Critical roles of the miR-17∼92 family in thymocyte development, leukemogenesis, and autoimmunity.

Thymocyte development requires precise control of PI3K-Akt signaling to promote proliferation and prevent leukemia and autoimmune disorders. Here, we show that ablating individual clusters of the miR-17∼92 family has a negligible effect on thymocyte development, while deleting the entire family severely impairs thymocyte proliferation and reduces thymic cellularity, phenocopying genetic deletion of Dicer. Mechanistically, miR-17∼92 expression is induced by Myc-mediated pre-T cell receptor (TCR) signaling, and miR-17∼92 promotes thymocyte proliferation by suppressing the translation of Pten. Retroviral expression of miR-17∼92 restores the proliferation and differentiation of Myc-deficient thymocytes. Conversely, partial deletion of the miR-17∼92 family significantly delays Myc-driven leukemogenesis. Intriguingly, thymocyte-specific transgenic miR-17∼92 expression does not cause leukemia or lymphoma but instead aggravates skin inflammation, while ablation of the miR-17∼92 family ameliorates skin inflammation. This study reveals intricate roles of the miR-17∼92 family in balancing thymocyte development, leukemogenesis, and autoimmunity and identifies those microRNAs (miRNAs) as potential therapeutic targets for leukemia and autoimmune diseases.

Immunomodulatory Function of Pien Tze Huang in T Cell-Mediated Anti-tumor Activity against B16-F10, MC38 and Hep1-6 Tumor Models.

OBJECTIVE: To investigate the anti-tumor effects of Pien Tze Huang (PZH) in mouse models of B16-F10 melanoma, MC38 colorectal cancer, Hep1-6 hepatocellular carcinoma and chemically induced hepatocellular carcinoma model. METHODS: Various tumor models, including B16-F10, MC38 and Hep1-6 tumor hypodermic inoculation models, B16-F10 and Hep1-6 pulmonary metastasis models, Hep1-6 orthotopic implantation model, and chemically induced hepatocellular carcinoma model, were utilized to evaluate the anti-tumor function of PZH. Tumor growth was assessed by measuring tumor size and weight of solid tumors isolated from C57BL/6 mice. For cell proliferation and death of tumor cells in vitro, as well as T cell activation markers, cytokine production and immune checkpoints analysis, single-cell suspensions were prepared from mouse spleen, lymph nodes, and tumors after PZH treatment. RESULTS: PZH demonstrated significant therapeutic efficacy in inhibiting tumor growth (P<0.01). Treatment with PZH resulted in a reduction in tumor size in subcutaneous MC38 colon adenocarcinoma and B16-F10 melanoma models, and decreased pulmonary metastasis of B16-F10 melanoma and Hep1-6 hepatoma (P<0.01). However, in vitro experiments showed that PZH only had slight impact on the cell proliferation and survival of tumor cells (P>0.05). Nevertheless, PZH exhibited a remarkable ability to enhance T cell activation and the production of interferon gamma, tumor necrosis factor alpha, and interleukin 2 in CD4+ T cells in vitro (P<0.01 or P<0.05). Importantly, PZH substantially inhibited T cell exhaustion and boosted cytokine production by tumor-infiltrating CD8+ T cells (P<0.01 or P<0.05). CONCLUSION: This study has confirmed a novel immunomodulatory function of PZH in T cell-mediated anti-tumor immunity, indicating that PZH holds promise as a potential therapeutic agent for cancer treatment.

MHC class II regulation of CD8+ T cell tolerance and implications in autoimmunity and cancer immunotherapy.

Major histocompatibility complex (MHC) class II-reactive CD8+ T cells are found in humans and animals, but little is known about their identity, development, and function. In this study, we discover a group of CD8+ T cells reactive to both MHC class I and II molecules in MHC class II-deficient mice. We clone their T cell receptors (TCRs) and analyze their development and function. In wild-type animals, thymocytes bearing those TCRs are purged by negative selection. In the absence of MHC class II, they develop into mature CD8+ T cells. When encountering MHC class II in the periphery, they undergo robust activation and proliferation, attack self-tissues, and cause lethal autoimmune diseases. In adoptive T cell therapy, those CD8+ T cells are able to efficiently control MHC class II-expressing tumors. This study opens the door to investigation of dual-reactive CD8+ T cells, their development and selection in the thymus, and the perils and promises when their normal development and selection are compromised.

