Acquisition of renogenic competence in the early mouse embryo and embryonic stem cells

Abstract

The acquisition of renogenic competence (the ability to give rise to kidney) during embryonic development is not yet fully understood. Clarifying the temporal and molecular aspects of this process is equally essential for understanding excretory system development and for devising methods for successful differentiation of embryonic stem cells (ESCs) to renal cells for disease modeling, toxicology screening and potential cell replacement therapies. In embryo development, the metanephric (permanent) kidney arises as a result of inductive interactions between two embryonic structures that arise in the intermediate mesoderm - the ureteric bud (UB, a diverticulum of the Wolffian duct) and the metanephric mesenchyme (MM). The UB develops into the collecting duct system and the MM undergoes an epithelial-to-mesenchymal transition to form the secretory units of the kidney - the nephrons. In this thesis, I used a tissue disaggregation-reaggregation method that allows the reconstruction of mouse organotypic kidney rudiments to place different embryonic cells in the environment of a developing kidney and assess their potential to integrate into kidney epithelia and differentiate to renal cells. First, the suitability of this method was evaluated and a quantitative assay for evaluating the numbers of test cells integrating in various renal compartments was developed. Second, the reaggregation method was used to characterise the renogenic potential of undifferentiated mouse ESCs, ESC-derived cells after Notch inhibition, and cells derived from the presumptive nephrogenic regions of embryos at various stages of development. ESCs are isolated from the inner cell mass of an embryo and have the potential to differentiate to any tissue of the body when injected into mouse blastocysts. Strategies have successfully been devised for ESC differentiation to many lineages, but very few studies reported any success with the differentiation of ESCs to a renal lineage. Undifferentiated ESCs showed a very good ability to form chimeric structures with developing kidney tubules (both nephrons and extending UBs). Nevertheless, the resulting structures were morphologically different from renal epithelia in most cases and integrated ESC-derived cells were not positive for several combinations of kidney markers. These results suggested that the influence of the niche was not sufficient for a successful ESC differentiation to renal cells. Treatment of ESC with an inhibitor of the Notch pathway to increase the proportion of mesodermal cells did not improve this outcome. On the basis of these results, it was speculated that the earliest lineage to which embryonic stem cells must be differentiated in order to become competent to make renal cells should first be identified. I addressed this by determining the developmental stage at which cells able to contribute to the formation of metanephric epithelia first appear in mouse embryo development. When mixed in embryonic kidney reaggregates labelled cells isolated from the nephrogenic regions of E9.5 embryos integrated into various renal compartments. These cells were seen in UBs, nephrons, glomeruli and the condensing mesenchyme. Marker expression studies showed that the exogenous E9.5 cells expressed an array of kidney markers - Pax2 in renal epithelia and the condensing mesenchyme, Wt1 in glomeruli and Six2 in the condensing mesenchyme. Furthermore, exogenous E9.5 cells also co-expressed Pax2/Wt1 in the condensing mesenchyme, Megalin/Ecadherin in the proximal tubule and Pax2/E-cadherin in renal epithelia. This provides evidence that challenges the existing model and suggests that some cells from the intermediate mesoderm at a stage where the metanephric blastema is yet formed are competent to contribute to kidney structures. Furthermore, experiments with E8.5 embryos showed that such a renocompetence could be acquired even before the specification of intermediate mesoderm. These findings contribute to our knowledge about kidney cell specification and provide valuable information to guide future attempts to develop an efficient method for deriving renal cells from ESCs. Furthermore, the reported ability of ESC-derived non-kidney cells to form chimeric structures with renal tubules provides a proof-of-principle that it might be possible to use exogenous types of cells for physiological support to injured kidney tubules, thus offering a possible novel approach for cell replacement therapies.