Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs.

BACKGROUND: There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. METHODS: The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. RESULTS: Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. CONCLUSIONS: This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

Stable insulin-secreting ducts formed by reprogramming of cells in the liver using a three-gene cocktail and a PPAR agonist.

With the long-term aim of developing a new type of therapy for diabetes, we have investigated the reprogramming of liver cells in normal mice toward a pancreatic phenotype using the gene combination Pdx1, Ngn3, MafA. CD1 mice were rendered diabetic with streptozotocin and given a single dose of Ad-PNM, an adenoviral vector containing all three genes. Ad-PNM induced hepatocytes of the liver to produce insulin, and the blood glucose became normalized. But over several weeks, the insulin-positive cells were lost and the blood glucose rose back to diabetic levels. Simultaneous administration of a peroxisome-proliferator-activated receptor agonist, WY14643, caused remission of diabetes at a lower dose of Ad-PNM and also caused the appearance of a population of insulin-secreting ductal structures in the liver. The insulin-positive ducts were stable and were able to relieve diabetes in the long term. We show that the effect of WY14643 is associated with the promotion of cell division of the ductal cells, which may increase their susceptibility to being reprogrammed toward a beta cell fate.

Inhibition of Hes1 activity in gall bladder epithelial cells promotes insulin expression and glucose responsiveness.

The biliary system has a close developmental relationship with the pancreas, evidenced by the natural occurrence of small numbers of biliary-derived beta-cells in the biliary system and by the replacement of biliary epithelium with pancreatic tissue in mice lacking the transcription factor Hes1. In normal pancreatic development, Hes1 is known to repress endocrine cell formation. Here we show that glucose-responsive insulin secretion can be induced in biliary epithelial cells when activity of the transcription factor Hes1 is antagonised. We describe a new culture system for adult murine gall bladder epithelial cells (GBECs), free from fibroblast contamination. We show that Hes1 is expressed both in adult murine gall bladder and in cultured GBECs. We have created a new dominant negative Hes1 (DeltaHes1) by removal of the DNA-binding domain, and show that it antagonises Hes1 function in vivo. When DeltaHes1 is introduced into the GBEC it causes expression of insulin RNA and protein. Furthermore, it confers upon the cells the ability to secrete insulin following exposure to increased external glucose. GBEC cultures are induced to express a wider range of mature beta cell markers when co-transduced with DeltaHes1 and the pancreatic transcription factor Pdx1. Introduction of DeltaHes1 and Pdx1 can therefore initiate a partial respecification of phenotype from biliary epithelial cell towards the pancreatic beta cell.

Different isoforms of the Wilms' tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus.

The Wilms' tumour suppressor gene WT1 encodes multiple isoforms of a transcription factor essential for correct mammalian urogenital development. Maintenance of the correct isoform ratio is critical. In humans, perturbation of this ratio causes Frasier syndrome, which is characterized by developmental defects of the kidney and urogenital tract. Different WT1 isoforms are thought to regulate transcription and participate in mRNA processing, functions reflected by a complex sub-nuclear distribution. However, the role of individual WT1 isoforms remains unclear and pathways leading to WT1 sub-nuclear localization are completely unknown. Here we use cells expressing green fluorescent protein-tagged WT1 to demonstrate that the two major WT1 isoforms occupy separate and dynamic intranuclear locations in which one isoform, WT1+KTS, preferentially associates with the nucleolus. The alternatively spliced zinc finger region is found to be critical for the initial sub-nuclear separation of WT1 isoforms, but interactions between different isoforms influence the sub-nuclear distribution of WT1. We illustrate how disruption of WT1 nuclear distribution might result in disease. This study contributes to the emerging picture of intranuclear protein trafficking.

Regulation of the Wilms' tumour suppressor (WT1) gene by an antisense RNA: a link with genomic imprinting?

Antisense transcripts are typically associated with the down-regulation of gene expression. In this issue, Moorwood et al. present evidence that an antisense RNA can enhance expression of the Wilms' tumour suppressor locus WT1. We suggest that the unusual function of the WT1 antisense RNA might relate to the recent discovery of an antisense transcript that is involved in regulating imprinted expression of the murine Igf2r gene, particularly since there is some evidence that the WT1 gene is regulated by genomic imprinting in humans.

StuAp is a sequence-specific transcription factor that regulates developmental complexity in Aspergillus nidulans.

The Aspergillus nidulans Stunted protein (StuAp) regulates multicellular complexity during asexual reproduction by moderating the core developmental program that directs differentiation of uninucleate, terminally differentiated spores from multinucleate, vegetative hyphae. StuAp is also required for ascosporogenesis and multicellular development during sexual reproduction. StuAp is a member of a family of fungal transcription factors that regulate development or cell cycle progression. Further, StuAp characterizes a sub-family possessing the conserved APSES domain. We demonstrate for the first time that the APSES domain is a sequence-specific DNA-binding domain that can be modeled as a basic helix-loop-helix (bHLH)-like structure. We have found that StuAp response elements (A/TCGCGT/ANA/C) are located upstream of both critical developmental regulatory genes and cell cycle genes in A.nidulans. StuAp is shown to act as a transcriptional repressor in A.nidulans, but as a weak activator in budding yeast. Our data suggest that the differentiation of pseudohyphal-like sterigmatal cells during multicellular conidiophore development requires correct StuAp-regulated expression of both developmental and cell cycle genes in A.nidulans. The budding pattern of sterigmata may involve processes related to those controlling pseudohyphal growth in budding yeast.