Gender-specific characteristics of hypertrophic response in cardiomyocytes derived from human embryonic stem cells

Introduction: Gender-specific phenotypes of the heart were reported with respect to both physiology and pathology. While most differences were associated with the sex hormones, differential expression of genes received special attention, particularly X-Y chromosomes’ genes. Methods: Here, we compared cardiogenesis by gene expression analysis of lineage specific markers and X-Y chromosomes’ genes, during in vitro differentiation of XY and XX human embryonic stem cells (hESC), in a hormone-free setup. Results: Downregulation of pluripotency marker (NANOG) and upregulation of cardiac mesoderm and progenitor markers (GATA4, TBX5, NKX2.5, ISL1) was remained temporally similar in differentiating XY and XX hESCs. Isoproterenol treatment of XY and XX hESC-derived cardiomyocytes (hESCCM) induced hypertrophy in a sex-specific manner, with female cardiomyocytes showing response at higher isoproterenol concentration and a later time point of differentiation. Interestingly, KDM5C as an X-linked gene, was markedly upregulated in both hypertrophied male and female cardiomyocytes. Conclusion: Collectively, our results indicated a temporally identical cardiogenesis, but more susceptibility of XY hESC-CM to hypertrophic stimulus in a hormone-free condition.

Reporters of Cancer Stem Cells as a Tool for Drug Discovery

In view of the importance of cancer stem cells (CSCs) in chemoresistance, metastasis and recurrence, the biology of CSCs were explored in detail. Based on that, several modalities were proposed to target them. In spite of the several clinical trials, a successful CSC-targeting drug is yet to be identified. The number of molecules screened and entered for clinical trial for CSC-targeting is comparatively low, compared to other drugs. The bottle neck is the lack of a high-throughput adaptable screening strategy for CSCs. This review is aimed to identify suitable reporters for CSCs that can be used to identify the heterogeneous CSC populations, including quiescent CSCs, proliferative CSCs, drug resistant CSCs and metastatic CSCs. Analysis of the tumor microenvironment regulating CSCs revealed that the factors in CSC-niche activates effector molecules that function as CSC markers, including pluripotency markers, CD133, ABCG2 and ALDH1A1. Among these factors OCT4, SOX2, NANOG, ABCG2 and ALDH1A1 are ideal for making reporters for CSCs. The pluripotency molecules, like OCT4, SOX2 and NANOG, regulate self-renewal, chemoresistance and metastasis. ABCG2 is a known regulator of drug resistance while ALDH1A1 modulates self-renewal, chemoresistance and metastasis. Considering the heterogeneity of CSCs, including a quiescent population and a proliferative population with metastatic ability, we propose the use of a combination of reporters. A dual reporter consisting of a pluripotency marker and a marker like ALDH1A1 will be useful in screening drugs that target CSCs.

A Case of Myoepithelial Hamartoma: Morphological Variation Supported by OCT4 Expression

In this report, we describe a patient with myoepithelial hamartoma, which is regarded as synonymous with adenomyosis and heterotopic pancreas. Endoscopy revealed a submucosal tumor in the antrum of the stomach. Subsequently, distal gastrectomy with Roux-en-Y reconstruction was performed. Histological findings of adenomyomatous lesion and heterotopic pancreatic tissue were observed in this lesion. The distribution of OCT4, which is a pluripotency marker, varied in each part.

Multiplexed live visualization of cell fate dynamics in hPSCs at single-cell resolution

ABSTRACTLive imaging can provide powerful insights into developmental and cellular processes but availability of multiplexable reporters has been limiting. Here we describe ORACLE, a cell fate reporter class in which fluorescent proteins fused with the nucleoporin POM121 are driven by promoters of transcription factors of interest. ORACLE’s nuclear rim localisation therefore enables multiplexing with conventional nuclear reporters. We applied ORACLE to investigate the dynamics of pluripotency exit at single-cell level, using human pluripotent stem cells (hPSCs) imaged by multi-day time-lapse high-content microscopy. Using an ORACLE-OCT4 pluripotency marker we reveal that G1 phase length and OCT4 level are strongly coupled and that spatial location in a colony impacts the timing of pluripotency exit. Combining ORACLE-OCT4 and an ORACLE-SOX1 early neuronal differentiation marker, we visualize in real-time the dynamics of cell fate transition between pluripotency and early neural fate, and show that pluripotency exit and differentiation onset are likely not tightly coupled in single-cells. Thus ORACLE is a powerful tool to enable quantitative studies of spatiotemporal cell fate control.

