C. Divieto

C. Paoletti

D. Nurzynska

F. Di Meglio

V. Chiono

English

PORTO@iris (Publications Open Repository TOrino - Politecnico di Torino)

18 October 2022POLITECNICO DI TORINORepository ISTITUZIONALEINFLUENCE OF 3D CULTURE MICROENVIRONMENT ON MICRORNA-MEDIATED DIRECT REPROGRAMMING OFHUMAN CARDIAC FIBROBLASTS INTO CARDIOMYOCYTES / Paoletti, C.; Divieto, C.; Di Meglio, F.; Nurzynska, D.;Chiono, V.. - (2020). ((Intervento presentato al convegno Bioprinting Winter School 2020 - Università degli Studi di Pavia.OriginalINFLUENCE OF 3D CULTURE MICROENVIRONMENT ON MICRORNA-MEDIATED DIRECTREPROGRAMMING OF HUMAN CARDIAC FIBROBLASTS INTO CARDIOMYOCYTESPublisher:PublishedDOI:Terms of use:openAccessPublisher copyright(Article begins on next page)This article is made available under terms and conditions as specified in the  corresponding bibliographic description inthe repositoryAvailability:This version is available at: 11583/2872178 since: 2021-02-22T11:58:20ZBioprinting Winter School 2020 - Università degli Studi di PaviaC. Paoletti1,2, C. Divieto3, F. Di Meglio4, D. Nurzynska4 and V. Chiono1,2POLITECNICO DI TORINO1 Politecnico di Torino, Department of Mechanical and Aerospace Engineering, 10129 Torino, Italy2POLITO Biomedlab, Politecnico di Torino, Torino, Italy3 Istituto Nazionale di Ricerca Metrologica, Advanced Materials Metrology and Life Science, Torino, Italy4 Università di Napoli Federico II, Department of Public Health, 80138 Napoli, Italycamilla.paoletti@polito.itINFLUENCE OF 3D CULTURE MICROENVIRONMENT ON MICRORNA-MEDIATEDDIRECT REPROGRAMMING OF HUMAN CARDIAC FIBROBLASTS INTOCARDIOMYOCYTESIntroduction & Aim of the workMyocardial infarction (MI) remains one of the leading causes of death worldwide. MI results in the loss of up to 1 billion of cardiomyocytes (CMs) and the formation of a fibrotic scar, mainly populated by cardiac fibroblasts,which impair normal cardiac function [1]. Direct reprogramming of human cardiac fibroblasts into CMs may hold a great potential for this purpose. Recently, direct reprogramming of murine fibroblasts into inducedcardiomyocytes (iCMs) using the combination of four different microRNAs (miR-1, 133, 208 and 499), named “miRcombo”, has been demonstrated in 2D monolayer culture. [2]. However, aiming at mimicking the physiologicalmicroenvironment, a more complex culture method is required in order to increase the reprogramming efficiency and maturation of iCMs in vitro. For this reason, 3D hydrogel culture have been shown to better mimic thenative cardiac tissue environment compared to classic tissue culture plate (TCP)[3].In this work, we investigated: miRcombo-mediated reprogramming oh human cardiac fibroblasts into iCMs in classic monolayer culture A 2D and 3D fibrin-based hydrogel role in enhancing the reprogramming efficiency of fibroblasts into iCMsCell culture and transfectionAHCFs (Lonza, CC-2903) were transfected withmiR-1, 133, 208 and 499 (mirVana) usingDharmaFECT1 (Dharmacon). NegmiR (mirVana)transfected AHCFs were used as control. After24 hours, medium was changed to completemedium.Fibroblast culture in fibrin-based hydrogelsAfter transfection, cells were detached by trypsin andcultured on the surface (2D) or embedded into 3D fibrinbased hydrogels composed of 5 mg/mL fibrinogen (Merck)and 50 u/mL trombin (Merck).Digital Droplet PCR (ddPCR)Digital Droplet PCR was performed according to manufacturer’s instructions (BIO-RAD). Results are reported as number of cDNA copies/µL of the gene of interestnormalized on GAPDH number of cDNA copies/µL.Calcium AnalysisCells were loaded with in 5 μM Fluo-4 AM (Invitrogen, codice) at 37 °C for 15 and 30minutes in modified Tyrode solution (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mMMgCl2 and 10mM glucose at pH 7.4) and analysed using Spinning disk microscope(Nikon)Flow CytometryCell were trypsinized and incubated with Cardiac Troponin T (Abcam) and Alexa-488(Abcam) antibodies.