Project description:Brain perivascular cells have been recently identified as new mesodermal cell type of the human brain. These cells reside in the perivascular niche and were shown to have mesodermal and â to a lesser extend â tissue-specific differentiation potential. Mesenchymal stem cells (MSCs) are widely discussed for the use in cell therapy in many neurological disorders. Therefore it is of importance to better understand the âintrinsicâ MSC population of the human brain. Here we systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation and compared these cells to fetal and adult human brain-derived NSCs and adult human bone marrow derived MSCs. We found that adult human brain pericytes can be isolated from hippocampal as well as cortical white matter, are â in contrast to adult human NSCs â easily expandable in monolayer cultures and show high similarities to human bone marrow-derived MSCs both regarding surface marker expression and whole transcriptome analysis. Human brain pericytes differentiated only in negligible amounts into neuroectodermal cell types using various differentiation conditions but efficiently differentiated into mesodermal progenies. Thus bone marrow-derived MSCs resemble human brain pericytes and might be therefore very interesting for possible autologous NPC-based treatment strategies, cell therapeutic approaches of neurological diseases. For the gene expression microarray analysis we used the Affymetrix U133A chips The whole procedure was performed following the manufacturer's standard protocol (Affymetrix, Santa Clara, CA). For the data processing, normalization was calculated with the GCRMA (GC content corrected Robust Multi-array Analysis) algorithm. Data post-processing and graphics was performed with in-house developed functions in Matlab. 17 samples were analyzed fNSC, Neural Stem Cell, 2 replicates ANPC-hip, adult Neuroprogenitor - Cell Hippocampus, 3 replicates ANPC-wm, adult Neuroprogenitor Cell - White Matter, 3 replicates ABPMC-hip, adult Brain Perivascular Mesodermal Cell - Hippocampus, 3 replicates ABPMC-wm, adult Brain Perivascular Mesodermal Cell - White Matter, 3 replicates MSC, Mesenchymal Stem Cell, 3 replicates
Project description:Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 16,015 nuclei in human adult testis. This dataset includes five samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Brain perivascular cells have been recently identified as new mesodermal cell type of the human brain. These cells reside in the perivascular niche and were shown to have mesodermal and – to a lesser extend – tissue-specific differentiation potential. Mesenchymal stem cells (MSCs) are widely discussed for the use in cell therapy in many neurological disorders. Therefore it is of importance to better understand the “intrinsic” MSC population of the human brain. Here we systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation and compared these cells to fetal and adult human brain-derived NSCs and adult human bone marrow derived MSCs. We found that adult human brain pericytes can be isolated from hippocampal as well as cortical white matter, are – in contrast to adult human NSCs – easily expandable in monolayer cultures and show high similarities to human bone marrow-derived MSCs both regarding surface marker expression and whole transcriptome analysis. Human brain pericytes differentiated only in negligible amounts into neuroectodermal cell types using various differentiation conditions but efficiently differentiated into mesodermal progenies. Thus bone marrow-derived MSCs resemble human brain pericytes and might be therefore very interesting for possible autologous NPC-based treatment strategies, cell therapeutic approaches of neurological diseases. For the gene expression microarray analysis we used the Affymetrix U133A chips The whole procedure was performed following the manufacturer's standard protocol (Affymetrix, Santa Clara, CA). For the data processing, normalization was calculated with the GCRMA (GC content corrected Robust Multi-array Analysis) algorithm. Data post-processing and graphics was performed with in-house developed functions in Matlab.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes