We also provide an immunofluorescence protocol for comparing, characterizing, and analyzing the differences between the healthy and diabetic organoids, and comment on additional steps for preparing the organoids for analysis by other techniques after differentiation. Organoids are then differentiated under healthy (normal insulin and glucose) and diabetic conditions (high insulin and glucose) over time, allowing for the study of the effects of pregestational diabetes on the developing human heart. The protocol starts with the generation of hPSC-derived embryoid bodies in a 96-well plate, followed by a small molecule–based three-step Wnt activation/inhibition/activation strategy. By using glucose and insulin to mimic the diabetic environment that the embryo faces in PGD, this system allows modeling critical features of PGD in a human system with relevant physiology, structure, and cell types. To bypass many of these technical and ethical limitations, we describe here a human pluripotent stem cell (hPSC)–based method to generate developmentally relevant self-organizing human heart organoids. Clinical practice precludes direct studies in developing human embryos, further highlighting the need for physiologically relevant models. While studies using animal models and cells in culture have demonstrated that PGD alters critical cellular and developmental processes, the mechanisms remain obscure, and it is unclear to what extent these models recapitulate PGD-induced CHD in humans. The prevalence of PGD-induced CHD is increasing due to the ongoing diabetes epidemic. PGD is challenging to manage clinically due to the extreme sensitivity of the developing embryo to glucose oscillations, and constitutes a critical health problem for the mother and the fetus. Maternal diabetes during the first trimester of pregnancy (pregestational diabetes, or PGD) is one of the most prominent factors contributing to CHD, and is present in a significant population of female patients with diabetes in reproductive age. Congenital heart defects (CHD) constitute the most common type of birth defect in humans.
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