Abstract
The future viability of ex vivo cell therapies will dependent largely on the optimization of culture processes leading to adequate cell production in a reasonable culture time. Toward this, we have previously reported that the megakaryocyte (MK) differentiation and the yield of MK and platelets produced ex vivo can be enhanced by culturing cord blood (CB) CD34+ cells at 39°C (Biotechnol Bioeng, 2004, 88). We are now working at better characterizing and identifying the mechanism(s) responsible for this phenomenon. First, we investigated whether this effect on cell expansion and differentiation was gradual, or whether it was rapidly induced. CB CD34+ cells were cultured in a media promoting MK differentiation at 39°C for various length of time before being placed at 37°C for a total of 14 days. Though it varied between samples, a minimum period of 4 days was required to observe most of the impacts, with the full qualitative and quantitative impacts obtained after an incubation of 7 days. Next, we analyzed the impact of the culture temperature on the cell cycle kinetics of CB cells. In short, cells grown at 39°C had a reduced mean doubling time during the first week of culture caused by a reduction in time spend in the G1 phase. This was evidenced by a, 1) decreased proportion of cells in G1 (P<0.05) and increased proportion in the S/G2/M phases (S/G2/M of 39.7±2.7% vs 37.3±1.9% (P=0.05)), 2) similar mitotic indexes (%G/M2, P=0.3), 3) comparable viability as determined by annexinV/PI staining (P=0.4) and, 4) increased BrdU incorporation in the first 5 days of culture (+6.9 ±0.7%, P<0.03 (n=2)). Next, we investigated whether transcription factors (TF) normally involved in MK differentiation could be implicated in the increased and accelerated MK differentiation observed at 39°C. The expression of 3 key MK TFs (NF-E2,GATA-1 and Fli-1) was assessed by quantitative PCR (Q-PCR) in cultures of CB CD34+ cells under conditions favoring MK differentiation, at 37 and 39°C. Although no major perturbations were detected, their maximal expression levels were reached faster at 39°C. These differences appear to be a consequence rather than the cause of the increased MK differentiation seen at 39°C, since these correlated with the earlier apparition of immature CD41+ MK and mature CD42+ MK in 39°C cultures. To identify other candidate genes, we used microarray analysis (Affymetrix, U133A 2.0) to compare the gene expression repertoire of CD41+-MK cultured at 39°C or 37°C for 7 days. Analysis of 2 experiments identified a total of 239 genes differentially expressed (116 upregulated). This gene list was corroborated by Q-PCR, which confirmed the changes in expression of 10 of 12 genes. Interestingly, PDGF-a, a cytokine reported to promote MK expansion, was among the genes whose expression was found increased at 39°C by 2.8-fold. Hence, we tested whether addition of PDGF to culture at 37°C could recapitulate the increased and/or accelerated MK differentiation kinetic seen at 39°C. However, PDGF failed to increase MK output at 37°C and had little impact on MK differentiation (n=3). In summary, our results link the increased CB cell expansion and accelerated differentiation kinetics observed at 39°C to a shortening of the G1 phase. Although culture of CB MK at 39°C leads to significant differences in gene expression profile, it had a minimal impact on key MK TFs. Present work is aimed at identifying the mechanism(s) responsible for the shorter time required for CB cells to progress or exit the G1 phase.
Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice