Selecting suitable reference genes for qPCR normalization: a comprehensive analysis in MCF-7 breast cancer 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.

An Effect of Culture Media on Epithelial Differentiation Markers in Breast Cancer Cell Lines MCF7, MDA-MB-436 and SkBr3.

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.

Cartilage-derived extracellular matrix extract promotes chondrocytic phenotype in three-dimensional tissue culture.

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.

Role of genetic factors on the effect of additional loading doses and two maintenance doses used to overcome clopidogrel hyporesponsiveness.

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 <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.

Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A.

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 (TGFß2), 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 TGFß2 and on several developmental genes linked to TGFß 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 TGFß2 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 TGFß2 as well as the expression of several TGFß2-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 TGFß2 and other genes involved in vertebrate early cardiovascular morphogenesis.

Transforming growth factor beta2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis.

Vitamin A-deficient (VAD) quail embryos lack the vitamin A-active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFbeta2 is involved in RA-regulated cardiovascular development. In VAD embryos TGFbeta2 mRNA and protein expression are greatly elevated. The expression of TGFbeta receptor II is also elevated in VAD embryos but is normalized by treatment with TGFbeta2-specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFbeta2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFbeta2 protein to normal quail embryos mimics the excessive TGFbeta2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFbeta2 has a major role in the retinoic acid-regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFbeta2 signals during vertebrate heart formation.

Retinoic acid is a negative physiological regulator of N-cadherin during early avian heart morphogenesis.

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.

Embryonic stem cell marker expression pattern in human mesenchymal stem cells derived from bone marrow, adipose tissue, heart and dermis.

Mesenchymal stem cells (MSCs) have been isolated from a variety of human tissues, e.g., bone marrow, adipose tissue, dermis, hair follicles, heart, liver, spleen, dental pulp. Due to their immunomodulatory and regenerative potential MSCs have shown promising results in preclinical and clinical studies for a variety of conditions, such as graft versus host disease (GvHD), Crohn's disease, osteogenesis imperfecta, cartilage damage and myocardial infarction. MSC cultures are composed of heterogeneous cell populations. Complications in defining MSC arise from the fact that different laboratories have employed different tissue sources, extraction, and cultivation methods. Although cell-surface antigens of MSCs have been extensively explored, there is no conclusive evidence that unique stem cells markers are associated with these adult cells. Therefore the aim of this study was to examine expression of embryonic stem cell markers Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4 in adult mesenchymal stem cell populations derived from bone marrow, adipose tissue, dermis and heart. Furthermore, we tested whether human mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions. We found that bone marrow MSCs express embryonic stem cell markers Oct4, Nanog, alkaline phosphatase and SSEA-4, adipose tissue and dermis MSCs express Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4, whereas heart MSCs express Oct4, Nanog, SOX2 and SSEA-4. Our results also indicate that human adult mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions during early passages, as shown by distinct germ layer and embryonic stem cell marker expression patterns. Studies are now needed to determine the functional role of embryonic stem cell markers Oct4, Nanog and SOX2 in adult human MSCs.

Characterization of human skin-derived mesenchymal stem cell proliferation rate in different growth conditions.

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.