INO80 function is required for mouse mammary gland development, but mutation alone may be insufficient for breast cancer.

The aberrant function of ATP-dependent chromatin remodeler INO80 has been implicated in multiple types of cancers by altering chromatin architecture and gene expression; however, the underlying mechanism of the functional involvement of INO80 mutation in cancer etiology, especially in breast cancer, remains unclear. In the present study, we have performed a weighted gene co-expression network analysis (WCGNA) to investigate links between INO80 expression and breast cancer sub-classification and progression. Our analysis revealed that INO80 repression is associated with differential responsiveness of estrogen receptors (ERs) depending upon breast cancer subtype, ER networks, and increased risk of breast carcinogenesis. To determine whether INO80 loss induces breast tumors, a conditional INO80-knockout (INO80 cKO) mouse model was generated using the Cre-loxP system. Phenotypic characterization revealed that INO80 cKO led to reduced branching and length of the mammary ducts at all stages. However, the INO80 cKO mouse model had unaltered lumen morphology and failed to spontaneously induce tumorigenesis in mammary gland tissue. Therefore, our study suggests that the aberrant function of INO80 is potentially associated with breast cancer by modulating gene expression. INO80 mutation alone is insufficient for breast tumorigenesis.

Alterations and Co-Occurrence of C-MYC, N-MYC, and L-MYC Expression are Related to Clinical Outcomes in Various Cancers.

Background and Objectives: MYC, also known as an oncogenic reprogramming factor, is a multifunctional transcription factor that maintains induced pluripotent stem cells (iPSCs). Although MYC is frequently upregulated in various cancers and is correlated with a poor prognosis, MYC is downregulated and correlated with a good prognosis in lung adenocarcinoma. MYC and two other MYC family genes, MYCN and MYCL, have similar structures and could contribute to tumorigenic conversion both in vitro and in vivo. Methods and Results: We systematically investigated whether MYC family genes act as prognostic factors in various human cancers. We first evaluated alterations in the expression of MYC family genes in various cancers using the Oncomine and The Cancer Genome Atlas (TCGA) database and their mutation and copy number alterations using the TCGA database with cBioPortal. Then, we investigated the association between the expression of MYC family genes and the prognosis of cancer patients using various prognosis databases. Multivariate analysis also confirmed that co-expression of MYC/MYCL/MYCN was significantly associated with the prognosis of lung, gastric, liver, and breast cancers. Conclusions: Taken together, our results demonstrate that the MYC family can function not only as an oncogene but also as a tumor suppressor gene in various cancers, which could be used to develop a novel approach to cancer treatment.

Label-free and non-destructive identification of naïve and primed embryonic stem cells based on differences in cellular metabolism.

Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging. This study reports an electrochemical method that enables simple, label-free, and non-destructive detection of naïve embryonic stem cells (ESCs) derived from mouse ESCs, based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)-glycolysis and oxidative phosphorylation (OXPHOS)-were blocked, and it was found that mitochondrial energy generation is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve-primed switchable metastable ESCs. The mouse PSCs derived from doxycycline-inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non-invasive and label-free manner. Thus, it can be useful in the development of stem cell-derived therapeutics.

Dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells.

