MicroRNA-Mediated Direct Reprogramming of Human Adult Fibroblasts Toward Cardiac Phenotype

open6Modulation of microRNA expression holds the promise to achieve direct reprogramming of fibroblasts into cardiomyocyte-like cells as a new strategy for myocardial regeneration after ischemic heart disease. Previous reports have shown that murine fibroblasts can be directly reprogrammed into induced cardiomyocytes (iCMs) by transient transfection with four microRNA mimics (miR-1, 133, 208, and 499, termed "miRcombo"). Hence, study on the effect of miRcombo transfection on adult human cardiac fibroblasts (AHCFs) deserves attention in the perspective of a future clinical translation of the approach. In this brief report, we studied for the first time whether miRcombo transient transfection of AHCFs by non-viral vectors might trigger direct reprogramming of AHCFs into cardiomyocyte-like cells. Initially, efficient miRNA delivery to cells was demonstrated through the use of a commercially available transfection agent (DharmaFECT1). Transient transfection of AHCFs with miRcombo was found to upregulate early cardiac transcription factors after 7 days post-transfection and cardiomyocyte specific marker cTnT after 15 days post-transfection, and to downregulate the expression of fibroblast markers at 15 days post-transfection. The percentage of cTnT-positive cells after 15 days from miRcombo transfection was ∼11%, as evaluated by flow cytometry. Furthermore, a relevant percentage of miRcombo-transfected AHCFs (∼38%) displayed spontaneous calcium transients at 30 days post-transfection. Results evidenced the role of miRcombo transfection on triggering the trans differentiation of AHCFs into iCMs. Although further investigations are needed to achieve iCM maturation, early findings from this study pave the way toward new advanced therapies for human cardiac regeneration.openPaoletti C; Divieto C; Tarricone G; Di Meglio F; Nurzynska D; Chiono VPaoletti, C; Divieto, C; Tarricone, G; Di Meglio, F; Nurzynska, D; Chiono,

uncertainty analysis of cell counting by metabolic assays

Cell counting is a fundamental procedure in living cell culture-based experiments and protocols in which the cell number quantification is required. The number of cells is one of the parameters necessary to investigate several cell culture features requiring to be monitored as function of time, such as cell viability, proliferation, growth, fitness and metabolism. Aim of this paper is contributing to declare a comprehensive uncertainty budget for cell counting through metabolic assays according to the EURACHEM/CITAC Guide Quantifying Uncertainty in Analytical Measurement

Diagnostic uncertainty in Alzheimer's disease : A bio-metrology perspective addressed to microRNA-based biomarkers

Uncertainties affecting diagnostic tests for Alzheimer's disease (AD) are discussed from a bio-metrological point of view. The estimated sensitivity and specificity of a test are analyzed in a numerical example as predictors of its performance in real cases application. Current trends in molecular diagnostic research are outlined in view of reducing the effects of uncertainty factors involved in AD diagnosis

Cardiac Tissue-like 3D Microenvironment Enhances Route towards Human Fibroblast Direct Reprogramming into Induced Cardiomyocytes by microRNAs

The restoration of cardiac functionality after myocardial infarction represents a major clinical challenge. Recently, we found that transient transfection with microRNA combination (miRcombo: miR-1, miR-133, miR-208 and 499) is able to trigger direct reprogramming of adult human cardiac fibroblasts (AHCFs) into induced cardiomyocytes (iCMs) in vitro. However, achieving efficient direct reprogramming still remains a challenge. The aim of this study was to investigate the influence of cardiac tissue-like biochemical and biophysical stimuli on direct reprogramming efficiency. Biomatrix (BM), a cardiac-like extracellular matrix (ECM), was produced by in vitro culture of AHCFs for 21 days, followed by decellularization. In a set of experiments, AHCFs were transfected with miRcombo and then cultured for 2 weeks on the surface of uncoated and BM-coated polystyrene (PS) dishes and fibrin hydrogels (2D hydrogel) or embedded into 3D fibrin hydrogels (3D hydrogel). Cell culturing on BM-coated PS dishes and in 3D hydrogels significantly improved direct reprogramming outcomes. Biochemical and biophysical cues were then combined in 3D fibrin hydrogels containing BM (3D BM hydrogel), resulting in a synergistic effect, triggering increased CM gene and cardiac troponin T expression in miRcombo-transfected AHCFs. Hence, biomimetic 3D culture environments may improve direct reprogramming of miRcombo-transfected AHCFs into iCMs, deserving further study

MicroRNA-Mediated Direct Reprogramming of Human Adult Fibroblasts Toward Cardiac Phenotype

Modulation of microRNA expression holds the promise to achieve direct reprogramming of fibroblasts into cardiomyocyte-like cells as a new strategy for myocardial regeneration after ischemic heart disease. Previous reports have shown that murine fibroblasts can be directly reprogrammed into induced cardiomyocytes (iCMs) by transient transfection with four microRNA mimics (miR-1, 133, 208, and 499, termed “miRcombo”). Hence, study on the effect of miRcombo transfection on adult human cardiac fibroblasts (AHCFs) deserves attention in the perspective of a future clinical translation of the approach. In this brief report, we studied for the first time whether miRcombo transient transfection of AHCFs by non-viral vectors might trigger direct reprogramming of AHCFs into cardiomyocyte-like cells. Initially, efficient miRNA delivery to cells was demonstrated through the use of a commercially available transfection agent (DharmaFECT1). Transient transfection of AHCFs with miRcombo was found to upregulate early cardiac transcription factors after 7 days post-transfection and cardiomyocyte specific marker cTnT after 15 days post-transfection, and to downregulate the expression of fibroblast markers at 15 days post-transfection. The percentage of cTnT-positive cells after 15 days from miRcombo transfection was ∼11%, as evaluated by flow cytometry. Furthermore, a relevant percentage of miRcombo-transfected AHCFs (∼38%) displayed spontaneous calcium transients at 30 days post-transfection. Results evidenced the role of miRcombo transfection on triggering the trans differentiation of AHCFs into iCMs. Although further investigations are needed to achieve iCM maturation, early findings from this study pave the way toward new advanced therapies for human cardiac regeneration

Cell elasticity change as a biomarker for cancer detection: An AFM metrological approach

The cancer diagnosis relies on morphological examination of human cells or tissue samples: This is considered the 'gold standard' for the diagnosis of malignancy in any organ system. However, diagnosis based on morphological examination can be difficult, recent studies recognized the cell elasticity changes as a marker for cancer detection. A metrological characterized AFM can be used to make reproducible elasticity measurements on biological samples. In this paper a method to accurately measure a cell elasticity change based on measurement of a stable and homogeneous material is proposed. Nanoindenter characteristics (nanoindenter with pyramidal tip and triangular shape) and indentation parameters (0.1 V as approaching point, 5 um/s as indentation speed and indentation load of 0.4 V) were found feasible for all tested samples. A modal cell elastic modulus of 0.5 kPa for lung tumor cell and a limit AFM measurement reproducibility of 4% was obtained