Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions

Abstract

Background: Research on mesenchymal stromal cells has created high expectations for a variety of therapeutic applications. Extensive propagation to yield enough mesenchymal stromal cells for therapy may result in replicative senescence and thus hamper long-term functionality in vivo. Highly variable proliferation rates of mesenchymal stromal cells in the course of long-term expansions under varying culture conditions may already indicate different propensity for cellular senescence. We hypothesized that senescence-associated regulated genes differ in mesenchymal stromal cells propagated under different culture conditions.

Design and methods: Human bone marrow-derived mesenchymal stromal cells were cultured either by serial passaging or by a two-step protocol in three different growth conditions. Culture media were supplemented with either fetal bovine serum in varying concentrations or pooled human platelet lysate.

Results: All mesenchymal stromal cell preparations revealed significant gene expression changes upon long-term culture. Especially genes involved in cell differentiation, apoptosis and cell death were up-regulated, whereas genes involved in mitosis and proliferation were down-regulated. Furthermore, overlapping senescence-associated gene expression changes were found in all mesenchymal stromal cell preparations.

Conclusions: Long-term cell growth induced similar gene expression changes in mesenchymal stromal cells independently of isolation and expansion conditions. In advance of therapeutic application, this panel of genes might offer a feasible approach to assessing mesenchymal stromal cell quality with regard to the state of replicative senescence.

Figure 1.

Growth curves of MSC. MSC were cultured in α-MEM supplemented with either 10% FBS or 10% pHPL. Cells were harvested between day 12 and 14 after reaching confluence and cumulative PD were calculated in relation to the initial CFU-F frequency until P2.

Figure 2.

Morphological analysis of MSC. MSC were cultured in medium with either 10% pHPL or 10% FBS as indicated. Microphotographs were taken from subconfluent MSC in P1 and after expansion until 38 to 39 cumulative PD. (Original magnification 100x; scale bar = 100 μm).

Figure 3.

Gene expression changes in MSC upon large scale expansion. MSC were harvested from initial colonies (P0) and after P1 and P2. Gene expression changes were analyzed in comparison to P0. In culture medium supplemented with FBS there were 30 expressed sequence tags (EST) that were significantly up-regulated and 44 EST that were significantly down-regulated (A; FDR < 5). In culture medium supplemented with pHPL 68 EST were up-regulated and 159 EST were down-regulated (B; FDR < 5). Many of these senescence-associated gene expression changes were consistent in the different culture media as indicated by the heat maps and by the overlap of significantly differentially expressed genes (C; HUGO name and ABI gene ID are provided).

Figure 4.

Gene expression changes in long-term culture of different MSC preparations. The log₂ ratios of senescence-associated gene expression changes in MSC-M1 were plotted against those of long-term culture of FBS-MSC (A) or pHPL-MSC (B). For this comparison we focused on genes that were present in both datasets and more than two-fold up-regulated or down-regulated. There was a moderate relation in senescence-associated gene expression changes of MSC-M1 and FBS-MSC (R = 0.49) and a clear correlation between MSC-M1 and pHPL-MSC (R = 0.84).

Figure 5.

Gene expression changes are continuously acquired during long-term culture. In the overlap of senescence-associated gene expression changes of FBS-MSC, pHPL-MSC and MSC-M1, there were eight genes that were differentially expressed by more than two-fold in each of the three datasets: PTPL1-associated RhoGAP 1 (PARG1), serpin peptidase inhibitor (SERPINE1), cyclin-dependent kinase inhibitor 2B (CDKN2B), netrin 4 (NTN4), toll interacting protein (TOLLIP) and brain-derived neurotrophic factor (BDNF) were senescence-induced. Minichromosome maintenance complex component 3 (MCM3) and pleiotrophin (PTN) were senescence-repressed. Analysis of microarray data revealed that most of these changes increase with every passage (A–C; for MSC-M1 data of serial passages of donor 1 were reanalyzed from our previous work). These gene expression changes were also validated by RT-PCR (D–F; mean of two biological replicas are presented).