Bioprocessing of human Pluripotent Stem Cells for cardiac cell therapy and pre-clinical research

• Autor(es): Correia, Cláudia Susana Pedreira
• Data: 2017
• Identificador Persistente: http://hdl.handle.net/10362/20446
• Origem: Repositório Institucional da UNL
• Assunto(s): Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química
• Tipo de Documento: Tese de doutoramento
• Idioma: Inglês
• Orientador(es): Alves, Paula; Serra, Margarida
• Contribuidor(es): RUN

Descrição

Cardiovascular diseases remain the leading cause of death worldwide. Some of these diseases, e.g. myocardial infarction (MI), are associated with a massive and permanent loss of cardiomyocytes (CMs), a non-proliferative and terminally differentiated cell population in the heart. Available pharmacological and interventional therapies are not suitable to amend the effects of this cell loss, mainly due to the limited regenerative capacity of the myocardium, and heart transplantation is limited by the number of compatible organs donated. Recently, human pluripotent stem cells (hPSCs), have emerged as attractive candidate cell sources to obtain CMs. Due to their inherent capacity to proliferate indefinitely and to differentiate into all mature cells of the human body, hPSCs constitute the unique cell source that can provide, ex-vivo, an unlimited number of functional CMs suitable for cell therapy and other applications including disease modeling and cardiotoxicity drug testing. Nonetheless, the complex nets of signaling pathways involved in cardiomyogenesis as well as the line-to-line variability compromise the effectiveness of the existing differentiation protocols to reproducibly produce high-quality CM from multiple hPSC lines. The immature phenotype of the produced hPSC-CMs and the lack of efficient methods for worldwide shipment of these cells also constrain the applicability of these cells in the clinic and industry. The main aim of this thesis was to devise robust, scalable and integrated approaches for the production and maturation of hPSC-CMs. The strategy consisted in exploring the impact of environmental factors, cell culture configuration, and metabolic substrate availability on bioprocess yields and cell’s quality using a set of “-omic” tools, namely transcriptomics, metabolomics and fluxomics, and cell characterization assays. In Chapter 1, the recent advances on the use of hPSC for cardiac cell therapy were reviewed. In Chapter 2, the effect of dissolved oxygen and bioreactor hydrodynamics on CM differentiation was explored. It was demonstrated that combining a hypoxia culture (4% O2 tension) with wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields. Chapter 3 focused on the development and characterization of a robust protocol for directed differentiation of hPSC towards CMs, suitable to generate CMs in both 2D monolayer and 3D aggregate culture formats. The culture of hPSC-cardiac progenitors as 3D aggregates revealed to be an efficient approach to improve CM enrichment and commitment. Although hPSC-CMs generated from both methods revealed to be highly glycolytic, 3D aggregate cultures showed slightly improved metabolic energetics (including increased TCA-cycle activity and ATP production). Chapter 4 assessed whether alterations in hPSC-CM culture medium composition to mimic in vivo substrate availability during cardiac development would induce hPSC-CM maturation in vitro. It was demonstrated that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing media promotes their maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to ventricular CMs, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics, within 10-20 days. The feasibility to cold store hPSC-CMs monolayers and aggregates in a fully-defined clinical compatible formulation was evaluated in Chapter 5. It was demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage–induced cell injury in 3D aggregates than in 2D monolayers, showing high cell recoveries, typical (ultra)structure and functionality after 7 days of storage. Chapter 6 consists of a general discussion, where the main scientific and technological outcomes of this thesis are outlined. Overall, by pursuing an holistic approach that brought together a quantitative molecular and metabolic characterization, this thesis provides novel insights into the interaction of metabolism and CM differentiation/maturation but also establishes robust and scalable methods for production, functional maturation and short-term storage of hPSC-CMs. This will pave the way for the widespread application of hPSC-CMs in clinical and preclinical applications.