Determination of Appropriate Stage of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes for Drug Screening and Pharmacological Evaluation In Vitro

Abstract

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) at different stages (approximate days 30, 60, and 90) were used to determine the appropriate stage for functional and morphological assessment of drug effects in vitro. The hiPS-CMs had spontaneous beating activity, and β-adrenergic function was comparable in all stages of differentiation. Microelectrode array analyses using ion channel blockers indicated that the electrophysiological properties of these ion channels were comparable at all differentiation stages. Ultrastructural analysis using electron microscopy showed that myofibrillar structures at days 60 and 90 were similarly distributed and more mature than that at day 30. Analysis of motion vectors in contracting cells showed that the velocity of contraction was the highest at day 90 and was the most mature among the three stages. Gene expression analysis demonstrated that expression of some genes related to myofilament and sarcoplasmic reticulum increased with maturation of morphological and contractile properties. In conclusion, day 30 cardiomyocytes are useful for basic screening such as the assessment of electrophysiological properties, and days 60 and 90 are the appropriate differentiation stage for morphological assays. For the assay of contractile function associated with subcellular components such as sarcoplasmic reticulum, day 90 cardiomyocytes are the most suitable.

Measuring the Contractile Forces of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Arrays of Microposts

“…Furthermore, we found that the average beat frequency of hiPSC-CMs on microposts coated with all three ECM proteins was approximately equal, and only varied from 0.9 to 1.1 Hz. These findings agree with previous measurements of the spontaneous beating rate for early-stage iPSC-derived cardiomyocytes, but these values are slightly higher than the spontaneous beating rate for adult cells [27,63,69]. However, these results should be confirmed with paced cells to assess the effect of pacing on cytoskeletal organization and maturation, as well as on the contractile properties of these cells.…”

“…The average twitch velocity of early-stage (20-40 days postdifferentiation) hiPSC-derived cardiomyocytes has previously been measured to be approximately 2 lm/s, based on optical edge detection, while the twitch velocity of midstage (40-60 days postdifferentiation) cells was approximately 3 lm/s [69]. Alternatively, using traction force microcopy, the average twitch velocity of midstage hESC-derived cardiomyocytes on a 4 kPa gel was measured to be approximately 6.9 lm/s [62].…”

Mechanical Stress Conditioning and Electrical Stimulation Promote Contractility and Force Maturation of Induced Pluripotent Stem Cell-Derived Human Cardiac Tissue

“…For example, Shinozawa et al used in vitro aging to show that, while day-30 cardiomyocytes already demonstrate basic electrophysiological properties, day-60 and -90 cardiomyocytes have more mature morphological and functional traits. 36 On the other hand, 3D topology has been shown to influence cell morphology, cellular junctions, and myofibril protein expression. 37 …”

Tissue engineering the cardiac microenvironment: Multicellular microphysiological systems for drug screening

“…The electrophysiological properties and force of contraction vary significantly from adult cardiomyocytes, raising questions on the validity of the drug response observed using hPSC-CM [12,25]. While a 2D monolayer of hPSC-CM expresses most of the ion channels that are of interest for drug screening [15], hPSC-CM change ion channel expression levels and develop subcellular structures such as sarcoplasmic reticulum over long culture periods (~100 days) [26–28]. The concern over hPSC-CM immaturity has further contributed to the development of microphysiological systems, as many strategies to mature hPSC-CM involve mimicking the native cardiac microenvironment, as recently reviewed [29].…”