ABSTRACT: We compare the transcriptome of two different clones of multipotent adult progenitor cells (MAPCs) using Affymetrix arrays. Keywords: mRNA expression profiling, oligonucleotide microarrays, stem cells Overall design: Three mRNA samples (biological replicates) per cell type taken at different passage number were compared. Two independently isolated MAPC clones were used.
Project description:We compare the transcriptome of two different clones of multipotent adult progenitor cells (MAPCs) using Affymetrix arrays. Experiment Overall Design: Three mRNA samples (biological replicates) per cell type taken at different passage number were compared. Two independently isolated MAPC clones were used.
Project description:We compare the transcriptome of embryonic stem cells (ESCs), adult stem cells with apparent greater differentiation potential such as multipotent adult progenitor cells (MAPCs), mesenchymal stem cells (MSCs) and neurospheres (NS). Mouse and rat MAPCs were used in this study and two different array platforms (Affymetrix and NIA) were used for mouse samples. Experiment Overall Design: Three mRNA samples (biological replicates) per cell type taken at different passage number were compared. Cell types include mouse ESCs, mouse MSCs and three clones isolated using mouse MAPC culture condition: mMAPC-1, mMAPC-2 and mClone-3. mMAPC-1 and mClone-3 were obtained from same bone marrow isolation, mMAPC-2 was obtained in a different bone marrow isolation. Two clones derived from same rat bone marrow using rat MAPC culture conditions were compared. Three mRNA samples (biological replicates) per clone were taken at different passage numbers
Project description:Gene Markers of Cellular Aging in Human Multipotent Stromal Cells in Culture Identifying gene markers of cellular aging as determined by cellular passaging of human multipotent stromal cells (MSCs) derived from bone marrow Repeated Measures Experiment; MSC from 6 different donors at 3 passages (passages 3, 5, & 7) with 3 technical replicates at each passage; a total of 54 microarrays
Project description:Multipotent adult resident cardiac stem cells (CSCs) originally were identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone distinguish CSCs from other c-kit-expressing cardiac cells because the adult heart contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has not been considered in recent c-kit-expressing cell fate mapping publications, which have equated the contribution of the whole heterogeneous c-kitpos population to cardiomyocyte generation in adulthood, which is minimal, to that of the CSCs, a result at odds with previous publications. To shed light on this issue, we have assessed the identity, abundancy and myogenic potential of true multipotent CSCs within the total c-kitpos cardiac cell cohort. Blood lineage-committed c-kitpos cells were removed by CD45 negative sorting to obtain a CD45negc-kitpos cell population (<10% of the total c-kitpos cells), which is enriched for cells that express c-kit at low levels and possess all properties of multipotent stem/progenitor cells in vitro. These characteristics are absent from the c-kitneg and the lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived CSC clones, representing 1-2% of total c-kitpos cells, when instructed by TGF-b/Wnt molecules, acquire full transcriptome expression, sarcomere organization, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes. Significantly, clonogenic CSCs have a potent cardio-regenerative/repair capacity in vivo after acute myocardial infarction. CSC myogenic regenerative capacity is dependent on cardiomyocyte commitment through activation of the SMAD2 pathway. Such regeneration was not apparent when freshly-isolated total c-kitpos cardiac cells were administered. In conclusion, only a very small fraction of cardiac c-kitpos cells (~1-2%) have the characteristics of multipotent CSCs but these exhibit robust myogenic properties. Overall design: RNA-seq data from CSCs, iCMs, neonatal (neoCMs) and adult cardiac myocytes (aCMs) (triplicated) have been analysed and compared. Also, mRNA was collected from clonogenic undifferentiated rat CSCs, beating colonies of CSC-derived iCMs, freshly isolated cardiomyocytes obtained from the hearts of 1-2 post-natal day and from 12 week-old Wistar rats. Please note that the datasets of CSC1,2 and 3 are from a biological triplicate of the same cardiac stem cell clone. In particular, the dataset of the sample entitled CSC1 has also been used in a different experiment with the title of cCSC4. This dataset was compared with three datasets respectively from three different cardiac stem cell clones (cCSC1, 2, and 3) for clonal identity analysis.
