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S. Geyh et al.
As a first read-out, we quantified the proliferative potential and the growth capacities of MSC. TGFβ1 sig- nificantly inhibited the proliferation and growth proper- ties of healthy MSC, as indicated by the absolute cell number and cumulative population doubling of the cells treated in this way being similar to those observed in pri- mary patient-derived MSC (Figure 3A,B and Online Supplementary Figure S6). This suppressive effect also per- sisted when healthy MSC were primed with TGFβ1 for 3 days and subsequently cultured in control media for another 3 days (Figure 3A,B). In line with these findings, microscopic analysis of healthy MSC exposed to TGFβ1 revealed a disorganized cellular architecture, which dif- fered sharply from the fibroblastoid feeder layer of the control MSC (Figure 3C). The inhibitory effects of TGFβ1 on growth and proliferation of MSC were abrogated by the TGFβ receptor I inhibitor SD-208 (Figure 3A-C).
In subsequent experiments we investigated the influ- ence of TGFβ1 on osteogenic differentiation, which has been previously shown to be significantly impaired in MDS- and AML-derived MSC.12,13 Using established cul- ture conditions and staining methods12,13 we found that TGFβ1 significantly impaired osteogenic differentiation capacity (Figure 4A-B, Online Supplementary Figure S7). In
agreement with this result, mRNA expression of the bone formation marker osteocalcin was significantly reduced after exposure to TGFβ1 (Figure 4D). Similar to its effect on MSC growth, SD-208 also abolished the TGFβ1-medi- ated suppression of osteogenic differentiation (Figure 4A- C). RNA sequencing of healthy MSC after incubation with TGFβ1 revealed a specific gene expression pattern substantially overlapping with the expression profile of primary patient-derived MSC (Figure 4E, Online Supplementary Figure S8 and Online Supplementary Table S7). In this regard, we decided to check whether the expression of PITX2, HOXB6 and TBX15, three genes physiologically involved in cell differentiation and skele- tal morphogenesis, and which we had previously linked to the impaired osteogenesis in MDS and AML,12,13 could also be affected in the TGFβ1-treated healthy MSC. Although only PITX2 showed a statistically significant change in the RNA sequencing experiment (fold change=3.57, q<0.001), the dysregulation of the three genes could be further verified by quantitative real-time PCR (Online Supplementary Figure S9). SD-208 again reverted the TGFβ1-induced gene expression changes, including those for the three marker genes. (Figure 4F and Online Supplementary Figure S9).
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Figure 3.Transforming growth factor β1 impairs growth properties of healthy mes- enchymal stromal cells. (A) Bar charts showing the absolute MSC numbers after 3 days pre-incubation of 2x105 MSC with the respective factors (10 ng/mL). HC: healthy control; DMSO: dimethylsulfoxide; w/o TGFβ1: healthy MSC were treated with TGFβ1 for 3 days, then the medium was changed and the MSC were cultured for 3 additional days without TGFβ1. (B) Bar charts depicting cumulative population dou- bling (CPD). (C) Representative micrographs showing the morphology of healthy MSC after incubation in the presence of the respective compounds. Scale bars represent 100 μm. For all experiments results are expressed as mean ± SEM. Asterisks indi- cate P-values **P<0.01, ***P<0.001.
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haematologica | 2018; 103(9)