Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

Abstract

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

Keywords: Cardiac Differentiation; Drug-selected cardiomyocyte; Matrix elasticity; Mechanical interferometry; Pluripotent Embryonic Stem Cell; Synchronization.

Figure 1.

Experimental design for ES stiffness culture. (A) Elastic modulus of PDMS substrates of varying base-to-curing-agent ratio were rheometrically characterized before and after culture in liquid media relative to 3 GPa standard TC plate. ES cells are formed into EBs via a hanging drop method, marking the onset of differentiation and (B) landed onto substrates. (C) EBs show similar adhesion profile to PDMS and tissue culture plates. All culture conditions show greater than 85% adhesion of individual EBs with no statistically significant difference between groups (p > 0.05).

Figure 2.

Undifferentiated ES cells show greatest cardiac differentiation on rigid substrates. (A) Timeline of culture and analysis of ES-derived cardiomyocytes from stiffness culture. (B) Fluorescent staining of cTnT (green) on adherent EBs shows organized striation patterns and is a suitable marker for analysis. (C) Representative FACS analysis profiles and (D) cumulative data of cTnT⁺ cells demonstrate a correlation between high rigidity and cardiac proliferation from a pluripotent state (n = 5). (E) qRT-PCR analysis of cultured cells for cTnT mRNA expression relative to GAPDH shows that the rigidity of the 2D substrate determines cardiac induction in both murine (n = 4) and human (n = 3) ES cell lines. FACS quantification of MF20⁺ cells in day 15 EB generated from indicated human ES cell lines (F). Representative FACS profiles show induction of MF20⁺ cardiomyocytes at day 15 of EB formation (G). All values are mean ±SD, where ∗ indicates p-value < 0.05 and ∗∗ indicates p-values < 0.01.

Figure 3.

Nkx2-5 drug-selected cardiomyocytes show greatest cardiac maturation on rigid substrates. (A) Nkx2-5^neoR/+ ES cells were formed into EBs, landed onto PDMS and/or TC plate substrates, and subjected to drug selection. After 6 days under neomycin selection, ES-derived cardiac foci showed greater maturity on rigid matrices via (B) cTnT mRNA expression relative to GAPDH (n = 3) and (C) beating rate (n = 3). Cardiac foci also demonstrated a higher expression of adult α-MHC relative to fetal β-MHC, verifying their mature cardiac phenotype (D). Expression analyses of MLC2v (ventricular myocytes) and SLN (atrial myocytes) indicate that rigid and soft environments induce ventricular and atrial myocytes, respectively (n = 3) (E). MLC2v and SLN expression levels are normalized to those of 50:1 and TC plate, respectively. All values are mean ± SD, where ∗ indicates p-value <0.05 and ∗∗ indicates p-value <0.01.

Figure 4.

Transplanted cardiomyocyte foci display synchrony with the neonatal cardiomyocyte feeder layer. Cardiac foci derived from drug-selection (EB) and stiffness culture were transplanted onto a monolayer of neonatal cardiomyocytes (CM) at day 16, as shown in figure 3(A). (A) Representative light microscopic image of EB cultured on CM. The M-mode image was obtained at the red line. (B), (C) After 2 days, M-mode image analysis of videos of co-cultured samples confirms synchronization of beating frequency between EB (B) and neonatal cardiomyocyte (C) layers. Beating is largely regular with minor arrhythmia. (D)–(F) MII analyses at the beating regions of EBs transplanted onto CM (D). EB and CM initially beat asynchronously (E), but become fully synchronized after 48 h in co-culture (F).