To address the potential roles of the ECM on indirect cardiac reprogramming, we selected natural ECM proteins that could be formed into 2-D hydrogels. Working with these tractable 2-D hydrogel systems allow several aspects of the extracellular matrix to be changed. For example, the mechanical properties of the matrix can be modulated by changing the protein concentration in the hydrogel and the identity of the matrix can be varied by incorporating more than one ECM protein in different ratios. The natural ECM proteins selected for this study were Matrigel, collagen I and fibrin. Prior studies on iPSC reprogramming had used Matrigel or collagen I to support the reprogramming process, so we reasoned that hydrogels fashioned from these two materials should support indirect cardiac reprogramming22,23. Indeed, Matrigel had been demonstrated to support indirect cardiac reprogramming by Efe et al.19 Fibrin, on the other hand, has never been used to support reprogramming. However, fibrin was selected based on studies that showed it could mitigate the deterioration of cardiac function when injected into the infarcted myocardium24,25. Potentially, the cardio-protective effects of fibrin might enhance cardiac regeneration if the reprogramming factors were introduced via a fibrin delivery system.

Cardiac reprogramming is more efficient on fibrin gels

We first performed a comparison between Matrigel, collagen I and fibrin to determine which of these substrates could support the indirect cardiac reprogramming process. Fibrin gels readily supported both the dedifferentiation (indicated by number of colonies/cm2, Fig. 1d) and cardiac differentiation (indicated by the % of contractile colonies, Fig. 1e) phases of the reprogramming process. Surprisingly, indirect reprogramming on Matrigel and collagen I gels in our hands was very poorly efficient, generating very small numbers of colonies (Fig. 1d) that were not contractile (Fig. 1e). When the fibrin gel concentrations were varied from 1.25 to 7.5 mg/mL, no significant differences in dedifferentiation or cardiac reprogramming efficiencies occurred (Fig. 1d, e). However, an increasing trend for cardiac reprogramming efficiency was observed with increasing fibrin gel concentration (Fig. 1e). Tissue culture plastic (TCP) coated with the various ECM proteins did not yield significant differences in terms of dedifferentiation efficiency (Fig. 1d), but only fibrinogen-coated TCP supported cardiac reprogramming (Fig. 1e).

Thrombin activated cell proliferation does not account for the enhanced cardiac reprogramming on fibrin gels

During the process of cardiac reprogramming, we observed that the MEFs were proliferating at different rates on the various gels. When the proliferation of MEFs cultured on 2.5 mg/mL gels of different identities was quantified, cells cultured on fibrin proliferated at a significantly higher rate than those cultured on either Matrigel or collagen I gels and also higher than those cultured on fibrinogen-coated TCP (Fig. 2a). A prior study with induced pluripotent stem cells (iPSCs) suggested that dedifferentiation is a stochastic process that can be accelerated by cell proliferation26 and so we investigated whether the more efficient cardiac reprogramming observed on fibrin gels could be explained by the enhanced rate of cell proliferation. Furthermore, it has been shown that thrombin in fibrin clots retains its activity and consequently enhances cell proliferation27,28. Therefore, we focused on a role for thrombin in the cardiac reprogramming process.

Figure 2 Cell proliferation is enhanced by thrombin in fibrin gels, but does not fully explain fibrin's superior support of indirect cardiac reprogramming. (a) Cell proliferation of MEFs determined by DNA concentration for cultures on fibrinogen-coated TCP ( ) and 2.5 mg/mL Matrigel ( ), fibrin ( ) and collagen I ( ) gels. The ANOVA test indicates significant differences across the different substrates (p < 0.0001). * Significance between TCP-fibrinogen or Matrigel or collagen I gels and fibrin gels (p < 0.05). # Significance between Matrigel or collagen I gels and TCP-fibrinogen (p < 0.05). Significance versus collagen I (p < 0.05). (b) Cell proliferation of MEFs cultured on TCP-fibrinogen supplemented with 1 U/mL thrombin ( ) and 2.5 mg/mL fibrin gels pre-soaked in PBS for 1 week ( ). The ANOVA test indicates significant differences across the different substrates (p < 0.0001). * Significance versus fibrin gels (p < 0.05). # Significance versus TCP-fibrinogen (p < 0.05). Significance versus TCP-fibrinogen supplemented with thrombin (p < 0.05). No significant differences in the total number of colonies per cm2 (c) and percentage of contractile colonies (d) were observed between fibrinogen-coated TCP and fibrinogen-coated TCP supplemented with thrombin conditions. No significant differences in the total number of colonies per cm2 (e) and percentage of contractile colonies (f) were observed between fibrin and PBS pre-soaked fibrin conditions. No significant differences for cell area at day 1 were observed across the different matrices (g), n = 100. Significant differences were however observed for the percentage of cells that initially adhered at day 1 (h) (p < 0.0001 via ANOVA). Matched symbols denote significant (p < 0.05) differences. Error bars represent ± s.e.m., n = 3 independent experiments unless indicated otherwise. Full size image