BACKGROUND: The requirement of the Mek1 inhibitor (iMek1) during naïve pluripotency maintenance results from the activation of the Mek1-Erk1/2 (Mek/Erk) signaling pathway upon leukemia inhibitory factor (LIF) stimulation. METHODS: Through a meta-analysis of previous genome-wide screening for negative regulators of naïve pluripotency, Ptpn11 (encoding the Shp2 protein, which serves both as a tyrosine phosphatase and putative adapter), was predicted as one of the key factors for the negative modulation of naïve pluripotency through LIF-dependent Jak/Stat3 signaling. Using an isogenic pair of naïve and primed mouse embryonic stem cells (mESCs), we demonstrated the differential role of Shp2 in naïve and primed pluripotency. RESULTS: Loss of Shp2 increased naïve pluripotency by promoting Jak/Stat3 signaling and disturbed in vivo differentiation potential. In sharp contrast, Shp2 depletion significantly impeded the self-renewal of ESCs under primed culture conditions, which was concurrent with a reduction in Mek/Erk signaling. Similarly, upon treatment with an allosteric Shp2 inhibitor (iShp2), the cells sustained Stat3 phosphorylation and decoupled Mek/Erk signaling, thus iShp2 can replace the use of iMek1 for maintenance of naïve ESCs. CONCLUSIONS: Taken together, our findings highlight the differential roles of Shp2 in naïve and primed pluripotency and propose the usage of iShp2 instead of iMek1 for the efficient maintenance and establishment of naïve pluripotency.

Role of Transcriptional and Epigenetic Regulation in Lymphatic Endothelial Cell Development.

The lymphatic system is critical for maintaining the homeostasis of lipids and interstitial fluid and regulating the immune cell development and functions. Developmental anomaly-induced lymphatic dysfunction is associated with various pathological conditions, including lymphedema, inflammation, and cancer. Most lymphatic endothelial cells (LECs) are derived from a subset of endothelial cells in the cardinal vein. However, recent studies have reported that the developmental origin of LECs is heterogeneous. Multiple regulatory mechanisms, including those mediated by signaling pathways, transcription factors, and epigenetic pathways, are involved in lymphatic development and functions. Recent studies have demonstrated that the epigenetic regulation of transcription is critical for embryonic LEC development and functions. In addition to the chromatin structures, epigenetic modifications may modulate transcriptional signatures during the development or differentiation of LECs. Therefore, the understanding of the epigenetic mechanisms involved in the development and function of the lymphatic system can aid in the management of various congenital or acquired lymphatic disorders. Future studies must determine the role of other epigenetic factors and changes in mammalian lymphatic development and function. Here, the recent findings on key factors involved in the development of the lymphatic system and their epigenetic regulation, LEC origins from different organs, and lymphatic diseases are reviewed.

Meiosis-specific cohesin complexes display essential and distinct roles in mitotic embryonic stem cell chromosomes.

BACKGROUND: Cohesin is a chromosome-associated SMC-kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. However, it remains unclear whether meiosis-specific cohesin complexes are functionally active in mitotic chromosomes. RESULTS: Through high-resolution 3D-structured illumination microscopy (3D-SIM) and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, α-kleisin REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that STAG3 is required for the efficient localization of REC8 to the nucleus by interacting with REC8. REC8-STAG3-containing cohesin regulates topological properties of chromosomes and maintains sister chromatid cohesion. Second, REC8-cohesin has additional sister chromatid cohesion roles in concert with mitotic RAD21-cohesin on ESC chromosomes. SIM imaging of REC8 and RAD21 co-staining revealed that the two types of α-kleisin subunits exhibited distinct loading patterns along ESC chromosomes. Third, knockdown of REC8 or RAD21-cohesin not only leads to higher rates of premature sister chromatid separation and delayed replication fork progression, which can cause proliferation and developmental defects, but also enhances chromosome compaction by hyperloading of retinoblastoma protein-condensin complexes from the prophase onward. CONCLUSIONS: Our findings indicate that the delicate balance between mitotic and meiotic cohesins may regulate ESC-specific chromosomal organization and the mitotic program.

Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus.

Cluster of differentiation 73 (CD73, also known as ecto-5'-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.

DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition.

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.

Inhibition of neural stem cell aging through the transient induction of reprogramming factors.

