Page 149 - Haematologica March 2020

Bone marrow niche dysfunction in ET
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Figure 1. Transcriptomic analysis revealed multiple abnormalities in bone marrow derived mesenchymal stromal cells (BM-MSC) from patients with JAK2V617F-positive essential thrombocythemia (ET). A. Heatmap of transcriptomic analysis from eight MSC samples (control, n=5; untreated patients with JAK2V617F-positive ET, n=3) demonstrated that MSC from patients with JAK2V617F-positive ET differed from those isolated from HD. Briefly, 1,195 genes were iden- tified with a cut-off of greater than 2.0-fold for gene expression change and P<0.05. B. GO analysis of genes enriched in terms of different function showed changes in gene sets related to the cell cycle, cell differentiation, proliferation, death, and aging. C. KEGG analysis was carried out to identify differential pathway enrichment between ET and control. Rich factor refers to the ratio of the number of genes differentially expressed in the pathway entry to the total number of genes in the pathway entry. A larger rich factor indicates a higher degree of enrichment. The q-value is the P-value after multiple-hypothesis test corrections, ranging from 0 to 1 (a value closer to zero indicates a more significant enrichment). The figure is plotted with the top 20 paths sorted according to the q value from small to large and shows enrichment of genes of inflammation pathways. D. GSEA using MSigDB identified differential gene enrichment between ET and the control. NES, Normal p and FDR q-values for each gene set are shown. The results revealed differential expression of genes involved in cell cycle, inflammatory responses, and hematopoietic sup- port. E. qPCR validation of relevant genes (control, n=16; untreated patients with JAK2V617F-positive ET, n=16). MSC used for gene analysis were isolated and expanded in vitro and identified according to the minimal criteria for defining multipotent mesenchymal stromal cells stated by the International Society for Cellular Therapy position at passage four.50 *P< 0.05; **P<0.01, ***P<0.001; ****P<0.0001. Data are presented as the mean or mean ± SEM. ET: essential thrombo- cythemia; HD: healthy donors; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: gene set enrichment analysis; MsigDB: Molecular Signatures Database; NES: normalized enrichment score; qPCR: quantitative real-time PCR; n: number of unique donors in each group; ns: not significant; SEM: stan- dard error of mean.
1B). Further analysis showed a prominent abundance of gene signatures associated with inflammation (Figure 1C). Gene Set Enrichment (GSEA) analysis confirmed signifi- cant enrichment of dysregulated genes involved in the cell cycle, inflammatory responses, and hematopoietic sup- port (Figure 1D). These changes, confirmed by qPCR (Figure 1E), suggested multiple functional defects in MSC of patients with JAK2V617F-positive ET.
BM-MSC from patients with JAK2V617F-positive ET show enhanced proliferation and attenuated apoptosis, senescence, and differentiation
To verify the results of the gene expression analyses, we performed Cell Counting Kit 8 (CCK-8) assays, which revealed that the MSC of the patients had higher prolifer- ative capacity than the MSC of the HD (Figure 2A). Furthermore, the apoptosis (Figure 2B) and senescence rates (Figure 2C) of the MSC derived from the patients
were lower. The adipogenic and osteogenic differentiation potentials of the ET MSC were also significantly lower (Figure 2D). In line with the above results, the majority of the ET MSC were in S and G2 phases (Figure 2E). Additionally, we detected an increase in the level of NES mRNA (Figure 2F) and the number of NES-positive cells (Figure 2 G-H) in the BM of the patients. No difference in immunophenotype or morphology was detected between the MSC of the HD and those of the patients (Online Supplementary Figure S1 A-B).
BM-MSC from patients with JAK2V617F-positive ET show insufficient capacity to support normal hematopoiesis
To assess the capacity of MSC to support hematopoiesis, we established a coculture setting involv- ing normal CD34-positive cells and MSC derived from the HD or patients with JAK2V617F-positive ET. The recov-
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