| Abstract |
Introduction
Mesenchymal stromal cells (MSCs) are important component of the bone marrow microenvironment which regulates the survival, proliferation and differentiation of hematopoietic stem cells, while their characterization is not complete. Considering the participation of marrow microenvironment in the development and progress of leukemia, one could apply the hypothesis that MSCs are involved in abnormal hematopoiesis. MSCs involvement in acute lymphoblastic leukemia in children has recently been addressed and there are indications that the proliferation rate is slower and the support of hematopoiesis in long term cultures is affected.
The aim of this study is the morphological, immunophenotypical and functional characterization of mesenchymal stromal cells from the bone marrow of children with acute lymphoblastic leukemia in the diagnosis of disease, in order to assess the possible effect of leukemia in biological and functional characteristics. There will further be compared with the relevant characteristics of MSC derived from different treatment phases, in order to evaluate the effect of treatment on these features.
Methodology
The samples of the study include bone marrow (BM) from children with acute lymphoblastic leukemia (ALL) at diagnosis, at the different phases of the treatment during the therapeutic protocol and at the end of chemotherapy after treatment completion. The control group consisted of BM samples from children with solid tumors without BM involvement.
The proliferation capacity of pluripotent mesenchymal cells was studied by cell doubling time (DT) calculation at all stages, after each trypsinization. The clonogenicity was evaluated by colony forming unit- fibroblast (CFU-F) formation. For the immunophenotypic profile the expression of surface antigens, relevant for mesenchymal and hematopoietic cells in various stages of culture was performed by flow cytometry. Apoptosis and cell cycle analysis were assessed at passages P2 and P4 with flow cytometry.
The ability of mesenchymal stromal cells to differentiate into adipocytes, chondrocytes and osteocytes was studied in passages P2 or P3 of cultures. Differentiation was confirmed by
histochemical analysis after staining with Oil Red O for adipocytes, von Kossa for osteocytes and Alcian Blue for chondrocytes.
SDF-1a and angiopoietin-1 levels in MSC supernatant at diagnosis and in different phases of treatment were assessed by ELISA.
For some of the above features the results were reviewed after classification of the patients into medium-risk group (MR) and high-risk group (HR), according to the prognostic factors for ALL outcome.
The results of the experiments are expressed as mean ± standard error of the mean (SEM) unless otherwise indicated. Differences between groups were assessed using the non-parametric Mann–Whitney U-test. p-values lower than 0.05 were considered as statistically significant. For the statistical analysis the statistical package SPSS, (v18.0) was used.
Results
Morphology:
BM MSCs from all groups were expanded up to the fifth passage and all displayed the characteristic spindle-shape morphology. No morphological differences were observed among cells of different groups.
Immunophenotypic analysis:
The immunophenotypic analysis was performed in mononuclear cells (D0) and in mesenchymal cells at passages P2 and P4. There were no differences in the expression of surface antigens among groups. At diagnosis, mesenchymal cells highly express the CD90, CD105, CD146, CD29, CD44, CD95 and CD73 markers, while there was no expression of the hematopoietic markers CD34, CD45 and CD14. The same profile remained in all phases of treatment, at the end of chemotherapy and in the control group.
Growth rate (DT ):
Mesenchymal cells in the fraction of mononuclear cells (D0) required several days at diagnosis to form layer. In the next passages, cell doubling time was similar in all groups (diagnosis, day 15 and 33 of therapy, consolidation therapy, maintenance and at the end of treatment). These results indicate that mesenchymal cells present in the fraction of mononuclear cells at diagnosis which, is mainly constituted of lymphoblasts, expanded more slowly compared to treatment phases and the control group, but this defect subsides with the progression of culture (more advanced P). It was found that the more advanced the passage was, the slower was the proliferation rate in all groups and there were no differences between the phases of treatment and the control group. The HR group numerically presented lower doubling time at diagnosis at the beginning of the culture and at the initial passages (P1 to P2) but the comparison of the DT between High (HR) and Medium (MR) risk patients, in each treatment group showed no statistically significant difference.
CFU - F development:
On day 0, the CFU-F formation at the time of diagnosis was impaired compared to the treatment phases, the end of chemotherapy and the control group. The number of CFU-F gradually increases from d15, but continues to lag behind until d33 compared with the other phases of treatment. This result derives from the lesser number of medium and large-sized colonies that grow. In the initial stages of culture there are more large-sized colonies, while in advanced stages of the population of CFU-F colonies consist, mainly, of small-sized colonies. The reduced CFU-F development at diagnosis was a constant finding observed at subsequent passages as well. With the progress of cultures, the number of colonies formed appears reduced at all treatment phases and at the control group and became statistically significant at the later passages (P1 vs P4 or P5). Comparing clonogenicity of the risk groups there was no statistically significant difference in the total number of CFU-F although in most cases MR were producing more colonies than HR.. MSC n the fraction of mononuclear cells (MNCs) in both risk groups developed fewer colonies atdiagnosis compared to other phases, finding that should be attributed to large-size colonies.
