An integrated statistical model for enhanced murine cardiomyocyte differentiation via optimized engagement of 3D extracellular matrices

Abstract

The extracellular matrix (ECM) impacts stem cell differentiation, but identifying formulations supportive of differentiation is challenging in 3D models. Prior efforts involving combinatorial ECM arrays seemed intuitively advantageous. We propose an alternative that suggests reducing sample size and technological burden can be beneficial and accessible when coupled to design of experiments approaches. We predict optimized ECM formulations could augment differentiation of cardiomyocytes derived in vitro. We employed native chemical ligation to polymerize 3D poly (ethylene glycol) hydrogels under mild conditions while entrapping various combinations of ECM and murine induced pluripotent stem cells. Systematic optimization for cardiomyocyte differentiation yielded a predicted solution of 61%, 24%, and 15% of collagen type I, laminin-111, and fibronectin, respectively. This solution was confirmed by increased numbers of cardiac troponin T, α-myosin heavy chain and α-sarcomeric actinin-expressing cells relative to suboptimum solutions. Cardiomyocytes of composites exhibited connexin43 expression, appropriate contractile kinetics and intracellular calcium handling. Further, adding a modulator of adhesion, thrombospondin-1, abrogated cardiomyocyte differentiation. Thus, the integrated biomaterial platform statistically identified an ECM formulation best supportive of cardiomyocyte differentiation. In future, this formulation could be coupled with biochemical stimulation to improve functional maturation of cardiomyocytes derived in vitro or transplanted in vivo.

Figure 1. Integrating a biomaterial platform with statistical approaches.

(a) Schematic representation showing production of ECM composites including several different ECM proteins at varied concentrations with single iPS cells via NCL of PEG precursors (thioester or Cys). We use three different ECM proteins representing fibrillar collagen type I, basement membrane laminin-111, and cell-ECM linking fibronectin, but a larger variety is technically feasible. (b) Statistical optimization of multiple ECM proteins can maximize the expression of cTnT by RSM, yielding a set of potentially optimal solutions. (c) The optimal formulation is validated by measuring the improvement of cTnT expression and the functionality of cardiomyocytes by imaging contractile kinetics and Ca²⁺ transients.

Figure 2. FEs inform the formulation space for three ECM proteins.

(a) A simple plot shows that multiple ECM proteins (detailed formulations in Table 1) promoted the expression of cTnT, compared to no ECM (−−−) or single ECM (+−−, −+−, or −−+). (b) Effect magnitudes of all main effects and interactions for the expression of cTnT (protein). Blue lines indicate significant probability values of 0.05. (c) Contour plots showing the interactions between FN and Col I, LN and Col I, and FN and LN. (d) ECM composites as delineated in Table 1 were also tested for the expression of Tnnt2 (gene). Results in (a,d), ANOVA Tukey’s HSD post hoc test, n = 3, *p < 0.05, mean ± S.D.

Figure 3. RSM of a formulation space modified to reflect adjusted levels (concentrations) of effects employed in the FE.

(a) Effect magnitudes of all main effects and two-factor interactions to fit response surface regressions. Blue lines indicate significant probability values of 0.05. (b) Contour plots showing the interactions between FN and Col I, LN and Col I, and FN and LN from the RSM, which are different from those of the FE in Fig. 2c and indicative of different ranges tested in RSM.

Figure 4. Validation of the optimized ECM formulation by the expression of sarcomeric proteins.

Enhancement of cTnT (a) αMHC (b) and αSA (c) expression was tested with experimental (opt) and control groups (PEG and +++, formulations in Supplementary Table 4). (d) Expression of Cx43 was visualized with MPLSM in 3D ECM composites. ANOVA Fisher’s LSD post hoc test, n = 7 (a) or n = 3 (b or c) **p < 0.01 and *p < 0.05, mean ± S.D. The higher and darker gradient wedge indicates higher concentrations or numbers of ECM proteins associated in the formulation.

Figure 5. Validation of the optimized formulation by assessing contractility and Ca²⁺ transients and by adhesion modulation.

(a) % cluster length was evaluated by utilizing high frame rate DIC. Representative contractility from ECM composites. (b) Fluo-4 AM (Ca²⁺ indicator) intensity (F) was normalized to the baseline fluorescence (F₀). Representative local Ca²⁺ transient from ECM composites. (c) Effect magnitudes of all main effects and interactions for the expression of cTnT (protein) with Col I, FN, and TSP1 (formulations in Table 3).