The transcription factor Zeb1 controls homeostasis and function of type 1 conventional dendritic cells.

Type 1 conventional dendritic cells (cDC1) are the most efficient cross-presenting cells that induce protective cytotoxic T cell response. However, the regulation of their homeostasis and function is incompletely understood. Here we observe a selective reduction of splenic cDC1 accompanied by excessive cell death in mice with Zeb1 deficiency in dendritic cells, rendering the mice more resistant to Listeria infection. Additionally, cDC1 from other sources of Zeb1-deficient mice display impaired cross-presentation of exogenous antigens, compromising antitumor CD8+ T cell responses. Mechanistically, Zeb1 represses the expression of microRNA-96/182 that target Cybb mRNA of NADPH oxidase Nox2, and consequently facilitates reactive-oxygen-species-dependent rupture of phagosomal membrane to allow antigen export to the cytosol. Cybb re-expression in Zeb1-deficient cDC1 fully restores the defective cross-presentation while microRNA-96/182 overexpression in Zeb1-sufficient cDC1 inhibits cross-presentation. Therefore, our results identify a Zeb1-microRNA-96/182-Cybb pathway that controls cross-presentation in cDC1 and uncover an essential role of Zeb1 in cDC1 homeostasis.

The miR-17∼92 miRNAs promote plasma cell differentiation by suppressing SOCS3-mediated NIK degradation.

The miR-17∼92 family microRNAs (miRNAs) play a key role in germinal center (GC) reaction through promoting T follicular helper (TFH) cell differentiation. It remains unclear whether they also have intrinsic functions in B cell differentiation and function. Here we show that mice with B cell-specific deletion of the miR-17∼92 family exhibit impaired GC reaction, plasma cell differentiation, and antibody production in response to protein antigen immunization and chronic viral infection. Employing CRISPR-mediated functional screening, we identify Socs3 as a key functional target of miR-17∼92 in regulating plasma cell differentiation. Mechanistically, SOCS3, whose expression is elevated in miR-17∼92 family-deficient B cells, interacts with NIK and promotes its ubiquitination and degradation, thereby impairing NF-κB signaling and plasma cell differentiation. This moderate increase in SOCS3 expression has little effect on IL-21-STAT3 signaling. Our study demonstrates differential sensitivity of two key signaling pathways to alterations in the protein level of an miRNA target gene.

Glycogen synthase kinase 3 controls T-cell exhaustion by regulating NFAT activation.

Cellular immunity mediated by CD8+ T cells plays an indispensable role in bacterial and viral clearance and cancers. However, persistent antigen stimulation of CD8+ T cells leads to an exhausted or dysfunctional cellular state characterized by the loss of effector function and high expression of inhibitory receptors during chronic viral infection and in tumors. Numerous studies have shown that glycogen synthase kinase 3 (GSK3) controls the function and development of immune cells, but whether GSK3 affects CD8+ T cells is not clearly elucidated. Here, we demonstrate that mice with deletion of Gsk3α and Gsk3ß in activated CD8+ T cells (DKO) exhibited decreased CTL differentiation and effector function during acute and chronic viral infection. In addition, DKO mice failed to control tumor growth due to the upregulated expression of inhibitory receptors and augmented T-cell exhaustion in tumor-infiltrating CD8+ T cells. Strikingly, anti-PD-1 immunotherapy substantially restored tumor rejection in DKO mice. Mechanistically, GSK3 regulates T-cell exhaustion by suppressing TCR-induced nuclear import of NFAT, thereby in turn dampening NFAT-mediated exhaustion-related gene expression, including TOX/TOX2 and PD-1. Thus, we uncovered the molecular mechanisms underlying GSK3 regulation of CTL differentiation and T-cell exhaustion in anti-tumor immune responses.

Dhx33 promotes B-cell growth and proliferation by controlling activation-induced rRNA upregulation.

