Saliva exposure reduces gingival keratinocyte growth on TiO2-coated titanium.

Bioactive, nanoporous TiO2-coating has been shown to enhance cell attachment on titanium implant surface. The aim of this study was to evaluate, whether the saliva proteins affect the epithelial cell adhesion on TiO2-coated and non-coated titanium. Grade V titanium discs were polished. Half of the discs were provided with TiO2-coating produced in sol with polycondensation method. Half of the TiO2-coated and non-coated discs were treated with pasteurized saliva for 30 min. After saliva treatment, the total protein amounts on surfaces were measured. Next, the hydrophilicity of discs were measured with water contact angle measurements. Further, the gingival keratinocyte adhesion strength was measured after 2 and 6 h of cultivation using serial trypsinization. In addition, cell growth and proliferation were measured after 1, 3, and 7 days of cell culture. Finally, cell morphology, spreading and adhesion protein signals were detected with high resolution confocal microscopy. As a result, in sol coated TiO2-surface had significantly higher hydrophilicity when compared to non-coated titanium, meanwhile both non-coated and TiO2-coated surfaces with saliva treatment had a significant increase in hydrophilicity. Importantly, the amounts of adhered saliva proteins were equal between TiO2-coated and non-coated surfaces. Adhesion strength against enzymatic detachment was weakest on non-coated titanium after saliva exposure. Cell proliferation and cell spreading were highest on TiO2-coated titanium, but saliva exposure significantly decreased cell proliferation and spreading on TiO2-coated surface. To conclude, even though saliva exposure makes titanium surfaces more hydrophilic, it seems to neutralize the bioactive TiO2-coating and decrease cell attachment to TiO2-coated surface.

Generation of three isogenic human induced pluripotent stem cell lines from normal neonate skin fibroblasts.

Characterized human induced pluripotent stem cell lines are important for basic research. Here, we report the establishment of three isogenic human induced pluripotent stem cell (hiPSC) lines generated from normal neonate male skin fibroblasts. Pluripotency was induced using the integration free Sendai virus reprogramming method. The pluripotency, identity, quality, and safety of the lines were confirmed to establish characterized human induced pluripotent stem cell lines to be used as normal control cell lines in future studies.

Focal adhesion formation of primary human gingival fibroblast on hydrothermally and in-sol-made TiO2 -coated titanium.

Optimal cell adhesion of the gingival fibroblasts to dental implants is important for maintaining good implant integration. The aim of this study was to discover, if the nanoporous TiO2 -coating on titanium alloy substrates is able to increase the cell adhesion of the human gingival fibroblasts (HGF). The study consisted of three differently produced titanium groups: hydrothermally produced TiO2 -coating (HT), novel TiO2 -coating made in sol (SOL), and noncoated control group. Primary HGF cells were initiated from gingival biopsies from patients having a third molar extraction. HGF were cultivated on titanium discs for 2 and 24 h to determine the initial attachment with confocal microscope. The cell spreading and adhesion protein signals were measured. In addition, expression of adhesion proteins vinculin, paxillin, and focal adhesion kinase (FAK) were measured after 3 days of cultivation by using Western Blotting. Higher protein levels of paxillin, vinculin, and FAK were induced on both coated discs compared to noncoated discs. The difference was statistically significant (p < 0.05) concerning expression of paxillin. The cell spreading was significantly larger on SOL discs after 2 and 24 h when comparing to noncoated controls. The confocal microscope analyses revealed significantly higher adhesion protein signals on both HT- and SOL-coated titanium compared to control group. This study showed, that both methods to produce TiO2 -coatings are able to increase HGF adhesion protein expression and cell spreading on titanium surface. Accordingly, the coatings can potentially improve the gingival attachment to titanium implant surfaces.

MASTL is enriched in cancerous and pluripotent stem cells and influences OCT1/OCT4 levels.

MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with ß3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-ß receptor II (TGFBR2), a key modulator of TGF-ß signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.

Epithelial cell attachment and adhesion protein expression on novel in sol TiO2 coated zirconia and titanium alloy surfaces.

