Modeling Hypertrophic Cardiomyopathy with Human Induced Pluripotent Stem Cells

Research of genetic cardiovascular diseases has lacked of good disease models because rodents, which are primarily used, differ greatly from humans. The ability to derive human induced pluripotent stem cells (hiPSCs) from patients carrying inherited cardiac diseases has revolutionized research in the cardiovascular field. The aim for this chapter is to review the current hiPSC reprogramming methods and methods for differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes. The chapter focuses on the published hiPSC models for hypertrophic cardiomyopathy (HCM) and discusses the challenges related to modeling this interesting disease using hiPSC technology

CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels

CytoSpectre user guide. This file contains the user guide of the software. (PDF 464 kb

Effects of cardioactive drugs on human induced pluripotent stem cell derived long QT syndrome cardiomyocytes

Human induced pluripotent stem cells (hiPSC) have enabled a major step forward in pathophysiologic studies of inherited diseases and may also prove to be valuable in in vitro drug testing. Long QT syndrome (LQTS), characterized by prolonged cardiac repolarization and risk of sudden death, may be inherited or result from adverse drug effects. Using a microelectrode array platform, we investigated the effects of six different drugs on the electrophysiological characteristics of human embryonic stem cell-derived cardiomyocytes as well as hiPSC-derived cardiomyocytes from control subjects and from patients with type 1 (LQT1) and type 2 (LQT2) of LQTS. At baseline the repolarization time was significantly longer in LQTS cells compared to controls. Isoprenaline increased the beating rate of all cell lines by 10-73 % but did not show any arrhythmic effects in any cell type. Different QT-interval prolonging drugs caused prolongation of cardiac repolarization by 3-13 % (cisapride), 10-20 % (erythromycin), 8-23 % (sotalol), 16-42 % (quinidine) and 12-27 % (E-4031), but we did not find any systematic differences in sensitivity between the control, LQT1 and LQT2 cell lines. Sotalol, quinidine and E-4031 also caused arrhythmic beats and beating arrests in some cases. In summary, the drug effects on these patient-specific cardiomyocytes appear to recapitulate clinical observations and provide further evidence that these cells can be applied for in vitro drug testing to probe their vulnerability to arrhythmia.Peer reviewe

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes

The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases; it is primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. This study aimed at assessing the issue of modelling the rare Finnish founder mutation in cardiomyocytes (CMs) differentiated from iPSCs; therefore, presenting the same cardiac abnormalities observed in the patients. To explore the abnormal functions in JPH2-HCM, skin fibroblasts from a Finnish patient with JPH2 p.(Thr161Lys) were reprogrammed into iPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. Finally, iPSC-CMs were evaluated for the morphological and functional characteristics associated with JPH2 mutation. JPH2-hiPSC-CMs displayed key HCM hallmarks (cellular hypertrophy, multi-nucleation, sarcomeric disarray). Moreover, JPH2-hiPSC-CMs exhibit a higher degree of arrhythmia and longer action potential duration associated with slower inactivation of calcium channels. Functional evaluation supported clinical observations, with differences in beating characteristics when compared with isogenic-hiPSC-CMs. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as a translationally relevant platform to study genetic cardiac diseases.publishedVersionPeer reviewe

Erittäin monikykyisten kantasolujen erilaistus sydänlihassoluiksi ja tautimallin kehittäminen hypertrofiseen kardiomyopatiaan