Upon recognition of foreign antigens, naïve B cells undergo rapid activation, growth, and proliferation. How B-cell growth and proliferation are coupled with activation remains poorly understood. Combining CRISPR/Cas9-mediated functional analysis and mouse genetics approaches, we found that Dhx33, an activation-induced RNA helicase, plays a critical role in coupling B-cell activation with growth and proliferation. Mutant mice with B-cell-specific deletion of Dhx33 exhibited impaired B-cell development, germinal center reactions, plasma cell differentiation, and antibody production. Dhx33-deficient B cells appeared normal in the steady state and early stage of activation but were retarded in growth and proliferation. Mechanistically, Dhx33 played an indispensable role in activation-induced upregulation of ribosomal DNA (rDNA) transcription. In the absence of Dhx33, activated B cells were compromised in their ability to ramp up 47S ribosomal RNA (rRNA) production and ribosome biogenesis, resulting in nucleolar stress, p53 accumulation, and cellular death. Our findings demonstrate an essential role for Dhx33 in coupling B-cell activation with growth and proliferation and suggest that Dhx33 inhibition is a potential therapy for lymphoma and antibody-mediated autoimmune diseases.

Myc inhibition tips the immune balance to promote antitumor immunity.

Aberrant expression of Myc is one of the most common oncogenic events in human cancers. Scores of Myc inhibitors are currently under development for treating Myc-driven cancers. In addition to directly targeting tumor cells, Myc inhibition has been shown to modulate the tumor microenvironment to promote tumor regression. However, the effect of Myc inhibition on immune cells in the tumor microenvironment remains poorly understood. Here, we show that the adaptive immune system plays a vital role in the antitumor effect of pharmacologic inhibition of Myc. Combining genetic and pharmacologic approaches, we found that Myc inhibition enhanced CD8 T cell function by suppressing the homeostasis of regulatory T (Treg) cells and the differentiation of resting Treg (rTreg) cells to activated Treg (aTreg) cells in tumors. Importantly, we demonstrated that different Myc expression levels confer differential sensitivity of T cell subsets to pharmacologic inhibition of Myc. Although ablation of the Myc gene has been shown to suppress CD8 T cell function, Treg cells, which express much less Myc protein than CD8 T cells, are more sensitive to Myc inhibitors. The differential sensitivity of CD8 T and Treg cells to Myc inhibitors resulted in enhanced CD8 T cell function upon Myc inhibition. Our findings revealed that Myc inhibitors can induce an antitumor immune response during tumor progression.

Sulfate oligosaccharide of Gracilaria lemaneiformis modulates type 1 immunity by restraining T cell activation.

Seaweeds are important "blue food" and some seaweeds have been demonstrated that have the immunoregulation activities and benefit for human health. However, which immune cell subsets are targeted and how immune processes are regulated by those seaweeds remain poorly understood. Here we identified sulfated oligosaccharide of Gracilaria lemaneiformis (GLSO) inhibits IFNγ production by T cells in ovalbumin (OVA) immunized mice and in vitro activation system of OVA-specific CD4+ T cells. Furthermore, GLSO predominantly targets T cells but not dendritic cells, and regulates the late stage of expression of T cell activation and differentiation markers. Mechanistically, results of transcriptomic and proteome analysis indicate that GLSO inhibits mTOR activity, glycolysis, cell cycle and DNA replication. Thus, GLSO has an immunomodulatory function in Th1 immune responses by restraining T cell activation. Our study unravels the anti-inflammatory mechanism of GLSO and provides a new insight for industrial development of seaweeds as health food.

Mitochondrial C1qbp promotes differentiation of effector CD8+ T cells via metabolic-epigenetic reprogramming.

