Reprogramming adult kidney epithelial cells to induced nephron progenitors

Hendry, Caroline Elizabeth (2010). Reprogramming adult kidney epithelial cells to induced nephron progenitors PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Hendry, Caroline Elizabeth
Thesis Title Reprogramming adult kidney epithelial cells to induced nephron progenitors
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2010-08
Thesis type PhD Thesis
Supervisor Melissa Little
Fiona Rae
Total pages 185
Total colour pages 52
Total black and white pages 133
Language eng
Subjects 1108 Medical Microbiology
Formatted abstract
The adult kidney has a limited capacity to repair. The only characterized nephron progenitor cell population that exists is the embryonic cap mesenchyme (CM), which is present only from E10.5 through to postnatal day 2 in the mouse. The CM is a self-renewing population that gives rise to all the cell types of the nephrons via a mesenchyme to epithelial transition until the cessation of nephrogenesis. Reprogramming adult kidney cells to a CM-like phenotype (induced nephron progenitors, iNPs) may represent an alternative to an endogenous kidney stem cell for cell therapy purposes. Adult kidney cells are likely to be amenable to reprogramming as they routinely undergo dramatic phenotypic changes when they undergo epithelial to mesenchyme transition (EMT) in the disease context and in vitro. This PhD project investigated whether adult kidney cells could be reprogrammed to iNPs via growth factor signalling, small molecules and nuclear reprogramming. Our results show that the human proximal tubular cell line HK2s can be reprogrammed to undergo EMT in response to TGF-β1, however this does reprogram the HK2s to iNPs based on in vitro assays of nephron potential (ie, the competence of the reprogrammed cells to integrate into an embryonic kidney and form nephrons). In contrast, we have found that treatment of HK2s with VPA, a histone deacetylase inhibitor associated with reprogramming, can induce partial reprogramming to iNPs. In this partial reprogramming event, E-cadherin expression levels were upregulated, thereby representing a possible epigenetic barrier restricting the cells from transitioning into the CM attractor state. In order to remove this barrier and to deepen the CM attractor state, we undertook a high-content lentivirus-mediated screen for reprogramming factors via sequential pooling of 15 CM, EMT or “stemness” lineage-instructive transcription factors. We identified one pool of transcription factors that, together with VPA treatment, lowered E-cadherin expression whilst inducing most elements of a genuine CM gene regulatory network (GRN). These putative iNPs behaved in a manner similar to CM when assayed for functional nephron potential in a recombination assay and an epithelial colony-forming assay . This is the first report demonstrating targeted reprogramming within the kidney field. These results concur with the observations of the field that reprogramming involves overcoming multiple epigenetic barriers in order to both destabilize the GRN specific to the initiating cells and fully activate the network required for the desired endpoint phenotype.
Keyword Kidney development
Dedifferentiation
Progenitor
Cap mesenchyme
Reprogramming

 
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