Abstract
Using a selective-amplification system, we have ex vivo expanded umbilical cord blood (UCB) derived lineage-negative− (Lin−: CD2−, CD3−, CD14−, CD16−, CD19−, CD24−, CD56−, CD66b−, and Glycophorin A−) cells. To assess at the molecular level the effect of the ex vivo expansion on properties of UCB Lin−-cells, we compared gene expression profiles between cells at day 0 and cells cultured in serum-free medium supplemented with rhFlt-3L, rhSCF and rhTPO (100ng/ml each) for 14 days with a reselection using the same lineage depletion antibody cocktail at the 7th day of culture. In this study, we utilized a human stem cell gene cDNA array (SuperArray Bioscience Corp.), which include genes encoding surface markers, growth factor/cytokines, extracellular matrix molecules and cell cycle regulators for embryonic, neural, mesodermal and hematopoietic stem/progenitor cells. Of the 266 total genes in the array, we detected 85 genes expressed above background in either of the two populations. Among these expressed genes, 19 were found only in the day_0 (pre-culture) population and 11 were found only in the day_14 cultured cells. Ex vivo expansion under these conditions had no significant effect on the majority of the genes, which include highly expressed homing-related (CXCR4, CD44) and cell adhesion (CD31) molecules. Further analysis revealed that, compared to the levels in the original day_0 Lin− population, 7 genes showed more than 3-fold increases and 13 genes showed more than 3-fold decreases in their levels of expression in the cultured day_14 cell population. The up-regulated genes included those involved in cell cycling, proliferation and migration or anti-apoptosis (cyclin E1, IGFR-2, alpha(4)beta(1)-integrin, Mdm2, Cystatin C, ALK-5). The down-regulated genes included those involved in embryological or neural development (GCMb, FGF11), a subset of integrin molecules (integrin alpha E, beta 3 and 5) and an inhibitor of BMP signaling (CER1). In conclusion, our results indicate that selective ex vivo expansion conditions promote expression of genes related to the growth and expansion of targeted hematopoietic precursor cells while retaining expression of homing-related genes. Further studies are slated to link, where possible, these transcriptional expression patterns with expression and functions of these genes at the protein and cellular levels.
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