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Normalization of gene expression using internal controls or reference genes (RGs) has been the method of choice for standardizing the technical variations in reverse transcription quantitative polymerase chain reactions (RT-qPCR). Conventionally, ACTB and GAPDH have been used as reference genes despite evidence from literature discouraging their use. Hence, in the present study we identified and investigated novel reference genes in SK-BR-3, an HER2-enriched breast cancer cell line. Transcriptomic data of 82 HER2-E breast cancer samples from TCGA database were analyzed to identify twelve novel genes with stable expression. Additionally, thirteen RGs from the literature were analyzed. The expression variations of the candidate genes were studied over five successive passages (p) in two parallel cultures S1 and S2 and in acute and chronic hypoxia using various algorithms. Finally, the most stable RGs were selected and validated for normalization of the expression of three genes of interest (GOIs) in normoxia and hypoxia. Our results indicate that HSP90AB1, DAD1, PFN1 and PUM1 can be used in any combination of three (triplets) for optimizing intra- and inter-assay gene expression differences in the SK-BR-3 cell line. Additionally, we discourage the use of conventional RGs (ACTB, GAPDH, RPL13A, RNA18S and RNA28S) as internal controls for RT-qPCR in SK-BR-3 cell line.

Background MCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study has not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to postulate an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies. Results The analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference genes while for culture A2, GAPDH-RNA28S were identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-CCSER2-PCBP1 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress. Conclusions The variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-CCSER2-PCBP1 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

Background MCF-7 cell line remains the most extensively studied patient derived model in breast cancer research, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, in the past have identified suitable reference genes in various breast cancer cell lines including MCF-7. However, over the course of identification of stable internal controls, a comparative analysis comprising these genes together in a single study have not been previously undertaken. Further, very little is known about how these identified reference genes are expressed in MCF-7 sub-clones and when the cell line is cultured over multiple passages (p), given the heterogenic expression behavior often associated with MCF-7 cell line. We investigated the expression dynamics of 12 previously reported and suggested endogenous reference genes using RT-qPCR, available algorithms (NormFinder, geNorm, BestKeeper etc.) and TCGA transcriptomic analysis within identically cultured two sub-clones (culture A1 and A2) of MCF-7 cell line cultured over multiple passages. Further candidate reference genes were used to normalize 4 genes of interest (2 simulated and 2 backed by qPCR data) to make an evidence-based recommendation of the least variable reference genes that could be used in MCF-7 cell line. Results The analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as least variable reference gene pair while for culture A2, GAPDH-RNA28S was recommended. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization with 3 genes and analyzing the combined dataset from culture A1 and A2 (supported by transcriptomic analysis), GAPDH-PCBP1-CCSER2 triplet was found to be least variable and hence potentially more well placed to handle any expression heterogeneity that may arise within sub-clones over multiple passages. Conclusions The variance in expression behavior amongst sub-clones shows the need for exercising caution while selecting and using reference genes for MCF-7. Further, using same reference genes amongst sub-clones can lead to misleading results arising from inaccurate normalization. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls in optimizing intra- and inter-assay gene expression differences.

Bone healing can be significantly improved by applying electrical stimuli in the injured region. Thus, electrically active scaffolds with 3D structure are of interest as bone graft substitute materials. Such materials can locally deliver electrical current to the cells in the bone defects and in the same time ensure space for new bone formation. Present study is focused on preparation of novel highly porous and electrically active TiO2-x ceramic scaffolds via polymer replica method. Scaffolds showed fully open and interconnected pore structure with porosity above 95%. Thermal treatment of the scaffolds under high vacuum conditions was realized to obtain nonstoichiometric TiO2-x scaffolds and as a result electrical conductivity significantly increased from ∼10−9 mS/m to ∼40 mS/m. In vitro studies confirmed that scaffolds are cytocompatible and enhances cell spreading. Thus, TiO2-x scaffolds holds a potential to be used in bone tissue regeneration as an electrical stimuli supplier enhancing bone healing process.

To target areas of the skeletal system which are clinically significant sites is a new strategy as the"local treatment of osteoporosis". Synthetic bioceramics implanted into critical sized bone defect of rats with experimental osteoporosis demonstrateds better effect to bone tissue repair in osteoporosis and/or osteoporosis status. Stem cell transplantation may improve bone mineral density in animal models of osteoporosis. An adequate blood supply of mesenchymal cells (MSCs and osteoprogenitors) is important for efficient bone regeneration. The concentration and quality of MSCs may vary significantly, depending on the individual (especially in older people), the cell obtaining sites and techniques used. Combination of BCP and stem cells are not studied on old experimental animals with double induced osteoporosis.

Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines (MCF7, SkBr3 and MDA-MB-436). Materials and methods: Cell lines were conditioned in three studied media (all containing 5% fetal bovine serum (FBS) + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines (MCF7, SkBr3), than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times.

Summary Introduction. Hydroxyapatite (HAp) scaffolds have become an alternative to autologous bone grafts in orthopedic surgery, because it is possible to fill larger scale bone defects and of the decreased operation time and complication risks at the graft donor site, a promising method in bone regeneration is to load bone marrow - mesenchymal stromal cells (BM-MSCs) on to a HAp scaffold. Aim of the Study. The aim of our study is to compare subcutaneous tissue reaction of experimental animals to implantation of HAp and HAp that is coated with BM-MSCs. Materials and Methods. Autologous BM-MSCs were cultivated from crista iliaca dextra biopsy, 4 HAp ceramic implants were coated with BM-MSC and implanted in subcutaneous tissue on one side and non-coated HAp implants on the other side of the spine of 4 rabbits. Immunohistochemical staining of BM-MSC and connective tissue included TUNEL assay, NFkBp105, HSP 70k, Wnt1, TNF-α, VEGF, MMP-2 antibodies, microscoped at 400X magnification and analyzed semiquantitatively. Results. Routine staining with haemotoxilin and eosin demonstrated formation of granulation tissue around the HAp implant, which was more distinct on the control side. There was an increased number of NFκB p105 positive cells (fibroblasts, myocytes, endotheliocytes) on the experimental side (+++), in comparison to the control side (++). HSP70 and apoptosis index was decreased on the experimental side (++ and 60%), in comparison to the control side (+++ and 70%). Number of MMP2 positive structures was increased on the control side in the fibrous capsule (++), in comparison to the experimental side, where the result varied (0 - +) Conclusions. Cultured BM-MSC activity is partly limited due to the apoptosis, which is probably induced by environmental factors. BM-MSC coated HAp implant stimulates secretion of proliferation marker NFkBp105 in subcutaneous tissue, decreases cell stress, apoptosis and tissue degradation (decreased HSP 70, apoptotic cell count and MMP 2) to improve the local tissue quality and proving BM-MSC tissue protective effect

The vitamin A-deficient (VAD) early avian embryo has a grossly abnormal cardiovascular system that is rescued by treating the embryo with the vitamin A-active form, retinoic acid (RA). Here we examine the role of N-cadherin (N-cad) in RA-regulated early cardiovascular morphogenesis. N-cad mRNA and protein are expressed globally in the presomite through HH14 normal and VAD quail embryos. The expression in VAD embryos prior to HH10 is significantly higher than that in normal embryos. Functional analyses of the N-cad overproducing VAD embryos reveal N-cad involvement in the RA-regulated cardiovascular development and suggest that N-cad expression may be mediated by Msx1. We provide evidence that in the early avian embryo, endogenous RA is a negative physiological regulator of N-cad. We hypothesize that a critical endogenous level of N-cad is needed for normal early cardiovascular morphogenesis to occur and that this level is ensured by stage-specific, developmentally regulated RA signaling.

This study investigated conditions for optimal in vitro propagation of human skin-derived mesenchymal stem cells (S-MSC). Forty primary skin-derived precursor cell (SKP) cultures were established from both male and female donors (age 29–65 years) and eight of them were randomly selected for in-depth characterization. Effects of basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), leukemia inhibiting factor (LIF) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) on S-MSC proliferation were investigated. Primary SKP cultures were >95% homogenous for CD90, CD73, and CD105 marker expression enabling to classify these cells as S-MSC. FGF-2 dose-dependent stimulation was observed in low serum medium only, whereas EGF neither stimulated S-MSC proliferation nor potentates the effect of FGF-2. Pronounced donor to donor differences among S-MSC cultures were observed in 3-day proliferation assay. This study demonstrates that homogenous S-MSC populations can be reproducibly isolated from individual donors of different age. Optimal cell culture conditions for in vitro propagation of S-MSC are B27 supplemented or low serum media with FGF-2 (4 ng/ml). EGF and LIF as well as db-cAMP are dispensable for S-MSC proliferation.

Cell transplantation is a promising regenerative therapy for cartilage degeneration. However, obtaining sufficient numbers of cells for this purpose is a challenge, due a lack of autologous donor tissue and the difficulty of culturing chondrocytes in vitro. Tissue engineering strategies that induce or maintain chondrocytic phenotype may solve these problems by (1) broadening the range of available donor tissue, and (2) facilitating the expansion of these cells while controlling phenotypic drift. In this study, bone marrow-derived mesenchymal stem cells (MSCs) and cartilage-derived cells (CDCs) were cultured on composite hydrogels containing agarose and homogenized cartilage extracellular matrix (ECM). MSCs cultured on agarose-ECM scaffolds did not show significant signs of chondrogenic differentiation in the absence of additional cues. However, CDCs cultured on agarose-ECM scaffolds proliferated more rapidly than their ECM-free counterparts and MSCs, while retaining chondrocytic morphology. These results were corroborated via expression of cartilage marker genes: in autologous constructs, SOX 9 expression was upregulated by 12.6 ± 5.3-fold, and COL II was upregulated by 2.0 ± 0.3-fold. Agarose-ECM composite hydrogels are therefore useful for expanding partially differentiated CDCs for applications in regenerative medicine.

