Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories

Abstract

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are being increasingly used to model human heart diseases. hiPSC-CMs generated by earlier aggregation-based methods (i.e., embryoid body) often lack functional sarcoplasmic reticulum (SR) Ca stores characteristic of mature mammalian CMs. Newer monolayer-based cardiac differentiation methods (i.e., Matrigel sandwich or small molecule-based differentiation) produce hiPSC-CMs of high purity and yield, but their Ca handling has not been comprehensively investigated. Here, we studied Ca handling and cytosolic Ca buffering properties of hiPSC-CMs generated independently from multiple hiPSC lines at Stanford University, Vanderbilt University and University of Wisconsin-Madison. hiPSC-CMs were cryopreserved at each university. Frozen aliquots were shipped, recovered from cryopreservation, plated at low density and compared 3-5days after plating with acutely-isolated adult rabbit and mouse ventricular CMs. Although hiPSC-CM cell volume was significantly smaller, cell capacitance to cell volume ratio and cytoplasmic Ca buffering were not different from rabbit-CMs. hiPSC-CMs from all three laboratories exhibited robust L-type Ca currents, twitch Ca transients and caffeine-releasable SR Ca stores comparable to adult CMs. Ca transport by sarcoendoplasmic reticulum Ca ATPase (SERCA) and Na/Ca exchanger (NCX) was similar in all hiPSC-CM lines, but slower compared to rabbit-CMs. However, the relative contribution of SERCA and NCX to Ca transport of hiPSC-CMs was comparable to rabbit-CMs. Ca handling maturity of hiPSC-CMs increased from 15 to 21days post-induction. We conclude that hiPSC-CMs generated independently from multiple iPSC lines using monolayer-based methods can be reproducibly recovered from cryopreservation and exhibit comparable and functional SR Ca handling.

Fig. 1

Surface area and volume of mouse-CM and hiPSC-CMs. A, representative examples of three-dimensional reconstruction of mouse-CM and hiPSC-CMs. B, averaged data. Values are mean±SEM. N=13-34 per group. ***P<0.001 vs mouse-CM.

Fig. 2

Ca handling of hiPSC-CMs and animal CMs. A, confocal images of a mouse-CMs and hiPSC-CMs loaded with low-affinity Ca indicator, Mag-Fluo-4 AM 5 μM to label intracellular Ca stores (i.e. SR). B-C, representative Ca transients (B) and averaged data (C) recorded in hiPSC-CMs and acutely isolated adult ventricular rabbit and mouse CMs. Cells were field-stimulated (0.5 Hz, 20 s train) in 2 mM Ca Tyrode's solution followed by caffeine spritz (10 mM, 5s) to estimate SR Ca content. Values are mean±SEM. N=22-27 per group. *P<0.05 vs. hiPSC-CM. (hiPSC-CM images (A) and example Ca transient records (B) shown are from Vanderbilt hiPSC-CMs).

Fig. 3

Cytoplamic Ca buffering properties of hiPSC-CMs and rabbit-CMs. A, representative cytoplasmic buffering curves generated using the Trafford method by plotting cytosolic [ca]_free against the change in total cytoplasmic Ca concentration (Δ[ca]_total) derived from the NCX current integral during caffeine spritz to release SR Ca in hiPSC-CMs (example trace from Stanford hiPSC-CM) and rabbit-CMs. B, averaged data. Values are mean±SEM. N=9-12 per group.

Fig. 4

L-type Ca currents of hiPSC-CM and mouse-CM. A, C, representative example of Ca currents from hiPSC-CM (Stanford, A) and mouse-CM (B). Voltage protocol shown below current records. B, D current-voltage relationships of peak Ca currents from hiPSC-CM (Stanford, light gray, n=6; Vanderbilt, gray, n=6; Wisconsin, black, n=5) and from mouse CM (n=6), respectively.

Fig. 5

Measurement of NCX and non-NCX Ca efflux. A, hiPSC-CM (example trace from Wisconsin hiPSC-CM) and animal CMs were exposed to Tyrode's solutions containing 10 mM caffeine to measure combined NCX and non-NCX mediated Ca efflux. B, to measure only non-NCX-mediated Ca efflux, CMs were exposed to Tyrode's solution lacking Na and Ca. The Ca transient decay rate (τ) was calculated for each cell and averaged for each group. N= 10-17 per group. *P<0.05 vs. animal-CMs.

Fig. 6

Relative contribution of Ca flux pathways in different CMs. K_SERCA, K_NCX, and K_non-NCX were calculated from rate constants of decay of field-stimulated and caffeine-induced Ca transients (see supplemental table 1). The individual rate constants were normalized by total flux rate (K_twitch), yielding the relative contribution of Ca flux pathways for the three hiPSC-CM lines, rabbit-CMs and mouse-CMs.

Fig. 7

Maturation of Ca handling in hiPSC-CMs. A-B, representative Ca transients (A), and average data (B) of field-stimulated (τ_twitch) and caffeine-induced Ca transients (τ_Caff, τ_Caff0Na) at day 15, 21, and 30 after hiPSC-CM induction. Data are from hiPSC-CMs generated at the University of Wisconsin. C, relative contribution of Ca fluxes calculated as in Fig. 5. N= 13-22 per group. *P<0.05 vs. 15 days.

Fig. 8

Reproducibility of Ca handling parameters in hiPSC-CMs. A, average data of field-stimulated (τ_twitch) and caffeine-induced Ca transients (τ_Caff, τ_Caff0Na) from two independent platings of each hiPSC-CM line. Values are mean±SEM, N= 13-22 per group. B, relative contribution of Ca fluxes calculated as in Fig. 5.