Highly sensitive amplicon-based transcript quantification by semi-conductor sequencing
Ontology highlight
ABSTRACT: We have adapted a commercial assay that employs mRNA quantification based on the frequency of PCR amplicons determined by next-generation to a high-throughput semi-conductor sequencing platform (Ion-Torrent Proton). We show parallel amplification of pathway derived transcript sets/genes in 12 reference RNA samples followed by sequence-based quantification covering a dynamic range of five orders of magnitude with low technical M-BM- variation. Expression of selected genes were profiled at four time points (0d, 10d, 20d, and 60d) during differentiation of induced pluripotent stem cells into cardiomyocytes, with three independent biological replicates at each time point.
Project description:Our purpose was to investigate genes and molecular mechanisms involved in patients with Leber congenital amaurosis (LCA). Fibroblasts from two unrelated clinically-identified patients (Coriell) were reprogrammed to pluripotency by retroviral transduction. These human induced Pluripotent Stem Cells (hiPSCs) were differentiated into neural stem cells (NSC) that mimicked the neural tube stage and retinal pigmented epithelial (RPE) cells that could be targeted by the disease. A genome wide transcriptome analysis was performed with Affymetrix Exon Array GeneChipM-BM-., comparing LCA-hiPSCs derivatives to controls. The aim was to identify differentially expressed genes which may be associated with early developmental defect before the establishment of mature retinal circuitry. We analyzed iPSC-derived retinal pigmented epithelial (RPE) cells from LCA patient's fibroblast (n=2) and iPSC-derivedretinal pigmented epithelial (RPE) cells from healthy people fibroblast (n=2). A total of 13 samples were analyzed : 9 RPE cells derived from iPSC LCA and 4 RPE cells derived from wild-type iPSC.
Project description:Regenerative medicine in aims to restore structure and function to tissues or organs damaged by time, disease or injury. Stem cells have great potential for tissue repair and regeneration, why they are intensely investigated in equine clinical research. However, before any type of stem cell can be applied in practice, it is crucial that the isolated stem cells have been definitively characterised by a set of specific functional or phenotypic markers. This project includes a surface mapping of equine mesenchymal (MSC) stem cell surface proteome.
Project description:To identify the consequences of mechanical stress of differentiated cardiac muscles from healthy- and PPCM individuals. RNA-sequencing data was obtained from 4 individuals from 2 families. Each family had one member with PPCM and one healthy. Individual 1 and 2 were sisters, one healthy, one with PPCM. Individual 3 and 4 were a healthy mother and PPCM daughter. From each patient 4, 6, 1 and 3 clones were derived, respectively. From each clone multiple pairs of cells were collected and either stretched or left static to a total of 40 pairs, with 2 unpaired static samples.
Project description:Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease in disease relevant cells, as well as a promising source for cell replacement therapies for degenerative diseases. However one of the crucial limitations before realizing the full promise of this “disease in a dish” approach has been the inability to do controlled experiments under genetically defined conditions. This is particularly relevant for disorders with long latency periods, such as Parkinson’s disease (PD), where in vitro phenotypes of patient-derived iPSCs are predicted to be subtle and susceptible to significant epistatic effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this key problem by generating isogenic pairs of disease and control human embryonic stem cells (hESCs) and hiPSCs lines that differ exclusively at a susceptibility variant for PD by modifying a single point mutation (A53T) in the ?-synuclein gene. The robust capability to genetically correct disease causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell replacement therapies using autologous cells. ZFN-mediated genome edited human iPS cells or ES cells were assayed for genomic variation
Project description:Human induced pluripotent stem cells (hiPSCs) represent an unlimited supply for the generation of human otic sensory progenitor cells (OSPCs) in vitro. To characterize and confirm the identity of enriched genes in early (day 6) and late (day 13) otic lineage cell populations obtained from undifferentiated hiPS cells (day 0), we used RNA-seq analysis.
Project description:Human pluripotent stem cells (hPSCs) such as embryonic stem cells and induced pluripotent stem cells are promising materials for cell-based regenerative therapies to heart diseases. However, until realization there are many hurdles such as high efficiency of cardiac differentiation of hPSCs and production of clinical-grade cardiac cells derived from hPSCs. Here, we show that a novel small molecule KY02111 robustly enhances differentiation to functional cardiomyocytes from hPSCs. To reveal how KY02111 function on promoting cardiac differentiation of hPSCs, we analyzed the gene expression profiles in KY02111-treated IMR90-1 hiPSCs using the microarray technique. At Day3 of cardiac differentiation from hiPSCs, KY02111 or DMSO was added in the culture and then the cell population was harvested after 12 or 24 hours for RNA extraction.
Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals. Undifferentiated aHDF- and PB-iPSCs from the same individuals (two Parkinson’s disease patients (PD #1 and PD #2) and one adult healthy donor (donor91))
Project description:3 healthy cells - 3 DMD cells - 7 time points (tissue-derived myoblasts, tissue-derived myotubes, hiPSCs, Day 3 of differentiation, Day 10 of differentiation, Day 17 of differentiation, Day 25 of differentiation) - 1 replicate per cell line for the tissue-derived samples and 3 biological replicates per cell line for the hiPSC-derived samples.