Ihmisen alkion kantasolut (hESC-solut) ja indusoidut kantasolut (hiPSC-solut) ovat erittäin monikykyisiä eli pluripotentteja kantasoluja. Pluripotentit kantasolut (hPSC-solut) pystyvät jakautumaan rajattomasti, minkä lisäksi niitä voidaan erilaistaa halutuksi solutyypiksi laboratorio-olosuhteissa. Tällä hetkellä pluripotentteja kantasoluja voidaan jo hyödyntää erilaisten tautien tutkimisessa sekä lääkekehityksessä. Ainutlaatuisten ominaisuuksiensa ansiosta erittäin monikykyisiä kantasoluja voidaan tulevaisuudessa käyttää erilaisista solupuutoksista tai solujen toimintojen häiriöistä johtuvien sairauksien hoitoon. Hypertrofinen kardiomyopatia (HCM) on periytyvä sydänlihassairaus, jossa sydämen kammioiden välinen seinämä paksuuntuu. Kudoksen liikakasvun vuoksi paksuuntunut kammiolihas rentoutuu huonosti, mikä voi aiheuttaa potilaille sydämen vajaatoiminta-oireita sekä pitkälle edettyään rytmihäiriöitä ja jopa äkkikuolemia. Tautiin ei ole olemassa parannusta, ja nykyiset hoitomenetelmät keskittyvät potilaiden oireiden hoitoon. Tyypillisesti taudin aiheuttavat geenimutaatiot sijaitsevat sarkomeeriproteiineja koodaavissa geeneissä. Suomessa kaksi valtamutaatiota kattaa noin 18% HCM-potilaista. Mutaatiot sijaitsevat myosiinia sitovassa proteiinissa (MYBPC3-Gln1061X) tai α-tropomyosiinissa (TPM1-Asp175Asn). Aikaisemmin perinnöllisiä sydänsairauksia on tutkittu joko eläinmallien tai aikuisen yksilön sydämestä eristettyjen solujen avulla. Eläinten sydänlihassolut poikkeavat paljon ihmisen sydänlihassoluista, ja aikuisesta yksilöstä eristettyjen sydänlihassolujen kasvattaminen laboratorio-olosuhteissa on vaativaa. hiPSC-soluteknologian avulla aikuisen yksilön jo täysin erilaistuneet solut voidaan muuttaa takaisin alkion kantasoluja muistuttaviksi pluripotenteiksi soluiksi. hiPSC-soluja voidaan tuottaa esimerkiksi perinnöllisiä sydänsairauksia sairastavien potilaiden ihosoluista. Nämä potilasspesifiset solut sisältävät saman geneettisen informaation kuin potilaan perimä, mukaan lukien sairauden aiheuttavan geenivirheen. Potilasspesifisiä hiPSC-soluja on mahdollista erilaistaa laboratoriossa sydänlihassoluiksi, joiden morfologisia ja toiminnallisia ominaisuuksia voidaan sen jälkeen tutkia tarkemmin. Tämä väitöskirja käsittelee hESC- ja hiPSC-solujen erilaistamista sydänlihassoluiksi sekä solumallin kehittämistä HCM-taudin mallintamista varten. Ensimmäisessä osatyössä hPSC-solulinjojen välillä havaittiin merkitseviä eroja niiden kyvyssä erilaistua sydänlihassoluiksi, minkä lisäksi hiPSC-solujen valmistustavan huomattiin vaikuttavan solujen erilaistumistehokkuuteen. Toisessa osatyössä hPSC-soluja kasvatettiin kolmessa eri kasvatusolosuhteessa, joiden havaittiin vaikuttavan solujen erilaistumispotentiaaliin merkitsevästi. Näin ollen sekä hiPSC-solujen valmistustapa että hPSC-solujen kasvatusmenetelmät tulisi optimoida tulevissa tutkimuksissa. Väitöskirjan kolmannessa osatyössä kehitettiin solumalleja HCM-taudin tutkimusta varten. Suomalaisia valtamutaatioita (TPM1-Asp175Asn tai MYBPC3-Gln1061X) kantavilta potilailta eristettiin fibroblasteja, jotka uudelleenohjelmoitiin hiPSC-soluiksi. Potilaiden hiPSC-soluista erilaistettujen sydänlihassolujen ominaisuuksia verrattiin keskenään sekä kontrollihenkilöiden hiPSC-soluista erilaistettujen sydänlihassolujen ominaisuuksiin. Tutkimuksessa havaittiin merkitseviä eroja sekä HCM-potilaiden ja kontrollihenkilöiden sydänlihassolujen välillä että kahden eri HCM-mutaation sisältävien sydänlihassolujen välillä. Tässä väitöskirjassa kehitettyjä HCM-solumalleja voidaan tulevaisuudessa käyttää tarkempien tautimekanismien tutkimisessa ja lääkekehityksessä.The derivation of human embryonic stem cells (hESCs) from developing embryos, as well