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Title: Embryonic stem cell-derived macrophages : a novel approach to developing anti-inflammatory macrophages for cell therapy
Author: Nkejabega, Noémie Caroline J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2013
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Ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) that is associated with high morbidity and mortality. Macrophages (Mφ) are multifunctional cells involved in the initiation, progression and resolution of kidney inflammation response. The enzyme hemeoxygenase-1 (HO-1) is upregulated in response to cell stress and metabolises heme-containing proteins to carbon monoxide (CO) and bilirubin that possess anti-apoptotic and anti-oxidant properties. Previous work has shown that Mφ can be used as therapeutic vectors with the administration of bone marrow-derived macrophages (BMDM) overexpressing HO-1 being protective in murine renal IRI (Ferenbach et al., 2010). However, these studies used primary BMDM that are inherently heterogeneous with the genetic manipulation achieved by adenoviral transduction that is not suitable for use in patients. Previous work has shown that pure Mφ populations may be generated from murine and human embryonic stem cells (ESC) in vitro, providing an essentially limitless source of Mφ that can be derived from genetically manipulated cells. This project has adopted an ESC approach to develop anti-inflammatory ESC-derived macrophages (ESDM) overexpressing HO-1 for therapeutic use in experimental models of AKI. We successfully generated Mφ from ESC and demonstrate that ESDM are comparable to BMDM in all properties that we tested. ESDM are large mononuclear cells and express Mφ cell surface markers (F4/80^high CD11b^high CD11c^high MHC class II^low). ESDM are phagocytic and produce pro-inflammatory mediators when activated by lipopolysaccharide and interferon-γ (LPS and IFNγ). ESC were then genetically manipulated to overexpress HO-1 under the control of the constitutive pCAG promoter. Our results suggest that a high level of constitutive HO-1 overexpression in ESC impacts on ESC number, self-renewal and Mφ differentiation. Thus, we decided to generate ESC that overexpress HO-1 under an inducible promoter using the A2lox.cre system. HO-1 inducible ESC exhibited significant HO-1 up- regulation and generated F4/80^high CD11b^high CD11c^int MHC II^low ESDM. However, HO -1 inducibility by doxycycline (Dox) was lost following both ESDM differentiation and RA induced differentiation of ESC. A similar loss of Dox-induced gene expression following ESDM differentiation or RA induced cell differentiation was found in ESC expressing a control eGFP transgene under the control of the same inducible promoter. In view of the difficulties encountered using the inducible systems, a pharmacological approach using treatment with the potent HO-1 inducer hemearginate (HA) was used to upregulate HO-1 expression in ESDM. HA treatment significantly upregulated HO-1 expression by ESDM. HA treatment modulated ESDM phagocytosis and the production and gene expression of both pro- and anti-inflammatory mediators (NO, TNFα, IL-10 and IL-6) following LPS and IFNγ stimulation. Intravenous injection of fluorescently labelled ESDM directly after inducing renal IRI resulted in the localisation of the ESDM to solid organs and preferential homing to the injured kidney at the 1 hour time point indicating their potential for use in cell therapy. In conclusion, we were able to generate functional Mφ from ESC. The differentiation of ESC to Mφ resulted in the loss of inducible gene expression and pharmacological HO-1 induction was used. HA treated ESDM present promising anti-inflammatory properties in vitro and home to the injured kidney in a model of murine IRI in vivo. Further experiments are still needed to determine if HA treated ESDM exert anti- inflammatory properties in vivo. However, our findings are a strong platform for future studies of the translational potential of using modified ESDM in cell therapy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available