Automatic Optimization of an in Silico Model of Human iPSC Derived Cardiomyocytes Recapitulating Calcium Handling Abnormalities

The growing importance of human induced pluripotent stem cell-derived cardiomyoyctes (hiPSC-CMs), as patient-specific and disease-specific models for studying cellular cardiac electrophysiology or for preliminary cardiotoxicity tests, generated better understanding of hiPSC-CM biophysical mechanisms and great amount of action potential and calcium transient data. In this paper, we propose a new hiPSC-CM in silico model, with particular attention to Ca2+ handling. We used (i) the hiPSC-CM Paci2013 model as starting point, (ii) a new dataset of Ca2+ transient measurements to tune the parameters of the inward and outward Ca2+ fluxes of sarcoplasmic reticulum, and (iii) an automatic parameter optimization to fit action potentials and Ca2+ transients. The Paci2018 model simulates, together with the typical hiPSC-CM spontaneous action potentials, more refined Ca2+ transients and delayed afterdepolarizations-like abnormalities, which the old Paci2013 was not able to predict due to its mathematical formulation. The Paci2018 model was validated against (i) the same current blocking experiments used to validate the Paci2013 model, and (ii) recently published data about effects of different extracellular ionic concentrations. In conclusion, we present a new and more versatile in silico model, which will provide a platform for modeling the effects of drugs or mutations that affect Ca2+ handling in hiPSC-CMs

Comprehensive in vitro Proarrhythmia Assay (CiPA): Pending issues for successful validation and implementation

International audienceIntroduction: The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use.Topics covered: Key talks delivered at the ‘CiPA on my mind’ session, held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS), are summarized. Issues and potential solutions relating to crucial constituents [e.g., biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes), study platforms, drug solutions, and data analysis] of CiPA core assays are critically examined.Discussion: In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy, systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories, it can be implemented as qualified regulatory procedure

Stem Cell Research on Cardiology

Even today, cardiovascular diseases are the main cause of death worldwide, and therapeutic approaches are very restricted. Due to the limited regenerative capabilities of terminally differentiated cardiomyocytes post injury, new strategies to treat cardiac patients are urgently needed. Post myocardial injury, resident fibroblasts begin to generate the extracellular matrix, resulting in fibrosis, and finally, cardiac failure. Recently, preclinical investigations and clinical trials raised hope in stem cell-based approaches, to be an effective therapy option for these diseases. So far, several types of stem cells have been identified to be promising candidates to be applied for treatment: cardiac progenitor cells, bone marrow derived stem cells, embryonic and induced pluripotent stem cells, as well as their descendants. Furthermore, the innovative techniques of direct cardiac reprogramming of cells offered promising options for cardiovascular research, in vitro and in vivo. Hereby, the investigation of underlying and associated mechanisms triggering the therapeutic effects of stem cell application is of particular importance to improve approaches for heart patients. This Special Issue of Cells provides the latest update in the rapidly developing field of regenerative medicine in cardiology

Quantification of Stem Cell Derived Cardiomyocyte Beating Mechanics using Video Microscopy Image Analysis

