Mesenchymal stromal cells (MSC) from JAK2+ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis

[EN]There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HDMSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cell

Transcriptomic characterization of human Mesenchymal Stromal/Stem Cells

[EN] The therapeutically applied population of Mesenchymal Stromal/Stem Cells, defined under the minimal criteria of the International Society of Cellular Therapy (ISCT), have still a defectively characterized phenotype difficult to distinguish from similar cell populations. This Doctoral Thesis has been formulated as a characterizing data-driven approach. Thus, the main scope is to improve the characterization of the phenotype of human mesenchymal stem/stromal cells. We have approached such scope through a deep comparative transcriptomic study applying high-throughput genome-wide analytic techniques: producing and integrating RNA sequencing and microarray data assays, with a meta-analysis of a large collection of public expression data. The research falls within the fields of cellular, molecular and systems biology, which entails two complementary approaches: the experimental, and the computational ones. First, we isolated, cultured and validated (following the ISCT criteria of immunophenotype and in-vitro tri-lineage differentiation) human MSCs from three tissue origins: placenta, bone marrow, and adipose tissue. Applying bioinformatics techniques, we then defined a gene expression signature common to all human tissue-MSCs, as well as the specific expression profiles associated to each tissue-MSC type. Based on theseachievements, we could come up with novel insights about the MSC phenotype that will lead to the drafting of new hypotheses. Also, we yielded a broad transcriptomic signature resource ready to serve as reference on upcoming identification studies

Mesenchymal stromal cells (MSC) from JAK2⁺ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis

<div><p>There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2⁺ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells.</p></div

Differential expression of genes related to hematopoiesis in MPN-MSC.

<p>RT-PCR was used to determine the expression level of the different genes associated with maintenance of hematopoiesis. GAPDH was used as housekeeping to normalize the results. In the scatter plot graphic are represented by the median and the range.</p

Clinical characterisitics of MPN patients.

<p>Clinical characterisitics of MPN patients.</p

Protein expression of NF-ƙB, ANGPT-1 and CXCL12 in MPN-MSC.

<p>(A) Western Blot of NF-ƙB and ANGPT-1 in MPN and HD-MSC. (B) Representative image of CXCL12 expression in the MSC by immunofluorescence. HD-MSC shows more CXCL12 (red) than the positive control (HeLa cells) and the MPN-MSC. In green shows tubulin. Scale: 0–50μm.</p

Characterization of BM-MSC from JAK2V617F patients and healthy donors.

<p>(A) The number of population doublings (PD) in each passage was calculated using the following equation: PD = log10(N)/log10(2), where N is the number of cells harvested at the end of the culture per the number of seeded cells, where isolated from HD and JAK2⁺ PV and ET patients BM-MSC (Passage1 to Passage 3). (B) Mean fluorescence intensity of positive surface marker expression of HD-MSC and MPN-MSC; HD-MSC n = 20 and JAK2-MSC n = 30. Values indicate the mean ± SEM. (C) In vitro multilineage differentiation assays performed in HD-, PV- and ET-MSC. Left-handed photos represent the negative controls (no induction medium applied). The photos of the middle show osteogenic differentiation detected by alkaline phosphatase activity. Right-handed photos show adipogenic differentiation detected fat staining with Oil-Red-O (20X).</p

Expression of NF-ƙB and ANGPT-1 and CXCL12 by real-time PCR in MSC cell lines (hTERT and HS5) after co-culture with UKE-1 cells.

<p>Results were normalized with the housekeeping gene GAPDH. Results are represented by the median and the interquartile range. (A) Angiopoetin 1. (B) NF-ƙB. (C) CXCL12. The control HS5 and hTERT represents the cells that were co-cultured for 72h with UKE cells (<i>transwell</i>) n = 10.</p

Capacity of MPN-MSC to support hematopoietic progenitor cells.

<p>(A) Selective protection of leukemic hematopoiesis by MPN-MSC. Left-handed graphic shows the total colony-forming unit (CFU-GM) from HD-CD34⁺ cells after 48h of culture with HD-MSC (n = 7) and MPN-MSC (n = 7), no differences were observed between groups. Middle-handed shows CFU-GM from JAK2V617F-CD34⁺ cells after culture with HD-MSC (n = 4) and MPN-MSC (n = 6), showing a significant increase when leukemic progenitor cells were cultured with MPN stroma. Right-handed graphic showed the results are expressed as the ratio between CFU-GM obtained with CD34⁺ cells that had been co-cultured with HD-MSC or MPN-MSC and CD34⁺ cells without MSC. The results are showed as absolute CFU-GM per 5000 CD34⁺ cells seeded in methylcellulose, after 14 days in culture. (B) Capacity of MPN-MSC to maintain HD-HPC in LTBMC. Total of CFU-GM from HD-CD34+ cells after 5 weeks in co-culture with HD-MSC (n = 7) and MPN-MSC (n = 7). Total BFU-e. Total number of colonies (CFU), results shown are expressed as the mean of CFU±SEM. * p˂0.05 ** p˂0.01.</p

Apoptosis and cell cycle analysis of BM-MSC.

<p>(A) Graph bar chart that represents the percentage of apoptotic cells (AnnexinV/7AAD positives), and representative FACS dotplot of annexinV/7AAD staining on BM-MSC.* p˂0.05. (B) Percentage of cells in G1, S and G2/M phase from HD-MSC, PV-MSC and ET-MSC. No differences were observed between groups.</p