Person:

Domian, Ibrahim

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.

Summary Stem cell-derived cardiomyocytes represent unique tools for cell- and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated. We reasoned that physiological metrics of engineered cardiac tissues offer a means of comparison. We built laminar myocardium engineered from cardiomyocytes that were differentiated from mouse embryonic stem cell-derived cardiac progenitors or harvested directly from neonatal mouse ventricles, and compared their anatomy and physiology in vitro. Tissues assembled from progenitor-derived myocytes and neonate myocytes demonstrated similar cytoskeletal architectures but different gap junction organization and electromechanical properties. Progenitor-derived myocardium had significantly less contractile stress and slower longitudinal conduction velocity than neonate-derived myocardium, indicating that the developmental state of the cardiomyocytes affects the electromechanical function of the resultant engineered tissue. These data suggest a need to establish performance metrics for future stem cell applications.

Summary The transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC.

Atrial natriuretic peptide (ANP) has a central role in regulating blood pressure in humans. Recently, microRNA 425 (miR-425) was found to regulate ANP production by binding to the mRNA of NPPA, the gene encoding ANP. mRNAs typically contain multiple predicted microRNA (miRNA)-binding sites, and binding of different miRNAs may independently or coordinately regulate the expression of any given mRNA. We used a multifaceted screening strategy that integrates bioinformatics, next-generation sequencing data, human genetic association data, and cellular models to identify additional functional NPPA-targeting miRNAs. Two novel miRNAs, miR-155 and miR-105, were found to modulate ANP production in human cardiomyocytes and target genetic variants whose minor alleles are associated with higher human plasma ANP levels. Both miR-15 and miR-105 repressed NPPA mRNA in an allele-specific manner, with the minor allele of each respective variant conferrin resistance to the miRNA either by disruption of miRNA base pairing or by creation of wobble base pairing. Moreover, miR-15 enhanced the repressive effects of miR-425 on ANP production in human cardiomyocytes. Our study combines computational genomic, and cellular tools to identify novel miRNA regulators of ANP production that could be targeted to raise ANP levels which may have applications for the treatment of hypertension or heart failure.

The immature phenotype of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) constrains their potential in cell therapy and drug testing. In this study, we report that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. Integrated “-Omics” analyses showed that addition of galactose to culture medium improves total oxidative capacity of the cells and ameliorates fatty acid oxidation avoiding the lipotoxicity that results from cell exposure to high fatty acid levels. This study provides an important link between substrate utilization and functional maturation of hPSC-CMs facilitating the application of this promising cell type in clinical and preclinical applications.

Aims Atrial natriuretic peptide (ANP), secreted primarily by atrial cardiomyocytes, decreases blood pressure by raising cyclic 3’,5’-guanosine monophosphate (cGMP) levels and inducing vasorelaxation, natriuresis, and diuresis. Raising the level of ANP has been shown to be an effective treatment for hypertension. To advance the future development of an anti-microRNA (miR) approach to increasing expression of ANP, we investigated the regulation of NPPA expression by two miRs: miR-425 and miR-155. We examined whether miR-425 and miR-155 have an additive effect on the expression and function of ANP. Methods and results Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were transfected with miR-425, miR-155, or a combination of the two miRs. Two days later, NPPA expression was measured using real time qPCR. Each of the miRs decreased NPPA expression over a wide range of concentrations, with a significant reduction at concentrations as low as 1 nM. The combination of miR-425 and miR-155 reduced NPPA expression to a greater extent than either miR-425 or miR-155 alone. An in vitro assay was developed to study the potential biological significance of the miR-induced decrease in NPPA expression. The cooperative effect of miR-425 and miR-155 on NPPA expression was associated with a significant decrease in cGMP levels. Conclusions: These data demonstrate that miR-425 and miR-155 regulate NPPA expression in a cooperative manner. Targeting both miRNAs with anti-miRs (possibly at submaximal concentrations) might prove to be a more effective strategy to modulate ANP levels, and thus blood pressure, than targeting either miRNA alone.