An adequate mucosal attachment is important when it comes to preventing peri-implant inflammation. The aim of this study was to compare epithelial cell adhesion and adhesion protein expression on in sol TiO2 -coated and non-coated zirconia and titanium alloy surfaces. Fifty-six zirconia and titanium discs were cut, and half of them were coated with bioactive TiO2 -coating. To study the epithelial cell attachment, human gingival keratinocytes were cultivated on discs for 1, 3, 6, and 24 h. The cell proliferation was detected by cultivating cells for 1, 3, and 7 days. In addition, the levels of adhesion proteins laminin y2, integrin α6, ß4, vinculin, and paxillin were detected with Western Blot method. Furthermore, high-resolution imaging of the actin cytoskeleton and focal adhesion proteins was established. Longer-term cell culture (1-7 days) revealed higher cell numbers on the coated zirconia and titanium discs compared to non-coated discs. The difference was statistically significant (p < .05) after 24 h on coated zirconia and after 3 and 7 days on coated titanium discs compared to non-coated discs. Clear induction in the protein levels of laminin y2 and integrin α6 were detected on both coated samples, meanwhile integrin ß4 were clearly induced on coated titanium alloy. The microscope evaluation showed significantly increased cell spreading on the coated discs. According to this study, the in sol induced TiO2 -coating increases keratinocyte attachment and the expression of adhesion proteins on coated zirconia and titanium in vitro. Consequently, the coating has potential to enhance the mucosal attachment on implant surfaces.

Kinase-Independent Functions of MASTL in Cancer: A New Perspective on MASTL Targeting.

Microtubule-associated serine/threonine kinase-like (MASTL; Greatwall) is a well-characterized kinase, whose catalytic role has been extensively studied in relation to cell-cycle acceleration. Importantly, MASTL has been implicated to play a substantial role in cancer progression and subsequent studies have shown that MASTL is a significant regulator of the cellular actomyosin cytoskeleton. Several kinases have non-catalytic properties, which are essential or even sufficient for their functions. Likewise, MASTL functions have been attributed both to kinase-dependent phosphorylation of downstream substrates, but also to kinase-independent regulation of the actomyosin contractile machinery. In this review, we aimed to highlight the catalytic and non-catalytic roles of MASTL in proliferation, migration, and invasion. Further, we discussed the implications of this dual role for therapeutic design.

MASTL promotes cell contractility and motility through kinase-independent signaling.

Microtubule-associated serine/threonine-protein kinase-like (MASTL) is a mitosis-accelerating kinase with emerging roles in cancer progression. However, possible cell cycle-independent mechanisms behind its oncogenicity remain ambiguous. Here, we identify MASTL as an activator of cell contractility and MRTF-A/SRF (myocardin-related transcription factor A/serum response factor) signaling. Depletion of MASTL increased cell spreading while reducing contractile actin stress fibers in normal and breast cancer cells and strongly impairing breast cancer cell motility and invasion. Transcriptome and proteome profiling revealed MASTL-regulated genes implicated in cell movement and actomyosin contraction, including Rho guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and MRTF-A target genes tropomyosin 4.2 (TPM4), vinculin (VCL), and nonmuscle myosin IIB (NM-2B, MYH10). Mechanistically, MASTL associated with MRTF-A and increased its nuclear retention and transcriptional activity. Importantly, MASTL kinase activity was not required for regulation of cell spreading or MRTF-A/SRF transcriptional activity. Taken together, we present a previously unknown kinase-independent role for MASTL as a regulator of cell adhesion, contractility, and MRTF-A/SRF activity.

Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin ß5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

SORLA regulates endosomal trafficking and oncogenic fitness of HER2.

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

A Strong Contractile Actin Fence and Large Adhesions Direct Human Pluripotent Colony Morphology and Adhesion.

Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.

RNA Polymerase III Subunit POLR3G Regulates Specific Subsets of PolyA+ and SmallRNA Transcriptomes and Splicing in Human Pluripotent Stem Cells.