First, we ascertained that the enhanced cell proliferation rate was indeed a result of the presence of thrombin by performing a proliferation assay of MEFs cultured on 2.5 mg/mL fibrin gels that had previously been soaked in PBS for a week and of MEFs cultured on fibrinogen-coated TCP with 1 U/mL thrombin supplementation. When compared with the fibrin data, PBS-soaked fibrin gels supported significantly lower rates of MEF proliferation due to the removal of thrombin by the PBS soak (Fig. 2b). Supplementing thrombin to the culture medium of MEFs growing on fibrinogen-coated TCP stimulated significant increases in cell proliferation, but not until day 7 (Fig. 2b). Thus, thrombin supplementation alone is not sufficient to explain the increased proliferation rates observed when culturing cells on fibrin gels. Furthermore, while thrombin supplementation also improved both the dedifferentiation and cardiogenic differentiation efficiencies of the reprogramming process when MEFs were cultured on fibrinogen-coated TCP, the differences observed +/− thrombin were not significant (Fig. 2c, d). Additionally, reprogramming on fibrin gels pre-soaked in PBS was not significantly different from reprogramming on the fibrin controls (Fig. 2e, f). Taken together, these data suggest that thrombin's effects on MEF proliferation are not the predominant determinant of the superior indirect cardiac reprogramming achieved on fibrin gels.

We also quantified the percentage of cells adherent to the gels and the spread cell areas on day 1 after initial seeding to determine if differences in these parameters correlate with the differential support of reprogramming observed in Fig. 1. Cells spread to the same degree on all of the different gels (Fig. 2g), thereby ruling out any potential effects from cell spreading. Quantification of initial cell adhesion revealed that a lower percentage of cells were adherent on day 1 on collagen I and fibrin soaked in PBS gels compared to the other gels (Fig. 2h). However, both Matrigel and collagen I gels support reprogramming to a much lower degree than fibrin gels (Fig. 1d, e), despite the fact that Matrigel and fibrin support cell adhesion equally (Fig. 2h). On the other hand, collagen I gels and fibrin gels soaked in PBS both show reduced adhesion relative to other conditions (Fig. 2h), but reprogramming on the fibrin + PBS soaked gels is equivalent to that on fibrin (Fig. 2e and 2f). Therefore, differences in initial adhesion did not correlate with the differences in reprogramming efficiencies.

Cardiac reprogramming is further enhanced by ascorbic acid

The results from the comparison of the various ECM gels validated fibrin as a supportive substrate for cardiac reprogramming. To further improve cardiac reprogramming on fibrin gels, we supplemented the media with ascorbic acid. Ascorbic acid had been used as a cardiogenic molecule in differentiating human embryonic stem cells29 and it was also used to improve the efficiency of induced pluripotent stem cell derivation30. We thus postulated that supplementing our reprogramming media with ascorbic acid might increase both the number of contractile and total colonies. Quantifying the number of colonies per cm2 and the percentage that were contractile revealed that ascorbic acid increased the number of contractile colonies, but not the total number of colonies (Fig. 3a, b). This implies that ascorbic acid enhances the cardiac differentiation phase of the two-step reprogramming process, rather than the initial dedifferentiation phase. For an individual substrate, enhancement achieved by ascorbic acid supplementation was only significant (compared to unsupplemented reprogramming) on 2.5 mg/mL and 7.5 mg/mL fibrin gels. However, ascorbic acid supplementation significantly enhanced cardiac reprogramming on fibrin gels across the board relative to that achieved on fibrinogen-coated TCP. Ascorbic acid supplementation on fibrin gels also resulted in larger contractile colonies (Supplementary movies S1 and S2) and all contractile colonies (with and without ascorbic acid supplementation) immunocytochemically stained for sarcomeric alpha-actinin and cardiac troponin I (Fig. 3c and d). Non-contractile colonies did not stain for these markers.

