Introduction: Heterozygous mutations in JAGGED1 (JAG1), encoding a ligand for Notch receptors, have been identified in 70 % of patients with Alagille syndrome (AGS), using PCR-SSCP (single strand conformation polymorphism). These mutations are located in the extracellular and transmembrane domains of the protein and 70 % of them lead to a premature termination codon (PTC). However, only few studies of the modified mRNAs have been performed. Methods: and aims: To improve the molecular diagnosis of AGS, we have performed RT-PCR analyses and sequencing from mRNAs of lymphoblastoid cell lines derived from 11 patients with no mutations identified by PCR-SSCP. In addition, we have studied the mutant transcript levels in 21 cell lines from AGS patients with already identified mutations, and from the tissues of a 23-week-old fetus with AGS. Results: 1/ in 3 of the patients without previously identified mutations, JAG1 mutations were found by RT-PCR analysis, 2/ in the 8 other patients, no mutation was found in the cDNA of JAG1, supporting the hypothesis of genetic heterogeneity, 3/ mutant transcripts were recovered from lymphoblastoid cells of all patients with missense mutations (5) or in-phase deletions (2), and from all but one of the 16 with PTC, 4/ mutant transcripts were present in tissues of the 23 week-old fetus, albeit in different relative amounts; this could originate from tissue specificity of nonsense mediated mRNA decay. Conclusion: We found that some JAGGED1 mutations not detected by PCR-SSCP were identified by RT-PCR analysis on lymphoblastoid cells with no treatment against the nonsense mediated mRNA decay, but that some AGS patients have no mutation in the cDNA of JAGGED1 supporting the hypothesis of genetic heterogeneity. Mutant transcripts were recovered whatever the mutations, even with PTC, suggesting that both haploinsufficiency and a dominant negative effect could be involved in molecular mechanisms of AGS.
An in vitro study of the behaviour of a human acute lymphoblastoid leukemia cell line (REH) towards the action of a mitogenic lectin of Robinia pseudoacacia was carried out. The results were compared with those a reference cell line (LHN13) established from normal human lymphocytes. In both cell lines, the lectin induces agglutination (measured by counting the number of aggregates as well as the number of cells in each aggregate) and decrease of growth (measured by counting the number of cells and the incorporation of tritiated thymidine into TCA-precipitable material per 10(6) cells). The agglutination and the decrease of growth are produced at the doses of 0.5 and 1 microgram/ml of culture medium and after 4 h of exposure of cells to the lectin, respectively. These effects increase progressively with higher doses of lectin and continues throughout the culture. However, the REH line is less sensitive than the LHN13 line to the effects of lectin. Both agglutination and growth decrease of REH as well as LHN13 cell lines by the lectin are reversible; this is confirmed by the fact that the monospecific anti-Robinia lectin serum suppresses these effects.
The vascular system is adapted to specific functions in different tissues and organs. Vascular endothelial cells are important elements of this adaptation, leading to the concept of 'specialized endothelial cells'. The phenotype of these cells is highly dependent on their specific microenvironment and when isolated and cultured, they lose their specific features after few passages, making models using such cells poorly predictive and irreproducible. We propose a new source of specialized endothelial cells based on cord blood circulating endothelial progenitors (EPCs). As prototype examples, we evaluated the capacity of EPCs to acquire properties characteristic of cerebral microvascular endothelial cells (blood-brain barrier (BBB)) or of arterial endothelial cells, in specific inducing culture conditions.First, we demonstrated that EPC-derived endothelial cells (EPDCs) co-cultured with astrocytes acquired several BBB phenotypic characteristics, such as restricted paracellular diffusion of hydrophilic solutes and the expression of tight junction proteins. Second, we observed that culture of the same EPDCs in a high concentration of VEGF resulted, through activation of Notch signaling, in an increase of expression of most arterial endothelial markers.We have thus demonstrated that in vitro culture of early passage human cord blood EPDCs under specific conditions can induce phenotypic changes towards BBB or arterial phenotypes, indicating that these EPDCs maintain enough plasticity to acquire characteristics of a variety of specialized phenotypes. We propose that this property of EPDCs might be exploited for producing specialized endothelial cells in culture to be used for drug testing and predictive in vitro assays.
Abstract Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133+KDR+ population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144+KDR+ population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144+KDR+ peak was shifted to day 7. Most of these cells, however, were CD31−, becoming CD31+ in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144+KDR+CD31− population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. Disclosure of potential conflicts of interest is found at the end of this article.