Early-activated CD8+ T cells increase both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). However, whether and how the augmentation of OXPHOS regulates differentiation of effector CD8+ T cell remains unclear. Here, we found that C1qbp was intrinsically required for such differentiation in antiviral and antitumor immune responses. Activated C1qbp-deficient CD8+ T cells failed to increase mitochondrial respiratory capacities, resulting in diminished acetyl­coenzyme A as well as elevated fumarate and 2-hydroxyglutarate. Consequently, hypoacetylation of H3K27 and hypermethylation of H3K27 and CpG sites were associated with transcriptional down-regulation of effector signature genes. The effector differentiation of C1qbp-sufficient or C1qbp-deficient CD8+ T cells was reversed by fumarate or a combination of histone deacetylase inhibitor and acetate. Therefore, these findings identify C1qbp as a pivotal positive regulator in the differentiation of effector CD8+ T cells and highlight a metabolic-epigenetic axis in this process.

Glycogen synthase kinase 3 drives thymocyte egress by suppressing ß-catenin activation of Akt.

Molecular pathways controlling emigration of mature thymocytes from thymus to the periphery remain incompletely understood. Here, we show that T cell­specific ablation of glycogen synthase kinase 3 (GSK3) led to severely impaired thymic egress. In the absence of GSK3, ß-catenin accumulated in the cytoplasm, where it associated with and activated Akt, leading to phosphorylation and degradation of Foxo1 and downregulation of Klf2 and S1P1 expression, thereby preventing emigration of thymocytes. A cytoplasmic membrane-localized ß-catenin excluded from the nucleus promoted Akt activation, suggesting a new function of ß-catenin independent of its role as a transcriptional activator. Furthermore, genetic ablation of ß-catenin, retroviral expression of a dominant negative Akt mutant, and transgenic expression of a constitutively active Foxo1 restored emigration of GSK3-deficient thymocytes. Our findings establish an essential role for GSK3 in thymocyte egress and reveal a previously unidentified signaling function of ß-catenin in the cytoplasm.

Ddb1 Is Essential for the Expansion of CD4+ Helper T Cells by Regulating Cell Cycle Progression and Cell Death.

Follicular helper T (TFH) cells are specialized CD4+ helper T cells that provide help to B cells in humoral immunity. However, the molecular mechanism underlying generation of TFH cells is incompletely understood. Here, we reported that Damage-specific DNA binding protein 1 (Ddb1) was required for expansion of CD4+ helper T cells including TFH and Th1 cells, germinal center response, and antibody response to acute viral infection. Ddb1 deficiency in activated CD4+ T cells resulted in cell cycle arrest at G2-M phase and increased cell death, due to accumulation of DNA damage and hyperactivation of ATM/ATR-Chk1 signaling. Moreover, mice with deletion of both Cul4a and Cul4b in activated CD4+ T cells phenocopied Ddb1-deficient mice, suggesting that E3 ligase-dependent function of Ddb1 was crucial for genome maintenance and helper T-cell generation. Therefore, our results indicate that Ddb1 is an essential positive regulator in the expansion of CD4+ helper T cells.

Diverse immunoglobulin gene usage and convergent epitope targeting in neutralizing antibody responses to SARS-CoV-2.

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population.

Sertoli-Leydig cell tumor in two siblings with DICER1 syndrome: A case report and literature review.

