Pluripotent stem cells (PSCs) can self-renew indefinitely in culture while maintaining their capacity to differentiate into any cell type of an organism, thus offering novel sources for drug screening, in vitro disease modelling, and cell replacement therapies. However, due to their sensitive nature, many PSC lines are still cultured using undefined components such as serum or serum-derived components, on either feeder cells or complex protein mixes such as Matrigel or gelatine. In order to fully realize the potential of these cells we need controlled, completely defined and xeno-free culturing conditions that maintain growth and survival of homogenous, non-differentiated colonies. This thesis focuses on the in vitro maintenance of both mouse and human PSCs, analysing the media and substrate requirements of these cells and linking them to the intracellular signalling pathways involved in the maintenance of pluripotency and self-renewal. Benchmarking of commercially available culture methods for PSCs has been performed, evaluating their capacity to maintain pluripotency and growth of undifferentiated PSCs over several passages and reporting new characteristics, like the tendency of mouse PSCs to grow as floating spheres in 2i medium, a novel media formulation that uses two inhibitors to hinder differentiation capacity and subsequently induce pure, undifferentiated cultures. The major finding in this thesis is the identification of Inter-α-Inhibitor (IαI) as a protein able to activate the previously described signal-transduction pathway Yes/YAP/TEAD in mouse PSCs and to induce transcription of the well-known stem cell transcription factors Nanog and Oct3/4. IαI is a serum protein found in high concentration in human serum that had been traditionally described as an extracellular matrix remodelling protein. For the first time, we describe IαI to have signalling capacity on PSCs. Moreover, IαI is demonstrated to induce attachment, growth and long-term survival of undifferentiated mouse and human PSCs when added to serum-free, chemically defined media. IαI is the first molecule described to date to induce attachment of human PSCs on uncoated, standard tissue-culture treated plastic, just by supplementation as a soluble molecule at the seeding step. Following this discovery, we evaluate a novel culture method using the completely defined, serum-free E8 medium supplemented with IαI (E8:IαI) for long-term propagation of four different human PSC lines and discover that IαI can indeed support long-term culture with maintained pluripotency, differentiation capacity, growth rate and genetic stability. Moreover, in contrast to the control culture method using a commercially available surface coating, IαI supplementation can support single cell passaging of human PSCs, and adapt feeder-dependent cultured human PSCs to E8:IαI with high efficiency. A mouse PSC line is also grown for over 20 passages in IαI with retained pluripotency, differentiation capacity and genetic stability. IαI is inexpensive to produce and derived from human plasma, and could therefore be produced in compliance with Good Manufacturing Practices. Ultimately, our group aims to develop and test large-scale, completely defined, xeno-free culturing methods for PSCs, suitable for pharmacological and medical applications.

Indiana University-Purdue University Indianapolis (IUPUI) / The extracellular matrix (ECM) provides a structural and signaling platform for cells that comprise various organs, playing a critical role in tissue maintenance, injury, and repair. Hyaluronan (also known as hyaluronic acid, HA) is a ubiquitous ECM polysaccharide consisting of a repeating disaccharide backbone that can be covalently modified by the heavy chains (HC) of the serum protein inter-alpha-inhibitor (IαI) during inflammation. Known as the only covalent modification of HA, the HC linking of HA is exclusively mediated by the inflammation-induced secreted enzyme TNFα-stimulated gene-6 (TSG-6). Mice deficient for HC-HA formation, due to the lack of either TSG-6 or IαI, display reduced survival during systemic lipopolysaccharide (LPS)-induced endotoxic shock and its associated acute lung injury. We therefore hypothesized that HC-HA should play an important protective role against acute lung injury induced by intratracheal LPS or Pseudomonas aeruginosa (PA) gram-negative bacteria. We also identified that lung instillation of LPS or PA caused rapid induction of lung parenchymal HC-HA that was largely cleared during resolution of injury, indicative of a high rate of HA turnover and remodeling during reversible lung injury. However, using TSG-6 knockout mice, we determined that HC-HA exerted minimal protective effects against intratracheal LPS or PA-induced acute lung injury. To better address the differential roles of HC-HA during systemic versus localized intratracheal exposure to LPS, we characterized and compared the induction of HC-HA in plasma and lung in these two models. While lung parenchymal HC-HA formed in both injury models, intravascular HC-HA and TSG-6 were exclusively induced during systemic LPS exposure and were associated with improved outcomes, including decreased number of circulating neutrophils and plasma TNFα levels. Our results suggest that LPS induces HC-HA formation in various tissues depending on the route of exposure and that the specific intravascular induction of HCHA during systemic LPS exposure may have a protective role during endotoxic shock.