Cardiomyocyte differentiation from human pluripotent stem cells

Kantasolutekniikan odotetaan luovan uusia hoitomuotoja vaikeisiin, kudostuhosta johtuviin sairauksiin, kuten sydänsairauksiin. Aikuisen ihmisen sydämen uusiutumiskyky on hyvin rajallinen, eikä sydän pysty korjautumaan itsestään. Tästä syystä esimerkiksi sydäninfarktin jälkeen sydämeen jää vaurioituneelle alueelle sen toimintaa heikentävä arpi. Ihmisen alkion kantasolut ja ihmisen uudelleenohjelmoidut kantasolut (iPS- solut) ovat pluripotentteja eli erittäin monikykyisiä kantasoluja, jotka pystyvät erilaistumaan periaatteessa kaikiksi kehon soluiksi. Näitä soluja voidaan monin eri menetelmin erilaistaa myös sydänlihassoluiksi eli kardiomyosyyteiksi. Erilaistettuja sydänlihassoluja ei pystytä vielä kliinisesti hyödyntämään, mutta niiden voidaan ajatella toimivan jo varsin pian solu- tai kudosmallina sydämen kehityksen ja toiminnan tutkimisessa sekä lääkekehityksessä. Ihmisen sydänlihassolumalli tehostaisi uusien lääkeainekandidaattimolekyylien turvallisuustestausta ja vähentäisi koe-eläinmallien tarvetta. Ihmisen iPS-solujen käyttö mahdollistaa potilasspesifisten kantasolulinjojen ja edelleen potilasspesifisten sydänlihassolujen aikaansaamisen. Täten vaikeiden geneettisten sydänsairauksien mallintaminen solutasolla on tulevaisuudessa mahdollista. Väitöskirjatutkimuksessani kuvataan sydänlihassolujen erilaistaminen sekä ihmisalkion kantasoluista että ihmisen iPS-soluista. Tutkimuksessani verrattiin eri kantasolulinjojen erilaistumiskykyä sekä eri kantasoluviljelyssä käytettävien tukisolutyyppien vaikutusta erilaistumiseen. Sydänerilaistumisen lisäksi ihmisalkion kantasolujen spontaania erilaistumista tutkittiin myös yleisemmällä tasolla. Tämän lisäksi erilaistetut sykkivät sydänlihassolut karakterisoitiin molekyylibiologisin ja elektrofysiologisin menetelmin. Tämän väitöskirjatutkimuksen tulokset osoittavat, että pluripotentit kantasolut erilaistuvat toiminnallisiksi sydänlihassoluiksi. Erilaistuneet solut sykkivät spontaanisti ja ilmentävät kardiomyosyyteille spesifisiä geenejä ja proteiineja. Erilaistumistehokkuus on kuitenkin matala ja tehokkuus vaihtelee eri kantasolulinjojen välillä. Erilaistetut sydänlihassolut ovat pääosin kammioperäisiä sydänlihassoluja, mutta erilaistunut solupopulaatio sisältää myös eteisperäisiä ja johtoratajärjestelmän solutyyppejä. Tämän lisäksi erilaistettujen sydänlihassolujen kypsyysaste vaihtelee, mutta osa soluista muistuttaa elektrofysiologisilta ominaisuuksiltaan aikuisen ihmisen sydänlihassoluja. Tutkimustulokset siis osoittavat, että toiminnallisten sydänlihassolujen tuottaminen on mahdollista, mutta kliinisten soluhoitojen toteutumiseksi tarvitaan vielä paljon lisätutkimuksia.The rapid development of stem cell technology has raised hopes for new and even revolutionary treatments for cardiac and other disorders with tissue damage. The adult human heart has very limited capability to regenerate and undergo extensive repair which is needed, for example, after myocardial infarction. Pluripotent stem cells, human embryonic stem cells (hESC) and human induced pluripotent stem (iPS) cells can be differentiated into cardiomyocytes by multiple methods. In spite of this development, therapeutic use of stem cell-derived cardiomyocytes is in its infancy. However, functional cardiomyocytes can be differentiated from stem cells and they are themselves very useful as a cardiac cell model. Development of human iPS technology has raised the hope for the potential use of differentiated cardiomyocytes even further. By this method, patient specific stem cell lines can be derived and therefore disease models for genetic illnesses can be obtained. The present thesis describes the differentiation of cardiomyocytes from pluripotent stem cells. The differentiation potential of several hESC lines and iPS cells was evaluated and the differentiated cells were characterized. Furthermore, the differentiation potential of hESC and iPS cells cultured on mouse and human feeder cells was monitored. Differentiation was performed by two differentiation methods, spontaneously in embroid bodies (EBs) and in co-culture with mouse visceral-endoderm-like cells (END-2 cells). In addition to the cardiac aspect, the formation of EBs and the differentiation of germ layers were evaluated in general. Differentiated cells were characterized by multiple molecular biology methods and their electrophysiological properties were also determined. Pluripotent stem cells can be differentiated into functional cardiomyocytes even though the differentiation efficiency is low and cell lines differ in their cardiac differentiation potential. The differentiated cells beat spontaneously and expressed specific cardiac markers. The populations of the differentiated cardiomyocytes were heterogenous, containing mainly ventricular cardiomyocytes with varying maturation states. However, some of the differentiated cells had relatively mature characteristics, resembling adult human cardiac phenotype