POLR3G is expressed at high levels in human pluripotent stem cells (hPSCs) and is required for maintenance of stem cell state through mechanisms not known in detail. To explore how POLR3G regulates stem cell state, we carried out deep-sequencing analysis of polyA+ and smallRNA transcriptomes present in hPSCs and regulated in POLR3G-dependent manner. Our data reveal that POLR3G regulates a specific subset of the hPSC transcriptome, including multiple transcript types, such as protein-coding genes, long intervening non-coding RNAs, microRNAs and small nucleolar RNAs, and affects RNA splicing. The primary function of POLR3G is in the maintenance rather than repression of transcription. The majority of POLR3G polyA+ transcriptome is regulated during differentiation, and the key pluripotency factors bind to the promoters of at least 30% of the POLR3G-regulated transcripts. Among the direct targets of POLR3G, POLG is potentially important in sustaining stem cell status in a POLR3G-dependent manner.

Mature Let-7 miRNAs fine tune expression of LIN28B in pluripotent human embryonic stem cells.

MicroRNAs (miRNA) are central regulators of diverse biological processes and are important in the regulation of stem cell self-renewal. One of the widely studied miRNA-protein regulators is the Lin28-Let-7 pair. In this study, we demonstrate that contrary to the well-established models of mouse ES cells (mESC) and transformed human cancer cells, the pluripotent state of human ES cells (hESC) involves expression of mature Let-7 family miRNAs with concurrent expression of all LIN28 proteins. We show that mature Let-7 miRNAs are regulated during hESC differentiation and have opposite expression profile with LIN28B. Moreover, mature Let-7 miRNAs fine tune the expression levels of LIN28B protein in pluripotent hESCs, whereas silencing of LIN28 proteins have no effect on mature Let-7 levels. These results bring novel information to the highly complex network of human pluripotency and suggest that maintenance of hESC pluripotency differs greatly from the mESCs in regard to LIN28-Let-7 regulation.

Epigenetic Silencing of the Key Antioxidant Enzyme Catalase in Karyotypically Abnormal Human Pluripotent Stem Cells.

Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells, however, the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase, a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues.

The L1TD1 protein interactome reveals the importance of post-transcriptional regulation in human pluripotency.

The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

Integrative genomics and transcriptomics analysis of human embryonic and induced pluripotent stem cells.

BACKGROUND: Human genomic variations, including single nucleotide polymorphisms (SNPs) and copy number variations (CNVs), are associated with several phenotypic traits varying from mild features to hereditary diseases. Several genome-wide studies have reported genomic variants that correlate with gene expression levels in various tissue and cell types. RESULTS: We studied human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) measuring the SNPs and CNVs with Affymetrix SNP 6 microarrays and expression values with Affymetrix Exon microarrays. We computed the linear relationships between SNPs and expression levels of exons, transcripts and genes, and the associations between gene CNVs and gene expression levels. Further, for a few of the resulted genes, the expression value was associated with both CNVs and SNPs. Our results revealed altogether 217 genes and 584 SNPs whose genomic alterations affect the transcriptome in the same cells. We analyzed the enriched pathways and gene ontologies within these groups of genes, and found out that the terms related to alternative splicing and development were enriched. CONCLUSIONS: Our results revealed that in the human pluripotent stem cells, the expression values of several genes, transcripts and exons were affected due to the genomic variation.

Continuous hypoxic culturing of human embryonic stem cells enhances SSEA-3 and MYC levels.

Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state.

Genetic and epigenetic stability of human pluripotent stem cells.

Studies using high-resolution genome-wide approaches have recently reported that genomic and epigenomic alterations frequently accumulate in human pluripotent cells. Detailed characterization of these changes is crucial for understanding the impact of these alterations on self-renewal and proliferation, and particularly on the developmental and malignant potential of the cells. Such knowledge is required for the optimized and safe use of pluripotent cells for therapeutic purposes, such as regenerative cellular therapies using differentiated derivatives of pluripotent cells.In this Review, we summarize the current knowledge of the genomic and epigenomic stability of pluripotent human cells and the implications for stem cell research.

High-throughput karyotyping of human pluripotent stem cells.

Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner.

RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation.

Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied, the role of cytoplasmic regulators is still poorly characterized. Here, we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11, FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore, we demonstrate that OCT4, SOX2, and NANOG all bind to the promoter of L1TD1. Moreover, L1TD1 is highly expressed in seminomas, and depletion of L1TD1 in these cancer cells influences self-renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus, we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness.