Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities. © 2013 Chan et al

The evolutionary significance of polyploidy

Polyploidy, or the duplication of entire genomes, has been observed in prokaryotic and eukaryotic organisms, and in somatic and germ cells. The consequences of polyploidization are complex and variable, and they differ greatly between systems (clonal or non-clonal) and species, but the process has often been considered to be an evolutionary 'dead end'. Here, we review the accumulating evidence that correlates polyploidization with environmental change or stress, and that has led to an increased recognition of its short-term adaptive potential. In addition, we discuss how, once polyploidy has been established, the unique retention profile of duplicated genes following whole-genome duplication might explain key longer-term evolutionary transitions and a general increase in biological complexity

Do Young Heroin Users in Madrid, Barcelona and Seville have Sufficient Knowledge of the Risk Factors for Unintentional Opioid Overdose?

To identify the self-perceived reasons for unintentional opioid overdose of young heroin users in three Spanish cities and their agreement with objective risk factors for overdose. Computer-Assisted Personal Interviews (CAPI) were held with 991 street-recruited current heroin users aged 18–30. The general reasons for overdose and the reasons for the last overdose suffered were explored with open-ended (OEQs) and pre-coded questions (PCQs). Limited knowledge of overdose risk factors was defined as mention of fewer than two objective risk factors for unintentional overdose in the OEQ. Univariate, bivariate, and logistic regression methods were used. 77.8% (Seville), 64.9% (Madrid) and 57.2% (Barcelona) of participants have limited knowledge of overdose risk factors. Residence in Seville and not having attended courses or meetings on overdoses were significantly associated with limited knowledge, after adjusting for other factors. The most frequently identified general reasons in OEQ or PCQ were using heroin in large amounts (66.8%), together with tranquilizers (62.0%), adulterated (60.7%), or purer than usual (57.6%). Most reasons were selected more frequently in PCQ than in OEQ, especially rapid injection of the entire dose and using heroin shortly after using tranquilizers or alcohol, by injection, or after a period of abstinence. The results were similar for overdoses suffered by participants. Most young heroin users do not have sufficient knowledge of overdose risk factors, especially the use of heroin by injection, after a period of abstinence, or together with alcohol or methadone. Specific informational or educational programs adapted to the local context are critically needed