Figure 3 Ascorbic acid further enhances indirect cardiac reprogramming on fibrin gels. (a) No significant differences in the total number of colonies per cm2 were found between cultures supplemented with ascorbic acid (+AA) versus those without (−AA) (mean ± s.e.m. for n = 3 independent experiments). (b) The number of contractile colonies increased with ascorbic acid supplementation when reprogramming was performed on fibrin gels. TCP substrates in both (a) and (b) were coated with fibrinogen. The ANOVA test indicates significant differences between treatment conditions (−AA vs. +AA, p < 0.0001) and between different protein concentrations (p = 0.0015). Matched symbols denote significant (p < 0.05) differences via pair-wise post-hoc analysis. Error bars represent ± s.e.m., n = 3 independent experiments. The reprogrammed MEFs were examined by immunocytochemical staining of cell nuclei (DAPI), cardiac troponin I (cTnI) (c) and sarcomeric alpha-actinin (α-actinin) (d). Higher magnification insets show characteristic cardiac striations. Scale bars 100 μm, inset scale bars 5 μm. Full size image

We used qPCR to examine the temporal expression profiles of selected pluripotent genes, early cardiac genes and late cardiac genes at discrete time points that included different phases of the reprogramming process. The expression levels for Oct4 were very high for transduced cells at early time points (day 4 and day 8) since we induced expression of Oct4 exogenously (Fig. 4a). However, high levels of Oct4 expression persisted for up to 8 days more (days 12 and 16), suggesting that expression of endogenous Oct4 had been stimulated. In contrast, the expression levels of endogenous Nanog gradually increased to a peak at day 12 and then decreased to sub-baseline levels by day 16 (Fig. 4a). The expression profiles of Oct4 and Nanog indicated that 7 days of OSKM transgene expression on fibrin gels was sufficient to induce dedifferentiation of the cells but incomplete reprogramming to an iPSC phenotype since the expression of Nanog was not sustained.

Figure 4 MEFs undergoing indirect cardiac reprogramming on fibrin gels in the presence of ascorbic acid supplementation exhibit differential gene expression profiles. The expression profiles of MEFs cultured on various fibrin gels or fibrinogen-coated TCP were examined via qPCR at multiple time points, with the levels of (a) pluripotent genes (Oct4 and Nanog), (b) early cardiac genes (Mesp1 and Gata4) and (c) late cardiac genes (Myl7, Myl2 and Tnnt2) assessed. * Significance versus TCP-fibrinogen. ANOVA indicates significant differences across groups in Mesp1 day 12 data (p = 0.03). Error bars represent ± s.e.m., n = 3 independent experiments. Full size image

The expression profiles of Mesp1, a cardiac mesoderm transcription factor and Gata4, a transcription factor involved in the formation of the heart tube, were also examined with qPCR. There was a sharp increase in expression of Mesp1 observed at day 12 (Fig. 4b), presumably due to the directed differentiation initiated at day 10 (Fig. 1c). The expression levels of Mesp1 at day 12 across the conditions were significantly different (p = 0.03); however the sample sizes were insufficient to determine significant pair-wise differences. At day 16, the levels of Mesp1 for fibrin gels dropped to baseline, whereas those for Gata4 at both day 12 and day 16 were above baseline (Fig. 4b). These expression profiles for Mesp1 and Gata4 recapitulate the sequential activation of transcription factors that occur during embryonic cardiac development31,32 suggesting that reprogramming on fibrin gels in the presence of ascorbic acid enhanced cardiogenesis on a molecular level. Surprisingly, we found that the expression levels of late cardiac genes such as Myl7, Myl2 and TnnT2 were elevated throughout the cardiac reprogramming process (Fig. 4c). This suggests that reprogramming on fibrin gels with ascorbic acid supplementation primes the dedifferentiated cells towards the cardiac lineage.

Incorporating collagen I into fibrin replaces ascorbic acid

Several groups have suggested that induction of collagen synthesis by ascorbic acid is a key determinant of cardiogenesis33,34. When the colonies that resulted from cardiac reprogramming on fibrin with ascorbic acid supplementation were stained for collagen I, the contractile colonies stained positive whereas non-contracting colonies were negative (Fig. 5a). We postulated that ascorbic acid supplementation may be replaced by incorporating a small amount of collagen I into the fibrin gels. We began by making 75% fibrin-25% collagen I composite gels of various total protein concentrations and performed the cardiac reprogramming process with MEFs on the composite gels. Comparing the data between the composite gels with that of fibrin gels supplemented with ascorbic acid, we observed that the dedifferentiation efficiencies (number of colonies/cm2) were similar if not identical in both cases (Fig. 5b). The composite gels showed a gradual increase in cardiogenic efficiency (% contractile colonies) with increasing total protein concentration; at 7.5 mg/mL total protein concentration, the composite gels achieved similar cardiogenic efficiencies similar to those achieved on pure fibrin gels supplemented with ascorbic acid (Fig. 5c).