RATIONALE: DICER1 syndrome is an autosomal-dominant tumor predisposition syndrome associated with numerous cancerous and noncancerous conditions. The most common sex cord-stromal tumor associated with DICER1 syndrome is Sertoli-Leydig cell tumor of the ovary (SLCT), which is extremely unusual and accounts for < 0.5% of all ovarian neoplasms. SLCT predominantly affects adolescents and young female adults. To date, there are only a few case reports of ovarian SLCT with underlying germline DICER1 mutations. The diagnosis and treatment of this rare malignancy remains challenging in the clinic mainly due to its rarity and varied presentation. PATIENT CONCERNS: A 21-year-old Chinese girl (proband) was admitted in hospital for experiencing a lower abdominal pain and irregular vaginal bleeding for half a year. She was initially diagnosed with abdominal cavity mass prior to surgical operation. The other 20-year-old patient is the younger sister of the proband, who was diagnosed with ovarian cysts and had irregular menstruation and amenorrhea for 4 months. The elder sister underwent an uncomplicated bilateral ovarian tumor resection. Given a high degree of malignancy, comprehensive staged fertility-preserving surgery, including left adnexectomy, omentectomy, pelvic, and para-aortic lymphadenectomy, was performed. Since the other patient requested to maintain her fertility, tumor resection was only conducted in the right ovary. DIAGNOSES: The elder sister was diagnosed as poorly differentiated SLCT accompanied with heterologous stage IC rhabdomyosarcoma (RMS) based on its typical pathology features and molecular characteristics from immunohistochemistry (IHC) staining. The younger sister was diagnosed as poorly differentiated SLCT. Targeted next-generation sequencing (NGS) detected DICER1 mutation in the plasma samples and postoperative tumor tissues of both patients. INTERVENTIONS: Both patients underwent surgical tumor resection, followed by combination chemotherapy with bleomycin, etoposide, and cisplatin for 4 cycles. OUTCOMES: Patients received the above clinical interventions but eventually died from disease recurrence. The elder sister died from disease relapse after one and a half years postsurgery. The younger sister had a relapse of the disease 1 year later, but she refused the comprehensive staged surgery and died from disease relapse quickly. LESSONS: Ovarian SLCT patients with DICER1 mutations and a family history have a high degree of malignancy and are associated with a poor prognosis. With ongoing research efforts on DICER1 mutations, genetic screening and counselling on a regular basis is recommended for predicting potential future cancer risk of individuals with DICER1 syndrome family history.

Bacterial magnetosomes-based nanocarriers for co-delivery of cancer therapeutics in vitro.

In recent times, co-delivery of therapeutics has emerged as a promising strategy for treating dreadful diseases such as cancer. MATERIALS AND METHODS: In this study, we developed a novel nanocarrier based on bacterial magnetosomes (BMs) that co-loaded with siRNA and doxorubicin (DOX) using polyethyleneimine (PEI) as a cross-linker (BMs/DP/siRNA). The delivery efficiency of siRNA as well as the pH-responsive release of DOX, and synergistic efficacy of these therapeutics in vitro were systematically investigated. RESULTS: The structure of DOX-PEI (DP) conjugates that synthesized via hydrazone bond formation was confirmed by 1H nuclear magnetic resonance (NMR). The in vitro release experiments showed that the DP conjugate (DOX-loading efficiency - 5.77%±0.08%) exhibited the long-term release behavior. Furthermore, the optimal BMs/DP/siRNA particle size of 107.2 nm and the zeta potential value of 31.1±1.0 mV facilitated enhanced cellular internalization efficiency. Moreover, the agarose gel electrophoresis showed that the co-delivery system could protect siRNA from degradation in serum and RNase A. In addition, the cytotoxicity assay showed that BMs/DP/siRNA could achieve an excellent synergistic effect compared to that of siRNA delivery alone. The acridine orange (AO)/ethidium bromide (EB) double staining assay also showed that BMs/DP/siRNA complex could induce cells in a stage of late apoptosis and nanocomplex located in the proximity of the nucleus. CONCLUSION: The combination of gene and chemotherapeutic drug using BMs is highly efficient, and the BMs/DP/siRNA would be a promising therapeutic strategy for the future therapeutics.

Evaluation of biomarkers for ecotoxicity assessment by dose-response dynamic models: Effects of nitrofurazone on antioxidant enzymes in the model ciliated protozoan Euplotes vannus.

Understanding dose-responses is crucial for determining the utility of biomarkers in ecotoxicity assessment. Nitrofurazone is a broad-spectrum antibiotic that is widely used in the aquaculture industry in China despite its detrimental effects on ecosystems. Potential dose-response models were examined for the effect of nitrofurazone on two antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), in the ciliated protozoan Euplotes vannus. This was achieved by measuring enzyme activity and gene expression profiling of SOD and GPx in ciliate cells exposed to nitrofurazone at doses ranging from 0 to 180mgl-1 for 6h, 12h, 18h and 24h. Dose-response dynamics were characterized by mathematical models. Results showed that: 1) dose-response patterns differed significantly among the tested endpoints, nitrofurazone concentrations and durations of exposure; 2) GPx activity was the best candidate biomarker because of its linear dose-response relationship; 3) SOD activity and mRNA relative expression levels of GPx and SOD are also candidate biomarkers but their dose-responses were non-linear and therefore more difficult to interpret; 4) partitioning the dose-response dynamic model by piecewise function can help to clarify the relationships between biological endpoints. This study demonstrates the utility of dynamic model analysis and the potential of antioxidant enzymes, in particular GPx activity, as a candidate biomarkers for environmental monitoring and risk assessment of nitrofurazone in the aquaculture industry.