«miRcombo»transient transfectionHuman adultcardicac fibroblasts2D hydrogel 3D hydrogelVs.iCMsResults and Future WorkMaterial and MethodsAHCF reprogramming into iCMs on monolayer culturei 2D vs. 3D culture systems Future DevelopmentsImage adapted from Castaldo et al [4]Fig.1A                                                                           Fig.2A  Fig.3A                                                                                  Fig.4A  Fig.1B Fig.2B Fig.3B We first evaluated if the combination of four microRNAs was able to induce theexpression of cardiomyocyte markers in AHCFs cultured in TCP. miRcombo-transfected AHCFs showed increased expression of cardiac transcription factors(TFs) after 7 days (Fig. 1A) and higher TnnT2 expression after 15 days of culturecompared to negmiR controls (Fig.2A).Then, we explored the effects of a fibrin 3D cultureenvironment in enhancing miRcombo-mediatedreprogramming of AHCFs after 15 days of culture. Toelucidate the role of fibrin hydrogel in cell reprogramming,cells were cultured on the surface of fibrin hydrogel (2D) orembedded into 3D fibrin hydrogel (3D). After 2 weeks, 3Dculture environment significantly enhanced mRNA levels of Tnnt2(Fig.1B), Tnni3 (Fig. 2B) and Myh6 (Fig.3B) cardiac markers in bothmiR combo and negmiR transfected AHCFs compared to 2Dhydrogels.References AcknowledgedmentsThis project has received funding from the European ResearchCouncil (ERC) under the European Union’s Horizon 2020 researchand innovation program (grant agreement No 772168).[1] Paoletti C et al. Cells 2018, 7, 114[ 2] Jayawardena TM et al. Circ Res. 2013, 110, 1465[3] Li Y et al. Sci. Rep. 2016, 6, 1[4] Castaldo et al. Biomed Res 2013Conclusions miRcombo transfection triggers the transion of AHCFs towards iCMs a 3D fibrin hydrogel was able to enhance miRcombo-mediated reprogramming of AHCFsinto induced CMs. Further investigations are in progress.Moreover, we evaluated reprograming efficiency using flow cytometry.Interestingly, after 15 days of culture, miRcombo-transfected cells exhibitednearly 11% of cTnT positive cells, whereas no positive cells were found innegmiR controls (Fig. 3A). After 4 weeks of culture, miRcombo transfected cellsalso showed calcium transient activity, while no calcium activity was found innegmiR controls (Fig.4A)http://www.biorecar.polito.itFuture developments will focus onunderstanding miRcombo-mediatedreprogramming in 3D fibrin basedhydrogel.Moreover, in order to recreate a 3Dstructure that resemble the physiologicalmicroenvironment, we will introducecardiac ECM proteins produced in vitro bycardiac fibroblasts.SEM images of a 3D fibrin hydrogel (a) and fibrin hydrogel blended with Biomatrix (b)(a)(b)

INFLUENCE OF 3D CULTURE MICROENVIRONMENT ON MICRORNA-MEDIATED DIRECT REPROGRAMMING OF HUMAN CARDIAC FIBROBLASTS INTO CARDIOMYOCYTES

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INFLUENCE OF 3D CULTURE MICROENVIRONMENT ON MICRORNA-MEDIATED DIRECT REPROGRAMMING OF HUMAN CARDIAC FIBROBLASTS INTO CARDIOMYOCYTES

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