Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes, ID1, BCL2L1 and HM13, expressed in human ES cells, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

BackgroundSelective maintenance of genomic epigenetic imprints during pre-implantation development is required for parental origin-specific expression of imprinted genes. The Kruppel-like zinc finger protein ZFP57 acts as a factor necessary for maintaining the DNA methylation memory at multiple imprinting control regions in early mouse embryos and embryonic stem (ES) cells. Maternal-zygotic deletion of ZFP57 in mice presents a highly penetrant phenotype with no animals surviving to birth. Additionally, several cases of human transient neonatal diabetes are associated with somatic mutations in the ZFP57 coding sequence.ResultsHere, we comprehensively map sequence-specific ZFP57 binding sites in an allele-specific manner using hybrid ES cell lines from reciprocal crosses between C57BL/6J and Cast/EiJ mice, assigning allele specificity to approximately two-thirds of all binding sites. While half of these are biallelic and include endogenous retrovirus (ERV) targets, the rest show monoallelic binding based either on parental origin or on genetic background of the allele. Parental-origin allele-specific binding is methylation-dependent and maps only to imprinting control differentially methylated regions (DMRs) established in the germline. We identify a novel imprinted gene, Fkbp6, which has a critical function in mouse male germ cell development. Genetic background-specific sequence differences also influence ZFP57 binding, as genetic variation that disrupts the consensus binding motif and its methylation is often associated with monoallelic expression of neighboring genes.ConclusionsThe work described here uncovers further roles for ZFP57-mediated regulation of genomic imprinting and identifies a novel mechanism for genetically determined monoallelic gene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0672-7) contains supplementary material, which is available to authorized users.

Proportion and pathways of the eddy-induced heat transport are critical in maintaining world’s ocean and climate states. However, an observation-based three-dimensional picture of how oceanic eddies contribute to the global heat transport is yet not quantitatively specified, particularly due to insufficiency of data. Here, using refined methods, we have achieved this goal by analyzing millions of high-quality Argo hydrographic profiles and high-resolution satellite altimetric data. We first presented the spatial differences of individual eddies by reconstructing 254 representative eddies all over the ocean, and then calculated heat fluxes associated with eddies in 5° × 5° boxes. It is revealed that all parameters of eddies vary significantly with both latitudes and longitudes, which is crucial in yielding spatially varying heat fluxes and transports. The eddies not only transport heat towards high latitudes (down-gradient), but also towards low latitudes (up-gradient), particularly at subsurface layers of mid-latitude northern Pacific Ocean and low-latitude Atlantic Ocean. The eddy heat transport is mainly confined in the upper 1000 m of the western part and mid-latitudes of the world’s ocean basins, coinciding with maximum meridional temperature gradients. It peaks at 0.8 PW and 0.3 PW (1 PW = 1015 W) at 45°S and 35°N, respectively, stronger than previous estimates based on model results, and accounts for about one half and one third of the estimated total oceanic heat transport at the same latitudes, respectively. In any location except for the areas associated with the Antarctic Circumpolar Current, the eddy stirring component is distinctly (1–10 times) larger than the eddy trapping component.

Resistance of breast cancer to human epidermal growth factor receptor 2 (HER2) inhibitors involves reprogramming of the kinome through HER2/HER3 signaling via the activation of multiple tyrosine kinases and transcriptional upregulation. The heterogeneity of induced kinases prevents kinase targeting by a single kinase inhibitor and presents a major challenge to the treatment of therapeutically recalcitrant HER2-positive breast cancers (HER2+ BCs). As a result, there is a critical need for effective treatment that attacks the aberrant kinome activation associated with resistance to HER2-targeted therapy. Here, we describe a novel treatment strategy that targets cyclin-dependent kinase 7 (CDK7) in HER2 inhibitor-resistant (HER2iR) breast cancer. We show that both HER2 inhibitor-sensitive (HER2iS) and HER2iR breast cancer cell lines exhibit high sensitivity to THZ1, a newly identified covalent inhibitor of the transcription regulatory kinase CDK7. CDK7 promotes cell cycle progression through inhibition of transcription, rather than via direct phosphorylation of classical CDK targets. The transcriptional kinase activity of CDK7 is regulated by HER2, and by the receptor tyrosine kinases activated in response to HER2 inhibition, as well as by the downstream SHP2 and PI3K/AKT pathways. A low dose of THZ1 displayed potent synergy with the HER2 inhibitor lapatinib in HER2iR BC cells in vitro. Dual HER2 and CDK7 inhibition induced tumor regression in two HER2iR BC xenograft models in vivo. Our data support the utilization of CDK7 inhibition as an additional therapeutic avenue that blocks the activation of genes engaged by multiple HER2iR kinases.