Differentiation capacity:
The ex vivo expanded mesenchymal cells differentiated successfully into the three cell types namely adipocytes (A), osteocytes (O), chondrocytes (C). At diagnosis the differantiation capacity was defective in comparison with the treatment phases of leukemia.
Study and analysis of cell cycle:
Cell cycle was studied at P2 and P4 by flow cytometry and, according to the results, the higher percentage of mesenchymal cells were found in the quiescence phase.
Study and analysis of apoptosis:
The study of apoptosis by flow cytometry was performed after staining with 7-AAD, at either P2 or P4. . According to our results, most of the MSC from childhood ALL are in quiescence. This finding confirms the stability of bone marrow MSCs under long-term culture expansion, both at the diagnosis of leukemia and the chemotherapy phases.
SDF -1a and Angio -1 expression:
SDF-1a in the MSC supernatants at diagnosis was variably expressed and did not differ compared to the treatment phases, and exhibited a more uniform expression in the rest treatment phases. Its levels were higher in the high-risk (HR) group compared to the medium-risk (MR) group.
As far as Ang-1 expression is concerned, in the two cell subpopulations of MNCs and MSCs, and the different treatment groups our results showed that, similar to SDF-1a, stromal cells secreted statistically significant higher amounts of this growth factor. No difference was found in the comparison of diagnosis with treatment groups. The Ang-1 levels of the MSC population were independent of the risk group as no difference was observed between HR and MR patients.
Discussion
Mesenchymal stromal cells support and regulate hematopoiesis, constituting a major component of the bone marrow microenvironment, although they represent only a small proportion of nucleated cells. The structure that represents the hematopoietic niche has a key role in the functional maintenance of hematopoietic stem cells by protecting them from harmful external factors. Maintaining homeostasis of the hematopoietic system requires a balance between self renewal, differentiation and apoptosis of hematopoietic cells and interaction with the bone marrow microenvironment. MSCs participate in the organization of the haematopoietic niche, exercising a key role in homing, mobilization and expansion of HSCs. Their presence is necessary for the installation and maintenance of HSCs and, moreover, they regulate the normal function of hematopoietic cells and maintain homeostasis in the niche, via secretion of growth factors. There are indications that haematological malignancies are associated with deregulation in the microenvironment of the bone marrow and that MSCs participate in the process of developing neoplasia by modifying the phenotype of tumours of the hematopoietic system.
Acute lymphoblastic leukemia is a heterogeneous disease of the hematopoietic tissue, as a result of accumulation of mutations and abnormal proliferation of progenitor B lymphocytes. It is the most common malignancy in children. The potential role or contribution of MSCs in the disease onset and the subsequent course of it and in the response to treatment is not yet studied. Also, there has been shown that mesenchymal bone marrow cells of children are not similar to those of adults.
The data obtained for the proliferation capacity, the clonogenicity and the trilineage differentiation ability of mesenchymal cells, suggest that the presence of blasts in the diagnosis of acute lymphoblastic leukemia affects these characteristics, while the application of chemotherapeutic agents during treatment has no effect.
The last decade the hypothesis that disturbances in the cell cycle regulation and the sequence of events of apoptosis lead to genetic instability, resulting in predisposition to malignant transformation, is gaining interest. In particular, there is the possibility of the involvement of deregulation between self renewal of blood cells and their ability to lead to apoptosis in pathogenesis, but also in the development of leukemia. Our results confirm the stability of bone marrow MSCs in long-term culture conditions, both in the diagnosis of leukemia and in treatment phases.
Finally, we evaluated the levels of SDF-1a and Ang-1, recently revealed as major regulators in the crosstalk between hematopoietic progenitors and their microenvironment. Data reporting the expression of SDF-1a by BM MSCs in patients with hematological malignancies are limited. SDF-1a in the supernatant of MSCs at diagnosis of ALL was slightly increased compared to that from treatment phases, although this difference was not statistically verified. Interestingly, high-risk patients exhibited higher levels compared to the medium-risk ones, a difference no longer occurring upon treatment initiation. We found that the lowest amount of Ang-1 was expressed in MSC culture supernatant from diagnosis, albeit not statistically differently from treatment phases.
In conclusion, biological characteristics and functional properties of MSCs are affected at the onset of leukemia. Most defects persist throughout passages. MSCs recover after treatment initiation and remission achievement and are not affected by chemotherapy. Their secretory profile remains unaltered by the disease. The summing of these data clearly indicates that any effect on MSCs from the leukemic clones in childhood ALL is transient and ceases upon treatment initiation.
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