Functional Analysis of Candidate Genes Associated with Congenital Heart Disease during Differentiation of Induced Pluripotent Stem Cells and in the Human Embryonic and Adult Heart at Single-Cell Resolution

Objectives: Genetic factors definitely play an essential role in the pathogenesis of congenital heart disease (CHD). We previously identified risk loci associated with CHD by genome-wide association study (GWAS). We now sought to identify the functional role of our candidate genes during cardiac differentiation of human induced pluripotent stem cells (iPSCs) and in the embryonic and adult human heart at single-cell resolution.

Methods: Relative expression of candidate genes during cardiac differentiation of human iPSCs was determined by qRT-PCR using β-ACTIN as a reference. Atrial and ventricular biopsies were obtained during surgery at our institute and were directly snap-frozen. Single nuclei were purified, and subsequently processed for RNAseq analysis according to the manufacturer's protocol. For embryonic human cells a previously published dataset was re-analyzed.

Result: In our previous GWAS, single nucleotide polymorphisms mapping to the MACROD2 and GOSR2 locus were associated with transposition of the great arteries and anomalies of thoracic arteries and veins, respectively. Further variants were proposed to modulate the expression of WNT3 and MSX1. During cardiac differentiation of human iPSCs expression of WNT3 and MSX1 peaked early at day 2 to 4. In contrast, the expression of MACROD2 gradually increased and reached its maximum at a later time-point on day 10, while the abundance of GOSR2 mRNA was similar at all time-points. In human embryonic cells, MACROD2 and GOSR2 were highly expressed in many cells of all developmental stages and anatomical regions of the heart. WNT3 and MSX1 were broadly expressed at a lower level and could be identified more concentrated in fibroblasts and myocytes. In adult human hearts, MACROD2 was strongly expressed in every cell type, while GOSR2-positive cells were completely absent. The distribution of WNT3 and MSX1 expressing cells in adult hearts was similar to the embryonic pattern, though the expression level was lower and MSX1 was virtually absent in myocytes.

Conclusion: The expression patterns of our candidate genes during cardiac differentiation of human-induced pluripotent stem cells in vitro and single-cell RNAseq analyses of embryonic and adult human hearts attribute an essential role to MACROD2, GOSR2, WNT3, and MSX1 at all developmental stages of human cardiogenesis.

No conflict of interest has been declared by the author(s).

Publication History

Article published online:
19 February 2021