DNA methylation plays an important role in the gene-silencing network of higher eukaryotes. We have analyzed the 21.5-kb maintenance methyltransferase (M-MTase) gene, met1, of the multicellular green alga Volvox carteri. The met1 transcript was detected only during the period when DNA replication and cell division are taking place. It encodes a 238 kDa protein containing eight C-terminal activity domains typical of M-MTases, plus upstream DNA-binding domains including the ProDom domain PD003757, which experimental analyses in animal systems have indicated is required for targeting the enzyme to DNA-replication foci. Several insertions of unknown function make Volvox Met1 the largest known member of the Met1/Dnmt1 family. Here we also show that several endogenous transposon families are CpG-methylated in Volvox, which we think causes them to be inactive. This view is supported by the observation that an in vitro CpG-methylated gene introduced into Volvox was maintained in the methylated and silent state over >100 generations. Thus, we believe that Met1 recognizes and perpetuates the in vitro methylation signal, and that the silencing machinery is then able to transduce such a methylation-only signal into a stable heterochromatic (and silent) state.

Background and objective: Additional loading doses and higher maintenance doses (MDs) have been used to overcome hyporesponsiveness of clopidogrel. We aimed to investigate whether genetic polymorphisms of two cytochromes (CYP2C19 and CYP2C9) and ABCB1 modify effect of such dose-adjustment strategy.Materials and methods: We enrolled 118 patients undergoing elective or acute percutaneous coronary intervention (PCI) with drug eluting stent (DES). Platelet reactivity index (PRI) was measured using the vasodilator-stimulated phosphoprotein (VASP) index and a cut-off value of ≥60% was defined as hyporesponsiveness. Polymorphism of two cytochromes (CYP2C19, CYP2C9) and gene ABCB1 were determined. In patients hyporesponsive to the initial LD the dose-adjustment was performed using up to 3 additional 600 mg LDs in order to achieve PRI &lt, 60%, and both 150 mg and 75 mg MD were tested at the follow-up.Results: Patients with at least one CYP2C19*2 allele had higher baseline PRI after the initial LD (78.2 ± 13.1 vs. 65.3 ± 19.5, P = 0.005). The PRI reduction with additional LD was significantly smaller in carriers of the CYP2C19*2 (25.2 ± 15.6 vs. 35.5 ± 16.8, P = 0.025) and similar trend was observed with subsequent additional LDs. Both MDs were less effective in presence of CYP2C19*2. Target PRI was, however, more frequently achieved with higher MD even in presence of CYP2C19*2 (in 70.6% vs. 23.5% of hyporesponders, P = 0.008). No such differences were observed for other polymorphisms.Conclusions: In patients hyporesponsive to a routine clopidogrel doses the potency of additional LD and higher MD of clopidogrel is compromised by presence of CYP2C19*2 allele. The dose-adjustment strategy is not affected by ABCB1 C3435T or CYP2C9 genotypes.

The vitamin A-deficient (VAD) early avian embryo has a grossly abnormal cardiovascular system that is rescued by treating the embryo with the vitamin A-active form, retinoic acid (RA). Here we examine the role of N-cadherin (N-cad) in RA-regulated early cardiovascular morphogenesis. N-cad mRNA and protein are expressed globally in the presomite through HH14 normal and VAD quail embryos. The expression in VAD embryos prior to HH10 is significantly higher than that in normal embryos. Functional analyses of the N-cad overproducing VAD embryos reveal N-cad involvement in the RA-regulated cardiovascular development and suggest that N-cad expression may be mediated by Msx1. We provide evidence that in the early avian embryo, endogenous RA is a negative physiological regulator of N-cad. We hypothesize that a critical endogenous level of N-cad is needed for normal early cardiovascular morphogenesis to occur and that this level is ensured by stage-specific, developmentally regulated RA signaling.

Background: Important knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGFβ2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGFβ2 and on several developmental genes linked to TGFβ signaling during heart morphogenesis. Results: We have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGFβ2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGFβ2 as well as the expression of several TGFβ2-linked developmental genes is regulated by RA. Conclusions: Our findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGFβ2 and other genes involved in vertebrate early cardiovascular morphogenesis.