Adult stem cells age during long-term in vitro culture, and neural stem cells (NSCs), which can self-renew and differentiate into neurons and glial cells, also display reduced differentiation potential after repeated passaging. However, the mechanistic details underlying this process remain unclear. In this study, we found that long-term in vitro culture of NSCs resulted in aging-related upregulation of inflammatory- and endoplasmic reticulum (ER) stress-related genes, including the proinflammatory cytokines interleukin (IL)1ß and IL6, the senescence-associated enzyme matrix metallopeptidase 13 (MMP13), and the ER stress-responsive transcription factor activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, the cyclic and transient induction of four reprogramming factors (POU domain, class 5, transcription factor 1, also known as octamer-binding transcription factor 4; SRY [sex determining region Y]-box 2; Kruppel-like factor 4; and myelocytomatosis oncogene; collectively referred to as OSKM) can inhibit NSC aging, as indicated by the decreased expression of the inflammatory and ER stress-related genes. We used ROSA-4F NSCs, which express OSKM from only one allele, to minimize the potential for full reprogramming or tumor formation during NSC rejuvenation. We expect that this novel rejuvenation method will enhance the potential of NSCs as a clinical approach to the treatment of neurological diseases.

Generation of brain organoids from mouse ESCs via teratoma formation.

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs), can differentiate into all cell types in the body; therefore, they are used in the study of development and regenerative medicine. Neural lineage differentiation from PSCs is the initial step to study neurodevelopment and in vitro disease modeling. Brain organoids, which are composed of neural stem cells (NSCs) and differentiated neural lineage cell population, are a powerful in vitro system to mimic the brain tissue. Here, we aimed to establish a new method to generate brain organoids efficiently in a mouse model. We applied the in vivo teratoma formation method as a new approach to generate brain organoids. We induced teratoma formation using Sox1-GFP transgenic ESCs, in which green fluorescence protein (GFP) is expressed under the control of the early NSC marker Sox1. Sox1-GFP-expressing early NSCs were isolated as clumps and further cultured to generate brain organoids. Sox1-GFP ESC-derived brain organoids, composed of multiple layers of distinct cellular components (ventricle, ventricular zone, and cortical layer), were formed within 3 weeks of in vitro culture. We also found that neighboring cells (Sox1-GFP-) surrounding the Sox1-GFP+ clumps are essential for the formation of brain organoids. Thus, in vivo and in vitro conjugated systems-initial commitment in vivo and further specialization in vitro-could be one of the promising platforms for organoid formation that are universally applicable.

Mitochondrial and Metabolic Dynamics of Endometrial Stromal Cells During the Endometrial Cycle.

The endometrial cycle in response to hormonal stimulation is essential for implantation. The female has endometrium that repeats this cycle through about half of a lifetime. The cycle includes three phases, proliferative, secretory, and menstrual, and each phase has distinct characteristics. The endometrial stromal cells (EnSCs) in each phase also have specialized characteristics, including cell cycle, morphologies, and cellular metabolic state. So we hypothesized that the cells in each phase have unique mitochondrial morphologies because they are generally linked to cellular metabolic state. To investigate the metabolic characteristics in each phase, we investigated the mitochondrial morphologies by transmission electron microscopy, oxygen consumption rate (OCR), and intracellular adenosine triphosphate (ATP) production. The decidualized EnSCs have shorter mitochondria than those in the proliferative phase. Besides, they also displayed distinct intracellular structural characteristics compared with the proliferative phase, such as ribosome-rich endoplasmic reticulum and increased formation of vesicles. OCR and luminescent ATP detection assay revealed that the basal respiration and ATP production in the decidualized EnSCs were lower than those in the proliferative phase. Thus, we concluded that morphological and intracellular structural changes were induced during the decidualization. Moreover, the decreased mitochondrial length was shown to correlate with decreased dependency on oxidative phosphorylation and ATP concentration in EnSCs.

Changes in the Expression of Mitochondrial Morphology-Related Genes during the Differentiation of Murine Embryonic Stem Cells.

During embryonic development, cells undergo changes in gene expression, signaling pathway activation/inactivation, metabolism, and intracellular organelle structures, which are mediated by mitochondria. Mitochondria continuously switch their morphology between elongated tubular and fragmented globular via mitochondrial fusion and fission. Mitochondrial fusion is mediated by proteins encoded by Mfn1, Mfn2, and Opa1, whereas mitochondrial fission is mediated by proteins encoded by Fis1 and Dnm1L. Here, we investigated the expression patterns of mitochondria-related genes during the differentiation of mouse embryonic stem cells (ESCs). Pluripotent ESCs maintain stemness in the presence of leukemia inhibitory factor (LIF) via the JAK-STAT3 pathway but lose pluripotency and differentiate in response to the withdrawal of LIF. We analyzed the expression levels of mitochondrial fusion- and fission-related genes during the differentiation of ESCs. We hypothesized that mitochondrial fusion genes would be overexpressed while the fission genes would be downregulated during the differentiation of ESCs. Though the mitochondria exhibited an elongated morphology in ESCs differentiating in response to LIF withdrawal, only the expression of Mfn2 was increased and that of Dnm1L was decreased as expected, the other exceptions being Mfn1, Opa1, and Fis1. Next, by comparing gene expression and mitochondrial morphology, we proposed an index that could precisely represent mitochondrial changes during the differentiation of pluripotent stem cells by analyzing the expression ratios of three fusion- and two fission-related genes. Surprisingly, increased Mfn2/Dnm1L ratio was correlated with elongation of mitochondria during the differentiation of ESCs. Moreover, application of this index to other specialized cell types revealed that neural stems cells (NSCs) and mouse embryonic fibroblasts (MEFs) showed increased Mfn2/Dnm1L ratio compared to ESCs. Thus, we suggest that the Mfn2/Dnm1L ratio could reflect changes in mitochondrial morphology according to the extent of differentiation.

Toxic Effects of Nonylphenol on Neonatal Testicular Development in Mouse Organ Culture.

Nonylphenol (NP) is an alkylphenol that is widely used in chemical manufacturing. Exposure to this toxic environmental contaminant has been shown to negatively affect the reproductive system. Herein, we evaluated the toxicity of NP in mouse testes, while using in vitro organ culture. Mouse testicular fragments (MTFs), derived from five-day postpartum neonatal mouse testes, were exposed to different concentrations of NP (1-50 µM) for 30 days. The results showed that NP impaired germ cell development and maintenance. Furthermore, NP significantly downregulated the transcript levels of both undifferentiated and differentiated germ cell marker genes relative to those in controls. In particular, a high dose of NP (50 µM) led to complete germ cell depletion and resulted in spermatogenic failure, despite the presence of Sertoli and Leydig cells. In addition, the mRNA expression of steroidogenic enzymes, such as steroidogenic acute regulatory protein (STAR), Cytochrome P450 Family 11 Subfamily A Member 1 (Cyp11α1), Cytochrome P450 17A1 (Cyp17α1), and androgen receptor (AR), increased with increasing concentration of NP. Conversely, the expression of estrogen receptor alpha (ESR1) and Cytochrome P450 family 19 subfamily A member 1 (Cyp19α1) in NP-exposed MTFs decreased when compared to that of the control. Taken together, this study demonstrates that NP has a negative effect on prepubertal spermatogenesis and germ cell maintenance and it disrupts steroidogenesis and induces hormonal imbalance in MTFs.

Anisotropic Platinum Nanoparticle-Induced Cytotoxicity, Apoptosis, Inflammatory Response, and Transcriptomic and Molecular Pathways in Human Acute Monocytic Leukemia Cells.

The thermoplasmonic properties of platinum nanoparticles (PtNPs) render them desirable for use in diagnosis, detection, therapy, and surgery. However, their toxicological effects and impact at the molecular level remain obscure. Nanotoxicology is mainly focused on the interactions of nanostructures with biological systems, particularly with an emphasis on elucidating the relationship between the physical and chemical properties such as size and shape. Therefore, we hypothesized whether these unique anisotropic nanoparticles could induce cytotoxicity similar to that of spherical nanoparticles and the mechanism involved. Thus, we synthesized unique and distinct anisotropic PtNPs using lycopene as a biological template and investigated their biological activities in model human acute monocytic leukemia (THP-1) macrophages. Exposure to PtNPs for 24 h dose-dependently decreased cell viability and proliferation. Levels of the cytotoxic markers lactate dehydrogenase and intracellular protease significantly and dose-dependently increased with PtNP concentration. Furthermore, cells incubated with PtNPs dose-dependently produced oxidative stress markers including reactive oxygen species (ROS), malondialdehyde, nitric oxide, and carbonylated protein. An imbalance in pro-oxidants and antioxidants was confirmed by significant decreases in reduced glutathione, thioredoxin, superoxide dismutase, and catalase levels against oxidative stress. The cell death mechanism was confirmed by mitochondrial dysfunction and decreased ATP levels, mitochondrial copy numbers, and PGC-1α expression. To further substantiate the mechanism of cell death mediated by endoplasmic reticulum stress (ERS), we determined the expression of the inositol-requiring enzyme (IRE1), (PKR-like ER kinase) PERK, activating transcription factor 6 (ATF6), and activating transcription factor 4 ATF4, the apoptotic markers p53, Bax, and caspase 3, and the anti-apoptotic marker Bcl-2. PtNPs could activate ERS and apoptosis mediated by mitochondria. A proinflammatory response to PtNPs was confirmed by significant upregulation of interleukin-1-beta (IL-1ß), interferon γ (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL-6). Transcriptomic and molecular pathway analyses of THP-1 cells incubated with the half maximal inhibitory concentration (IC50) of PtNPs revealed the altered expression of genes involved in protein misfolding, mitochondrial function, protein synthesis, inflammatory responses, and transcription regulation. We applied transcriptomic analyses to investigate anisotropic PtNP-induced toxicity for further mechanistic studies. Isotropic nanoparticles are specifically used to inhibit non-specific cellular uptake, leading to enhanced in vivo bio-distribution and increased targeting capabilities due to the higher radius of curvature. These characteristics of anisotropic nanoparticles could enable the technology as an attractive platform for nanomedicine in biomedical applications.

Epigenetic priming by Dot1l in lymphatic endothelial progenitors ensures normal lymphatic development and function.

Proper functioning of the lymphatic system is required for normal immune responses, fluid balance, and lipid reabsorption. Multiple regulatory mechanisms are employed to ensure the correct formation and function of lymphatic vessels; however, the epigenetic modulators and mechanisms involved in this process are poorly understood. Here, we assess the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Genetic ablation of Dot1l in Tie2(+) endothelial cells (ECs), but not in Lyve1(+) or Prox1(+) lymphatic endothelial cells (LECs) or Vav1(+) definitive hematopoietic stem cells, leads to catastrophic lymphatic anomalies, including skin edema, blood-lymphatic mixing, and underdeveloped lymphatic valves and vessels in multiple organs. Remarkably, targeted Dot1l loss in Tie2(+) ECs leads to fully penetrant lymphatic aplasia, whereas Dot1l overexpression in the same cells results in partially hyperplastic lymphatics in the mesentery. Genetic studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation. Mechanistically, inactivation of Dot1l causes a reduction of both H3K79me2 levels and the expression of genes important for LEC development and function. Thus, our study establishes that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.

Derivation of primitive neural stem cells from human-induced pluripotent stem cells.

Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogene-free hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes. These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders.

Mitochondrial Dynamics in Stem Cells and Differentiation.

Mitochondria are highly dynamic organelles that continuously change their shape. Their main function is adenosine triphosphate (ATP) production; however, they are additionally involved in a variety of cellular phenomena, such as apoptosis, cell cycle, proliferation, differentiation, reprogramming, and aging. The change in mitochondrial morphology is closely related to the functionality of mitochondria. Normal mitochondrial dynamics are critical for cellular function, embryonic development, and tissue formation. Thus, defects in proteins involved in mitochondrial dynamics that control mitochondrial fusion and fission can affect cellular differentiation, proliferation, cellular reprogramming, and aging. Here, we review the processes and proteins involved in mitochondrial dynamics and their various associated cellular phenomena.

SIRT2 is required for efficient reprogramming of mouse embryonic fibroblasts toward pluripotency.

The role of sirtuins (SIRTs) in cancer biology has been the focus of recent research. The similarities between underlying pathways involved in the induction of pluripotent stem cells and transformation of cancer cells revealed the role of SIRTs in cellular reprogramming. Seven SIRTs have been identified in mammals and downregulation of SIRT2 was found to facilitate the generation of primed pluripotent stem cells, such as human induced pluripotent stem cells. Herein, we evaluated the role of SIRT2 in naive pluripotent stem cell generation using murine cells. We found that absolute depletion of SIRT2 in mouse embryonic fibroblasts resulted in a notable reduction in reprogramming efficiency. SIRT2 depletion not only upregulated elements of the INK4/ARF locus, which in turn had an antiproliferative effect, but also significantly altered the expression of proteins related to the PI3K/Akt and Hippo pathways, which are important signaling pathways for stemness. Thus, this study demonstrated that SIRT2 is required for cellular reprogramming to naive states of pluripotency in contrast to primed pluripotency states.

Suppression of the ERK-SRF axis facilitates somatic cell reprogramming.

The molecular mechanism underlying the initiation of somatic cell reprogramming into induced pluripotent stem cells (iPSCs) has not been well described. Thus, we generated single-cell-derived clones by using a combination of drug-inducible vectors encoding transcription factors (Oct4, Sox2, Klf4 and Myc) and a single-cell expansion strategy. This system achieved a high reprogramming efficiency after metabolic and epigenetic remodeling. Functional analyses of the cloned cells revealed that extracellular signal-regulated kinase (ERK) signaling was downregulated at an early stage of reprogramming and that its inhibition was a driving force for iPSC formation. Among the reprogramming factors, Myc predominantly induced ERK suppression. ERK inhibition upregulated the conversion of somatic cells into iPSCs through concomitant suppression of serum response factor (SRF). Conversely, SRF activation suppressed the reprogramming induced by ERK inhibition and negatively regulated embryonic pluripotency by inducing differentiation via upregulation of immediate early genes, such as c-Jun, c-Fos and EGR1. These data reveal that suppression of the ERK-SRF axis is an initial molecular event that facilitates iPSC formation and may be a useful surrogate marker for cellular reprogramming.

Platelet-derived growth factor receptor-alpha positive cardiac progenitor cells derived from multipotent germline stem cells are capable of cardiomyogenesis in vitro and in vivo.

Cardiac cell therapy has the potential to revolutionize treatment of heart diseases, but its success hinders on the development of a stem cell therapy capable of efficiently producing functionally differentiated cardiomyocytes. A key to unlocking the therapeutic application of stem cells lies in understanding the molecular mechanisms that govern the differentiation process. Here we report that a population of platelet-derived growth factor receptor alpha (PDGFRA) cells derived from mouse multipotent germline stem cells (mGSCs) were capable of undergoing cardiomyogenesis in vitro. Cells derived in vitro from PDGFRA positive mGSCs express significantly higher levels of cardiac marker proteins compared to PDGFRA negative mGSCs. Using Pdgfra shRNAs to investigate the dependence of Pdgfra on cardiomyocyte differentiation, we observed that Pdgfra silencing inhibited cardiac differentiation. In a rat myocardial infarction (MI) model, transplantation of a PDGFRAenriched cell population into the rat heart readily underwent functional differentiation into cardiomyocytes and reduced areas of fibrosis associated with MI injury. Together, these results suggest that mGSCs may provide a unique source of cardiac stem/progenitor cells for future regenerative therapy of damaged heart tissue.