Multiple signaling pathways, which may display overlapping or redundant functions, are involved in generating cardiac progenitors and other cell lineages [57,64,65,66,67,68,69]. All issues considered, it is tempting to propose that the supplementation of exogenous cardiogenic instructive molecules at key moments during early embryonic stages may compensate for defective pathways and/or lineage decisions of cardiovascular progenitors to mitigate CHDs. To date, only a few experimental reports on animal models and stem cells support this idea. For instance, prolonged hypoxia causes DNA damage, premature senescence, impaired angiogenesis, and fibrosis associated with the upregulation of TGF-β1 expression in human fetuses with HLHS [201]. In vitro, hypoxia exposure of a human HLHS-derived iPSC disease cell model promotes the differentiation into cardiac fibroblasts instead of cardiac progenitors, cardiomyocytes, and endothelial cells [201]. Inhibition of TGF-β1 activity by its antagonist compound SB431542, in HLHS-derived iPSC exposed to hypoxia, prevented senescence and promoted genomic stability [201], suggesting that early interventions to inhibit TGF-β1 could improve ventricular growth and overcome pathways dysregulated in HLHS. IUGR is another gestational defect that hinders fetal growth in the uterus. IUGR is associated with increased placental vascular resistance, which forces the workload onto the fetal heart and increases the risk of cardiovascular disease [202]. Maternal administration of IGF-1 and IGF-2, which are known to stimulate placental and fetal growth in animal models, showed promising potential to treat IUGR in cases where downregulation of IGF-1 receptors in the placenta is not observed [202,203].

Emerging evidence suggests that the beneficial effects of stem cells in medical applications may primarily result from paracrine signaling molecules and the release of extracellular vesicles, rather than from the direct integration of stem cells or their derived cells into the target tissue [204]. Exosomes, which can be isolated and characterized by well-established approaches [205,206], present advantages such as scalable production, easy storage, consistent morphology and function, compliance with regulatory standards, and possible reduction in the variability of outcomes associated with cell therapies [207]. The therapeutic potential of exosomes and secretomes derived from hormonally primed human endometrial epithelial cells was tested in mouse models, and proven to enhance embryonic growth, development, and implantation [181]. This study also suggested that dysfunctions of the endometrial secretomes, which hinder implantation in cases of infertility, can be amended through the supply of exogenous exosomes. Also, maternal diabetic pregnancies in mouse models showed a higher occurrence of neural tube defects associated with exosomes produced from vascular progenitor cells expressing FLK1 derived from mesoderm cells lacking Survivin [208]. Interestingly, delivery into the amniotic cavity of Survivin-enriched vascular progenitor exosomes prevented neural tube defects in diabetic pregnancies [208], demonstrating the capacity of modified exosomes delivered in utero to limit neuronal pathology. Thus, exosomes are a promising non-cellular alternative to cells for clinical applications to correct embryonic defects. Exosomes can also be engineered to carry specific cargo, such as miRNAs or even gene-editing tools more suitable for the correction of genetic abnormalities associated with CHDs [176,209]. Notably, exosomes derived from the cardiac progenitors of children undergoing reconstructive heart surgeries showed promising effects in promoting angiogenesis, reducing fibrosis, resolving hypertrophy, and improving cardiac function in a rat model of heart arrhythmias caused by ischemia-reperfusion injury [39], showing the cardioprotective effect of exosomes from infant cardiac cells. Interestingly, exosomes from neonatal progenitors improved cardiac function regardless of oxygen levels, while exosomes from older children were only reparative in hypoxic conditions. Therefore, further research is needed to determine the most suitable cell sources and culture conditions for the production and modification of exosomes that would yield the best personalized clinical outcomes in the treatment of CHDs [40,208].

