Committed Hemopoietic Progenitors, Not Stem Cells, are the Principal Responders to Hox Gene Transduction

ABSTRACTAs hemopoietic stem cells differentiate, their proliferative lifespan shortens by unknown mechanisms. Homeobox cluster (Hox) genes have been implicated by their enhancement of self-renewal when transduced into hemopoietic cells, but gene deletions have been inconclusive because of functional redundancy. Here we enforced HOXB4 expression in purified precursor stages, and compared responses of early stages expressing the endogenous genes with later stages that did not. Contrary to the prevalent view that transduced Hox genes enhance the self-renewal of hemopoietic stem cells, stem cells or their multipotent progeny expressing the endogenous genes showed little response. Instead, immortalization, extensive self-renewal and acquired reconstituting potential occurred in committed erythroid and myeloid progenitors where the endogenous genes were shutting down. The results change our understanding of the stages affected by exogenous HOX proteins and point to shutdown of the endogenous genes as a principal determinant of the shortened clonal lifespans of committed progenitor cells.

Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence

Much has been learned about the mechanisms underlying cellular senescence. The pathways leading to senescence appear to vary, depending on the cell type and cell culture conditions. In this respect, little is known about senescence of human peritoneal mesothelial cells (HPMC). Previous studies have significantly differed in the reported proliferative lifespan of HPMC. Therefore, in the present study, we have examined how HPMC enter state of senescence under conditions typically used for HPMC culture. HPMC were isolated from omentum and grown into senescence. The cultures were assessed for the growth rate, the presence of senescence markers, activation of cell-cycle inhibitors, and the oxidative stress. HPMC were found to reach, on average, six population doublings before senescence. The terminal growth arrest was associated with decreased expression of Ki67 antigen, increased percentage of cells in the G1 phase, reduced early population doubling level cDNA-1 mRNA expression, and the presence of senescence-associated β-galactosidase. Compared with early-passage cells, the late-passage HPMC exhibited increased expression of p16INK4abut not of p21Cip1. In addition, these cells generated more reactive oxygen species and displayed increased presence of oxidatively modified DNA (8-hydroxy-2′-deoxyguanosine). These results demonstrate that early onset of senescence in omentum-derived HPMC may be associated with oxidative stress-induced upregulation of p16INK4a.

A Better Strategy To Extend the Proliferative Lifespan of Human T-Cells In Vitro.

Telomerase maintains telomere length by adding TTAGGG repeats to the 3′-ends of chromosomes. In human T-cells telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control their lifespan. To understand the effects of culture parameters on telomerase activity and cell lifespan is of critical importance to improve T-cell expansion. Here, we investigated the influence of culture conditions and of stimulation on lifespan, clonogenicity (number of positive wells), cell cycle and telomerase activity in T-cells in vitro. Isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured in RPMI 1640 medium containing IL-2, different sera (10%) (fetal calf serum (FCS), AB serum and autologous serum) and stimulated with phytohaemagglutinin (PHA) and irradiated allogeneic mononuclear feeder cells or anti-CD3/CD28 beads. T-cells were counted and passaged weekly until senescence. Expanded T-cells were analyzed for cell cycle by DNA staining with propidium iodide (PI) at various time points after stimulation and telomerase activity was measured using the telomerase PCR ELISA. Clonogenicity was determined by limiting dilution using different culture conditions. The proliferative lifespan of T-cells expanded with PHA/feeder cells and autologous serum from two different donors was significantly increased compared to T-cells stimulated with PHA/feeder cells and AB serum (44.7 vs. 14.2 PDs; 42.4 vs. 27.4 PDs). In contrast, no or only little difference was found for T-cells expanded with anti-CD3/CD28 beads and autologous or AB serum (8.7 vs. 8.1 PDs; 15.5 vs. 9.6 PDs). Most likely the latter findings reflect the loss of CD28 expression on T-cells. Furthermore, the use of autologous serum also increased the clonogenicity to about 4-fold compared to the use of AB serum or FCS without any signs for differences in the fractions of cycling cells. Interestingly, T-cells cultured with autologous serum exhibited a higher telomerase activity at day 3 after stimulation and a reduced decline of telomerase activity at day 6 and 9 compared to cultures with AB serum. Our results demonstrate that the use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity of human T-cells in vitro. In addition, T-cells cultured in medium containing autologous serum exhibit higher and prolonged telomerase activity compared to cultures with AB serum. Further studies are ongoing to elucidate the underlying factors involved in those observations. Nevertheless, we suggest that autologous serum together with PHA stimulation and feeder cells is superior to expand human T-cells especially for clinical applications where large numbers of specific T-cells are required for adoptive transfer strategies in cancer patients with for example severe viral infections.