MiR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition

During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-Type depends on complex molecular interactions in space and time. MicroRNAs represent a key control level allowing the flexibility and stability needed f

Combined analysis of microRNA and mRNA signatures in human hematopoietic stem and progenitor cells using a novel microarray quantification system

MicroRNAs (miRNAs), short non-coding RNAs of ~ 21 to 23 nucleotides in length, regulate target mRNAs post-transcriptionally. They play an important role in many different cellular, developmental, and physiological processes including hematopoiesis. It was the aim of this study to characterize the miRNA expression in human hematopoietic stem and progenitor cells. Hematopoietic stem cells (HSCs) have the ability to generate all different kinds of blood cells and express a number of specific surface markers such as CD34 or CD133. As CD133+ cells appear to be ancestral to CD34+ cells, the miRNA profile of these cells could broaden the knowledge of the miRNA role in the differentiation of CD133+ cells. To analyze the function of miRNAs in HSCs, a miRNA microarray platform (miRXploreTM) was developed. The platform was used, inter alia, to validate sequencing data of Small RNA libraries. Standard array experiments measure relative expression levels. However, absolute expression levels in terms of copy numbers per cell are also highly relevant. Therefore, a method for absolute quantification of miRNAs that relies on a universal reference – an equimolar pool of about 1000 synthetic miRNAs of known concentration – was developed. To characterize the role of miRNAs in hematopoiesis, different bone marrow subpopulations, namely CD133+, CD34+CD133– and CD34–CD133– cells, were isolated by magnetic cell separation. The analysis revealed 18 significantly differentially expressed miRNAs between CD133+ and CD34+CD133– cells, that could be validated via qRT-PCR and Solexa sequencing. To further analyze the role of the differentially expressed miRNAs in CD133+ stem cells, mRNA expression profiles were generated and the coexpression of bioinformatically predicted miRNA-mRNA pairs was examined. Luciferase assays were established to validate the predicted targets under physiological conditions. The miRNA-mRNA interactions that could be validated were miR 142-3p and CD133, miR-29a and FZD5 as well as miR-29a and TPM1. TPM1, an actin binding protein, and FZD5, a receptor of the Wnt-signaling pathway, play a role in the remodelling of the cytoskeleton. Further analysis of the predicted miRNA targets revealed that the miRNA targets are enriched for Gene Ontoloy categories related to stem cell-relevant processes. The differentially expressed miRNAs probably prevent differentiation of CD133+ cells and have an anti-apoptotic effect. Furthermore, first experiments were performed to analyze the influence of the differentially expressed miRNAs on the cultivation of CD133+ cells. In vitro expansion of CD133+ cells has turned out to be difficult as most of the tested culture supplements can induce proliferation but are unable to prevent differentiation. The addition of miRNAs to the culture medium could lead to an expansion of CD133+ cells without losing the primitive phenotype. Therefore, the influence of miRNA transfections on CD133+ cells was analyzed in a first step. In conclusion, the generated miRNA signature of CD133+ is the first comprehensive characterization of hematopoietic progenitor cells on miRNA level and will be highly relevant for the application of miRNAs in the field of regenerative medicine

MicroRNAs are shaping the hematopoietic landscape

Hematopoiesis is regulated by microRNAs (miRNAs). These small regulatory RNAs are master regulators of developmental processes that modulate expression of several target genes post-transcriptionally. Various miRNAs are up-regulated at specific stages during hematopoietic development and the functional relevance of miRNAs has been proven at many different stages of lineage specification. Knockout of specific miRNAs can produce dramatic phenotypes leading to severe hematopoietic defects. Furthermore, several studies demonstrated that specific miRNAs are differentially expressed in hematopoietic stem cells. However, the emerging picture is extremely complex due to differences between species, cell type dependent variation in miRNA expression and differential expression of diverse target genes that are involved in various regulatory networks. There is also evidence that miRNAs play a role in cellular aging or in the inter-cellular crosstalk between hematopoietic cells and their microenvironment. The field is rapidly evolving due to new profiling tools and deep sequencing technology. The expression profiles of miRNAs are of diagnostic relevance for classification of different diseases. Recent reports on the generation of induced pluripotent stem cells with miRNAs have fuelled the hope that specific miRNAs and culture conditions facilitate directed differentiation or culture expansion of the hematopoietic stem cell pool. This review summarizes our current knowledge about miRNA expression in hematopoietic stem and progenitor cells, and their role in the hematopoietic stem cell niche

