to generate offspring, which carried Oct4 promoter-driven GFP reporter (OG2 x 129 mice). Mouse embryonic fibroblasts (MEFs) were obtained from E13.5 embryos of OG2 x 129 mice. The mice were housed in the pathogen-free facility of the Laboratory Animal Center of Zhejiang University and fed a normal chow diet. The animal-associated procedures used in this study were approved by the Experimental Animal Ethics Committee of Zhejiang University.

MEFs and human embryonic kidney (HEK) 293T cells were maintained in DMEM containing 10% FBS and 1× penicillin/streptomycin (MEF medium). Mouse ESCs and ciPSCs were maintained on CF1 feeder cells in mESC medium containing DMEM, 10% KSR, 10% FBS, 1% NEAA, 0.055 mM 2-mercaptoethanol (β-ME), 1% P/S, 1,000 U mL−1 LIF, and the small molecules CHIR99021 (3 µM) and PD0325901 (0.2 µM). All cells were maintained in a humidified incubator at 37 °C and 5% CO2.

About 4 × 105 MEFs (at passage 3) were seeded onto one 24-well plate. After 24h, MEF medium was changed to Stage 1 medium: DMEM supplemented with 10% KSR, 10% FBS, 1% NEAA, 1% P/S, 0.055 mM β-ME, 20 ng mL−1 bFGF, and the small molecules VPA (0.5 mM), CHIR99021 (20 μM), 616452 (10 μM), parnate (5 μM), forskolin (50 μM), AM580 (0.5 μM), EPZ004777 (5 μM), and vitamin C (Vc) (250 μM). Stage 1 medium was changed every 4 d. On d12, the medium was switched to Stage 2 medium: DMEM supplemented with 10% KSR, 10% FBS, 1% NEAA, 1% P/S, 0.055 mM β-ME, 20 ng mL−1 bFGF, and the small molecules VPA (0.5 mM), CHIR99021 (10 μM), 616452, Parnate (5 μM), Forskolin (10 μM), AM580 (0.5 μM), DZNep (0.05 μM), 5-aza-dC (0.5 μM), SGC0946 (5 μM), and Vc (250 μM). Stage 2 medium was changed every 4 d for 12 d. On d24, Stage 3 medium was applied. Stage 3 medium contains 47% DMEM/F12, 47% neurobasal medium, 1× N2 supplement, 1× B27 supplement, 1% NEAA, 1% P/S, 0.055 mM β-ME, 1,000 U mL−1 LIF, and the small molecules CHIR99021 (3 µM) and PD0325901 (0.2 µM). Stage 3 medium was changed every 4 d for 12 to 16 d. Mouse ciPSC lines were generated from GFP+ colonies with typical iPSC morphology. The information of reagents is listed in SI Appendix, Table S1.

All experiments were performed with three replicates, and the data were presented as mean ± SD. Statistical significance was calculated using two-tailed Student’s t test, which was presented as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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