Project description:Adult neural progenitor cells (aNPCs) are a potential autologous cell source for cell replacement in neurologic diseases such as Parkinson’s disease or stroke or for cell-based gene therapy for neurometabolic diseases. Easy accessibility, long-term expandability and detailed characterization of NPC properties are important requisites for their future translational/clinical applications. aNPC can be isolated from different regions of the adult human brain including the accessible subcortical white matter (aNPCWM), but systematic studies comparing long-term expanded aNPCWM with aNPC from neurogenic brain regions to check for their NPC characteristics and performance are not available. Freshly isolated cells from subcortical white matter and hippocampus (aNPCHIP) expressed oligodendrocyte progenitor cell (OPC) markers such as A2B5, NG2 and OLIG2 in ~20% of cells but no neural stem cell (NSC) markers such CD133 (Prominin1), NESTIN, SOX2 or PAX6. The EGF receptor (EGFR) protein was expressed in 18% of aNPCWM and 7% of aNPCHIP, but only a small fraction of 1 cell out 694 cells from white matter and only 1 out of 1,331 hippocampal cells were able to generate neurospheres. Studies comparing subcortical aNPCWM with their hippocampal counterparts showed that both NPC types expressed mainly markers of glial origin such as NG2, A2B5 and OLIG2, and the NSC/NPC marker Nestin, but no pericyte markers. Both NPC types were able to produce fully mature neurons, astrocytes and oligodendrocytes in comparable amounts to fetal NSC. Whole transcriptome analyses finally confirmed the strong similarity of aNPCWM with aNPCHIP. Our data show that aNPCWM are multipotent NPC with long-term expandability capacity similar to NPC from hippocampus making them an easily accessible source for possible autologous NPC-based treatment strategies. Isolation and propagation of multipotent NPCs. Adult human hippocampal (hip) and subcortical white matter (wm) tissue was obtained from routine epilepsy surgery procedures following informed consent of the subjects. All procedures were in accordance with the Helsinki convention and approved by the Ethical Committee of the University of Dresden (No. 47032006). All subjects underwent high-resolution magnetic resonance imaging excluding tumors and were screened for the presence of infectious disease. In all cases the neuropathological examinations did not reveal evidences for tumor formation. Gene expression Single-channel oligonucleotide microarray analysis. For the gene expression microarray analysis we used the Affimetrix U133A chips containing 22.215 probe sets representing at least 12.905 individual genes. The whole procedure was performed following the manufacturer’s standard protocol (Affimetrix, Santa Clara, CA). For the data processing, normalization was calculated with the GCRMA (GC content corrected Robust Multi-array Analysis) algorithm. fNSC: Human fetal neural stem cells, 2 biological rep aNPChip: Human adult neural progenitor cells isolated from hippocampus, 3 biological rep aNPCwm: Human adult neural progenitor cells isolated from white matter, 2 biological rep
Project description:The aim of the study was to characterize the molecular mechanism involved in TGF-ß mediated smooth muscle formation in vitro. We employed rat bone marrow derived Oct4 expressing clones of multipotent adult progenitor cells (rMAPC). We subjected these cells to differentiation towards smooth muscle cell as previously reported using TGF-ß1. The differentiation process reuires 6 days with media change every 2 days followed by RNA harvest. RNA was isolated using commercially available kits (Qiagen RNA easy micro kit). RNA integrity and quality was assessed prior to labeling and hybridization. As a control RNA from rat aortic smooth muscle cells was commercially obtained. Two biological replicate clones of rMAPC cells were used for the differentiation to smooth muscle like cells. The RNA was harvested at days 0, 2, 4 and 6 in triplicates. The RNA from primary smooth muscle cells was commercially obtained and was used in duplicates as control.
Project description:Global Expression profiling of mutant mouse multipotent progenitors was performed. In order to mitigate the impact of the studied driver mutations on cell surface phenotypes, we performed transcriptome analysis on a homogeneous population of purified lineage negative, Sca-1/Kit positive multipotent progenitor cells (MPPs, cell surface profile: Lin-/CD34+/Flt3+/CD48+/CD150-). Aprroximately 1000 multipotent progenitors were isolated from wild type or mutant Npm1cA/+, NrasG12D, Npm1cA/+;NrasG12D, Flt3ITD/+ and Npm1cA/+;Flt3ITD/+ murine bone marrow cells. Total RNA extracted and mRNA amplified using
Project description:The mechanisms underlying the specification of oligodendrocyte fate from multipotent neural progenitor cells (NPCs) in developing human brain are unknown. In this study, we sought to identify antigens sufficient to distinguish NPCs free from oligodendrocyte progenitor cells (OPCs). We investigated the potential overlap of NPC and OPC antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFaR antigens. Surprisingly, we found that CD133, but not A2B5, was capable of enriching for OLIG2 expression, Sox10 enhancer activity, and oligodendrocyte potential. As a subpopulation of CD133- positive cells expressed CD140a, we asked whether CD133 enriched bone fide NPCs regardless of CD140a expression. We found that CD133+CD140a- cells were highly enriched for neurosphere initiating cells and were multipotent. Importantly, when analyzed immediately following isolation, CD133+CD140a- NPCs lacked the capacity to generate oligodendrocytes. In contrast, CD133+CD140a+ cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Gene expression analysis further confirmed the stem cell nature of CD133+CD140a- cells. As human CD133+ cells comprised both NPCs and OPCs, CD133 expression alone cannot be considered a specific marker of the stem cell phenotype, but rather comprises a heterogeneous mix of glial restricted as well as multipotent neural precursors. In contrast, CD133/CD140a-based FACS permits the separation of defined progenitor populations and the study of neural stem and oligodendrocyte fate specification in the human brain. 12 samples, 4 groups (FACS-sorted cell populations),3 replicates in each group, each replicate is from a separate patient sample
Project description:We used comparative transcriptome analysis to determine the relationship between rodent Multipotent Adult Progenitor Cells (MAPCs) and pluripotent embryonic stem cells (ESCs), or lineage restricted mesenchymal stem cells (MSCs) and neurospheres (NS). This comparison revealed a unique gene expression profile of MAPCs, that express transcripts of early endodermal and mesodermal but fewer ectodermal genes. In addition, MAPCs, but not MSCs or NS, express a number of genes expressed in ESCs including Oct-4 (Pou5f1) and other genes previously identified as ES cell associated transcripts and/or genes involved in maintenance of ESC selfrenewal capacity and pluripotency. However MAPCs do not express some key genes involved in maintaining selfrenewal and pluripotency in ESCs, including Nanog and Sox-2. Because MSC-like cells isolated under MAPC conditions are virtually identical to MSCs isolated in the presence of high serum, the MAPC signature is cell-type specific and not merely the result of differing culture conditions. Finally, this unique molecular signature was seen irrespective of the microarray platform used, and was highly similar for both mouse and rat MAPCs. Keywords: cell type comparison design Two biological replicates were used for hybridizations. cRNA from MAPCs, ESCs, and NS were added to a universal reference cRNA.