Cardiac Differentiation of Pluripotent Stem Cells

The ability of human pluripotent stem cells to differentiate towards the cardiac lineage has attracted significant interest, initially with a strong focus on regenerative medicine. The ultimate goal to repair the heart by cardiomyocyte replacement has, however, proven challenging. Human cardiac differentiation has been difficult to control, but methods are improving, and the process, to a certain extent, can be manipulated and directed. The stem cell-derived cardiomyocytes described to date exhibit rather immature functional and structural characteristics compared to adult cardiomyocytes. Thus, a future challenge will be to develop strategies to reach a higher degree of cardiomyocyte maturation in vitro, to isolate cardiomyocytes from the heterogeneous pool of differentiating cells, as well as to guide the differentiation into the desired subtype, that is, ventricular, atrial, and pacemaker cells. In this paper, we will discuss the strategies for the generation of cardiomyocytes from pluripotent stem cells and their characteristics, as well as highlight some applications for the cells

Sex differences in heart : from basics to clinics

Sex differences exist in the structure and function of human heart. The patterns of ventricular repolarization in normal electrocardiograms (ECG) differ in men and women: men ECG pattern displays higher T-wave amplitude and increased ST angle. Generally, women have longer QT duration because of reduced repolarization reserve, and thus, women are more susceptible for the occurrence of torsades de pointes associated with drugs prolonging ventricular repolarization. Sex differences are also observed in the prevalence, penetrance and symptom severity, and also in the prognosis of cardiovascular disease. Generally, women live longer, have less clinical symptoms of cardiac diseases, and later onset of symptoms than men. Sex hormones also play an important role in regulating ventricular repolarization, suggesting that hormones directly influence various cellular functions and adrenergic regulation. From the clinical perspective, sex-based differences in heart physiology are widely recognized, but in daily practice, cardiac diseases are often underdiagnosed and untreated in the women. The underlying mechanisms of sex differences are, however, poorly understood. Here, we summarize sex-dependent differences in normal cardiac physiology, role of sex hormones, and differences in drug responses. Furthermore, we also discuss the importance of human induced pluripotent stem cell-derived cardiomyocytes in further understanding the mechanism of differences in women and men.publishedVersionPeer reviewe

Arrhythmia mechanisms in human induced pluripotent stem cell-derived cardiomyocytes

Despite major efforts by clinicians and researchers, cardiac arrhythmia remains a leading cause of morbidity and mortality in the world. Experimental work has relied on combining high-throughput strategies with standard molecular and electrophysiological studies, which are, to a great extent, based on the use of animal models. As this poses major challenges for translation, the progress in the development of novel antiarrhythmic agents and clinical care has been mostly disappointing. Recently, the advent of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has opened new avenues for both basic cardiac research and drug discovery: now there is an unlimited source of CMs of human origin, both from healthy individuals and patients with cardiac diseases. Understanding arrhythmic mechanisms is one the main use-cases of hiPSC-CMs, in addition to pharmacological cardiotoxicity and efficacy testing, in vitro disease modeling, developing patient-specific models and personalized drugs, and regenerative medicine. Here, we review the advances that the hiPSC-based modeling systems have brought so far regarding the understanding of both arrhythmogenic triggers and substrates, while also briefly speculating about the possibilities in the future.publishedVersionPeer reviewe

Modeling of LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on β-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca2+ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease

Building blocks of microphysiological system to model physiology and pathophysiology of human heart

Microphysiological systems (MPS) are drawing increasing interest from academia and from biomedical industry due to their improved capability to capture human physiology. MPS offer an advanced in vitro platform that can be used to study human organ and tissue level functions in health and in diseased states more accurately than traditional single cell cultures or even animal models. Key features in MPS include microenvironmental control and monitoring as well as high biological complexity of the target tissue. To reach these qualities, cross-disciplinary collaboration from multiple fields of science is required to build MPS. Here, we review different areas of expertise and describe essential building blocks of heart MPS including relevant cardiac cell types, supporting matrix, mechanical stimulation, functional measurements, and computational modelling. The review presents current methods in cardiac MPS and provides insights for future MPS development with improved recapitulation of human physiology.Peer reviewe