Cystic renal-epithelial derived induced pluripotent stem cells from polycystic kidney disease patients.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient-specific iPSCs to study the function of PKD1 in kidney development and cyst formation in vitro. Somatic mutations are proposed to be the initiating event of cyst formation, and therefore, iPSCs were derived from cystic renal epithelial cells rather than fibroblasts. Mutation analysis of the ADPKD iPSCs revealed germline mutations in PKD1 but no additional somatic mutations in PKD1/PKD2. Although several somatic mutations in other genes implicated in ADPKD were identified in cystic renal epithelial cells, only few of these mutations were present in iPSCs, indicating a heterogeneous mutational landscape, and possibly in vitro cell selection before and during the reprogramming process. Whole-genome DNA methylation analysis indicated that iPSCs derived from renal epithelial cells maintain a kidney-specific DNA methylation memory. In addition, comparison of PKD1+/- and control iPSCs revealed differences in DNA methylation associated with the disease history. In conclusion, we generated and characterized iPSCs derived from cystic and healthy control renal epithelial cells, which can be used for in vitro modeling of kidney development in general and cystogenesis in particular.

The probability to initiate X chromosome inactivation is determined by the X to autosomal ratio and X chromosome specific allelic properties.

BACKGROUND: In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X ratio A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI. METHODOLOGY/PRINCIPAL FINDINGS: To obtain more insight in the role of the XratioA ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X ratio A ratios, provides evidence that the X ratio A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X ratio A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator. CONCLUSIONS: The present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X ratio A ratio. This finding supports the presence of an X-encoded activator of the XCI process.

X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator.

Female mammalian cells achieve dosage compensation of X-encoded genes by X chromosome inactivation (XCI). This process is thought to involve X chromosome counting and choice. To explore how this process is initiated, we analyzed XCI in tetraploid XXXX, XXXY, and XXYY embryonic stem cells and found that every X chromosome within a single nucleus has an independent probability to initiate XCI. This finding suggests a stochastic mechanism directing XCI counting and choice. The probability is directly proportional to the X chromosome:ploidy ratio, indicating the presence of an X-encoded activator of XCI, that itself is inactivated by the XCI process. Deletion of a region including Xist, Tsix, and Xite still results in XCI on the remaining wild-type X chromosome in female cells. This result supports a stochastic model in which each X chromosome in a nucleus initiates XCI independently and positions an X-encoded trans-acting XCI-activator outside the deleted region.