Figure 5 Incorporation of collagen I into fibrin gels replaces ascorbic acid (AA) supplementation. (a) Colonies were examined by immunocytochemical staining for collagen I. Contractile colonies stained positive for collagen I, whereas non-contractile colonies did not. Total number of colonies per cm2 (b) and percentage of contractile colonies (c) for reprogramming on fibrin-25% collagen I composite gels compared to fibrin gels with AA supplementation. The reprogramming efficiency on fibrin-25% collagen I composite gels approached that of fibrin gels + AA at 7.5 mg/mL total protein concentration. TCP substrates were coated with either fibrinogen or a 75:25 fibrinogen-collagen I solution. The total numbers of colonies (b) were not significantly different between the +AA and +25% collagen I conditions via ANOVA (p = 0.77). However, the differences in the % contractile colonies (c) were significant between the +AA and +25% collagen I conditions and across protein concentrations via ANOVA (p < 0.0001). Matched symbols denote significant (p < 0.05) differences via pair-wise post-hoc analysis. Increasing the percentage of collagen I in 2.5 mg/mL total protein concentration composite gels did not increase the total number of colonies per cm2 (d), but did increased the percentage of contractile colonies (e). The reprogramming efficiency of MEFs cultured on 2.5 mg/mL composite gels containing 75% collagen I approached that achieved on pure fibrin gels + AA. ANOVA on (d) (p = 0.0006) and (e) (p < 0.0001) indicate significant differences across groups. Matched symbols denote significant (p < 0.05) differences via pair-wise post-hoc analysis. (f) Percentage of contractile colonies obtained from MEFs reprogrammed on various hydrogels composed of 1.25, 2.5 and 7.5 mg/mL total protein concentration. The data suggest an optimal collagen I concentration for the composite gels. Line plot is a Giddings fit of the mean values at a collagen I concentration. Error bars represent ± s.e.m., n = 3 independent experiments. Full size image

We also made fibrin-collagen I composite gels with a constant 2.5 mg/mL total protein concentration and varied the percentage of collagen I in those gels. Dedifferentiation efficiencies were similar and appeared not to depend on the amount of collagen I (Fig. 5d). However, for a fixed total protein concentration, the cardiogenic efficiency increased as the percentage of collagen I increased (Fig. 5e). In fact, the efficiency of cardiac reprogramming achieved on 2.5 mg/mL total protein concentration containing 75% collagen I and 25% fibrin was similar to that achieved with ascorbic acid supplementation. Coupled with the results in Fig. 5c, these data suggested that the total amount of collagen I in the composite gels, rather than the amount relative to fibrin, determined the efficiency of cardiogenic reprogramming on the composite gels. To further test this idea, we also made fibrin-collagen I composite gels with a constant 7.5 mg/mL total protein concentration and again varied the percentage of collagen I. We then combined the data from this experiment with those from the other experimental conditions (1.25 mg/mL and 2.5 mg/mL total protein concentrations) on a single graph (Fig. 5f). These data illustrate that there is an optimal concentration of collagen I (~1.8 mg/mL) that makes the gels highly cardiogenic. Concentrations above and below this optimum decrease the cardiogenic efficiency of the gels.

Given these data showing the important role of collagen I in our improved reprogramming efficiencies, we returned to our earlier observation that MEFs on collagen I gels did not undergo reprogramming. Based on anecdotal observations, we postulated that large traction forces exerted by the MEFs on collagen I resulted in matrix remodeling and compaction, which in turn impeded reprogramming (or at least our ability to assess it). To address this hypothesis, a reprogramming experiment in which the collagen I gels were crosslinked with a 0.25% glutaraldehyde solution was performed. In this experiment, preventing the cells from compacting the collagen I matrix resulted in improved dedifferentiation and cardiogenesis. However, the dedifferentiation and cardiogenic efficiencies of MEFs cultured on these glutaraldehyde crosslinked collagen I gels were still significantly below that of ascorbic acid supplemented fibrin gels (Fig. 6). This may be due to the glutaraldehyde crosslinking destroying adhesion sites or signaling domains in collagen I and consequently inactivating these active sites. The fibrin-collagen I composite gels may provide an alternative method of crosslinking collagen I that preserves the biological activity of the collagen molecule. This could potentially explain both the high dedifferentiation and cardiogenic efficiencies observed with the fibrin-collagen I composite gels.

