Extracellular factors for preservation and delivery of stromal cells

Abstract

Modulating the immune response after a myocardial infarction seems like an appropriate strategy for reducing myocardial fibrosis. Mesenchymal Stromal Cells are immunomodulatory and have thus gained interest, but have so far not achieved the desired clinical outcomes. This is believed to due to the loss of their immunomodulatory and proliferative capacity during expansion and poor cell survival and retention upon delivery to the myocardium. The use of extracellular factors such as extracellular matrices, paracrine factors, nutrients as well as manipulation gas composition during culture might be used to overcome some of these shortcomings, which is further explored in this thesis.

We demonstrated in Study I, that encapsulation of human cells by thermos-responsive microcapsules, which upon exposure to physiological temperature partially decompose and enable release of the cells. The hydrogel combination of agarose, gelatin and fibrinogen provided both thermos-responsive features and attachment points for the cells, preventing cell death. However, additional components can be used to support the encapsulated cells while retaining the thermo-responsiveness. In order to discover such components, we developed an in vitro model to study the cell- and extracellular matrix dynamics making use of the organ’s extracellular matrix and define anatomical regions that are capable of retaining the desired phenotype of the cell. To generate such a syngeneic model, naïve stromal cells were isolated from fetal rat hearts, and cultured on decellularized extracellular matrix sections of adult rat hearts. We found that when culturing cells with pericyte-like characteristics on the matrices, the surface marker expressions of CD146 and PDGFR-β were depending on the matrix composition, and especially of laminin alpha 4. Cells expressing CD146 were mainly located to the atrioventricular junction and to the perivascular niche, while PDGFR-β expression was more widespread. Since CD146 is also a potency marker for Mesenchymal Stromal Cells, these results indicate a matrix dependent niche for naïve stromal cells. These findings were next verified by immunohistochemistry of the native rat heart, where CD146 populations were mainly found in the atrioventricular and perivascular niche.

In Study III, we explored the preferred metabolism of adult and fetal MSCs. It is known that proliferating stem-, progenitor cells utilize glycolysis, even in presence of oxygen. Therefore, we wanted to explore the metabolic profiles of human fetal (naïve) and MSCs during culture in either hypoxia 3% (close to physiological oxygen tension) or normoxia 20%. Adult MSCs grown in hypoxia retained oxidative phosphorylation and increased glycolytic activity, adapting a progenitor metabolic profile while in normoxia the adult MSCs down-regulated glycolysis and adapted an adult, or differentiated cell metabolic profile. Fetal MSCs demonstrated preserved oxidative phosphorylation and glycolytic activity regardless of oxygen tension, thus exhibiting a stem-, progenitor metabolic profile. The findings from these studies might help in designing future culture protocols and delivery systems for cell therapies.