Ascorbic Acid: A New Player of Epigenetic Regulation in LPS-gingivalis Treated Human Periodontal Ligament Stem Cells

Periodontitis is usually sustained from microorganism of oral cavity, like Porphyromonas gingivalis (P. gingivalis). Periodontal disease is an infectious disease that afflicts a large number of people. Researches are investigating on the mesenchymal stem cells (MSCs) response to inflammatory events in combination with antioxidant substances. In particular, ascorbic acid (AA) increased cell proliferation, upregulated the cells pluripotency marker expression, provide a protection from inflammation, and induced the regeneration of periodontal ligament tissue. The purpose of the present research was to investigate the effects of AA in primary culture of human periodontal ligament stem cells (hPDLSCs) exposed to P. gingivalis lipopolysaccharide (LPS-G). The effect of AA on hPDLSCs exposed to LPS-G was determined through the cell proliferation assay. The molecules involved in the inflammatory pathway and epigenetic regulation have been identified using immunofluorescence and Western blot analyses. miR-210 level was quantified by qRT-PCR, and the ROS generation was finally studied. Cells co-treated with LPS-G and AA showed a restoration in terms of cell proliferation. The expression of NFκB, MyD88, and p300 was upregulated in LPS-G exposed cells, while the expression was attenuated in the co-treatment with AA. DNMT1 expression is attenuated in the cells exposed to the inflammatory stimulus. The level of miR-210 was reduced in stimulated cells, while the expression was evident in the hPDLSCs co-treated with LPS-G and AA. In conclusion, the AA could enhance a protective effect in in vitro periodontitis model, downregulating the inflammatory pathway and ROS generation and modulating the miR-210 level.

Embryonic disc formation following post-hatching bovine embryo development in vitro

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

The Effects of Platelet-Rich and Platelet-Poor Plasma on Biological Characteristics of BM-MSCs In Vitro

Platelet-rich plasma (PRP) and its byproduct platelet-poor plasma (PPP) are rich sources of cytokines in tissue damage repair. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have received more and more attention for their ability to treat multiple diseases. The purpose of our study was to investigate the biologic action of PPP and PRP on BM-MSCs. The adipogenic potential of BM-MSCs revealed no obvious change, but the osteogenic ability of BM-MSCs was enhanced after treated with PRP. CCK8 assays and cell colony formation assays showed that PRP promoted cell proliferation, while this effect of PPP was not obvious. No obvious difference was found in cell cycle and apoptosis of BM-MSCs between PRP and PPP treatment. Expression of β-galactosidase, a biological marker of senescence, was decreased upon PRP treatment which indicated that PRP provided significant protection against cellular senescence. The migratory capacity of BM-MSCs was detected by scratch and transwell assays. The results indicated that PRP could affect the migration ability of BM-MSCs. From immunofluorescence detection and western blot, we demonstrated that the level of epithelial-mesenchymal transition-related proteins was changed and several pluripotency marker genes, including Sox2, Sall4, Oct4, and Nanog, were increased. Finally, the expression of the key signal pathway such as PI3K/AKT was examined. Our findings suggested that PRP promoted cell migration of BM-MSCs via stimulating the signaling pathway of PI3K/AKT.

Derivation of ringed seal (Phoca hispida) tripotent induced pluripotent stem-like cells

Induced pluripotent stem (iPS) cells have been produced just for a few species among order Carnivora: snow leopard, Bengal tiger, serval, jaguar, cat, dog, ferret, and American mink. For the first time, we derived the ringed seal (Phoca hispida) iPS-like cells. We had shown the expression of pluripotency marker gene Rex1. Ringed seal iPS-like cells were able to differentiate into derivatives of endoderm (expression of AFP), mesoderm (adipocytes and osteocytes), and trophectoderm (expression of Cdx2). To confirm pluripotency, we need to differentiate cells into ectoderm cell types, for instance into neurons.