Research Plan

The overall aim of this project is to define, isolate and expand renal stem cells such that they can be used to either repair or regenerate a kidney. The research program is divided into two sections; I) Characterisation of markers for and regulators of renal progenitor cells, and II) Manipulation of stem cell populations to adopt a renal fate and use of stem cells for renal regeneration and repair.

Figure 1: Example of a mouse cDNA array fabricated and interrogated at the IMB.

Section 1. Characterization of markers for and regulators of renal progenitors
To define a renal stem cell or drive an ES cell towards a renal fate, it is first critical to define the population/s required. Individual projects within Section 1 will involve a series of expression profiling experiments carried out to record the temporal transcriptional program of metanephric development (Figure 1). Each project is aimed at both defining the expression pattern of renal development and identifying secreted proteins and cell surface markers that may be of value to Section 2.

Figure 2: Computational pipeline for predicting secreted molecules and membrane organization of all mammalian gene products (R.Teasdale)

To support all of Section 1, a bioinformatic pipeline is being established to define all mammalian secreted and transmembrane proteins (Figure 2). The bioinformatic classification of gene products will be overlaid onto the expression profiling data. Reverse transfection microarrays will be used to confirm sub-cellular localizations in a high throughput fashion.

Project 1: Identification of secreted factors involved in the induction of renal development

Identification of UB tip-specific novel growth factors which induce nephron induction and novel UB tip specific receptors that transduce branching signals from the MM. The former may be useful in experimental induction of stem cells towards a renal fate. The latter may assist in the isolation of UB progenitors. The aim of this project will be to profile ureteric tree versus renal mesenchyme. Profiling will also be performed on tree branch versus tip. These studies will identify growth factors being produced by the tree that may be important in instructing the mesenchyme to form nephrons. Hox7b-GFP transgenic mice will be sued to allow the separation of tree from mesenchyme using FACs and laser microcapture. c-ret antibodies will be used to identify tip from branch of the ureteric tree. Growth factors from mesenchyme that may direct branching will also be identified. Novel factors will be ectopically expressed and their ability to drive mesenchymal differentiation in explants or induce tubule formation from mIMCD3 cells in vitro will be assessed. The ability of these factors to effect ES cell-metanephric mesenchyme differentiation will be assessed in Project 4.

Figure 3. GFP expression confined to the ureteric bud compartment of the developing mouse kidney in Hox7b transgenic mice (derived by Prof. Frank Constatini).

Project 2: Identification of renal progenitor markers to assist in the identification and isolation of renal stem cell populations.

In this project, a complete temporal expression analysis of the developing kidney from 10.5dpc to postnatal will be examined. Upon this temporal framework, spatial information from Project 1 and 3 can be placed. Specific A versus B profiling will also be performed between 10.5dpc renal mesenchyme and adjacent intermediate mesoderm that will not become kidney. This project particularly seeks to identify transmembrane markers of potential progenitor cell populations in the kidney. As describe previously, the membrane organization of all mammalian genes will be assessed computationally. Genes believed to encode trans-membrane proteins and are found to be expressed in the kidney, based on expression data obtained in projects 1 and 2, will be further analyzed. In situ hybridization will be used to assess the spatial expression pattern of these potential cell surface markers in the kidney. Antibodies will be made to lead markers and these will be used to isolate different cell populations. Finally, the potential of these populations to repair damaged kidneys will be assessed in a variety of explant and engrafting assays.

Project 3: Expression profiling of renal sub-compartments, including the interstitial cells, tissue macrophages and podocytes, to identify specific markers of the endpoints of renal differentiation.

In addition to the temporal expression profiling of kidney development, a series of profiling experiments are to be undertaken to define expression markers of specific cell types and regions of the kidney. Specific cell types are to be generated by primary culture methods (renal interstital cells) and cell sorting of cell specific GFP-tagged cells form the kidneys of different transgenic mice (renal macrophages, cap condensates, podocytes). This data will provide an expanded set of expression markers for specific cell types and differentiation states for cells that make up the mammalian kidney. It will also provide an excellent recourse for cell specific expression markers that can be used in Project 4.

Figure 4: Differentiation protocol for ES cells

Section 2. Manipulation of stem cell populations to adopt a renal fate

Project 4: Examination of the potential for ES cells to be differentiated into the necessary lineages for renal de novo generation or repair.

In this project we will attempt to direct murine or human embryonal stem cells towards a renal fate using a variety of inducing conditions. In the case of murine ES cells, this will be an adaptation of the mesodermal induction process used in embryoid body (EB) formation (Figure 5). As tagged murine ES cells can be generated, this will be the most insightful approach. Information from Section 1 will provide i) markers for which progress towards a renal fate can be monitored using wholemount in situ hybridisation (Figure 6), ii) growth factors which may assist in the process and iii) cell surface markers which can facilitate the isolation and enrichment of the desired cell types from mixed progenitor populations. We will also use human embryonal cells to more crudely assess this renal potential. Both of these can be used to test novel growth factors isolated in Projects 1 and 2.

Figure 5: Localized expression of Lim1 in murine EBs by WISH