Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes

Abstract

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions.

Keywords: Cardiomyocytes; Combinatorial polymer; Maturation; Myosin light chain-2v; Troponin I; iPSC.

Fig. 1. Characterization of combinatorial copolymer matrices, and their effect on iPSC-CM contractility

(a) Copolymer structure represented as x%PEG-y%PCL-z%cPCL where x, y and z represent the respective molar ratios of the subunits (PEG, PCL and cPCL). (b) Surface hydrophilicity, modulus and degradation of combinatorial polymers with different molar ratios of subunits. (c) Surface plot image on each polymer matrix represents the contractile activity of iPSC-CMs at the 15-day point after seeding. iPSC-CMs maintained on 90%PCL-10%cPCL and 4%PEG-96%PCL exhibited stronger contractile movement, compared to two controls (Glass and 100% PCL). (d) Parts (1) and (2) show the phase-contrast and fluorescent images (single cell size) from fluorescence beads (0.75um) mixed with Matrigel after culturing an iPSC-CM on glass for 15 days. Scale bar indicates 20μm. Part (3) is an example of a displacement field image of fluorescence beads calculated by the particle image velocimetry (PIV) algorithm. Part (4) illustrates an example of a traction force field image calculated using the PIV result from part (3) by the Fourier transform traction cytometry (FTTC) method. (e) Traction force generated from individual iPSC-CMs on Matrigel after 15-day culture on glass, 4%PEG-96%PCL, and 4%PEG-86%PCL-10%cPCL. *p<0.05.

Fig. 2. Effects of combinatorial polymer matrix on mitochondria functions of iPSC-CMs

(a) Mitochondrial function of iPSC-CMs maintained on each polymer matrix was assessed by tetramethyl rhodamine methylester (TMRM), a fluorescent probe of inner mitochondrial membrane potential (red). Scale bar is 100 μm. (b) A higher intensity of TMRM staining was seen on iPSC-CMs maintained on 4%PEG-96%PCL compared to glass and other matrices. *p<0.05: compared to other substrates. (c) Immunostaining of iPSC-CMs for mitofusin-2 (Mfn2), which is involved in the mitochondrial maturation, revealed higher level of Mfn2 expression in iPSC-CMs maintained on 4%PEG-96%PCL compared to 100%PCL. *p<0.05: compared to PCL as a polymer control. Scale bar is 100 μm.

Fig. 3. Effects of polymer matrices on maturation of iPSC-CMs

(a) Immunofluorescence staining for α-actinin and myosin light chain-2v (MLC-2v; encoded by MYL2). The iPSC-CMs maintained on 4%PEG-96%PCL exhibited clear sarcomere structures with distinct z-lines and myofilament structures. Arrow indicates an undefined sarcomere structures. Scale bar is 100 μm. (b) MYL2 expression is dramatically higher in iPSC-CMs on 4%PEG-96%PCL than other test matrices at 30 days after re-plating. *p<0.001 compared to other matrices. (c) FACS analysis showed the expression pattern of myosin light chain 2 isoforms, myosin light chain-2a (atrial) and -2v (ventricular). It determined higher expression of MLC-2v, a reported marker of ventricular maturation in iPSC-CMs maintained on 4%PEG-96%PCL for 30 days, compared to the other test substrates.

Fig. 4. Troponin I isoform transition from ssTnI to cTnI

(a) Western blotting for ssTnI and cTnI. “60D” indicates iPSC-CMs maintained on traditional substrate (Matrigel) for 60 days after cardiac differentiation. (b) Ratio of cTnI/ssTnI protein expressed under each condition. Black bar represents percentage of cTnI with respect to the total Troponin I protein amount. White bar represents percentage of ssTnI with respect to the total Troponin I protein amount. (c) Representative iPSC-CM images of cTnI immunofluorescence in iPSC-CMs on glass, 4%PEG-96%PCL, and 4%PEG-86%PCL-10%cPCL. Scale bar indicates 100um.

Fig. 5. RNA-sequencing expression profiles of iPSC-CMs maintained on 4%PEG-96%PCL versus 4%PEG-86%PCL-10%cPCL

(a) iPSC-CMs maintained on 4%PEG-96%PCL expressed higher levels of sarcomere genes (MYH7, TCAP, MYH14, TNNI3, ACTN2, MYL3, MYL4, and MYL2) compared to cells maintained on 4%PEG-86%PCL-10%cPCL. Of note, MYL2 (encoding MLC-2v) expression was increased by over 8-fold in 4%PEG-96%PCL iPSC-CMs (p-value of 5×10⁻⁵). (b) iPSC-CMs maintained on 4%PEG-96%PCL expressed higher levels of genes associated with calcium handling and ion fluxes (ATP2A1, SCN5A, CACNA1, PLN, TRDN, RRAD, P2RX1, and CASQ2). Notably, expression of CASQ2, a marker of mature sarcoplasmic reticulum, was increased by over 8-fold in 4%PEG-96%PCL iPSC-CMs. (c) iPSC-CMs maintained on either polymer matrix exhibited dramatically different expression profiles of integrin subunits which mediate cell-matrix interaction. For instance, maintaining cells on 4%PEG-96%PCL significantly enhanced the expression of ITGA7 (p-value of 0.005) whereas 4%PEG-86%PCL-10%cPCL increased the expression of ITGB8 and ITGB4 (p-value of 0.002 and 5×10⁻⁵, respectively). (d) iPSC-CMs maintained on 4%PEG-96%PCL expressed much higher levels of the intermediate filament genes (MYOM3, SGCA, NRAP, TRIM63, XIRP1, SNTA1, MYOZ2, ITGBIBP3, and MYOM2).

Fig. 6. Quantitative PCR analyses of the impact of different substrates on gene expression of iPSC-CMs

Relative expression of the representative genes associated with the (a) sarcomere, (b) calcium handling, (c) voltage-gated potassium channel and the sarcomeric mitochondrial creatine kinase2, and (d) integrins were determined by qRT-PCR. Distinct matrices, notably the 4%PEG-96%PCL copolymer, exert differential effects on iPSC-CM maturation, and that these matrix-dependent influences involve distinct subset of integrins and intermediate filaments that serve as mechanical link to the myofibrils. *p<0.05 compared to 4%PEG-86%PCL-10%cPCL