Development of optimal media for cardiovascular models

The concentration of serum in the medium had a significant impact on the functionality and structure of cardiovascular models. In both cardiovascular models, a universal medium was developed to primarily support CM functionality and maturation and secondarily to induce adequate vascular-like network formation. CM cultured in medium with low serum concentrations (0-5 %) have been shown to maintain the cell number, phenotype and contractile properties (Li, 2002). Our results showed that optimal medium for rat cardiovascular model was serum-free medium with angiogenic growth factors VEGF and FGF-2. Since serum was not needed to support NRC contractility, our results suggest that in vitro models with different primary cell types can be maintained in serum-free conditions that supports the functionality of the cells. This might be due to beneficial microenvironment similar to native tissue. Moreover, although FGF-2 have been shown to increase the

expression of dedifferentiation markers, such as atrial natriuretic factor, α-smooth muscle-actin and myofibrillar organization, in adult rat cardiomyocytes (Montessuit et al., 2004) we detected more mature phenotype of NRC in the presence of vascular-like network compared to NCR in monoculture.

In human cardiovascular model, VEGF and FGF-2 were excluded from the medium due to unknown effects on hPSC-CM differentiation and maturation. In addition, 5

% concentration of FBS was needed to maintain physiologically relevant phenotype and growth of the human CM. hPSC-CM have been, however, successfully cultured in serum-free medium at least in monoculture (Stancescu et al., 2015). Hence, further optimization of the medium components might be needed for human cardiovascular model especially when combining vasculogenesis-angiogenesis model with hPSC-CM.

6.2.2 Rat cardiovascular model

The importance of endothelial cells and hASC to the viability of primary CM has been reported earlier (Sadat et al., 2007, Narmoneva et al., 2004). However, the effects of vascular-like network on CM viability and contractility without artificial scaffold was studied here for the first time. Our results showed that the contractility of NRC could be maintained twice as long in culture with vascular-like network compared to NRC monoculture. The increased functionality provides a significant advantage for the model and enables long term use or repeated dose toxicity studies.

We detected that vascular-like network provides mechanical support and inductive growth factors for cardiovascular model thus acting as a natural scaffold. Hence, our results highlight the importance of relevant microenvironment and supportive, essential cell types for the viability and functionality of in vitro models as suggested also by others (Suter-Dick et al., 2015, Knight & Przyborski, 2014). This should be taken into consideration in the development of all in vitro cell- and tissue models including tissue constructs aimed for regenerative medicine. Since the angiogenesis model possessed more stable platform with parallel co-localization of NRC, it was chosen for further development of completely human cell based cardiovascular model.

The microelectrode array (MEA) is a robust, widely used method to analyse the electrophysiological properties of CM. Our results showed that rat cardiovascular model conducted the electrical signal and electrophysiology of the construct was detectable with MEA. This is essential if the model is to be used for screening of cardiotoxic compounds that often affect on the electrophysiological properties of CM. In addition, vascular-like structures in cardiovascular model might assist in the formation of cell-cell contacts and gap junctions between the cardiomyocytes thus enhancing the conduction of electrical signals. Due to critical differences in electrophysiological properties of human and rat cardiomyocytes, especially in the response to rapid delayed rectifier potassium current (Hirt et al., 2014), chemical tests were not performed to rat cardiovascular model. However, proof-of-concept on the cardiovascular model was received.

6.2.3 Human cardiovascular model

Myocardium is composed of cardiomyocytes and non-myocytes embedded in ECM guiding cellular organization and facilitating efficient cell contraction and electrical transmission of the cells (van Spreeuwel et al., 2014). Previous in vitro studies have shown that cell alignment improves CM calcium handling and contractile properties when compared to randomly oriented CM monocultures (Pong et al., 2011, Feinberg et al., 2012). However, since the use of scaffolds in cardiac constructs has been associated with reduced cell-cell contacts as well as incorrect deposition and alignment of extracellular matrix (Norotte et al., 2009), we used vascular-like network to support and orientate CM. Genetic analysis showed that stable tubule structures were formed before seeding of CM thus providing a supportive platform for CM orientation and maturation in human cardiovascular model. In addition, our result demonstrated that seeding of CM to stable vascular-like network orientates the cells mostly parallel but also surrounding the vascular structures thus resembling the architecture of in vivo myocardium.

Although regression of vascular structures after seeding of CM was seen in the absence of VEGF and FGF-2, it did not seem to have effect on the human CM viability or contractility as detected in the MEA and calcium metabolism measurements. These results suggests that cell-cell as well as cell-matrix interactions,

instead of completely developed vascular structures, are critical for CM viability and contractility.

