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haematologica | 2018; 103(9)
TGFβ1-mediated inhibition of MDS- and AML-derived MSC
entire bone marrow microenvironment and, in particular, mesenchymal stromal cells (MSC).3,4 In this context, it was demonstrated in several mouse models that genetic per- turbation of mesenchymal cells can induce MDS and pro- mote the emergence of AML.5,6 Vice versa, malignant myeloid cells can also act on niche elements such as MSC leading to a self-reinforcing mechanism supporting leukemic cells at the expense of normal hematopoiesis.7-9 Investigating the pathogenic role of MSC in humans, we and others have recently shown that primary MSC from the bone marrow of MDS and AML patients are struc- turally, genetically and functionally altered.4,10-15 For instance, we found that these MSC have impaired growth capacity and a decreased ability to undergo osteogenic dif- ferentiation, accompanied by a specific methylation signa- ture. Along with impaired stromal support of healthy HSPC this suggests that MSC alterations contribute sub- stantially to the inadequate hematopoiesis in MDS and AML.12,13 Furthermore, these findings imply significantly deregulated bi-directional crosstalk between malignant myeloid cells and MSC. In the current analysis, we used discovery-based strategies, such as RNA sequencing, fol- lowed by candidate-testing approaches. We identified transforming growth factor β (TGFβ) signaling as a rele- vant mechanism responsible for the functional inhibition of MSC in MDS and AML and showed that this was phar- macologically reversible upon TGFβ blockage.
Methods
Patients, healthy controls and cell preparation
Bone marrow samples were obtained from a total of 28 patients with newly-diagnosed MDS or AML (median age 59 years; range, 25-89 years) and 16 age- and sex-matched healthy controls (median age 68 years; range, 39-86 years; P=0.14). Details of the patients’ characteristics are given in Online Supplementary Table S1. The study was approved by our local institutional review board (approval number: 4777) and all indi- viduals gave written informed consent to participation in it.
MSC were derived from the mononuclear cell fraction of the specimens and cultured as described previously.12,13 All experi- ments were carried out using MSC derived from passages 3–4. Furthermore, CD34+ HSPC were obtained from the bone mar- row of healthy controls by density gradient separation and sub- sequent immunomagnetic selection (Miltenyi Biotec, Bergisch Gladbach, Germany) as described elsewhere.16
RNA sequencing
Transcriptome sequencing libraries were prepared from isolat- ed total RNA using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), and single-read 50 bp sequenc- ing was performed in a HiSeq-2000 device (Illumina). Reads were then trimmed by removing stretches of bases with a qual- ity score <30 at their ends, and subsequently mapped using Tophat2.0.6 against the hg19 assembly of the human genome.17 Finally, differential expression was quantified using DESeq2 and Cuffdiff 2.0, and subjected to diverse testing corrections.18,19 Genes with a q-value <0.05 were considered differentially expressed. Principal component analysis plots and heat maps were created in R using the FactoMineR and pheatmap pack- ages, respectively. For gene set enrichment analysis (GSEA, Broad Institute, Boston, MA, USA),20 the fragments per kilobase of transcript per million mapped reads (FPKM) values obtained from Cuffdiff 2.0 for the different samples were compared either
with the gene sets contained in the Molecular Signature Database or with self-made gene lists. The Signal2Noise metric and 1000 gene set-based permutations were applied to all analy- ses. Ingenuity pathway analysis software (Qiagen, Hilden, Germany) was used to predict the potential extrinsic factors capable of generating the different RNA sequencing data sets.
Cell culture conditions and reagents
Healthy MSC were cultured in Dulbecco modified Eagle medium low glucose supplemented with 30% fetal bovine serum and 1% penicillin/streptomycin/L-glutamine (all from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in the presence of TGFβ1 (10 ng/mL, PeproTech, Rocky Hill, NJ, USA) and/or SD-208 (0.5 μM, Tocris/R&D Systems, MN, USA), an established small molecule pyridopyrimidine inhibitor of the TGFβ receptor I kinase.21-23 The inhibitor was diluted in dimethyl sulfoxide (10 mM). The concentration of TGFβ1 used in our experiments is within the range of TGFβ1 levels previously detected in patients with MDS24,25 and has demonstrated inhibitory effects on healthy and MDS-derived hematopoietic cells.26-28 Furthermore, it was previously shown that TGFβ1 at this concentration mediates pathophysiologically relevant effects in other myeloid malignancies such as AML, chronic myeloid leukemia and myeloproliferative syndromes.9,29 Depending on the experimental setting, incubation time ranged from 3 to 28 days corresponding to the duration of the culture of MSC investigated by RNA sequencing. Detailed information is given in the legends of the respective figures.
Subsequently, to investigate the effects of TGFβ1 on the func- tionality of cells, pre-incubated MSC underwent the phenotypic and functional analyses described below.
Phenotypic and functional characterization of mesenchymal stromal cells
The morphology and growth properties of primary and pre- incubated MSC were characterized by light microscopy. For quantification of growth potential, absolute cell numbers and cumulative population doubling were determined as described elsewhere.12,13 Furthermore, surface expression of established stromal cell markers CD73, CD90 and CD105,30 hematopoietic antigens CD34 and CD45 as well as Jagged1 were measured by flow cytometry using a FACSCalibur (BD Biosciences, Heidelberg, Germany) and FCS Express V3 software (De Novo Software, Los Angeles, CA, USA) for data analysis. Antibody specifications are provided in Online Supplementary Table S2. Osteogenic differentiation capacity was investigated and visual- ized by Alizarin red staining as reported elsewhere.12,13 In addi- tion, mRNA expression of markers of osteogenesis, osterix and osteocalcin, and other candidate genes was measured by quan- titative polymerase chain reaction (PCR) on a StepOne Plus Realtime PCR Cycler (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) as described before.12,13
Statistical analysis
Statistical analyses were performed using Prism 5.01 (GraphPad Software Inc., La Jolla, USA). For inter-individual
Long-term culture-initiating cell assay
After pre-incubation with TGFβ1 or its antagonist, SD-208, 0.8x106-1.2x106 MSC were cultured on 96-well plates (Costar, Corning, USA) and irradiated with 30 Gray using Gulmay RS225 X-ray equipment. Subsequently, 6x103 healthy CD34+ cells were plated on these MSC feeder layers and then further processed using the same conditions and reagents as in our previous work.12,13
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