Engineered cardiomyocytes and uses thereof
A cardiomyocyte, engineering technology, applied in the fields of cell biology, pluripotent stem cells and cell differentiation, can solve problems such as unclear gene regulatory network interactions
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Embodiment 1
[0241] Example 1: Generation of patient-specific iPS cells and functionally engineered cardiomyocytes
[0242] A heterozygous c.886G>A mutation in human GATA4 is associated with 100% permeable atrial or ventricular septal defect, AVSD, and pulmonary stenosis (PS) (Figures 1 and 2) (Garg et al., 2003). Mutant GATA4 translates into a G296S missense substitution flanking the zinc finger domain, a region involved in DNA binding and protein-protein interactions (Fig. 1, lower panel). identified several GATA4 G296S patients who developed tardive cardiomyopathy in their juvenile years. It is characterized by reduced left ventricular systolic function and abnormal right ventricular echocardiographic findings with deep trabeculation and papillary muscle thickening in the left ventricle (Figure 3). Profound trabecular formation is typical of a condition known as noncompaction, which is thought to reflect failure of ventricular myocyte maturation (Hutchins and Schaefer, 2012). This cli...
Embodiment 2
[0245] Example 2: Impaired contractility, calcium regulation, metabolic activity of GATA4 mutant engineered cardiomyocytes
[0246] Production of cTnT with wild-type (WT), G296S, and CRISPR-corrected isogenic IPS cells + D32-cardiomyocytes (Fig. 19), although the mutant line showed a slight delay in the onset of spontaneous contraction (Fig. S3A-B). To more precisely model the contractile properties of mature cardiomyocytes, a physiological micropatterning platform for single iPS-derived cardiomyocytes was designed (Fig. 20) (Ribeiro et al., 2015). Compared with 70% of wild-type cardiomyocytes, only 50% of patterned G296S cardiomyocytes accurately responded to electrical pacing at 1 Hz; although wild-type cells did not respond to pacing at frequencies above 1 Hz, 20% of G296S cardiomyocytes were able to beat at this faster rate (Figure 21). G296S cardiomyocytes also had significantly reduced contractile force production / cell motility and shortened contraction times (Figures...
Embodiment 3
[0248] Example 3: Attenuation of the Cardiac Gene Program in Mutant CPCs and Cardiomyocytes
[0249]RNA-seq was performed on isogenic iPS cells at day 7 during cardiac differentiation into CPCs, contracting cardiomyocytes before (D15) and after lactate purification (D32) ( FIG. 28 ). The LASSO regression algorithm (Roost et al., 2015) accurately predicted that our iWT cardiomyocyte data represented the cardiac transcriptome (0.6-1) rather than other human tissues, whereas the G296S transcriptome from CPC, D15-cardiomyocytes and D32-cardiomyocytes Consistently low cardiac score (<0.6) (Figure 29). During at least one of the 3 time points, 2228 genes were differentially expressed in G296S cells with significant dynamic changes from CPC to mature cardiomyocytes (Fig. 30,31). There were 38 genes involved in the Wnt-PCP pathway, or vasculature, endocardium, cardiac development and cardiac progenitor cell differentiation that were consistently downregulated or upregulated (Figures ...
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