Renal ischemia/reperfusion injury (IRI) is the major cause of acute renal failure (ARF) and is still associated with high morbidity and mortality. The enzyme hemeoxygenase-1 (HO-1) is upregulated in response to various cell stresses such as hypoxia. HO-1 metabolises haem-containing proteins derived from injured cells etc to carbon monoxide (CO) and biliverdin (rapidly converted to bilirubin by biliverdin reductase). CO inhibits platelet aggregation and exerts anti-apoptotic effects whilst bilirubin is a powerful anti-oxidant. Our recent work suggests that deficient upregulation of HO-1 plays a key role in the increased susceptibility of aged mice to ARF following renal IRI (manuscript under review at JASN). Also, the administration of bone marrow-derived macrophages (BMDM) induced to overexpress HO-1 by adenoviral transduction is protective in murine renal IRI (revised manuscript under review, Molecular Therapy). This project has adopted an embryonic stem (ES) cell approach to develop ES cell-derived macrophages (ESDM) overexpressing HO-1 for therapeutic use in experimental models of ARF.

An ES cell line (E14 IV) was induced to form embryoid bodies and the non-adherent cells harvested and cultured in the presence of M-CSF and IL-3 for 7 days to form ESDM. The phenotype of ESDM was compared to BMDM and bone marrow derived dendritic cells (BMDC) by assessment of morphology, expression of cell surface markers (F4/80, CD11b, CD11c and MHC Class II) by flow cytometry and evaluation of their capacity to phagocytose fluorescent latex beads. ES cells with constitutive overexpression of HO-1 were generated by cloning HO-1 cDNA into a pCAG vector, which randomly integrated into the ES cell genome. HO-1 expression level was determined by western blot analysis and ESDM generated from selected HO-1^HI and HO-1^LOW expressing ES cell clones.

We successfully generated functional ESDM in vitro. ESDM were characterised by a large mononuclear cell morphology. ESDM exhibit a cell surface phenotype comparable to BMDM rather than BMDC and are F4/80^HICD11b^HICD11c^LOWMHC class II^LOW. ESDM are significantly phagocytic and readily ingest latex beads. However, cell count and microscopic analysis indicated that constitutive overexpression of HO-1 in ES cells using the pCAG system resulted in the generation of very few ESDM. Current work is examining whether the detrimental effect of HO-1 overexpression is upon ES cell self renewal or the process of macrophage differentiation.

Our work demonstrates that functional macrophages may be generated from ES cells. Since the randomly inserted pCAG vector may inadvertently silence key genes such as those involved in ES cell self-renewal and/or macrophage differentiation, our future work will use a more precise and targeted tetracycline inducible system that integrates at the HPRT locus. Also, studies of ESDM localisation to injured kidneys are in progress.