Until recently, the studying of human cardiac cells had been a difficult and to some extent dangerous task due to the risks involved in cardiac biopsies. Induced pluripotent stem cell technology enables the conversion of human adult cells to stem cells, which can be further differentiated to cardiac cells. These cells have the same genotype as the patient from whom they were derived, allowing the studying of genetic cardiac diseases, as well as the cardiac safety and efficacy screening of pharmaceutical agents using human cardiac cells instead of animal cell models. Using the stem cell derived cardiac cells in these studies, however, requires novel and specialized measurement methods for understanding the functioning of these cells.Long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT) are genetic cardiac diseases, which can induce deadly arrhythmias. The induced pluripotent stem cell derived cardiac cells allow the studying of these diseases in laboratory conditions. A greater understanding of these diseases is important for prevention of sudden cardiac death, more accurate diagnosis, and development of possible treatment options. In order to understand the functioning of these cells, new methods are sought after. Traditionally, the electrical function of these cells are measured. However, the primary function of the cardiac cells is to beat in order to pump blood for circulation. The methods to quantify this mechanical function, the contraction and relaxation movement of cells, has been in lesser focus.The main objective of this work is to develop a measurement method, which allows the in vitro quantification of biomechanics of single human cardiac cells using video microscopy. The method uses digital image correlation to determine movement occurring in cardiac cells during contractile movement. The method is implemented in a software tool, which enables the characterization and parametrization of the cardiomyocyte beating function. The beating function itself can be affected by environmental factors, pharmacological agents and cardiac disease.Here, the quantification of mechanical function is performed using digital image correlation to estimate displacement between subsequent video frames. Velocity vector fields can then be used to calculate signals that characterize the contraction and relaxation movement. We estimate its accuracy in cardiac cell studies using artificial data sets and its feasibility with concurrent electrical measurements. Cardiac diseases are studied by quantifying beating mechanics from Long QT and CPVT specific cell lines. Traditional electrophysiological measurements are used for validation and comparison. The interaction between calcium and contraction is studied with a simultaneous measurement of biomechanics and calcium imaging.This thesis resulted a new and accessible analysis method capable of measuring cardiomyocyte biomechanics. This method was determined to be non-toxic and minimally invasive, and found capable to be automated for high-throughput analysis. Due to not harming the cells, repeated measurements are enabled. Using the method, we observed for the first time abnormal beating phenotypes in two long QT associated mutations in single cardiomyocytes. Further, we demonstrated a concurrent calcium and motion measurement without background corrections. This provided also evidence that this combined analysis could be particularly useful in some cardiac disease cases. The methods and results shown in the thesis represent key early advances in the field.The method was implemented in a software tool, which enabled cell biologists to use it different stages of cardiomyocyte studies. Overall, the results of the thesis represent an accessible method of studying cardiomyocyte biomechanics, which improves the understanding of contraction-calcium coupling and paves way for high-throughput analysis of cardiomyocytes in genetic cardiac disease and pharmacological research.Viime aikoihin asti ihmisen sydämen solujen tutkiminen on ollut vaikeaa ja vaarallista, sillä näytepalojen ottamiseen sydämestä liittyy paljon