MicroRNA expression profiles of serum from patients before and after chemotherapy

AbstractRecovery of the blood and immune system after chemotherapy requires proliferation of hematopoietic stem and progenitor cells (HPSCs). It has been shown that systemically released factors in serum after chemotherapy stimulate HSPC expansion in vitro. We wondered if microRNAs (miRNAs) circulating in serum could account for this effect. Therefore, we compared the miRNA expression profiles of serum from patients with hematologic malignancies before and after chemotherapy. In addition to a general decrease in miRNA expression after chemotherapy, we found 23 miRNAs to be significantly differentially expressed in serum before versus after chemotherapy. The miRNA microarray data are available at NCBI's Gene Expression Omnibus (GEO) Series accession number GSE57570. Here, we provide a detailed protocol of the miRNA microarray and data analysis

RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5'- and 3'-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs

Corrigendum to “Factor VII activating protease (FSAP) exerts anti-inflammatory and anti-fibrotic effects in liver fibrosis in mice and men” [J Hepatol 2013;58:104-111]

This study aims to estimate the health risks and economic losses due to the effects of air pollution, with a focus on traffic controllers, and to estimate the associated costs through the use of work loss days (WLD) as an indicator of morbidity in São Paulo from 2000 to 2007. the association between traffic controllers' absenteeism and air pollution was determined by generalized linear models (GLM). the increase in relative risk for WLD was 2.08 (95% CI: 2.04-2.12) per 10 mu g/m(3) PM10, which in monetary terms represented 9,430 USD/year, equivalent to 133 absences per 1,308 traffic controllers annually that are attributable to air pollution (accumulated total cost was USD 75,439, which was 19% of the company's operational expenses during the period). These results were extrapolated for the economically active population, and we found that air pollution resulted in 129,832 absences/year and a cost of USD 6,472,686 (77% related to lost wages) per 3,555,237 workers. the estimated values are relevant for planning environmental policies, and are sufficient to promote corrective and preventive actions to avoid this externality.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Faculty of Medicine of the University of São Paulo (FMUSP)Federal University of São Paulo (UNIFESP)Universidade Federal de São Paulo UNIFESP, Dept Ciencias Exatas & Terra, Setor Engn Quim, BR-09972270 Diadema, SP, BrazilUniv São Paulo FMUSP, Fac Med, BR-01246903 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ciencias Exatas & Terra, Setor Engn Quim, BR-09972270 Diadema, SP, BrazilWeb of Scienc

miR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition

During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-type depends on complex molecular interactions in space and time. MicroRNAs represent a key control level allowing the flexibility and stability needed for this process. Insight into the role of this regulatory pathway in the brain is still limited. We performed a sequential experimental approach using postnatal olfactory bulb neurogenesis in mice, starting from global expression analyses to the investigation of functional interactions between defined microRNAs and their targets. Deep sequencing of small RNAs extracted from defined compartments of the postnatal neurogenic system demonstrated that the miR-200 family is specifically induced during late neuronal differentiation stages. Using in vivo strategies we interfered with the entire miR-200 family in loss- and gain-of-function settings, showing a role of miR-200 in neuronal maturation. This function is mediated by targeting the transcription factor Zeb2. Interestingly, so far functional interaction between miR-200 and Zeb2 has been exclusively reported in cancer or cultured stem cells. Our data demonstrate that this regulatory interaction is also active during normal neurogenesis.status: publishe