Telomere length, c-myc and mad-1 expression could represent prognosis markers of myelodysplastic syndrome.

Telomere dysfunction might generate genomic instability leading to the progression of myelodysplastic syndromes (MDS) into acute myeloid leukemia (AML). We investigated telomere length (TL), telomerase activity (TA) and hTERT, c-myc, mad1, and p53 expression in the bone marrow of patients with MDS (n=109), AML (n=47) and in controls (n=24). TL was lower in MDS patients than in controls (p<0.001) and higher in L-MDS (low, intermediate-1 IPSS, p<0.01) respect H-MDS (high, intermediate-2 IPSS, p<0.01) patients. Mad-1 expression was higher in MDS patients than in controls (p<0.01), c-myc expression was highest in AML and in H-MDS patients. Our results show that the telomere dynamics might be useful for stratifying patients according to a risk scoring system.

Prognostic role of immunohistochemical analysis of 5 mc in myelodysplastic syndromes.

BACKGROUND: Aberrant DNA methylation at CpG islands within promoters is increasingly recognised as a common event in human cancers and has been associated with the silencing of important tumour suppressor genes. Epigenetic therapy using hypomethylating agents has demonstrated clinical effectiveness; the drugs azacitidine and decitabine have been approved for the treatment of MDS. METHOD: We investigated the association between global DNA methylation and clinical outcome in MDS. We evaluated 134 MDS bone marrow trephine biopsies (BMTB) by immunohistochemistry and compared the results with those from an age-matched group of normal BMTB. Immunohistochemistry was performed on paraffin-embedded sections using the anti-5-methylcytosine (5mc) antibody. RESULTS: Our results showed that the 5mc immunostaining score (M-score) of patients with MDS was higher than those of normal controls and that overall survival significantly correlated with global DNA methylation, age and IPSS score. Therefore, we found that patients with high levels of methylation had a shorter median overall survival (OS) compared with patients with lower levels. These immunohistochemistry results were confirmed by analysing global DNA methylation on LINE-1 sequences using the COBRA method and pyrosequencing. CONCLUSION: This study reports that global DNA methylation detected by immunohistochemistry predicts OS in MDS.

Molecular and functional characterization of human bone marrow adipocytes.

Adipocytes are a cell population largely located in the human bone marrow cavity. In this specific microenvironment where adipocytes can interact with a variety of different cells, the role of fat is mainly unknown. To our knowledge, this report is the first to characterize mature adipocytes isolated from human bone marrow (BM-A) molecularly and functionally to better understand their roles into the hematopoietic microenvironment. Healthy BM-A were isolated after collagenase digestion and filtration. We studied the morphology of BM-A, their gene expression and immunophenotypic profile and their functional ability in the hematopoietic microenvironment, comparing them with adipocytes derived from adipose tissue (AT-A). BM-A showed a unilocular lipid morphology similar to AT-A and did not lose their morphology in culture; they showed a comparable pattern of stem cell-surface antigens to AT-A. In line with these observations, molecular data showed that BM-A expressed some embryonic stem cells genes, such as Oct4, KLf4, c-myc, Gata4, Tbx1, and Sox17, whereas they did not express the stem cell markers Sox2 and Nanog. Moreover, BM-A had long telomeres that were similar to bone marrow mesenchymal stem cells. Notably, BM-A supported the survival and differentiation of hematopoietic stem cells in long-term cultures. These results showed that BM-A are stromal cells with a gene expression pattern that distinguished them from AT-A. BM-A showed stem cell properties through their hematopoietic supporting function, which was certainly linked to their role in the maintenance of the bone marrow microenvironment. Depending on specific demands, BM-A may acquire different functions based on their local environment.

Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. Here, we studied the gene expression and functional properties of mature adipocytes isolated from human omental and subcutaneous fat tissues. We also focused on dedifferentiated adipocytes in culture and their morphologies and functional changes with respect to mature adipocytes, stromal-vascular fraction (SVF)-derived mesenchymal stem cells (MSCs) and bone marrow (BM)-derived MSCs. Isolated mature adipocytes expressed stem cell and reprogramming genes. They replicated in culture after assuming a fibroblast-like shape and expanded similarly to SVF- and BM-derived MSCs. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the typical mesenchymal morphology and immunophenotype, expressed stem cell genes and differentiated into multilineage cells. Moreover, during the dedifferentiation process, we showed changes in the epigenetic status of mature adipocytes, which led dedifferentiated adipocytes to display a similar DNA methylation condition to BM-derived MSCs. Like SVF- and BM-derived MSCs, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature adipocytes stimulated their growth. Furthermore, dedifferentiated adipocytes maintained the survival and complete differentiation characteristic of hematopoietic stem cells. This is the first study that in addition to characterizing isolated and dedifferentiated adipocytes also reports on the immunoregulatory and hematopoietic supporting functions of these cells. This structural and functional characterization might have clinical applications of both mature and dedifferentiated adipocytes in such fields, as regenerative medicine.

Gene expression profile of cytokines in patients with chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation with reduced conditioning.

There are no reliable markers useful to predict the onset or the evolution of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT), although several candidate biomarkers have been identified from limited hypothesis-driven studies. In this study we evaluated 14 patients who received a reduced intensity conditioning HSCT. Seven patients had cGVHD, whereas 7 never developed cGVHD during the period of observation. The expression of 114 cytokines in immunoselected cell populations was explored by microarray analysis and 11 cytokines were selected for further evaluation by real-time PCR. Differential gene expression measurements showed a significant up-regulation for INFγ (interferon, gamma) in CD8+ and for TNFSF3 (tumor necrosis factor superfamily, member 3) and for TNFSF10 (tumor necrosis factor superfamily, member 10) in CD14+ cell population when comparing cGVHD with control samples. The expression levels were significantly decreased for TNFSF10 in CD8+ cell population and for TNFSF12 (tumor necrosis factor superfamily, member 12) and for PDGFß (platelet-derived growth factor, beta) in CD4+. Our data seem to suggest that different immune populations can play a role in cGVHD pathogenesis and the early detection of gene expression profile in these patients could be useful in the monitoring of GVHD. We hypothesized that PDGFß down-regulation could represent a negative feedback to compensate for enhanced expression of its receptor recently reported.

Human mesenchymal stem cells from chorionic villi and amniotic fluid are not susceptible to transformation after extensive in vitro expansion.

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. Increasing evidence suggests that MSCs isolated from fetal tissues are more plastic and grow faster than adult MSCs. In this study, we characterized human mesenchymal progenitor cells from chorionic villi (CV) and amniotic fluid (AF) isolated during the first and second trimesters, respectively, and compared them with adult bone marrow-derived MSCs (BM). We evaluated 10 CV, 10 AF, and 6 BM samples expanded until the MSCs reached senescence. We used discarded cells from prenatal analyses for all the experiments. To evaluate the replicative stability of these cells, we studied the telomerase activity, hTERT gene transcription, and telomere length in these cells. Spontaneous chromosomal alterations were excluded by cytogenetic analysis. We studied the expression of c-myc and p53, tumor-associated genes, at different passage in culture and the capacity of these cells to grow in an anchorage-independent manner by using soft agar assay. We isolated homogeneous populations of spindle-shaped CV, AF, and BM cells expressing mesenchymal immunophenotypic markers throughout the period of expansion. CV cells achieved 14 ± 0.9 logs of expansion in 118 days and AF cells achieved 21 ± 0.9 logs in 118 days, while BM cells achieved 11 × 0.4 logs in 84 days. Despite their high proliferation capacity, fetal MSCs showed no telomerase activity, no hTERT and c-myc transcriptions, and maintained long, stable telomeres. A constant expression level of p53 and a normal karyotype were preserved throughout long-term expansion, suggesting the safety of fetal MSCs. In conclusion, our results indicate that fetal MSCs could be an alternative, more accessible resource for cell therapy and regenerative medicine.