Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Rationale: The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery.

Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs.

Methods and results: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa).

Conclusions: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Keywords: excitation contraction coupling; matrigel; myocytes, cardiac; pluripotent stem cells; stem cells.

Figure 1. Overview of Matrigel mattress method

A, Schematic of cardiac induction and Matrigel mattress method plating protocol. B, Matrigel mattress method work flow. C, Matrigel mattresses plated on Delta TPG Dish (Fisher Scientific), white arrows indicate edge of each mattress, blue dye included to visualize mattress. D, hiPSC-CMs plated on control (left panel) and mattress (right panel). Scale bar 50 μm. White arrows indicate edge of mattress platform.

Figure 2. Matrigel mattress morphometry and contractility

A, Immunofluorescence staining of cardiac myocyte (CM) structural marker α-actinin (green) and nucleus (blue, DAPI) for control human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) (left), mattress hiPSC-CM (center) and rabbit-CM (right). Scale bar 20 μm. 63X. B, Cell morphometry measuring circularity index (left) and sarcomere length (right) are plotted for indicated CMs. Error bars, s.d. (n = 9 – 48 cells per group). C, Representative control and mattress hiPSC-CM Ca transients (upper panel) and contraction traces (lower panel), spontaneously contracting. D, Representative contraction traces for indicated CM. (0.2 Hz). E, Contractility assessment measuring resting cell length (left), cell shortening (center) and cell shortening kinetics (right) are plotted. Error bars, s.d. (n = 6 – 20 cells per group) **** P< 0.0001 vs hiPSC-CM. *P<0.05 vs mouse-CM. n.s., Not significant.

Figure 3. Pharmacological analysis

(A, D and H) Representative Ca transients (upper panel) and contraction traces (lower panel) treated with indicated compound. Contraction and Ca concentration-response curves. (B and C) Extracellular Ca (Hill slope = 18.7 and 2.6 respectively). (E and F) Verapamil (Hill slope = −1.8 and −0.9 respectively). (I and J) EMD57033 (Hill slope = 1.1). (n = 5 – 20 cells per group). EC₅₀, effective concentration 50 % of max response. Veh, vehicle.