Human iPSC-derived renal organoids engineered to report oxidative stress can predict drug-induced toxicity.

Advances in regenerative medicine have led to the construction of many types of organoids, which reproduce important aspects of endogenous organs but may be limited or disorganized in nature. While their usefulness for restoring function remains unclear, they have undoubted usefulness in research, diagnostics, and toxicology. In toxicology, there is an urgent need for better models for human kidneys. We used human iPS-cell (hiPSC)-derived renal organoids to identify HMOX1 as a useful marker of toxic stress via the oxidative stress pathway, and then constructed an HMOX1 reporter in hiPSCs. We used two forms of hiPSC-derived HMOX1-reporter renal organoids to probe their ability to detect nephrotoxicants in a panel of blind-coded compounds. Our results highlight the potential usefulness, and some limitations, of HMOX1-reporter renal organoids as screening tools. The results may guide development of similar stress-reporting organoid assays for other stem-cell-derived organs and tissues.

Calcium/NFAT signalling promotes early nephrogenesis.

A number of Wnt genes are expressed during, and are known to be essential for, early kidney development. It is typically assumed that their products will act through the canonical ß-catenin signalling pathway. We have found evidence that suggests canonical Wnt signalling is not active in the early nephrogenic metanephric mesenchyme, but instead provide expressional and functional evidence that implicates the non-canonical Calcium/NFAT Wnt signalling pathway in nephrogenesis. Members of the NFAT (Nuclear Factor Activated in T cells) transcription factor gene family are expressed throughout murine kidney morphogenesis and NFATc3 is localised to the developing nephrons. Treatment of kidney rudiments with Cyclosporin A (CSA), an inhibitor of Calcium/NFAT signalling, decreases nephron formation--a phenotype similar to that in Wnt4(-/-) embryos. Treatment of Wnt4(-/-) kidneys with Ionomycin, an activator of the pathway, partially rescues the phenotype. We propose that the non-canonical Calcium/NFAT Wnt signalling pathway plays an important role in early mammalian renal development and is required for complete MET during nephrogenesis, potentially acting downstream of Wnt4.

Wt1 and retinoic acid signaling are essential for stellate cell development and liver morphogenesis.

Previous studies of knock-out mouse embryos have shown that the Wilms' tumor suppressor gene (Wt1) is indispensable for the development of kidneys, gonads, heart, adrenals and spleen. Using OPT (Optical Projection Tomography) we have found a new role for Wt1 in mouse liver development. In the absence of Wt1, the liver is reduced in size, and shows lobing abnormalities. In normal embryos, coelomic cells expressing Wt1, GATA-4, RALDH2 and RXRalpha delaminate from the surface of the liver, intermingle with the hepatoblasts and incorporate to the sinusoidal walls. Some of these cells express desmin, suggesting a contribution to the stellate cell population. Other cells, keeping high levels of RXRalpha immunoreactivity, are negative for stellate or smooth muscle cell markers. However, coelomic cells lining the liver of Wt1-null embryos show decreased or absent RALDH2 expression, the population of cells expressing high levels of RXRalpha is much reduced and the proliferation of hepatoblasts and RXRalpha-positive cells is significantly decreased. On the other hand, the expression of smooth muscle cell specific alpha-actin increases throughout the liver, suggesting an accelerated and probably anomalous differentiation of stellate cell progenitors. We describe a similar retardation of liver growth in RXRalpha-null mice as well as in chick embryos after inhibition of retinoic acid synthesis. We propose that Wt1 expression in cells delaminating from the coelomic epithelium is essential for the expansion of the progenitor population of liver stellate cells and for liver morphogenesis. Mechanistically, at least part of this effect is mediated via the retinoic acid signaling pathway.

Smad4 haploinsufficiency in mouse models for intestinal cancer.

