Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

The transcription factor OCT6 promotes the dissolution of the naïve pluripotent state by repressing Nanog and activating a formative state gene regulatory network.

In the mouse embryo, the transition from the preimplantation to the postimplantation epiblast is governed by changes in the gene regulatory network (GRN) that lead to transcriptional, epigenetic, and functional changes. This transition can be faithfully recapitulated in vitro by the differentiation of mouse embryonic stem cells (mESCs) to epiblast-like cells (EpiLCs), that reside in naïve and formative states of pluripotency, respectively. However, the GRN that drives this conversion is not fully elucidated. Here we demonstrate that the transcription factor OCT6 is a key driver of this process. Firstly, we show that Oct6 is not expressed in mESCs but is rapidly induced as cells exit the naïve pluripotent state. By deleting Oct6 in mESCs, we find that knockout cells fail to acquire the typical morphological changes associated with the formative state when induced to differentiate. Additionally, the key naïve pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb were expressed at higher levels than in wild-type cells, indicating an incomplete dismantling of the naïve pluripotency GRN. Conversely, premature expression of Oct6 in naïve cells triggered a rapid morphological transformation mirroring differentiation, that was accompanied by the upregulation of the endogenous Oct6 as well as the formative genes Sox3, Zic2/3, Foxp1, Dnmt3A and FGF5. Strikingly, we found that OCT6 represses Nanog in a bistable manner and that this regulation is at the transcriptional level. Moreover, our findings also reveal that Oct6 is repressed by NANOG. Collectively, our results establish OCT6 as a key TF in the dissolution of the naïve pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as an important toggle mediating the transition to the formative state.

Establishment of Induced Pancreatic Stem Cells by Yes-Associated Protein 1.

Recently, we and others generated induced tissue-specific stem/progenitor (iTS/iTP) cells. The advantages of iTS/iTP cells compared with induced pluripotent stem (iPS) cells are (1) easier generation, (2) efficient differentiation, and (3) no teratomas formation. In this study, we generated mouse induced pancreatic stem cells (iTS-P cells) by the plasmid vector expressing Yes-associated protein 1 (YAP). The iTS-P YAP9 cells expressed Foxa2 (endoderm marker) and Pdx1 (pancreatic marker) while the expressions of Oct3/4 and Nanog (marker of embryonic stem [ES] cells) in iTS-P YAP9 cells was significantly lower compared with those in ES cells. The iTS-P YAP9 cells efficiently differentiated into insulin-expressing cells compared with ES cells. The ability to generate autologous iTS cells may be applied to diverse applications of regenerative medicine.

TATA-binding associated factors have distinct roles during early mammalian development.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

Early neural specification of stem cells is mediated by a set of SOX2-dependent neural-associated enhancers.

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Interlinked bi-stable switches govern the cell fate commitment of embryonic stem cells.

The development of embryonic stem (ES) cells to extraembryonic trophectoderm and primitive endoderm lineages manifests distinct steady-state expression patterns of two key transcription factors-Oct4 and Nanog. How dynamically such kind of steady-state expressions are maintained remains elusive. Herein, we demonstrate that steady-state dynamics involving two bistable switches which are interlinked via a stepwise (Oct4) and a mushroom-like (Nanog) manner orchestrate the fate specification of ES cells. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feedforward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future.

Complex regulatory networks influence pluripotent cell state transitions in human iPSCs.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.

In Vivo Reprogramming Using Yamanaka Factors in the CNS: A Scoping Review.

Central nervous system diseases, particularly neurodegenerative disorders, pose significant challenges in medicine. These conditions, characterized by progressive neuronal loss, have remained largely incurable, exacting a heavy toll on individuals and society. In recent years, in vivo reprogramming using Yamanaka factors has emerged as a promising approach for central nervous system regeneration. This technique involves introducing transcription factors, such as Oct4, Sox2, Klf4, and c-Myc, into adult cells to induce their conversion into neurons. This review summarizes the current state of in vivo reprogramming research in the central nervous system, focusing on the use of Yamanaka factors. In vivo reprogramming using Yamanaka factors has shown promising results in several animal models of central nervous system diseases. Studies have demonstrated that this approach can promote the generation of new neurons, improve functional outcomes, and reduce scar formation. However, there are still several challenges that need to be addressed before this approach can be translated into clinical practice. These challenges include optimizing the efficiency of reprogramming, understanding the cell of origin for each transcription factor, and developing methods for reprogramming in non-subventricular zone areas. Further research is needed to overcome the remaining challenges, but this approach has the potential to revolutionize the way we treat central nervous system disorders.

