The Use Of Induced Pluripotent Stem Sells (iPSCs) And Mesenchymal Stem Cells (MSCs) To Study The Genetic Basis Of Human DiseasesI. The Use Of Mesenchymal Stem Cells (MSCs) For Treatment Of Diabetic Wound In Nude Mice.II. The Use Of Induced Pluripotent Stem Cells (iPSCs) In Modeling Neutrophil Defects Resulting From A Single-Gene Mutation

Abstract

Objectives: The aim of this thesis was to evaluate the potential of new technologies, including mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), to understand the molecular basis of human diseases. These technologies were evaluated for their ability to restore diabetes-induced defects in wound tissue repair (MSCs) and to generate mature neutrophils after in vitro differentiation of iPSCs. The latter used iPSCs from a patient with impaired WASp (Wiskott Aldrich syndrome protein) signalling. Other techniques evaluated were differentiation of the myeloid cell line, PLB-985 (expressing exogenous genes) into mature neutrophils and new advances in metabolomics, to identify altered neutrophil function in human diseases. Methods: The potential of MSCs and oral vitamin C to generate factors that could promote healing of diabetic wounds, was measured by RT-PCR for eight genes associated with either angiogenesis or extracellular matrix production, after incubation under normoglycaemic and hyperglycaemic conditions with and without vitamin C. The angiogenic effects of the MSC secretome on wound healing was measured using a tubular formation assay (in vitro) and a nude mice diabetic wound model (in vivo). The bilateral full-skin thickness wounds were created in an in vivo wound model using diabetic nude mice. Oral vitamin C (1.5 g/L) was administered in combination with topical MSC treatment (MSCs 1x 106 cells per wound). The capillary density was measured under in vivo fluorescent microscopy. WAS dermal fibroblasts were reprogrammed using retrovirus transfection, and the corrected-WAS-iPSCs were differentiated into the neutrophil-like cells via the formation of iPS-sacs (containing haematopoietic progenitor cells derived from iPSCs). Neutrophil (from WAS patients and healthy controls) chemotaxis was measured using transwell migration towards N-formylmethionine-leucyl-phenylalanine (fMLP). PLB-985 and KCL-22 cells were differentiated into neutrophil-like cells using RPMI-1640 media containing N,N-dimethyl formamide, sodium pyruvate, all-trans retinoic acid, human AB serum and dimethyl sulfoxide. Morphology was assessed by cytospin. PLB-985 cells were transduced with enhanced green fluorescent protein (EGFP)-tagged Myeloid Cell Leukaemia-1 (Mcl-1), sub-cloned into a pLVX-TetOne-Puro system. 1H NMR metabolomics was carried out using protocols optimised for neutrophils as part of this thesis. The NMR analyses were also optimised to identify neutrophil metabolites and allow the comparison from resting and activated states and in health and disease (rheumatoid arthritis patients). Results: Upregulation of angiogenic genes, vascular endothelial growth factor-α (mVEGF-α) and platelet-derived growth factor-BB (mPDGF-BB), in response to TGF-β1 in MSCs was lower following incubation under hyperglycemia (compared to normoglycaemic controls), but vitamin C treatment re-sensitised the MSC response to TGF-β1. A diabetic mouse model showed that administration of oral vitamin C, as an adjunct to MSC therapy, resulted in accelerated wound healing that was associated with increased capillary density. Preliminary experiments with WAS neutrophils showed significantly lower rates of chemotaxis towards fMLP compared to healthy controls. iPSCs from WAS fibroblasts were cultured and differentiated into neutrophil-like cells. PLB-985 cells, transfected with Mcl-1:EGFP in pLVX-TetOne-Puro system were generated. Nuclear magnetic resonance (NMR) metabolomics identified metabolites and pathways altered during in vitro activation with PMA (including metabolites of NADPH synthesis and inhibitors of reactive oxygen species (ROS)) and in vivo activation in rheumatoid arthritis identified metabolites of the ketosis pathway, citrullination pathway and tryptophan metabolism. Conclusions: A number of technologies have been evaluated to study the molecular basis of human disease, including metabolic (diabetes mellitus) and genetic (WAS) diseases. Vitamin C modulated the secretome of MSCs, increasing angiogenesis and accelerating wound healing, providing a potential new approach for designing adjuncts to existing therapies. Neutrophils from WAS patients demonstrated chemotactic defects, and the potential of WAS-iPSCs to differentiate into neutrophil-like cells was demonstrated. The approach could be applied in further studies to study genetic defects of leukocyte function. PLB-985 cells transduced with EGFP-tagged Mcl-1 in an inducible expression vector, was developed as a cell-line model of neutrophil differentiation, to facilitate further studies into the role of the Mcl-1 gene in regulating neutrophil survival. Protocols for human neutrophil metabolomics, using 1H NMR spectroscopy were developed and applied to the study of in vitro and in vivo activated neutrophils. The results demonstrated the potential of metabolomics for future studies of human diseases.