riskejä. Menetelmä indusoitujen pluripotenttien kantasolujen tuottamiseen sallii aikuisten solujen muuntamisen takaisin kantasolumuotoon, josta ne voidaan vielä erilaistaa sydänsoluiksi. Näillä soluilla on sama geeniperimä kuin potilaalla, josta ne on johdettu. Tämä luo mahdollisuuden tutkia geneettisiä sydänsairauksia, ja sallii lääkeaineiden sydänturvallisuuden ja tehokkuuden tutkimisen käyttäen ihmisen sydänsoluja eläinkokeiden sijaan. Kantasolupohjaisten sydänsolujen käyttäminen näissä tutkimuksissa kuitenkin vaatii uusia ja erityisiä mittausmenetelmiä solujen toiminnan ymmärtämiseksi.Pitkä QT-syndrooma (LQTS) ja katekolamiiniherkkä polymorfinen kammiotakykardia (CPVT) ovat perinnöllisiä sydänsairauksia, jotka voivat aiheuttaa kuolemaan johtavia rytmihäiriöitä. Indusoiduista pluripotenteista kantasoluista johdettujen sydänsolujen avulla voidaan tutkia näitä sairauksia laboratorio-oloissa. Ymmärtämällä paremmin näitä sairauksia voidaan saavuttaa tarkempia diagnooseja ja kehittää mahdollisia uusia hoitomuotoja sydänperäisten äkkikuolemien estämiseksi. Uusia mittausmenetelmiä tarvitaan, jotta näiden solujen toimintaa voidaan tutkia. Näiden solujen toiminnallisuutta on perinteisesti tutkittu mittaamalla niiden sähköistä toimintaa. Sydänsolujen pääasiallinen tehtävä on kuitenkin mekaaninen: pumpata verta sydämestä verenkiertoon. Tätä solujen supistumista ja rentoutumista mittaavia menetelmiä on tutkittu vähemmän.Tämän väitöskirjan päämäärä on kehittää mittausmenetelmä, jolla voidaan määrittää yksittäisten ihmisen sydänsolujen biomekaniikkaa in vitro videomikroskopiaa käyttäen. Menetelmä käyttää digitaalista kuvien korrelaatiota määrittämään sydänsoluissa supistusliikkeen aikana tapahtuvan liikkeen. Menetelmää käytetään ohjelmistotyökalussa, jolla voidaan karakterisoida ja parametrisoida sydänsolun syketoimintaa. Syketoimintaan voi vaikuttaa niin ympäristötekijät, lääkeaineet kuin sydänsairaudetkin.Tässä väitöskirjassa sydänsolujen mekaanista toimintaa mitataan videomikroskopian avulla määrittämällä liikettä videon peräkkäisistä kuvista digitaalista kuvakorrelaatiota käyttäen. Saaduista nopeusvektorikentistä lasketaan supistumista ja rentoutumista kuvaavia signaaleja. Arvoimme sen tarkkuutta sydänsolututkimuksissa käyttäen keinotekoisia tietoaineistoja ja sen soveltuvuutta yhtäaikaisilla sähköisillä mittauksilla. Tutkimme perinnöllisiä sydänsairauksia (LQTS ja CPVT) mittaamalla sykinnän mekaniikkaa yksittäisistä sydänsoluista, jotka ovat johdettu näitä sairauksia kantavien potilaiden kantasolulinjoista. Perinteisiä sähköfysiologisia mittauksia käytetään menetelmän validointiin ja vertailuun. Kalsiumin ja sykinnän vuorovaikutusta tutkitaan yhtäaikaisella biomekaniikan ja kalsiumaineenvaihdunnan mittauksella.Tämän väitöskirjan tuloksena saatiin aikaan uusi ja helposti lähestyttävä menetelmä sydänlihassolujen biomekaniikan tutkimiseen. Menetelmä ei ole soluille haitallinen ja se vaikuttaa solujen toimintaan perinteisiin menetelmiin verrattuna vain vähän. Se on automatisoitavissa suuria näytemääriä varten. Koska se ei vahingoita soluja, mittaukset voidaan myös toistaa samoilla soluilla. Tätä menetelmää käyttäen havaitsimme ensimmäisinä kahdesta eri LQT1-mutaatiota kantavista potilaista johdetuissa yksittäisissä sydänsoluissa poikkeavia sykintätyyppejä. Lisäksi, osoitimme yhtäaikaisen kalsiumin ja liikkeen mittauksen olevan mahdollinen ilman laskennallisia taustan korjauksia ja havaitsimme, että näin yhdistetystä analyysista voi olla erityistä hyötyä sydänsairauksien tutkimisessa. Väitöskirjassa esitetyt menetelmät ja tulokset edustavat alan tärkeitä ensimmäisiä edistysaskelia.Tätä menetelmää käytettiin väitöskirjan ohella tehdyssä ohjelmistotyökalussa, jota voidaan käyttää sydänlihassolujen tutkimuksen eri vaiheissa. Väitöskirjan tuloksena syntynyt helposti lähestyttävä menetelmä sallii sydänlihassolujen biomekaniikan analyysin. Sen avulla voidaan myös ymmärtää paremmin supistusliikkeen ja kalsiumin kytkentää. Kokonaisuutena, väitöskirja luo pohjaa sydänlihassolujen suurten näytemäärien analyysille sydänsairauksien ja lääkeaineiden tutkimuksessa