A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma.

BACKGROUND: Increasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins. METHODS: The GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms. RESULTS: PDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription. CONCLUSIONS: Our research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

Cellular plasticity in reprogramming, rejuvenation and tumorigenesis: a pioneer TF perspective.

The multistep process of in vivo reprogramming, mediated by the transcription factors (TFs) Oct4, Sox2, Klf4, and c-Myc (OSKM), holds great promise for the development of rejuvenating and regenerative strategies. However, most of the approaches developed so far are accompanied by a persistent risk of tumorigenicity. Here, we review the groundbreaking effects of in vivo reprogramming with a particular focus on rejuvenation and regeneration. We discuss how the activity of pioneer TFs generates cellular plasticity that may be critical for inducing not only reprogramming and regeneration, but also cancer initiation. Finally, we highlight how a better understanding of the uncoupled control of cellular identity, plasticity, and aging during reprogramming might pave the way to the development of rejuvenating/regenerating strategies in a nontumorigenic manner.

Highly cooperative chimeric super-SOX induces naive pluripotency across species.

Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

POU5F1 promotes the proliferation, migration, and invasion of gastric cancer cells by reducing the ubiquitination level of TRAF6.

POU5F1 plays an important role in maintaining the cancer stem cell (CSC) -like properties of gastric cancer (GC) cells. The impact of POU5F1 on the proliferation and metastasis of GC was examined, along with the potential of ATRA as a specific therapeutic agent for GC. The dysregulation of POU5F1 expression in GC tissues was analyzed using public databases and bioinformatics techniques, and the disparity in POU5F1 expression between normal gastric tissues and GC tissues was further assessed through western blot, RT-qPCR, and immunohistochemistry. The present study aimed to investigate the impact of POU5F1 on the proliferation, migration, and invasion of GC cells through both in vivo and in vitro experiments. Additionally, the effects of ATRA on the proliferation, migration, and invasion of GC cells were examined using in vivo and in vitro approaches. Our findings revealed a significant upregulation of POU5F1 in GC tissues, which was found to be associated with a poorer prognosis in patients with GC. Moreover, POU5F1 was observed to enhance the proliferation, migration, and invasion of GC cells in vitro, as well as promote subcutaneous tumor growth and lung metastasis of GC cells in vivo. The overexpression of POU5F1 mechanistically triggers the process of Epithelial-mesenchymal transition (EMT) by down-regulating E-Cadherin and up-regulating N-Cadherin and VIM. POU5F1 hinders the ubiquitination of TRAF6 through negative regulation of TRIM59, thereby facilitating the activation of the NF-κB pathway. Furthermore, the administration of ATRA effectively impedes the proliferation, migration, and invasion of GC cells by suppressing the expression of POU5F1. The upregulation of POU5F1 elicits EMT, fosters the initiation of the NF-κB signaling pathway in GC cells, and stimulates the proliferation, invasion, and metastasis of GC cells. All-trans retinoic acid (ATRA) can impede these POU5F1-induced effects, thereby potentially serving as an adjunctive therapeutic approach for GC.

A paradigm for post-embryonic Oct4 re-expression: E7-induced hydroxymethylation regulates Oct4 expression in cervical cancer.

The Octamer-binding transcription factor-4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post-embryonic re-expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus (HPV)-related than HPV-unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4-transcriptional output and Oct4-related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4-protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl-binding proteins (MBD2 and MBD3), are determinants of Oct4-driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the Oct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re-expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re-expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.

Expression and Functional Analysis of core stemness factors OSKM (OCT4, SOX2, KLF4, and MYC) in Pan-cancer.

