Project description:Endothelial cells of xenotransplanted gene-edited porcine kidneys are important due to the initial and prolonged exposure to host immune cells. To better study this cell population kidney endothelial cell lines (KECs) were generated by double CD31+ sorting. In order to ensure the endothelial identity of the cells RNA-seq was performed on KECs and the concomitant CD31- population from the initial sort. Analysis of gene expression in KECs showed a depletion of markers associated with tubular identity (SLC34A1, SLC2A2, SLC12A1), and enrichment for markers of endothelial (PECAM1, CDH5, VWF) and kidney endothelial (EHD3, HOX87, HOX89) identity.
Project description:A subset of cells in cellular populations may be preferentially reprogrammed into pluripotent stem cells due to their innate gene expression characteristics. SSEA-1+ sorted cells were shown to be more amenable to reprogramming than other cells in porcine fetal fibroblast cell lines. This microarray data reveals which genes are unique to the SSEA1+ sorted cells and helps to give them an identity. Porcine fetal fibroblasts were subjected to Magnetic Activated Cell Sorting using anti-SSEA-1. Five biological replicates were performed resulting in 5 SSEA-1 positive fractions and 5 SSEA-1 negative fractions which were analyzed.
Project description:A major challenge in realizing regenerative treatment using freshly isolated Adipose Derived Regenerative Cells (ADRCs) is the cellular heterogeneity and donor variability. Ex vivo expanded ADRCs (=ASCs) are available at larger quantities and represent a more homogeneous population suitable for allogenic use. Emerging pre-clinical and clinical data however suggest similar, or even superior efficacy of ADRCs as compared to ASCs for indications involving (micro)vascular deficiency. Since CD31+ ADRCs lack in cultured ASCs, we hypothesized that these cells account for improvement of vascular function in the heterogenous ADRCs. Herein, we found that in patients with erectile dysfunction (ED), the number of injected CD31+ ADRCs correlates positively with erectile function 12 months after one bolus of autologous ADRCs. Comprehensive in vitro and ex vivo analyses confirmed superior pro-angiogenic and paracrine effects of human CD31+ enriched ADRCs compared to the corresponding CD31- and parent ADRCs. CD31+, CD31- and ADRCs were co-cultured in aortic ring-, as well as in ED-relevant corpus cavernousum tube formation assays, both showing a significantly higher ability of the CD31+ ADRCs to support tube development. This effect was corroborated using conditioned medium (CM), while quantitative mass spectrometric analysis suggested that this is explained by secretory pro-angiogenic proteins such as DKK3, ANGPT2, ANAX2 and VIM, all being enriched in CD31+ADRC CM. To gain further specification of the ADRC subpopulations expressing these proteins, single-cell RNA sequencing was performed and showed that transcripts of the upregulated and secreted proteins were present in 9 endothelial ADRC subsets including endothelial progenitor cells in the heterogenous non-cultured ADRCs. Our data thus suggest that the vascular benefit of using ADRCs in regenerative medicine is dictated by CD31+ ADRCs, which likely represent an improved alternative to heterogenous ADRCs as well as ASCs when aiming for vascular repair in cell therapy.
Project description:A subset of cells in cellular populations may be preferentially reprogrammed into pluripotent stem cells due to their innate gene expression characteristics. SSEA-1+ sorted cells were shown to be more amenable to reprogramming than other cells in porcine fetal fibroblast cell lines. This microarray data reveals which genes are unique to the SSEA1+ sorted cells and helps to give them an identity.
Project description:In order to assess whether there were differences in gene expression between the polyclonal CD31+ and the polyclonal CD31- cell populations of adipose-derived adult stem cells (ADASCs), we wanted to identify possible genes that were differentially expressed between these two cell populations. To that end, RNA was isolated from polyclonal CD31+ and the polyclonal CD31- ADASCs from three different donors and analyzed using the Affymetrix Microarray HG-U133A. Then, using the Affymetrix program MAS 5.0 we performed three comparisons and could identify differentially expressed transcripts common between the three donors, using the Affymetrix program DMT 3.0.
Project description:MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3' untranslated region (3' UTR) of target mRNAs. MiRNAs participate in the regulation of myogenesis, and identification of the complete set of miRNAs expressed in muscles is likely to significantly increase our understanding of muscle growth and development. To determine the identity and abundance of miRNA in porcine skeletal muscle, we applied a deep sequencing approach. This allowed us to identify the sequences and relative expression levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads for the most abundant miRNAs. Moreover, we identified numerous examples of mature miRNAs that were derived from opposite sides of the same predicted precursor stem-loop structures, and also observed length and sequence heterogeneity at the 5' and 3' ends. Furthermore, KEGG pathway analysis suggested that highly expressed miRNAs are involved in skeletal muscle development and regeneration, signal transduction, cell-cell and cell-extracellular matrix communication and neural development and function. Examination of small RNA profiles in 7 isolates of porcine muscle The raw sequences were trimmed to 30 nucleotides, and it was set as a requirement that any sequence must appear at least three times and be present in at least two of the seven libraries. All identical reads within a library were grouped and converted into unique sequences. Reads containing Ns or long tracks (M-BM-!M-CM-^]8) of As were removed and the sequences were trimmed for adaptor-sequences. To annotate the unique sequences, a Decypher Tera-BLASTN Search was performed against a database of mature miRNAs obtained from miRBase (release 12.0). Hits with a match of 16 or more nucleotides to a miRNA from the database were gathered, and the count of each miRNA was normalized to the total number of sequence reads per lane. The outcome of this procedure can be seen in the Aarhus_University_GBI_FC208D2AAXX_blast_mirbase table below.
Project description:MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3' untranslated region (3' UTR) of target mRNAs. MiRNAs participate in the regulation of myogenesis, and identification of the complete set of miRNAs expressed in muscles is likely to significantly increase our understanding of muscle growth and development. To determine the identity and abundance of miRNA in porcine skeletal muscle, we applied a deep sequencing approach. This allowed us to identify the sequences and relative expression levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads for the most abundant miRNAs. Moreover, we identified numerous examples of mature miRNAs that were derived from opposite sides of the same predicted precursor stem-loop structures, and also observed length and sequence heterogeneity at the 5' and 3' ends. Furthermore, KEGG pathway analysis suggested that highly expressed miRNAs are involved in skeletal muscle development and regeneration, signal transduction, cell-cell and cell-extracellular matrix communication and neural development and function.
Project description:The atypical chemokine receptor 4 (ACKR4)-expressing spleen endothelial cells (ECs) form a three-dimensional sinusoidal network connected via shunts to the marginal sinus and tightly surrounds the outer perimeter of the marginal zone. To better define this new vascular compartment within the red pulp of the spleen, were sorted CD31+ACKR4+ and CD31+ACKR4- EC populations, routinely yielding 92-98% cell purity and analyzed their transcriptional profiles.