|Public on Nov 13, 2008
|Direct contact with mesenchymal stromal cells impacts CD133+ hematopoietic stem cells during ex-vivo expansion
|Expression profiling by array
|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).
|Alakel N, Jing D, Muller K, Bornhauser M, Ehninger G, Ordemann R
|Nov 12, 2008
|Last update date
|Feb 22, 2018
|university hospital Dresden
|Agilent-014850 Whole Human Genome Microarray 4x44K G4112F (Feature Number version)
|Non-adherent cells pool vs. Adherent cells pool