The dedifferentiation process of tumorigenesis and somatic cell reprogramming has some commonness and differences, which is the key question to cancer therapeutic strategy and stem cell applications. To further explore the commonalities and variance between carcinogenesis and induced pluripotent stem cell reprogramming, we investigated the role of stemness factors OSKM (OCT4, SOX2, KLF4, and MYC) in the pan-cancer process using public clinical data. Expression of OSKM in human pan-cancer was analyzed via the Genotype Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) database based on the RNA-seq data of tissues. The correlation of expression between OSKM genes was analyzed via the Tumor Immune Evaluation Resource (TIMER) database, while the STRING tool was used to construct the protein-protein interaction network for OSKM. Prognostic impact of OSKM in pan-cancer was analyzed by Cox proportional hazards regression model. The relationships between OSKM and tumor stemness, tumor microenvironment and immune checkpoint and were performed by Sangerbox platform using Pearson correlation analysis. Our results showed that OSKM were universally expressed and significantly altered in tumors compared with adjacent normal tissues in most tumor types. In addition, correlation analysis revealed the relevance of OSKM genes to patient prognosis, cancer cell stemness, tumor microenvironment or immune checkpoint. However, there is little similarity between these genes in terms of how they function in each cancer type. This study elucidates the different roles of core stemness factors OSKM in pan-cancer, offering potential therapeutic targets for novel anti-cancer strategies and knowledge to minimize the potential carcinogenic effects during stem cell transplantation.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat.

Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

Application of the Yamanaka Transcription Factors Oct4, Sox2, Klf4, and c-Myc from the Laboratory to the Clinic.

The transcription factors Oct4, Sox2, Klf4, and c-Myc enable the reprogramming of somatic cells into induced pluripotent cells. Reprogramming generates newly differentiated cells for potential therapies in cancer, neurodegenerative diseases, and rejuvenation processes. In cancer therapies, these transcription factors lead to a reduction in the size and aggressiveness of certain tumors, such as sarcomas, and in neurodegenerative diseases, they enable the production of dopaminergic cells in Parkinson's disease, the replacement of affected neuronal cells in olivopontocerebellar atrophy, and the regeneration of the optic nerve. However, there are limitations, such as an increased risk of cancer development when using Klf4 and c-Myc and the occurrence of abnormal dyskinesias in the medium term, possibly generated by the uncontrolled growth of differentiated dopaminergic cells and the impairment of the survival of the new cells. Therefore, the Yamanaka transcription factors have shown therapeutic potential through cell reprogramming for some carcinomas, neurodegenerative diseases, and rejuvenation. However, the limitations found in the studies require further investigation before the use of these transcription factors in humans.

Hepatitis B Virus x Protein Increases Cellular OCT3/4 and MYC and Facilitates Cellular Reprogramming.

Hepatitis B virus x (HBx) is a multifunctional protein coded by the Hepatitis B virus that is involved in various cellular processes such as proliferation, cell survival/apoptosis, and histone methylation. HBx was reported to be associated with liver "cancer stem cells." The stemness inducing properties of HBx could also facilitate the generation of pluripotent stem cells from somatic cells. It is well established that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using a cocktail of transcription factors called Yamanaka's factors (YFs) (OCT4, SOX2, KLF4, and MYC). The reprogramming process proceeds step-by-step with reprogramming factor chromatin interactions, transcription, and chromatin states changing during transitions. HBx is a "broad spectrum trans-activator" and therefore could facilitate these transitions. We electroporated low passage and high passage (difficult to reprogram) fibroblasts using YFs with and without HBx and evaluated the reprogramming efficiency. We also investigated the tri-lineage and terminal differentiation potential of iPSC derived using HBx. We found that the addition of HBx to YF improves iPSC derivation, and it increases the efficiency of iPSC generation from "difficult or hard-to-reprogram samples" such as high passage/senescent fibroblasts. Further, we show that HBx can substitute the key transcription factor MYC in the YF cocktail to generate iPSC. The cellular levels of OCT3/4 and MYC were increased in HBx expressing cells. Our results have practical value in improving the efficiency of pluripotent stem cell derivation from "difficult to reprogram" somatic cells, in addition to providing some insights into the mechanisms of liver carcinogenesis in chronic hepatitis B. To conclude, HBx improves the reprogramming efficiency of YFs. HBx increases the cellular levels of OCT3/4 and MYC.