Fluorescent gene reporters in human pluripotent stem cells : as model for studying human heart development and cardiomyocyte differentiation

It is critical to gain knowledge in the underlying mechanisms that control human cardiovascular developm ent, which helps us to understand the onset of congenital cardiovascular diseases, and to develop optimal culture methods for efficient in vitro cardiomyocyte differentiation from hPSCs, which are of interest for final translational applications including screening and efficacy assays for disease modelling, drug discovery and development, personalized medicine, and perhaps the regeneration of cardiovascular tissues for therapeutic purposes. In this thesis, we show how genetic manipulation of human pluripotent stem cells (hPSCs), resulting in the genomic integration of a fluorescent protein encoding sequence at the locus of a key cardiac transcription factor, allows us to visualize and isolate early pre-cardiac progenitors subpopulations, and to study the molecular mechanisms involved in their further differentiation to cells of the cardiac lineage, including smooth muscle cells, endothelial cells, and cardiomyocyte subtypes.Rembrandt Institute for Cardiovascular Research Nederlandse Hartstichting Stichting ProefdiervrijLUMC / Geneeskund

Bioprocessing of human Pluripotent Stem Cells for cardiac cell therapy and pre-clinical research

Cardiovascular diseases remain the leading cause of death worldwide. Some of these diseases, e.g. myocardial infarction (MI), are associated with a massive and permanent loss of cardiomyocytes (CMs), a non-proliferative and terminally differentiated cell population in the heart. Available pharmacological and interventional therapies are not suitable to amend the effects of this cell loss, mainly due to the limited regenerative capacity of the myocardium, and heart transplantation is limited by the number of compatible organs donated. Recently, human pluripotent stem cells (hPSCs), have emerged as attractive candidate cell sources to obtain CMs. Due to their inherent capacity to proliferate indefinitely and to differentiate into all mature cells of the human body, hPSCs constitute the unique cell source that can provide, ex-vivo, an unlimited number of functional CMs suitable for cell therapy and other applications including disease modeling and cardiotoxicity drug testing. Nonetheless, the complex nets of signaling pathways involved in cardiomyogenesis as well as the line-to-line variability compromise the effectiveness of the existing differentiation protocols to reproducibly produce high-quality CM from multiple hPSC lines. The immature phenotype of the produced hPSC-CMs and the lack of efficient methods for worldwide shipment of these cells also constrain the applicability of these cells in the clinic and industry. The main aim of this thesis was to devise robust, scalable and integrated approaches for the production and maturation of hPSC-CMs. The strategy consisted in exploring the impact of environmental factors, cell culture configuration, and metabolic substrate availability on bioprocess yields and cell’s quality using a set of “-omic” tools, namely transcriptomics, metabolomics and fluxomics, and cell characterization assays. In Chapter 1, the recent advances on the use of hPSC for cardiac cell therapy were reviewed. In Chapter 2, the effect of dissolved oxygen and bioreactor hydrodynamics on CM differentiation was explored. It was demonstrated that combining a hypoxia culture (4% O2 tension) with wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields. Chapter 3 focused on the development and characterization of a robust protocol for directed differentiation of hPSC towards CMs, suitable to generate CMs in both 2D monolayer and 3D aggregate culture formats. The culture of hPSC-cardiac progenitors as 3D aggregates revealed to be an efficient approach to improve CM enrichment and commitment. Although hPSC-CMs generated from both methods revealed to be highly glycolytic, 3D aggregate cultures showed slightly improved metabolic energetics (including increased TCA-cycle activity and ATP production). Chapter 4 assessed whether alterations in hPSC-CM culture medium composition to mimic in vivo substrate availability during cardiac development would induce hPSC-CM maturation in vitro. It was demonstrated that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing media promotes their maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to ventricular CMs, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics, within 10-20 days. The feasibility to cold store hPSC-CMs monolayers and aggregates in a fully-defined clinical compatible formulation was evaluated in Chapter 5. It was demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage–induced cell injury in 3D aggregates than in 2D monolayers, showing high cell recoveries, typical (ultra)structure and functionality after 7 days of storage. Chapter 6 consists of a general discussion, where the main scientific and technological outcomes of this thesis are outlined. Overall, by pursuing an holistic approach that brought together a quantitative molecular and metabolic characterization, this thesis provides novel insights into the interaction of metabolism and CM differentiation/maturation but also establishes robust and scalable methods for production, functional maturation and short-term storage of hPSC-CMs. This will pave the way for the widespread application of hPSC-CMs in clinical and preclinical applications

Cellular models for fundamental and applied biomedical research

Cell culture models play an important role in biomedical research and will continue to do so given the growing opposition to vivisection and the limited predictive value of animal models for human disease. Moreover, cell culture models can be easily established to mimic physiological or pathological processes, which is difficult to accomplish using in silico models. While non-cellular in vitro models are highly suitable for studying simple biochemical processes, cell culture models recapitulate many of the complex regulatory circuits governing protein activity in vivo and hence allow investigation of diverse physiological processes. Also, cell culture models offer the possibility to address fundamental research questions in a much more simplified, specific and controllable manner than can be achieved using in vivo models. CSCLUMC / Geneeskund