Although immature rodent cardiomyocytes have been used as model systems for decades, mature human cardiomyocytes are likely to reflect the physiology of the adult heart more closely and present more relevant in vitro model system for humans.

In addition, electrical and mechanical properties of hPSC-CM should resemble native myocardium. (Yang et al., 2014) One of the main objective of this thesis was to develop cardiovascular model using hPSC-CM to mimic adult human heart.

However, it is widely acknowledged that hPSC-CM possess fetal-like characteristics that may limit their use as a relevant test systems (Robertson et al., 2013). Long-term culture, 3D tissue engineering, electric stimulation and modulation of substrate stiffness are suggested to contribute to the maturation of hPSC-CM (Yang et al., 2014). Therefore, we were interested to know whether vascular-like network with 3D properties enhances the maturation of hPSC-CM and improves the utilization of hPSC-CM as a models of adult human heart.

We detected that in culture with vascular-like structures hPSC-CM obtained more mature phenotype compared to CM monoculture. Cardiac gene expression analysis confirmed the structural maturation in cardiovascular model. These results suggest a significant improvement in the structural maturation of fetal-like hPSC-CM towards more adult phenotype in co-culture with vascular-like structures. With the orientating and maturating benefits, the present cardiovascular construct provides more advanced in vitro model for adult human cardiotoxicity assessment compared to CM monoculture.

6.2.3.1 Electrophysiological properties of human cardiovascular model (IV)

The measurement of electrophysiological parameters is essential in characterizing critical ion channel properties of cardiomyocytes (Finlayson et al., 2004). In this study, Ca²⁺ imaging and MEA, two critical methods to assess functionality of cardiomyocytes, were used to study the electrophysiological properties and calcium metabolism of human cardiovascular model.

Since several clinically successful drugs may inhibit hERG channel and lead to potentially fatal long QT syndrome (Finlayson et al., 2004), our especial interest was in detecting drug-induced QT prolongation in human cardiovascular model. hERG blocker E-4031 exposure was performed to cardiovascular model to analyze the effects of an antiarrhythmic drug to field potential duration including the QT interval. The MEA measurements showed that E-4031 increased significantly the field potential duration and arrhythmogenicity of the cardiovascular model. Similar effect was detected in CM monoculture as has already been reported (Caspi et al., 2009, Braam et al., 2008). These results indicate that the in vitro cardiovascular model is a responsive test system and that drugs affecting to field potential duration and rhythmicity can be tested with this system.

Although the major ionic currents normally present in adult CM are expressed also in hPSC-CM (Robertson et al., 2013), differences are seen in the number of cardiac ion channel and calcium handling genes (Synnergren et al., 2007). Our genetic analysis showed that in the presence of vascular-like network the expression of transcripts of sodium and calcium channels were increased in the cardiovascular model. This in an important finding since intracellular calcium handling and sarcolemmal ion channels have been shown to play critical role in functional maturation of CM and contribute to their electrical properties (Kim et al., 2010).

These results support the earlier findings and confirm the maturation of CM in human cardiovascular model thus bringing the model one step closer towards adult characteristics.

Since intracellular Ca²⁺ is the key regulator of cardiac contractility, alterations in cardiomyocyte Ca²⁺ regulation may be critical in mechanical dysfunction and in arrhythmia (Bers, 2000). Calcium imaging analysis showed that Ca²⁺ transients were detectable and adrenaline responses evident in the cardiovascular model. As in native heart, adrenaline was shown to increase the diastolic Ca²⁺ level in cardiovascular construct. Interestingly, we detected that adrenaline increased significantly the beating frequency of cardiovascular model when compared to CM monoculture as detected both in MEA and Ca²⁺ cycling measurements. Neighboring non-myocytes could enhance the electrical maturation of CM in cardiovascular construct, especially through sarcolemmal ion channel development, as also suggested by Kim et al.

(2010) (Kim et al., 2010).

These result showing more sensitive response to adrenaline in cardiovascular model compared to CM monoculture was confirmed by genetic analysis. In CM, adrenaline binds to β1-adrenoreceptors and mediates the catecholamine-induced activation of the cells. We detected a higher expression of the transcripts of β1-adrenoreceptors in cardiovascular model suggesting that due to higher number of β1-adrenoreceptors, adrenaline increases the beating frequency more in the model compared to CM monoculture. Since it is crucial to provide the CM the microenvironment that supports their viability, adhesion and structural maturation (Pfannkuche et al., 2010b), the present cardiovascular construct could serve as a more mature and responsive test systems in cardiac safety and efficacy assessment.

However, more extensive characterization with cardiotoxic and antiarrhythmic drugs is needed before entering into validation.