The Smad4(+/E6sad) mouse carries a null mutation in the endogenous Smad4 gene resulting in serrated adenomas and mixed polyposis of the upper gastrointestinal (GI) tract with 100% penetrance. Here, we show by loss of heterozygosity (LOH) analysis and immunohistochemistry (IHC) that, although the majority of the tumors appear at 9 months of age, somatic loss of the wild-type Smad4 allele occurs only at later stages of tumor progression. Hence, haploinsufficiency underlies Smad4-driven tumor initiation in the GI tract. As both the Apc and Smad4 tumor suppressor genes map to mouse chromosome 18, we have bred Smad4(+/E6sad) with the Apc(+/1638N) model to generate two distinct compound heterozygous lines carrying both mutations either in cis (CAS) or in trans (TAS). Strikingly, both models show increased tumor multiplicities when compared with the single mutant littermates, although CAS mice are more severely affected and became moribund at only 5-6 weeks of age. Phenotypic and molecular analyses indicate that Smad4 haploinsufficiency is sufficient to significantly affect tumor initiation and progression both prior to and upon loss of Apc function. Moreover, complete loss of Smad4 strongly enhances Apc-driven tumor formation.

X-ray imaging for palaeontology.

Few may be aware that X-ray imaging is used in palaeontology and has been used since as early as 1896. The X-raying, preparation and exposure of Hunsrück slate fossils are described. Hospital X-ray machines are used by the author in his work. An X-ray is vital to provide evidence that preparation of a slate is worthwhile as well as to facilitate preparation even if there is little external sign of what lies within. The beauty of the X-ray exposure is an added bonus.

A targeted mouse Brca1 mutation removing the last BRCT repeat results in apoptosis and embryonic lethality at the headfold stage.

A mouse model with a targeted mutation in the 3' end of the endogenous Brca1 gene, Brca1(1700T), was generated to compare the phenotypic consequences of truncated Brca1 proteins with other mutant Brca1 models reported in the literature to date. Mice heterozygous for the Brca1(1700T) mutation do not show any predisposition to tumorigenesis. Treatment of these mice with ionizing radiation or breeding with Apc, Msh-2 or Tp53 mutant mouse models did not show any change in the tumor phenotype. Like other Brca1 mouse models, the Brca1(1700T) mutation is embryonic lethal in homozygous state. However, homozygous Brca1(1700T) embryos reach the headfold stage but are delayed in their development and fail to turn. Thus, in contrast to Brca1(null) models, the mutant embryos do not undergo growth arrest leading to a developmental block at 6.5 dpc, but continue to proliferate and differentiate until 9.5 dpc. Homozygous embryos die between 9.5-10.5 dpc due to massive apoptosis throughout the embryo. These results indicate that a C-terminal truncating Brca1 mutation removing the last BRCT repeat has a different effect on normal cell function than does the complete absence of Brca1.

Human BRCA1 gene rescues the embryonic lethality of Brca1 mutant mice.

Half of all familial breast cancers are due to mutation in the BRCA1 gene. However, despite its importance, attempts to model BRCA1-induced disease in the mouse have been disappointing. Heterozygous Brca1 knockout mice do not develop mammary tumors and homozygous knockout mice die during embryogenesis from ill-defined causes. Sequence analysis has shown that the coding region, genomic organization, and regulatory sequences of the human and mouse genes are not well conserved. This has raised the question of whether the mouse can serve as an effective model for functional analysis of the human BRCA1 gene. To address this question we have introduced a bacterial artificial chromosome containing the human BRCA1 gene into the germline of Brca1 knockout mice. Surprisingly, we have found that the embryonic lethality of Brca1 knockout mice is rescued by the human transgene. We also show that expression of human BRCA1 transgene mirrors the endogenous murine gene. Our "humanized" transgenic mice can serve as a model system for functional analyses of the human BRCA1 gene. Published 2001 Wiley-Liss, Inc.

Oct-4 regulates alternative platelet-derived growth factor alpha receptor gene promoter in human embryonal carcinoma cells.

