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control condition of patient-derived MSC cultured in the absence of SD-208 (Figure 6A-C). In addition, treatment of MDS- and AML-derived MSC with SD-208 substan- tially increased these cells’ support of healthy CD34+ cells, as reflected by their 2.1-fold higher LTC-IC frequen- cy in comparison to that of untreated MDS- and AML- derived MSC (Online Supplementary Figure S12, P=0.078). These findings clearly indicate that TGFβ1 inhibits osteogenic differentiation and hematopoietic support capacity of MSC in human MDS and AML and that this effect can be pharmacologically reverted by SD-208.
Discussion
In our previous work we demonstrated that MSC from patients with MDS and AML exhibit impaired growth and osteogenic differentiation capacity as well as altered expression of hematopoietic signaling molecules such as Angiopoietin-1, Kit-ligand and Jagged1. Comparable with recent results from Bhagat et al.,31 these structural and functional deficits were associated with a specific methy- lation pattern and resulted in impaired stromal support for HSPC.12,13 These findings were compatible with confir- matory results published by other groups in the last
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years.4,11,14,15,32,33 and directly linked alterations of MSC to the pathophysiology of MDS and AML.
To elucidate the underlying molecular mechanisms, in this study we performed RNA sequencing of MSC from patients with early MDS, advanced MDS and AML. Our data analysis defined a specific common molecular profile which clearly separated MSC of these myeloid diseases from those of healthy controls. Pathway analyses further identified an enrichment of genes involved in general developmental processes, cellular senescence and in par- ticular genes essential for skeletal morphogenesis, includ- ing the three candidate genes PITX2, HOXB6 and TBX15 that are commonly deregulated in MDS- and AML- derived MSC.12,13 On the molecular level these results pro- vide an excellent explanation for the phenotypic and functional alterations of MDS- and AML-derived MSC that were previously reported by our group and by oth- ers.11-14
Additional findings suggested an ongoing stromal response to an inflammatory environment. This is in line with the results from two other groups who also reported an inflammatory stress response in MSC derived from patients with low-risk MDS.10,14 Our data now expand this finding to MSC from patients with advanced MDS and AML, and suggest that the pro-inflammatory signal-
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Figure 5. Impaired hematopoietic support capacities of healthy mesenchymal stromal cells exposed to transforming growth factor β1. Healthy MSC (n=5) were pre-incubated with TGFβ1 and/or SD-208 for up to 28 days. Medium was changed every 3 days and supplemented with TGFβ1 and/or SD-208 at a concentration ranging from 5 ng/mL to 10 ng/mL (SD-208: from 0.25 μM to 0.5 μM). Cells were harvested on days 7, 14, 21, and 28. (A) Bar charts illustrate protein expression of Jagged1 as measured by flow cytometry at the given time points. The mean fluorescence intensity43 is shown. HC: healthy control; DMSO: dimethylsulfoxide. (B) mRNA expression of Angiopoietin-1 (Angpt1) and Kit-ligand (Kitlg) was measured by quantitative real-time PCR after 7 days of incubation. (C) Bar charts showing LTC- IC frequencies of healthy CD34+ HSPC cultured on healthy MSC which had been previously incubated with TGFβ1 and/or SD-208 for 28 days. For all experiments results are expressed as mean ± SEM. Asterisks indicate P-values *P<0.05, **P<0.01, ***P<0.001.
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haematologica | 2018; 103(9)