Figure 6 Indirect cardiac reprogramming is attainable, albeit poor, on crosslinked-collagen I gels. The total number of colonies per cm2 (a) and percentage of contractile colonies (b) show that reprogramming efficiencies on crosslinked-collagen I gels do not approach that fibrin with AA supplementation. ANOVA on (a) and (b) indicates significant differences between fibrin + AA and crosslinked collagen I groups (p < 0.0001 for a and p = 0.0019 for b). Matched symbols denote significant (p < 0.05) differences via pair-wise post-hoc analysis. Error bars represent ± s.e.m., n = 3 independent experiments. Full size image

Differences in matrix rigidity and microstructure are not sufficient to explain the enhanced cardiac reprogramming observed on fibrin gels

We investigated if the enhanced cardiac reprogramming observed on fibrin and fibrin-collagen I gels relative to pure collagen gels was simply due to differences in the mechanical properties of the gels. Quantification of the shear elastic moduli of the fibrin and collagen I gels showed how changing total protein concentration affected gel mechanical properties and that there were in fact significant differences in the shear moduli values of the fibrin and collagen I gels for a particular protein concentration (Fig. 7a). However, despite very significant differences in the differential abilities of fibrin and collagen to support reprogramming (Fig. 1d, e), the ranges of shear moduli for the different concentrations of fibrin (9.5–212.4 Pa) and collagen I (3.6–283.9 Pa) gels used in this study were quite similar. Moreover, cardiogenic reprogramming efficiencies on fibrin gels in either the absence or presence of ascorbic acid did not vary significantly as a function of protein concentration (Fig. 3b), even though these gels possessed a five fold difference in shear modulus (Fig. 7a). Finally, the cardiogenic reprogramming efficiency on fibrin-collagen I composite gels varied with composition (Fig. 5e) whereas the shear moduli of these gels are within the same order of magnitude (Fig. 7b).

Figure 7 Matrix modulus and microstructure do not account for differences in indirect cardiac reprogramming observed on different substrates. (a) Shear modulus of fibrin and collagen I gels of various total protein concentrations. ANOVA indicates significant differences between fibrin and collagen I gels (p = 0.0002). Matched symbols denote significant (p < 0.05) differences via pair-wise post-hoc analysis.(b) Shear modulus of 2.5 mg/mL fibrin-collagen I composite gels with various percentages of collagen incorporated. ANOVA indicates significant differences between groups (p < 0.0001). Matched symbols denote significant (p < 0.05) differences via pair-wise post-hoc analysis. Error bars represent ± s.e.m., n = 3 independent experiments. (c) Matrix microstructure examined by scanning electron microscope of the various 2.5 mg/mL fibrin-collagen I composite gels. Full size image

Collagen I, fibrin and fibrin-collagen I gels are all fibrillar in nature and their microstructures and mechanical properties are intimately linked to the final protein concentrations of the gels. Therefore, changing the total protein concentrations and the different collagen I to fibrin ratios in the composite gel not only affects the mechanical properties of the gels, but also the microstructural features. Qualitative assessment of the microstructures of fibrin, collagen I and various fibrin-collagen I composite gels (all of 2.5 mg/mL total protein) revealed them to be similar (Fig. 7c), incongruous with the significant differences and cardiogenic reprogramming efficiencies (Fig. 5e). Collectively, these data suggest that matrix rigidity and microstructure are insufficient to explain the observed differences in cardiac reprogramming as a function of material identity and composition, at least for the range of materials used here.

Traction forces modulate dedifferentiation during cardiac reprogramming

Previous studies have shown that cells cultured on different matrices exert different levels of traction forces35. We hypothesized that the response to matrix factors observed during cardiac reprogramming is mediated by traction forces. To address this hypothesis, we co-transduced tetracycline-inducible RhoA mutant vectors (Fig. 8a) with the tetracycline-inducible OSKM vector to modulate the traction forces exerted by a cell during the reprogramming process. The constitutively-active RhoA mutant (RhoA-Q63L) increases traction forces in a cell when expressed and conversely the dominant-negative RhoA mutant (RhoA-T19N) decreases traction forces when expressed36. A tetracycline inducible eGFP control vector was used to account for any non-specific effects of additional lentiviral infection. Efforts to reprogram cells expressing the RhoA-Q63L mutant showed that increasing the traction forces exerted by a cell significantly decreased dedifferentiation efficiency. On the other hand, decreasing cell traction, with the RhoA-T19N mutant, slightly increased dedifferentiation efficiency (Fig. 8b). Cardiogenic efficiency however was not significantly affected by the modulation of traction forces during the dedifferentiation phase (Fig. 8c). Furthermore, ectopic expression of the RhoA mutants did not significantly influence proliferation rates (Fig. 8d), thus suggesting that the observed differences in dedifferentiation efficiency induced by the RhoA mutants (Fig. 8b) were not due to differences in proliferation.