Human Pluripotent Stem Cells to Model Congenital Heart Disease
Congenital heart disease (CHD) is the most common cause of neonatal mortality related to birth defects. Etiology is multifactorial including genetic and/or environmental causes. The genetic etiology is known in less than 20 % cases. Animal studies have identified genes involved in cardiac development. However, generating cardiac phenotypes usually requires complete gene knockdown in animal models which does not reflect the haplo-insufficient model commonly seen in human CHD. Human pluripotent stem cells which include human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) provide a unique in vitro platform to study human “disease in a dish” by providing a renewable resource of cells that can be differentiated into virtually any somatic cell type in the body. This chapter will discuss the use of human pluripotent stem cells to model human CHD.
KeywordsHuman embryonic stem cells Induced pluripotent stem cells Williams syndrome Hypoplastic left heart syndrome Fetal reprogramming
Human embryoni c stem cells (hESC) can give rise to all three germ layers – ectoderm, endoderm, and mesoderm – and can be used to generate differentiated cells of different lineages . The Nobel prize-winning discovery by Yamanaka of the ability to reprogram somatic cells to induced pluripotent stem cells (iPSC) using specific reprogramming factors  uncovered a whole new field of research focused on the use of iPSCs to model human disease, perform drug screens, and explore strategies for autologous cell-based therapies in the future. Reprogramming protocols include ectopic expression of four transcription factors [2, 3] that induce reprogramming of somatic cells into an embryonic state. Viral integration-free protocols are also used albeit are less efficient. These cells can then be expanded and differentiated into several somatic cell types including cardiac lineages such as cardiomyocytes, vascular smooth muscles cells (SMCs), and endothelial cells. The process of cardiac differentiation of hESCs and hiPSCs recapitulates cardiac embryogenesis thereby providing a unique opportunity to explore the impact of gene or environmental defects on early cardiac development and gain novel insights into disease mechanisms . Strategies for modeling cardiac malformations are discussed.
45.2 Modeling Fetal Cardiac Reprogramming in Hypoplastic Left Heart Syndrome (HLHS)
HLHS is one of the m ost severe cardiac malformations characterized by poor growth of left-sided cardiac structures. This is commonly associated with endocardial fibroelastosis (EFE). The mechanism of LV growth failure and fibrosis is poorly understood. We studied 29 normal and 30 HLHS fetal hearts during second trimester . We found increased nuclear expression of hypoxia-inducible factor 1α (HIF1α) in fetal HLHS compared to normal LVs, a central hypoxia-responsive gene that promotes activation of angiogenic, metabolic, and other genes to facilitate cardiac adaptation to hypoxia. However, expression of vascular endothelial growth factor (VEGF) was downregulated. The failure of hypoxia-induced angiogenesis was likely related to cell senescence as shown by DNA damage (nuclear γH2AX activation and p53 upregulation) and of cell senescence (β-galactosidase upregulation). Senescent cells, although functional, do not produce growth factors essential for the survival and proliferation of stem/progenitor cells thereby compromising tissue renewal capacity. Not surprisingly, HLHS hearts showed fewer cardiac progenitor markers, as well as reduced differentiated cardiomyocyte and endothelial cells. DNA damage was most prominent in endothelial cells followed by myocytes, with SMCs being least susceptible. Additionally, there was increased transforming growth factor (TGFβ1) expression, increased myofibroblast transformation, and increased interstitial and perivascular fibrosis in fetal HLHS compared to controls. Together this suggested that the fetal LV may be susceptible to chronic hypoxia or reduced blood flow (a phenomenon that occurs in HLHS due to reduced antegrade flow through the diminutive ascending aorta) resulting in DNA damage and cell senescenc e and consequent loss of cell replication and growth capacity as well as fibrosis.
These findings have several implications. They suggest that antenatal intervention for HLHS may be more effective in promoting LV growth if performed before irreversible tissue injury. However, complementary strategies to provide missing growth factors and/or inhibit TGFβ1 either pre- or postnatally may be needed to promote LV growth and ameliorate progressive fibrosis.
45.3 hiPSCs to Model Williams-Beuren Syndrome (WBS)
45.4 Future Directions and Clinical Applications
These studies provide proof of principle that hESCs and iPSCs can generate in vitro models to study CHD. However, the cardiac lineages generated using this approach are relatively immature, i.e., fetal stage. While fetal stage cells may be well suited to study developmental cardiac disorders , maturation protocols that generate more functionally mature lineages may be more useful to study late-onset disease phenotypes and accurately evaluate drug responses [11, 12, 13, 14]. Our study further suggests that the technology can be expanded to study not just genetic influences, particularly in the rapidly emerging era of genome editing , but also environ mental teratogens (toxins, chemicals, drugs, infections) to define the mechanisms by which they impact fetal cardiac development or differentiation. This may facilitate delineating the combined role of genetic and environmental factors in CHD causation in the near future . The ability to differentiate pluripotent stem cells into many different organ or cell types may allow the study not only of cardiac but also of extracardiac phenotypes particularly in syndromic disorders as recently shown in a patient with Timothy syndrome [17, 18].
In summary, pluripotent stem cell-derived models are revolutionizing our understanding of disease pathogenesis and are positioned to expedite drug screening and discovery particularly for rare cardiac disorders with a genetic basis for which no therapies are available and where clinical studies are challenging. The technology provides a renewable source of functional cardiomyocytes and other cardiac lineages with genetic and epigenetic variation that are likely to be more human relevant. While the use of these cells for in vivo therapies is several years away, this platform is well positioned to study the molecular underpinnings of genetic cardiac disorders and help identify new therapies for personalized care of the affected child.
The work was funded by the Canadian Institute of Health Research (MOP 126146), SickKids Labatt Family Heart Centre Innovations fund, and the Ontario Ministry of Research and Innovation GL2 award.
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