Reproducible measurements of human mesenchymal stem cells counting and proliferation in 3D scaffolds for regenerative medicine

Human mesenchymal stem cells are a good candidate to repair and to regenerate tissues for regenerative medicine applications. Their use in combination with 3D scaffolds has been largely studied in vitro to characterize their properties and differentiation potential prior to apply them in vivo. One of the most important clues in vitro is given by their proliferation trend, leading to information about their viability, their wellness, their interaction with scaffolds, etc. In order to measure the proliferation of hMSCs on scaffolds for regenerative medicine, it is important to adopt accurate counting methods in both research and diagnostic studies. This work aims to develop a reproducible method for hMSCs proliferation measurement in 3D cell cultures on coralline scaffolds (Biocoral®). Results demonstrated that: proliferation curves obtained in this work are reproducible at different initial cell densities on several scaffolds cultured with hMSC in long term experiments (3 weeks)

Direct Reprogramming of Adult Human Cardiac Fibroblasts into Induced Cardiomyocytes Using miRcombo

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) through microRNAs (miRNAs) is a new emerging strategy for myocardial regeneration after ischemic heart disease. Previous studies have reported that murine fibroblasts can be directly reprogrammed into iCMs by transient transfection with four miRNAs (miRs-1, 133, 208 and 499 – termed “miRcombo”). While advancement in the knowledge of direct cell reprogramming molecular mechanism is in progress, it is important to investigate if this strategy may be translated to humans. Recently, we demonstrated that miRcombo transfection is able to induce direct reprogramming of adult human cardiac fibroblasts (AHCFs) into iCMs. Although additional studies are needed to achieve iCM maturation, our early findings pave the way toward new therapeutic strategies for cardiac regeneration in humans. This chapter describes methods for inducing direct reprogramming of AHCFs into iCMs through miRcombo transient transfection, showing experiments to perform for assessing iCM generatio

Uncertainty in cell confluency measurements

Pharmaceutical industries have declared their need of metrology in the cellular field, to improve new drugs developing time and costs by high-content screening technologies. Cell viability and proliferation tests largely use confluency of cells on a bi-dimensional (2D) surface as a biological measurand. The confluency is measured from images of 2D surface acquired via microscopy techniques. The plethora of algorithms already in use aims at recognizing objects from images and identifies a threshold to distinguish objects from the background. The reference method is the visual assessment from an operator and any objective uncertainty estimation is not yet available. A method to estimate the image analysis contribution to confluency uncertainty is here proposed. A maximum and a minimum threshold are identified from a visual assessment of the free edge of the cells. An application to a fluorescence microscopy image of 2D of PT-45 cell cultures is reported. Results shows that the method can be a promising solution to associate an uncertainty to cell confluency measurements to enhance reliability and efficiency of high-content screening technologies

Direct cell reprogramming as a new emerging strategy in cardiac regeneration

Myocardial infarction (MI) is the current leading cause of mortality in the industrialised world. It is due to the irreversible death of billions of cardiomyocytes, secondary to a condition of ischemia. This leads to the formation of a stiff fibrotic tissue, mainly populated by cardiac fibroblasts (CFs). Currently, the only available therapy addressing the irreversible loss of functional cardiomyocytes is heart transplantation. Different tissue engineering approaches and cell therapies are under investigation, aimed at recovering myocardial contractility. Main issues in these strategies are the poor grafting and survival ability of implanted cells as well as the limited endogenous regenerative potential of adult heart. A new strategy is now emerging based on direct reprogramming of CFs into induced cardiomyocytes (iCMs) using transcriptional factors and/ or microRNAs (miRNAs) (miR-combo) [2-4]. Proof of concepts results of in vitro and in vivo conversion of mouse CFs into iCMs have been published and in vitro direct reprogramming of human CFs has also been reported [1-3]. However, such strategy is still an immature approach: reprogramming efficiency is low and partially reprogrammed non-beating cardiomyocytes have been generally obtained. Recently, in vitro direct reprogramming efficiency of mouse CFs cultured in 3D fibrin hydrogels using miR-combo has resulted significantly increased compared to 2D culture systems [4]. Based on these preliminary results, in this work we studied the miR-combo mediated reprogramming efficiency of human dermal and cardiac fibroblasts cultured on hydrogel matrices, including fibrin, fibrin/laminin, fibrin/fibronectin and fibrin/cardiac biomatrix [5], by analysing cell morphology, cell viability, change in gene expression (PCR analysis) and presence of markers of trans-differentiation by immunohistochemistry. The 3D biomimetic hydrogels were able to increase reprogramming efficiency respect to 2D culture environment, both at a genetic and protein level, with an enhancement in the expression of cardiac genes and cardiac proteins such as cardiac troponin I and alpha sarcomeric actinin. [1] J.A. Batty et al. Eur. J. Heart Failure 2016; 18: 145 [2] T.M. Jayawardena et al. Circ. Res. 2012; 110: 1465-1473. [3] T.M. Jayawardena et al. Circ. Res. 2015; 116:418-24. [4] Y. Li et al. Scientific Reports 2016; 6: 38815. [5] C. Castaldo et al. Biomed Res Int. 2013; 2013: 352370. ERC-CoG 2017 BIORECAR project is acknowledge

In vitro mechanical stimulation to reproduce the pathological hallmarks of human cardiac fibrosis on a beating chip and predict the efficacy of drugs and advanced therapies

Cardiac fibrosis is one of the main causes of heart failure, significantly contributing to mortality. The discovery and development of effective therapies able to heal fibrotic pathological symptoms thus remain of paramount importance. Micro-physiological systems (MPS) are recently introduced as promising platforms able to accelerate this finding. Here a 3D in vitro model of human cardiac fibrosis, named uScar, is developed by imposing a cyclic mechanical stimulation to human atrial cardiac fibroblasts (AHCFs) cultured in a 3D beating heart-on-chip and exploited to screen drugs and advanced therapeutics. The sole provision of a cyclic 10% uniaxial strain at 1 Hz to the microtissues is sufficient to trigger fibrotic traits, inducing a consistent fibroblast-to-myofibroblast transition and an enhanced expression and production of extracellular matrix (ECM) proteins. Standard of care anti-fibrotic drugs (i.e., Pirfenidone and Tranilast) are confirmed to be efficient in preventing the onset of fibrotic traits in uScar. Conversely, the mechanical stimulation applied to the microtissues limit the ability of a miRNA therapy to directly reprogram fibroblasts into cardiomyocytes (CMs), despite its proved efficacy in 2D models. Such results demonstrate the importance of incorporating in vivo-like stimulations to generate more representative 3D in vitro models able to predict the efficacy of therapies in patients