Project description:MSC-adherent hematopoietic stem and progenotir cells (HSPC) express adhesion-associated genes at higher levels than non-adherent cells while cell-cycle and differentiation-associated genes are not significantly changed between the two cell populations. We used microarray to confirm identity of MSC-adherent and non-adherent cord blood-derived HSPCs and to exclude that cell cycle and differentiation affect adhesive capacity. CD34 positive cells were isolated from human cord blood (not older than 24h), expanded for 3 days and assayed for the adhesion to MSC. The adherent and non-adherent live CD34+ cells were sorted and total RNA was extracted.
Project description:MSC-adherent hematopoietic stem and progenotir cells (HSPC) express adhesion-associated genes at higher levels than non-adherent cells while cell-cycle and differentiation-associated genes are not significantly changed between the two cell populations. We used microarray to confirm identity of MSC-adherent and non-adherent cord blood-derived HSPCs and to exclude that cell cycle and differentiation affect adhesive capacity.
Project description:Direct contact with mesenchymal stromal impacts on migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex-vivo expansion Objective: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133+ hematopoietic stem cells (HSCs) in terms of expansion potential differentiation, migratory capacity and gene expression profile. Methods: CD133+ purified HSCs were cultured for 7 days on subconfluent MSCs supplemented with growth factor containing medium. After ex-vivo expansion, non-adherent and adherent cells were collected and analyzed separately. Results: The adherent cells were found to have a more immature phenotype compared to the non-adherent fraction. CXCR4 was up regulated in the adherent fraction which was associated with a higher migration capacity towards a SDF-1 gradient. CFU-GM and LTC-IC assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell cycle control, motility, self-renewal and apoptosis were expressed at a higher level in the adherent fraction. Conclusion: Adhesion and direct cell-cell contact with a MSC feeder layer supports ex-vivo expansion, migratory potential and stemness of CD133+ HSCs. Keywords: co-culture hematopoietic stem cells (HSCs) on mesenchymal stromal cells (MSCs) Non-adherent and adherent fractions from three independent experiments were isolated and collected. Cells were stabilized in PreProtct™ buffer (Miltenyi Biotec, Germany) and stored at -80ºC. Samples were shipped to Miltenyi Biotec (Bergisch Gladbach, Germany) a Whole Human Genome expression analysis. Briefly RNA was extracted and overall quality of total RNA samples was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). RNA samples were amplified and labelled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies). Non-adherent samples were pooled as “Non-adherent pool” and adherent samples were pooled as “adherent pool” and labelled with Cy3 and Cy5, respectively. Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner and the microarray image files were processed with The Agilent Feature Extraction Software (FES).
Project description:Direct contact with mesenchymal stromal impacts on migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex-vivo expansion Objective: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133+ hematopoietic stem cells (HSCs) in terms of expansion potential differentiation, migratory capacity and gene expression profile. Methods: CD133+ purified HSCs were cultured for 7 days on subconfluent MSCs supplemented with growth factor containing medium. After ex-vivo expansion, non-adherent and adherent cells were collected and analyzed separately. Results: The adherent cells were found to have a more immature phenotype compared to the non-adherent fraction. CXCR4 was up regulated in the adherent fraction which was associated with a higher migration capacity towards a SDF-1 gradient. CFU-GM and LTC-IC assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell cycle control, motility, self-renewal and apoptosis were expressed at a higher level in the adherent fraction. Conclusion: Adhesion and direct cell-cell contact with a MSC feeder layer supports ex-vivo expansion, migratory potential and stemness of CD133+ HSCs. Keywords: co-culture hematopoietic stem cells (HSCs) on mesenchymal stromal cells (MSCs)
Project description:Mononuclear cells were isolated from granulocyte colony stimulating factor mobilised peripheral blood. CD34+ cells were selected and separated into adherent and non-adherent cells. Adherent and non-adherent samples were amplified and labelled using two IVT cycles, and gene expression profiling was performed by hybridisation to Affymetrix Human Genome U133 Plus2.0 Arrays.
Project description:To understand the mechanism by which AML-EVs may enforce quiescence, we performed tandem mass tag (TMT) proteomic profiling of in vitro cultured c-Kit+ hematopoietic stem and progenitor cells (HSPC) (to obtain the minimum required amount of protein lysates) treated with extracellular vesicles (EVs) for 48 hours from human HL-60 or Molm-14 cell cultures versus controls.
Project description:This is a mathematical model describing the hematopoietic lineages with leukemia lineages, as controlled by end-product negative feedback inhibition. Variables include hematopoietic stem cells, progenitor cells, terminally differentiated HSCs, leukemia stem cells, and terminally differentiated leukemia stem cells.