Biotechnological approaches to cardiac differentiation of human induced pluripotent stem cells

ABSTRACT The heart can be considered the most important organ of our body, as it supplies nutrients to all the cells. When affected from injuries or diseases, the heart function is hampered, as the damaged area is substituted by a fibrotic scar instead of functional tissue. Understanding the mechanisms leading to heart failure and finding a cure for cardiac diseases represents a major challenge of modern medicine, since they are the leading cause of death and disability in Western world. Being the heart a vital organ it is difficult to have access to its cells, especially in humans. In order to model it or find therapeutic strategies many approaches and cell sources have been studied. For example cardiac stem cells, skeletal myoblasts, bone marrow-derived cells and peripheral blood mononuclear cells have been tested in pre-clinical and clinical trials, without significant tissue regeneration. Human pluripotent stem cells (hPSC) are thought to be the most promising cell type in the field, thanks to their unlimited capacity of self-renewal and retention of differentiation potency. Induced pluripotent stem cells (iPSC) are pluripotent cells derived through reprogramming from adult cells, easily accessible from patients, like keratinocytes. iPSC can be differentiated to cardiac cells, through stage-specific protocols that reproduce embryonic development, offering a very useful platform for modelling diseases of patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, properly mimicking cardiac tissue is very complex, since not only the correct cardiac cell type has to be reproduced, but also its overall cellular composition, architecture and biophysical functions. In order to study these aspects, we applied biotechnological strategies such as the use of transgenic cell lines for obtaining pure and scalable differentiated cells to be cultured in a 3D scaffold with a perfusion bioreactor. Although it is well known that iPSC can give rise to cardiomyocytes in vitro, not every cell line can be efficiently differentiated. Thus, a cell line-specific differentiation protocol has to be identified and optimized. We finally identified a fast and efficient stage-specific differentiation protocol suitable for the iPSC lines used in this work, derived from human keratinocytes. With this protocol, we can reproducibly obtain close to 50% cardiomyocytes after 15 days of differentiation. One important feature of currently available differentiation protocols is that the target cell type is obtained among a heterogeneous cell population. To track the cardiac population of interest we generated transgenic cell lines where the reporter protein GFP follows the expression of different genes specific for stages of differentiation, such as T (Brachyury) for mesoderm; NKX2.5 for cardiac progenitors; and MHC for cardiomyocytes. Moreover, cardiomyocytes obtained from hPSC using currently available differentiation protocols are typically immature, mostly resembling embryonic or fetal cardiomyocytes, arguably because of the lack of mechanical and electrical stimuli that only a 3D environment can provide. In order to create a piece of tissue in 3D we used a collagen and elastin-based scaffold, to mimic the structural proteins of endogenous extracellular matrix. We also built a perfusion bioreactor to culture the construct. After initial validation with primary cultures of rat neonatal cardiomyocytes, we tested iPSC-derived cardiac cells at different stages of differentiation. While early mesoderm or cardiac progenitors could not survive in our system, iPSC differentiated to cardiomyocytes, could be retained and maintained alive within the scaffold for at least 4 days. In conclusion, in this work we combined biotechnological tools in order to obtain a test platform for studying the mechanisms underlying cardiac differentiation, maturation, as well as providing valuable in vitro systems for disease modelling, drug screening of patient-specific heart muscle cells and cell therapy.