Project description:For the complete cure of tumors, it is essential to eliminate cancer-initiating cells (CICs). Immunohistochemically, most tumor cells were CD20 and/or CD138 positive in clinical samples of lymphoplasmacytic lymphoma (LPL), and CD20- CD138- cells were hardly detected. Therefore, useful positive markers expressing in a candidate of CICs of LPL are necessary. First, we performed gene expression microarray analysis between CD20- CD138- and CD20+ CD138+ subpopulations using sorted Waldenstrom macroglobulinemia cell line (MWCL-1).
Project description:For the complete cure of tumors, it is essential to eliminate cancer-initiating cells (CICs). Immunohistochemically, most tumor cells were CD20 and/or CD138 positive in clinical samples of lymphoplasmacytic lymphoma (LPL), and CD20- CD138- cells were hardly detected. Therefore, useful positive markers expressing in a candidate of CICs of LPL are necessary. First, we performed gene expression microarray analysis between CD20- CD138- and CD20+ CD138+ subpopulations using sorted Waldenstrom macroglobulinemia cell line (MWCL-1). Two dye-swap experiment was performed onto Human GE 4Ã44K V2 arrays (G4845A; Agilent Technologies).
Project description:Determination of expression levels of light chain V genes in peripheral blood B cells after FACS sorting for two populations of B cells (CD20+CD138-IgKappa+IgLambda- and CD20+CD138-IgKappa-IgLambda+). Analysis was performed on healthy individuals and SLE patients with analysis performed using several models.
Project description:Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described more than 30 years ago, the phenotype of MM-CSC is still a matter of debate, especially with respect to the expression of syndecan- 1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 surface expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are also phenotypically interconvertible. Overall, our results differ from previously published data which attribute a B-cell phenotype to MM-CSC and urge the need to explore more reliable markers to discriminate true clonogenic myeloma cells. To compare the gene expression profile of the subpopulations CD138++ and CD138low within the MM cell line, RPMI-8226. RNA was isolated from CD138++ (n=3) and CD138low (n=3) RPMI-8226 cells. RNA was hybridized to Human Gene 1.0 ST array (Affymetrix) according to Affymetrix protocols [Gutierrez, 2005, 402]. Microarray data were normalized using the Robust Multichip Analysis (RMA) algorithm implemented in the Affymetrix Expression Console. Data analysis was carried out using DNA-Chip Analyzer software (DChip). The comparison criteria used in DChip analysis were fold change E/BM-bM-^IM-%2 or B/EM-bM-^IM-%2, mean difference E-B>100 or B-E>100 and the lower 90% confidence bound of fold-change was used
Project description:Determination of expression levels of light chain V genes in peripheral blood B cells after FACS sorting for two populations of B cells (CD20+CD138-IgKappa+IgLambda- and CD20+CD138-IgKappa-IgLambda+). Analysis was performed on healthy individuals and SLE patients with analysis performed using several models. Dual channel hybridization with experimental samples detected on red channel and reference sample detected on green channel. Two replicate hybridizations.
Project description:Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described more than 30 years ago, the phenotype of MM-CSC is still a matter of debate, especially with respect to the expression of syndecan- 1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 surface expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are also phenotypically interconvertible. Overall, our results differ from previously published data which attribute a B-cell phenotype to MM-CSC and urge the need to explore more reliable markers to discriminate true clonogenic myeloma cells.
Project description:Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described more than 30 years ago, the phenotype of MM-CSC is still a matter of debate, especially with respect to the expression of syndecan- 1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 surface expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are also phenotypically interconvertible. Overall, our results differ from previously published data which attribute a B-cell phenotype to MM-CSC and urge the need to explore more reliable markers to discriminate true clonogenic myeloma cells.
Project description:Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described more than 30 years ago, the phenotype of MM-CSC is still a matter of debate, especially with respect to the expression of syndecan- 1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 surface expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are also phenotypically interconvertible. Overall, our results differ from previously published data which attribute a B-cell phenotype to MM-CSC and urge the need to explore more reliable markers to discriminate true clonogenic myeloma cells. To compare the Copy Number Abnormalities of the subpopulations CD138++ and CD138low within the MM cell line, RPMI-8226. DNA was isolated from CD138++ (n=3) and CD138low (n=3) RPMI-8226 cells. The detection of CNA was investigated using the CytoScan HD array (Affymetrix, Santa Clara, CA, USA) which includes more than 2.6 million copy number markers and 750.000 SNPs. Briefly, genomic DNA was digested with Nsp I restriction endonuclease, ligated to adaptors that recognize the cohesive four base pair overhangs and amplified by PCR. PCR product was purified and fragmented with DNAse I and then end-labeled using terminal deoxynucleotidyl transferase and hybridized to the CytoScan HD array. The arrays were processed using the Fluidics Station 450, GeneChip Scanner 3000 7 G and AGCC (AffymetrixGeneChip Command Console Software). For the analysis we employed the AffymetrixM-BM-. Chromosome Analysis Suite (ChAS) Software v2.0 and Nexus Copy Number Software Version 7.0 (BioDiscovery, El Segundo, CA, USA). The complete data set was analyzed by visual inspection using ChAS software. CNA were reported when the three following criteria were achieved: minimum of 50 markers per segment, 200 Kb minimum genomic sizes and 50% overlap with known copy number variants (Database of Genomic Variants).
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
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.