Comprehensive Gene and microRNA Expression Profiling Reveals a Role for microRNAs in Human Liver Development

BACKGROUND AND AIMS: microRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. miRNAs have been implicated in regulating gene expression in embryonic developmental processes, including proliferation and differentiation. The liver is a multifunctional organ, which undergoes rapid changes during the developmental period and relies on tightly-regulated gene expression. Little is known regarding the complex expression patterns of both mRNAs and miRNAs during the early stages of human liver development, and the role of miRNAs in the regulation of this process has not been studied. The aim of this work was to study the impact of miRNAs on gene expression during early human liver development. METHODS: Global gene and miRNA expression were profiled in adult and in 9-12w human embryonic livers, using high-density microarrays and quantitative RT-PCR. RESULTS: Embryonic liver samples exhibited a gene expression profile that differentiated upon progression in the developmental process, and revealed multiple regulated genes. miRNA expression profiling revealed four major expression patterns that correlated with the known function of regulated miRNAs. Comparison of the expression of the most regulated miRNAs to that of their putative targets using a novel algorithm revealed a significant anti-correlation for several miRNAs, and identified the most active miRNAs in embryonic and in adult liver. Furthermore, our algorithm facilitated the identification of TGFbeta-R1 as a novel target gene of let-7. CONCLUSIONS: Our results uncover multiple regulated miRNAs and genes throughout human liver development, and our algorithm assists in identification of novel miRNA targets with potential roles in liver development

Transcriptomics Comparison between Porcine Adipose and Bone Marrow Mesenchymal Stem Cells during In Vitro Osteogenic and Adipogenic Differentiation

Bone-marrow mesenchymal stem cells (BMSC) are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC). The abundance and ease of harvest make the adipose-derived stem cells (ASC) an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ≤0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition, functional analysis data might indicate differences in therapeutic application

Clinical-scale isolation of the total Aspergillus fumigatus-reactive T-helper cell repertoire for adoptive transfer

Background aims. Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective. Methods. We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immuno-magnetically isolating them via the transiently up-regulated activation marker, CD137. Results. In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From 2 x 10(9) peripheral blood mononuclear cells, we isolated 27 x 10(3)-318 x 10(3) Aspergillus-specific T-helper cells. Frequency among total T cells was increased, on average, by 200-fold. Specific studies indicate specificity and functionality: After non-specific in vitro expansion and re-stimulation with different antigens, we observed strong cytokine responses to A. fumigatus and some other fungi including Candida albicans, but none to unrelated antigens. Discussion. Our technology isolates naturally occurring Aspergillus-specific T-helper cells within 2 days of identifying the clinical indication. Rapid adoptive transfer of Aspergillus-specific T cells may be quite feasible; the clinical benefit remains to be demonstrated. A manufacturing license as an advanced-therapy medicinal product was received and a clinical trial in post-transplantation invasive aspergillosis patients approved. The product is dosed at 5 x 10E3/kg T cells (single intravenous injection), of which at least 10% must be A. fumigatus specific

Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1

In contrast to the deregulated hepatocellular division that is a feature of many hepatic diseases and malignancies, physiologic liver growth during embryonic development and after partial hepatectomy (PH) in adults is characterized by tightly controlled cell proliferation. We used forward genetic screening in zebrafish to test the hypothesis that a similar genetic program governs physiologic liver growth during hepatogenesis and regeneration after PH. We identified the uhrf1 gene, a cell cycle regulator and transcriptional activator of top2a expression, as required for hepatic outgrowth and embryonic survival. By developing a methodology to perform PH on adult zebrafish, we found that liver regeneration inuhrf1(+/−) adult animals is impaired.uhrf1 transcript levels dramatically increase after PH in both mice, and zebrafish and top2a is not up-regulated in uhrf1(+/−) livers after PH. This indicates that uhrf1 is required for physiologic liver growth in both embryos and adults and illustrates that zebrafish livers regenerate