Manipulating transcription factors in human induced pluripotent cell-derived cells to enhance the production and the maturation of red blood cells

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs

The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

Background: Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. Methodology/Principal Findings: We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. Conclusions/Significance: Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers

Encapsulation of environmentally-friendly biocides in silica nanosystems for multifunctional coatings

In cultural heritage field, significant research efforts have been recently made to improve the efficacy of anti-vegetative treatments and to reduce the environmental impact caused by biocides high concentration. According to the pro-ecological approach, this work reports a novel approach based on the encapsulation/incorporation of environmentally-friendly biocides in different silica nanosystems in order to control the development of biological patinas on outdoor cultural heritage. Two different green biocides have been selected and tested in silica nanosystems: the zosteric acid sodium salt (ZS), a natural antifoulant compound produced by Zostera marina (eelgrass), and the usnic acid (UA), a secondary metabolite produced by some saxicolous lichens. ZS was previously successfully encapsulated but never entrapped in mesoporous silica; instead, UA is, for the first time, encapsulated and in situ entrapped into the silica nanosystems in order to control the release over time. Both silica nanosystems have been characterized as far as their dimensions and superficial properties and loading capability. The antifouling activity was assessed against microorganisms from biopatinas colonising the Aurelian Walls in Rome. Our results have shown that the two nanosystems have complementary properties, thus it is possible to tune the antifouling efficiency by combining the two in different proportions

Comparative ecologic relationships of saturated fat, sucrose, food groups, and a Mediterranean food pattern score to 50-year coronary heart disease mortality rates among 16 cohorts of the Seven Countries Study

Background/objectives: We studied the ecologic relationships of food groups, macronutrients, eating patterns, and an a priori food pattern score (Mediterranean Adequacy Index: MAI) with long-term CHD mortality rates in the Seven Countries Study. Subjects/methods: Sixteen cohorts (12,763 men aged 40–59 years) were enrolled in the 1960s in seven countries (US, Finland, The Netherlands, Italy, Greece, former Yugoslavia: Croatia/Serbia, Japan). Dietary surveys were carried out at baseline and only in a subsample of each cohort. The average food consumption of each cohort was chemically analyzed for individual fatty acids and carbohydrates. Results: Ecologic correlations of diet were computed across cohorts for 50-year CHD mortality rates; 97% of men had died in cohorts with 50-year follow-up. CHD death rates ranged 6.7-fold among cohorts. At baseline, hard fat was greatest in northern Europe, olive oil in Greece, meat in the US, sweet products in northern Europe and the US, and fish in Japan. The MAI was high in Mediterranean and Japanese cohorts. The 50-year CHD mortality rates of the cohorts were closely positively ecologically correlated (r = 0.68–0.92) with average consumption of hard fat, sweet products, animal foods, saturated fat, and sucrose, but not with naturally occurring sugars. Vegetable foods, starch, and the a priori pattern MAI were inversely correlated (r = −0.59 to −0.91) with CHD mortality rates. Conclusions: Long-term CHD mortality rates had statistically significant ecologic correlations with several aspects of diet consumed in the 1960s, the traditional Mediterranean and Japanese patterns being rich in vegetable foods, and low in sweet products and animal foods.</p