Expression of the platelet-derived growth factor alpha-receptor (PDGFalphaR) gene is tightly controlled in mammalian embryogenesis. A well established model system to study human embryogenesis is the embryonal carcinoma cell line Tera2. We have shown previously that retinoic acid-differentiated Tera2 cells express two PDGFalphaR transcripts of 6.4 kilobase pairs (kb) (encoding the full-length receptor) and 3.0 kb, respectively, whereas in contrast, undifferentiated Tera2 cells express PDFGalphaR transcripts of 1.5 kb and 5.0 kb. Here we show that this switch in PDGFalphaR expression pattern during differentiation of Tera2 cells results from alternative promoter use. In undifferentiated cells, a second promoter is used, which is located in intron 12 of the PDGFalphaR gene. Functional analysis shows that this promoter contains a consensus octamer motif, which can be bound by the POU domain transcription factor Oct-4. Oct-4 is expressed in undifferentiated Tera2 cells but not in retinoic acid-induced differentiated cells. Mutation of the octamer motif decreases promoter activity, while ectopic expression of Oct-4 in differentiated Tera2 cells specifically enhances the activity of this PDGFalphaR promoter. Therefore, we suggest that an important aspect in the maintenance of the undifferentiated state of human embryonal carcinoma cells results from Oct-4 expression, which thereupon activates this PDGFalphaR promoter.

pMel17 is recognised by monoclonal antibodies NKI-beteb, HMB-45 and HMB-50 and by anti-melanoma CTL.

Recently, we cloned the cDNA encoding the melanocyte lineage-specific antigen gp100 and demonstrated that gp100 is recognised by three different monoclonal antibodies (MAbs) used to diagnose malignant melanoma. In addition, we showed that tumour-infiltrating lymphocytes (TIL 1200) from a melanoma patient reacted specifically with cells transfected with the gp100 cDNA. Molecular characterisation of the gp100 cDNA revealed that the gp100 antigen is highly homologous, but not identical, to another melanocyte-specific protein, pMel17. Here, we report that cells transfected with pMel17 cDNA also react with all three MAbs used to diagnose malignant melanoma, NKI-beteb, HMB-45 and HMB-50. Moreover, pMel17 transfectants are specifically lysed by TIL1200. These data demonstrate that antigenic processing of both gp100 and pMel17 give rise to peptides seen by anti-melanoma cytotoxic T lymphocytes (CTL) and are therefore potential targets for immunotherapy of malignant melanoma.

Use of transformation to construct antigenic hybrids of the class 1 outer membrane protein in Neisseria meningitidis.

The class 1 protein of Neisseria meningitidis is an important component of candidate outer membrane vaccines against meningococcal meningitis. This porin protein contains two variable regions which determine subtype specificity and provide binding sites for bactericidal monoclonal antibodies. To determine the contribution of each of these variable regions in the induction of bactericidal antibodies, a set of isogenic strains differing only in their class 1 epitopes was constructed. This was done by transformation of meningococcal strain H44/76 with cloned class 1 genes and selection of the desired epitope combinations in a colony blot with subtype-specific monoclonal antibodies. When used for the immunization of mice, outer membrane complexes induced bactericidal antibodies only against meningococcal strains sharing at least one of their class 1 epitopes. The results demonstrate that the P1.2 and P1.16 epitopes, normally located in the fourth exposed loop of the protein, efficiently induce bactericidal antibodies independently of the particular sequence in the first variable region. The P1.5 and P1.7 epitopes, normally located in the first exposed loop, were found to induce lower bactericidal titers. Hybrid class 1 outer membrane proteins were constructed by inserting oligonucleotides encoding the P1.7 and P1.16 epitopes into the porA gene. In this way, we obtained a set of strains which carry the P1.5 epitope in loop 1, P1.2 in loop 4, and P1.7 and P1.16 (separately or in combination) in either loop 5 or loop 6. The additional epitopes were found to be exposed at the cell surface. Outer membrane complexes from several of these strains were found to induce a bactericidal response in mice against the inserted epitopes. These results demonstrate that it is feasible to construct meningococcal strains carrying multivalent class 1 proteins in which multiple subtype-specific epitopes are present in different cell surface-exposed loops.