as the more recent discovery of human induced pluripotent stem cells (hiPSCs), has revolutionized the research of genetic diseases, also in the cardiovascular field. These cells are collectively called human pluripotent stem cells (hPSCs), and they possess the ability to self-renew and differentiate into all cell types of the human body. Thus, hPSCs represent a potential source of cells for future regenerative medicine applications and in vitro modeling of genetic diseases as well as a platform for drug screening. hPSCs can be cultured under laboratory conditions and differentiated into desired cell types. In vitro culture methods for hPSCs have improved markedly in recent years, from the use of animal-derived feeder cell layers and serum-containing medium to more defined feeder cell-free culture methods, including protein or synthetic attachment matrices and defined cell culture medium compositions. Current cardiac differentiation methods are based on EB (embryoid body) formation, co-culturing hPSCs with mouse endodermal-like cells (END-2), or to more guided monolayer cardiac differentiation applying various growth factors in feeder cell-free cultures. Hypertrophic cardiomyopathy (HCM) is one the most common genetic cardiovascular diseases, with a prevalence of 1:500 in the general population. In HCM, typically the interventricular septum (IVS) is thickened, which may lead to progressive heart failure and sudden cardiac death (SCD). The mutations that lead to HCM are typically located in the genes encoding sarcomeric proteins. In Finland, two founder mutations account for approximately 18% of Finnish HCM cases. These mutations are located either in the myosin-binding protein C (MYBPC3-Gln1061X) or in α-tropomyosin (TPM1-Asp175Asn) genes. Conventionally, cardiomyopathies have been studied using animal models, primarily rodents, or human tissue samples collected from end-stage HCM patients from surgical myectomies. The properties of rodent cardiomyocytes differ greatly from human cardiomyocytes, and cardiomyocytes derived from adults rapidly lose their typical properties, e.g., beating, when cultured in vitro. Using hiPSC technology, we are now able to reprogram patient somatic cells into hiPSCs that contain the same genetic information, including mutations, as the patient. These patient-specific hiPSCs can be differentiated into cardiomyocytes, and their morphological and electrophysiological properties can be studied in vitro. The first two aims of this thesis were to compare the cardiac differentiation efficiencies of various hESC and hiPSC lines and to evaluate the effects of hPSC culture methods on the cardiac differentiation efficiencies of hPSCs. We observed marked variations in the cardiac differentiation potential of individual hPSC lines. Furthermore, hESCs were more efficient in producing cardiomyocytes than hiPSCs. This phenomenon could partially be explained by the use of integrating retroviruses in hiPSC production. Furthermore, culture conditions had a significant effect on the cardiac differentiation potential of hPSCs, revealing the superiority of mouse feeder cell layer-based methods over the tested feeder cell-free culture method. The third aim was to develop cell models for studying HCM in vitro using patient-specific hiPSCs. hiPSC-derived HCM cardiomyocytes exhibited the HCM phenotype but simultaneously revealed significant differences between cardiomyocytes carrying the MYBPC3-Gln1061X or TPM1-Asp175Asn mutations. The hiPSC-derived in vitro models established in this thesis represent a valuable tool to study the pathophysiological mechanisms of HCM, drug screening and potentially optimize the drug treatments in a mutation-specific manner