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1.

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8(-/Y) ) model, we found that deletion of phf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8(+/Y) ) and ph8(-/Y) ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8(-/Y) by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8(-/Y) ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells 2016;34:1527-1540.

Recognizing the importance of exposure-dose-response dynamics for ecotoxicity assessment: nitrofurazone-induced antioxidase activity and mRNA expression in model protozoan Euplotes vannus.

The equivocality of dose-response relationships has, in practice, hampered the application of biomarkers as a means to evaluate environmental risk, yet this important issue has not yet been fully recognized or explored. This paper evaluates the potential of antioxidant enzymes in the ciliated protozoan Euplotes vannus for use as biomarkers. Dose-response dynamics, together with both the enzyme activity and the gene expression of the antioxidant enzymes, superoxide dismutase, and glutathione peroxidase, were investigated when E. vannus were exposed to graded doses of nitrofurazone for several discrete durations. Mathematical models were explored to characterize the dose-response profiles and, specifically, to identify any equivocality in terms of endpoint. Significant differences were found in both enzyme activity and messenger RNA (mRNA) expression in the E. vannus treated with nitrofurazone, and the interactions between exposure dosage and duration were significant. Correlations between enzyme activity, mRNA expression, and nitrofurazone dose varied with exposure duration. Particularly, the dose-responses showed different dynamics depending on either endpoint or exposure duration. Our findings suggest that both the enzyme activity and the gene expression of the tested antioxidant enzymes can be used as biomarkers for ecotoxicological assessment on the premise of ascertaining appropriate dosage scope, exposure duration, endpoint, etc., which can be achieved by using dose-response dynamics.

Expression profiles of histone lysine demethylases during cardiomyocyte differentiation of mouse embryonic stem cells.

AIM: Histone lysine demethylases (KDMs) control the lineage commitments of stem cells. However, the KDMs involved in the determination of the cardiomyogenic lineage are not fully defined. The aim of this study was to investigate the expression profiles of KDMs during the cardiac differentiation of mouse embryonic stem cells (mESCs). METHODS: An in vitro cardiac differentiation system of mESCs with Brachyury (a mesodermal specific marker) and Flk-1(+)/Cxcr4(+) (dual cell surface biomarkers) selection was used. The expression profiles of KDMs during differentiation were analyzed using Q-PCR. To understand the contributions of KDMs to cardiomyogenesis, the mESCs on differentiation d 3.5 were sorted by FACS into Brachyury(+) cells and Brachyury(-) cells, and mESCs on d 5.5 were sorted into Flk-1(+)/Cxcr4(+) and Flk-1(-)/Cxcr4(-) cells. RESULTS: mESCs were differentiated into spontaneously beating cardiomyocytes that were visible in embryoid bodies (EBs) on d 7. On d 12-14, all EBs developed spontaneously beating cardiomyocytes. Among the 16 KDMs examined, the expression levels of Phf8, Jarid1a, Jhdm1d, Utx, and Jmjd3 were increased by nearly 2-6-fold on d 14 compared with those on d 0. Brachyury(+) cells showed higher expression levels of Jmjd3, Jmjd2a and Jhdm1d than Brachyury(-) cells. A higher level of Jmjd3 was detected in Flk-1(+)/Cxcr4(+) cells, whereas the level of Jmjd2c was lower in both Brachyury(+) cells and Flk-1(+)/Cxcr4(+) cells. CONCLUSION: KDMs may play important roles during cardiomyogenesis of mESCs. Our results provide a clue for further exploring the roles of KDMs in the cardiac lineage commitment of mESCs and the potential interference of cardiomyogenesis.