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.Differentiation of stem cells into specific lineages during development is tightly regulated by the local microenvironment including growth factors, extracellular matrix (ECM) proteins, and surrounding cells over time 1,2 . Stem cells taken outside the body are similarly dependent on the microenvironment and the past two decades have witnessed a surge in knowledge and corresponding technology to best manipulate stem cell state in a culture dish 2 . To date, the primary focus is manipulation of growth factor and small molecule delivery 3,4 . Progress in this area has been catalyzed by improved understanding of the genetic events that cause specification and the soluble factors that can stimulate these genetic events. In addition, it is technologically straightforward to add soluble components to a 2D culture system. More challenging is the identification and presentation of multiple insoluble components, namely ECM proteins, in 3D.Differentiation of most cell types would benefit from the identification of defined, ECM formulations to enhance specification. Cardiomyocyte differentiation is an intriguing test case. Exogenous ECM proteins are required both to promote survival of cardiomyocytes ex vivo 5 and to implement all cardiomyocyte differentiation protocols employed to date 2 . While the soluble components required to drive cardiomyocyte differentiation have become

Distilling complexity to advance cardiac tissue engineering

“…In human tissue constructs, velocity of action potential propagation improved with increasing amounts of virtually pure CMs in the initial cell composition, whereas contractile force output was optimal for CM populations with purities of 60 to 80% (12). Overall, the presence of fibroblasts or other stromal cells in iPSC-derived cardiac tissues is found to be beneficial to CM maturation, via engagement of extracellular matrix proteins (13) and establishment of intercellular contacts, while excess of non-myocytes may compromise conduction and integration.…”

Cardiac Extracellular Matrix Modification as a Therapeutic Approach

“…Then by applying a “design of experiments” statistical approach to the platform, systematic optimization of the ECM ratios led to the identification of a formulation of cECM proteins optimized for cardiomyocyte differentiation. The theoretical formulation was confirmed experimentally via immunophenotyping and functional analyses [37]. Natural multicomponent ECMs derived from tissues have also been used in 3D to influence cell differentiation toward cardiomyocytes.…”

Laminin promotes differentiation of rat embryonic stem cells into cardiomyocytes by activating the integrin/FAK/PI3K p85 pathway

“…To further identify the optimal differentiation conditions for rESCs to cardiomyocytes, we first compared the effects of gelatin and laminin, which have been suggested to facilitate cardiomyocyte differentiation as supportive ECMs. 26,27 Most of the rEBs generated by the hanging droplet methodology ( Figure 2A) and cultured in gelatin coated dishes fail to attach ( Figure 2B). We found that laminin-induced rESC could induce the cardiomyocytes to beat on day 8 of differentiation, and the medium harbouring Laminin/differentiation-IMDM-FBS (DIF) produced 55.3% of beating rEBs derived from rESCs ( Figure 2E) Interestingly, rEBs cultured in laminin-coated dishes attached and proliferated well ( Figure 2B).…”