Like in many diseases, the early correction of a CHD is likely to result in a better clinical outcome, through early normalization of cardiogenesis. Reports on stem cell differentiation and heart development have suggested that cell positioning and functions within the heart may be determined prior to gastrulation, through precise signaling pathways and environmental cues [57,64,65,66,67,68,69]. Therefore, early interventions aiming to target the blastocyst at the pre-gastrulation/pre-implantation stage, very early embryonic states, may reduce the occurrence and severity of CHDs. Interestingly, pioneering studies on Inhibitors of Differentiation (ID) genes have revealed that double knockout of any pair of these genes in mouse models results in prominent cardiac defects and midgestational lethality of the embryos [210,211]. However, the injection of wild-type ESCs, either in ID-null blastocysts to form a chimera, or intraperitoneally in females prior to conception, rescued cardiac malformations and viability of ID-null embryos through upregulation of IGF-1 and WNT5A, without incorporation of wild-type ESCs into ID-null embryos [210,211]. Similarly, a chimeric embryo formation, using wild-type ESCs, improved the morphogenetic defects of RA signaling-deficient embryos by increasing RA expression [196]. Interestingly, transient exogenous WNT5A and WNT11, supplied at the 1-cell stage, efficiently rescued the viability and cardiac defects of CITED2-depleted zebrafish embryos and cardiac differentiation of mouse ESCs [212]. Dysfunctions of CITED2, a transcriptional modulator, have been widely associated with zebrafish, mouse, and human CHDs, as well as with embryonic lethality [55,212,213,214,215,216,217,218,219,220]. At the cellular level, CITED2 regulates the expressions of numerous genes involved in early cardiogenesis, such as BRACHYURY, MESP1, ISL1, GATA4, TBX5, MEF2C, NODAL, LEFTY1/2, PITX2C, VEGFA, WNT5A, and WNT11, among others [212,213,214]. Thus, the supplementation of WNT5A and WNT11 at the blastocyst stage in utero holds potential for rescuing CHDs associated with CITED2 dysfunction in mammals, since the function of CITED2 is conserved across vertebrates [212,214,216,221]. This strategy may also prevent CHDs triggered by the dysfunction of many genes other than CITED2, such as CITED2-target cardiogenic genes, including WNT5A and WNT11.

Moreover, WNT5A and WNT11 trigger many congenital cardiac anomalies, and contribute to DiGeorge syndrome, when defective [222,223,224,225,226,227,228]. Indeed, the WNT5A and WNT11 proteins are central for proper gestation and successful pregnancy, and their expressions are naturally highly increased and localized in the uterine luminal epithelium prior to and during blastocyst attachment to the uterus [229,230]. These proteins also promote embryonic uterine implantation and survival [229], as well as placental growth [230]. WNT5A and WNT11 are also important in late gastrulation, for the regulation of the anterior–posterior axis elongation, notochord extension, and proper patterning of the neural tube and somites [231]. In early mouse gastrulation, WNT11 is initially expressed in endoderm progenitors, and later, during mid-gastrulation, it plays a role in the formation of the embryonic and extraembryonic endothelia, as well as the formation of the endocardium in all chambers of the developing heart [232]. During late gastrulation, WNT11 showed successive waves of expression in different regions of the myocardium, important to originate left ventricle precursors (FHF progenitors) from E7.0–8.0, right ventricle progenitors (SHF progenitors) from E8.0–9.0, and the superior wall of the OFT from E8.5–10.5 (also SHF progenitors) [232]. Other studies have implicated WNT5A and WNT11 in the development of cardiac progenitors in vitro and in vivo [233,234,235,236], proper fetal hematopoiesis [237,238], kidney development [239], and guidance of sympathetic neurons to their innervation targets in vivo [240], among other processes. Altogether, the broad range of effects exhibited by WNT5A and WNT11 during early development emphasizes their high potential in limiting CHDs and other birth abnormalities when supplied exogenously at early embryonic stages. Another recent study highlighted the role of a subset of human amniotic epithelial cells (AECs) in mesoderm formation at E8.0–9.0 during early post-implantation stages [241]. Interestingly, impaired mesoderm formation and lethality due to loss of ISL1 expression in non-human primate embryos, or in human AEC differentiation in vitro due to the decrease in BMP4 expression, was partially restored through supplementation of BMP4 [241]. Overall, these findings demonstrate the potential for biomolecules, such as WNT5A/WNT11, BMP4, and inhibitors of the TGF-β pathway (SB431542, for instance), to reduce CHDs when administered at very early stages of development.