Integrative Studies from the Embryo to Scalable Multipronged Generation of hESC-derived Myocardial Progenitors for Heart Repair

Advanced heart failure is a major unmet clinical need arising from the loss of viable and functional cardiac muscle leading and is a major cause of mortality and morbidity. To date, while there have been early suggestions of small therapeutic benefits of regenerative cardiovascular approaches, there has not been evidence for robust regeneration of heart muscle tissue without genetic manipulation [1] highlighting the need for new approaches. A central challenge for stem cell-based regenerative therapy, as well as for the development of novel human drug discovery assays, is therefore the generation of large numbers of human cardiac myocytes for downstream clinical applications. Embryonic Stem Cells (ESCs) and induced Pluripotent Stem (iPS) cells represent a renewable potentially patient-specific source of cardiac progenitors and cardiomyocytes that can be used to generate cardiac tissue renewable potentially patient-specific source of cardiac progenitors and cardiomyocytes that can be used to generate cardiac tissue for in vitro drug screening and serve as a basis for regenerative cardiovascular medicine.
We have identified a secreted protein containing collagen and calcium binding EGF-like domains (Ccbe1) in differential screen designed to identify new genes preferentially expressed in the cardiac precursors [2]. We demonstrated that Ccbe1 is expressed in progenitors from the first and second heart fields as well as the proepicardium of embryonic mice [3]. In humans, CCBE1 mutations have been associated with Hennekam syndrome, a disorder characterized by abnormal lymphatic system development as well as multiple congenital heart defects [4, 5]. Recent data from our lab demonstrates that Ccbe1 is expressed in mouse ESC-derived cardiac progenitors, and knockdown of Ccbe1 during ESC differentiation results in a decreased number of cardiac progenitors.
Conversely, supplementing the ESC differentiation medium with Ccbe1 results in increased formation of mature cardiomyocytes. Collectively, these results argue that Ccbe1 is required for the normal formation and maintenance of mouse ESC-derived cardiac progenitors and ultimately functional cardiac myocytes. As a diffusible secreted protein, Ccbe1 therefore represents an important tool that should allow us to direct the formation of mammalian cardiac progenitors from a renewable stem cell source without the need of genetic manipulation. Accordingly, we propose to exploit these properties of Ccbe1 in a validated xeno-free bioprocesses to reproducibly generate large numbers of functional cardiac myocytes from embryonic stem cells differentiating in vitro. The group responsible for setting up the xeno-free system using Ccbe1 to direct cardiac differentiation, iBET, has shown that human ESCs exhibit improved cell growth and retain their stemness in a 3D scaffold microenvironment [6, 7]. Then, the directed differentiation into specific cell lineages can be subsequently induced by the use of chemically defined media and it is therefore at this stage that Ccbe1 will be used to direct hESCs differentiation towards relevant cardiac lineages.
Despite the recent progress in understanding the biology of Ccbe1, the downstream cellular processes and signaling pathways are not well understood. Herein we propose to delineate the temporal and spatial localization of Ccbe1 during mammalian heart development. We also propose to define the role of Ccbe1 in regulating cell proliferation, cell survival and cell migration in the developing heart [8, 9]. Lastly, in light with its known extracellular localization and genetic and protein interactions [10, 11], we propose several experiments where we aim to understand how the Ccbe1 protein is processed and thereby establish its active
domains and their molecular mechanism.
The current multidisciplinary collaborative proposal integrates the scientific and technological expertise and reagents of three laboratories in Portugal and at Harvard to bring the promise of developmental and stem cell biology, protein chemistry, and bioreactor technology to generate a robust xeno-free scalable system for the scalable production of human cardiac myocytes from a renewable patient-specific cell source for regenerative cardiovascular medicine.