Mouse epiblast stem cells (EpiSCs) derived from postimplantation embryos are developmentally and functionally different from embryonic stem cells (ESCs) generated from blastocysts. EpiSCs require Activin A and FGF2 signaling for self-renewal, similar to human ESCs (hESCs), while mouse ESCs require LIF and BMP4. Unlike ESCs, EpiSCs have undergone X-inactivation, similar to the tendency of hESCs. The shared self-renewal and X-inactivation properties of EpiSCs and hESCs suggest that they have an epigenetic state distinct from ESCs. This hypothesis predicts that EpiSCs would have monoallelic expression of most imprinted genes, like that observed in hESCs. Here, we confirm this prediction. By contrast, we find that mouse induced pluripotent stem cells (iPSCs) tend to lose imprinting similar to mouse ESCs. These findings reveal that iPSCs have an epigenetic status associated with their pluripotent state rather than their developmental origin. Our results also reinforce the view that hESCs and EpiSCs are in vitro counterparts, sharing an epigenetic status distinct from ESCs and iPSCs.

Most pituitary adenomas (PAs) are considered benign tumors, but approximately 0.2% can present metastasis and are classified as pituitary carcinomas (PCs). Refractory PAs lie between benign adenomas and true malignant PC and are defined as aggressive-invasive PAs characterized by a high Ki-67 index, rapid growth, frequent recurrence, and resistance to conventional treatments, including temozolomide. It is notoriously difficult to manage refractory PAs and PC because of the limited therapeutic options. As a promising therapeutic approach, cancer immunotherapy has been experimentally used for the treatment of many tumors, including pituitary tumors. The purpose of this review is to report the progress of immunotherapy in pituitary tumors, including refractory PAs and PCs. The tumor immune microenvironment has been recognized as a key contributor to tumorigenesis, progression, and prognosis. One study indicated that the number of CD68+ macrophages was positively correlated with tumor size and Knosp classification grade for tumor invasiveness. The infiltration of CD4+ and CD8+ T cells was relatively scant in these adenomas, but pituitary growth hormone (GH) adenomas exhibited significantly more CD4+ and CD8+ T cells than non-GH adenomas. These results suggest an association of CD68+ macrophage infiltration with an increase in pituitary tumor size and invasiveness. Another study suggested that a lower number of CD8+ lymphocytes is associated with cavernous sinus invasion and resistance to treatment with first-generation somatostatin analogs in acromegaly patients, highlighting a potential role of the tumor immune microenvironment in determining the prognosis of somatotroph pituitary tumors. Preclinical studies have indicated that widely varying degrees of programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs) are found among different subtypes. Functional PAs and aggressive PAs express significantly higher levels of PD-L1 and TILs than other subtypes, indicating that PD-1 blockade might be a promising alternative therapy for patients with aggressive PAs. PD-L1 transcript and protein levels were found to be significantly increased in functioning (GH and prolactin-expressing) pituitary tumors compared to nonfunctioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary tumors harbored higher levels of PD-L1 mRNA than recurrent tumors. These findings suggest the possibility of considering checkpoint blockade immunotherapy for functioning pituitary tumors refractory to conventional management. Animal models of Cushing’s disease also demonstrated PD-L1 and TIL expression in cultured tumors and murine models, as well as the effectiveness of checkpoint blockade therapy in reducing the tumor mass, decreasing hormone secretion, and increasing the survival rate. Clinical studies show that immunotherapy may be an effective treatment in patients with pituitary tumors. One corticotroph carcinoma patient showed a significant reduction in hormone levels and shrinkage of the tumor size of primary and metastatic lesions immediately after investigational treatment with ipilimumab and nivolumab. However, another patient with corticotroph adenoma progressed rapidly after four cycles of anti-PD-1 (pembrolizumab) treatment. To date, there are two registered clinical trials of immunotherapy for pituitary tumors. One of them is the phase II clinical trial of nivolumab combined with ipilimumab for patients with aggressive pituitary tumors (NCT04042753). The other one is also a phase II clinical trial of the combination of nivolumab and ipilimumab for rare tumors, including pituitary tumors (NCT02834013). Both clinical trials are in the stage of recruiting patients and have not been completed. In summary, the results from preclinical research and clinical studies indicated that immunotherapy might be a promising alternative therapy for PCs and refractory PAs resistant to conventional treatments. The combination of immunotherapy and radiotherapy or temozolomide may have synergistic effects compared to a single treatment. More preclinical and clinical studies are needed to further indicate the exact efficacy of immunotherapy in pituitary tumors.

Pituitary adenomas are classified as typical or atypical, invasive or noninvasive, and aggressive or nonaggressive based on pathological features, radiological findings, and clinical behavior. Only pituitary tumors with cerebrospinal and/or systemic metastasis are considered malignant carcinomas. However, some pituitary adenomas with high Ki-67 indexes exhibit aggressive behaviors, such as rapid growth, early and frequent recurrence, and resistance to conventional treatment, even in the absence of metastasis. Novel terminology is needed to define these tumors. Here, we propose the use of the term “refractory pituitary adenoma” to define malignant pituitary tumors exhibiting 1) a high Ki-67 index and rapid growth, 2) early and high frequency of recurrence, 3) resistance to conventional treatments and/or salvage treatment with temozolomide (TMZ), 4) poor prognosis, 5) and a lack of cerebrospinal or systemic metastases. To illustrate the utility of this refractory pituitary adenoma classification and the difficulty in managing disease in these patients, we examined twelve clinical cases. Correctly identifying refractory pituitary adenomas is crucial for improving patient prognoses. Early identification might encourage the early use of aggressive therapeutic strategies to prevent or delay recurrence.