Regenerative strategies with human amniotic fluid stem cells for neonatal disorders

Cell therapeutic approaches are fast emerging in human medicine, potentially enabling novel therapies for patients. Stem cells could be clinically translated for the replacement or repair of damaged or diseased structures, or the restoration of a normal organ’s function.
In this doctoral research, we explored the use of amniotic fluid-derived mesenchymal stem cells (AF-MSC) as a cell therapeutic tool in a perinatal setting, focusing on two conditions, namely bronchopulmonary dysplasia (BPD) and soft tissue defects (e.g. the diaphragmatic defect in congenital diaphragmatic hernia; CDH). The reasoning behind exploring the potential of the amniotic fluid as a stem cell source was threefold. Firstly, the use of AF-MSC enables autologous application of cells, which eliminates the risk for immunological reactions. Secondly, AF-MSC are easier to obtain than their adult MSC counterparts. AF can be collected by amniocentesis or even at the time of birth, and multiple efficient AF-MSC isolation and expansion protocols are described. Finally, the AF is a source of both potent immune modulators (MSC) and of tissue-specific progenitor cells (from the fetal lung, kidney, amniotic membranes,…). Clonal derivation and characterization of these progenitor cells enables the design of organ or disease-specific treatment strategies.
In the first part of this PhD project, we aimed to understand the potential of cell therapeutic approaches in the context of an acquired lung disease (BPD). In Chapter 2, we systematically reviewed the literature on cell-based therapy in BPD animal models. We identified a total of 47 interventions, with newborn mice/rats raised in a hyperoxic environment as a model for BPD in nearly all interventions. Several stem cell types –especially bone marrow and umbilical cord blood derived mesenchymal stem cells- and their conditioned medium yielded beneficial effects. Meta-analysis of the results was impeded by a lack of standardized outcome measures. In addition, most studies used only histology as an outcome parameter, without investigating mechanistic or long-term outcomes, which we consider pivotal to move forward with this research. In Chapter 3, we assessed the potential of clonal AF-MSC to modulate BPD-like injury. For this we employed a hyperoxia-induced preterm rabbit model of BPD, as we believe human pulmonary developmental and therapeutic aspects are better represented in the rabbit. We assessed both naïve AF-MSC and AF-MSC genetically modified to overexpress vascular endothelial growth factor (VEGF), which is a crucial factor in distal airway development. Naïve AF-MSC reduced lung inflammation but failed to prevent hyperoxia-induced damage, whereas VEGF-overexpressing AF-MSC had a beneficial effect on both events. As such, AF-MSC prove to be a suitable delivery vehicle for the targeted delivery of bioactive agents. AF-MSC are a heterogeneous collection of MSC derived from multiple developing organs including the fetal skin, kidneys, lungs, amniotic membranes, etc. The moderate response induced by naïve AF-MSC
in rabbits with hyperoxia-induced lung injury, stimulated us to search for AF-MSC with a pulmonary progenitor phenotype as such cells may be more potent for lung regeneration. In Chapter 4, we determined that one quarter of clonal AF-MSC lines isolated between 15-26 weeks of gestation has lung characteristics, based on their specific marker expression and in vivo differentiation capacity. These characteristics can be maintained by culturing them on fetal lung extracellular matrix coated dishes. This justifies further investigations into their therapeutic potential for perinatal lung diseases.
In the second part of this thesis we focused on the potential of AF-MSC to influence the host response to a biomaterial by paracrine modulation. Engineering biomaterials with AF-MSC would enable the delivery of a patient-specific construct for early postnatal diaphragmatic regeneration in CDH patients. In Chapter 5 and 6, we determined in rats the host response to small intestinal submucosa and electrospun polylactic acid matrices and the potential of AF-MSC to modulate this response. PLA matrices engineered with AF-MSC had a better inflammatory profile and displayed better remodeling compared to the unseeded matrix. However, this effect was not observed with the SIS matrix, which was overall well tolerated by the animals. As compared to PLA, implantation of SIS was accompanied by a modest inflammatory response and hence a minimal release of inflammatory cytokines and chemokines that could potentially activate the transplanted MSC.
In Chapter 7, we summarize the lessons learned from the research reported in this thesis and speculate on the future perspectives for cell therapy for neonatal disorders. We acknowledge the limitations of this type of clonal AF-MSC, but believe that our research provides a strong basis to continue exploring the potential of fetal MSC for therapeutic applications.