Establishing and optimizing feeder cell-free culture methods for human embryonic stem cells

Background and Aims: Human embryonic stem cells (hESCs) are pluripotent cells and thus provide a promising cell source for clinical applications of regenerative medicine. Currently hESCs are cultured on fibroblast feeder cell layers, which provide necessary cell-cell interactions for the attachment and soluble factors enabling the undifferentiated growth of hESCs. However, culturing of feeder cells is expensive and laborious. In addition, xeno-products, used in feeder cell and hESC cultures could transmit animal pathogens to hESCs, and cause rejections when transplanted to patients. Therefore there is a need to develop xeno- and feeder cell-free culturing methods for hESCs. The first aim of this research project was to set up and compare two commercial xeno-products containing feeder cell-free culturing methods for hESCs. The second aim was to optimize a novel, defined, serum- and xeno-free Reges medium, developed in Regea, into feeder cell-free conditions. Methods: Regea 06/015 hESC line was cultured in mTeSRTM1 medium on MatrigelTM attachment matrix and in STEMPRO® medium on CELLStartTM attachment matrix. Human ESCs were characterized by the expression of typical genes and proteins for undifferentiated and differentiated hESCs. Gene expressions were analyzed by quantitative real time PCR and protein expressions by immunocytochemistry and fluorescence-activated cell sorter (FACS). The pluripotency of hESCs was studied by the expression of genes from different germ layers in embryoid bodies. Human ESCs were also karyotyped. Total 15 different combinations of Reges media were tested in Reges optimization experiments. Results: Both of the commercial feeder cell-free methods and none of the tested Reges media compositions supported the undifferentiated growth of hESCs. Conclusions: Although both of the commercial feeder cell-free methods supported the undifferentiated growth of hESCs, they both contained xeno-products and thus are not optimal methods for culturing hESCs. However, a functional feeder cell-free method could ease the workload related to the preparation of feeder cells. Despite none of the Reges media supported the undifferentiated growth of hESCs, the results provided valuable information and showed a direction to further studies

Establishing and optimizing feeder cell-free culture methods for human embryonic stem cells

Background and Aims: Human embryonic stem cells (hESCs) are pluripotent cells and thus provide a promising cell source for clinical applications of regenerative medicine. Currently hESCs are cultured on fibroblast feeder cell layers, which provide necessary cell-cell interactions for the attachment and soluble factors enabling the undifferentiated growth of hESCs. However, culturing of feeder cells is expensive and laborious. In addition, xeno-products, used in feeder cell and hESC cultures could transmit animal pathogens to hESCs, and cause rejections when transplanted to patients. Therefore there is a need to develop xeno- and feeder cell-free culturing methods for hESCs. The first aim of this research project was to set up and compare two commercial xeno-products containing feeder cell-free culturing methods for hESCs. The second aim was to optimize a novel, defined, serum- and xeno-free Reges medium, developed in Regea, into feeder cell-free conditions. Methods: Regea 06/015 hESC line was cultured in mTeSRTM1 medium on MatrigelTM attachment matrix and in STEMPRO® medium on CELLStartTM attachment matrix. Human ESCs were characterized by the expression of typical genes and proteins for undifferentiated and differentiated hESCs. Gene expressions were analyzed by quantitative real time PCR and protein expressions by immunocytochemistry and fluorescence-activated cell sorter (FACS). The pluripotency of hESCs was studied by the expression of genes from different germ layers in embryoid bodies. Human ESCs were also karyotyped. Total 15 different combinations of Reges media were tested in Reges optimization experiments. Results: Both of the commercial feeder cell-free methods and none of the tested Reges media compositions supported the undifferentiated growth of hESCs. Conclusions: Although both of the commercial feeder cell-free methods supported the undifferentiated growth of hESCs, they both contained xeno-products and thus are not optimal methods for culturing hESCs. However, a functional feeder cell-free method could ease the workload related to the preparation of feeder cells. Despite none of the Reges media supported the undifferentiated growth of hESCs, the results provided valuable information and showed a direction to further studies