Alterations in the Physiology of Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Patients with Leukemia
Authors: Sorokina Tamara , Shipounova Irina , Bigildeev Alexey , Petinati Nataliya , Drize Nina I. , Turkina Anna , Chelysheva Ekaterina , Shukhov Oleg , Kuzmina Larisa A. , Parovichnikova Elena N. , Savchenko Valeriy G 
Published: October 2019

Abstract
Introduction
In acute and chronic leukemia, the changes in the basic properties of multipotent mesenchymal stromal cells (MSCs), including morphology, immunomodulatory abilities, and the expression of various genes, were described. The aim of the investigation was to study the ability of MSCs derived from the bone marrow (BM) of patients with acute lymphoblastic (ALL), myeloid (AML) leukemia and chronic myeloid leukemia (CML) to maintain normal hematopoietic progenitor cells.
Methods
The study included 14 patients with ALL, 25 with AML and 15 with CML. All work was conducted in accordance with the Declaration of Helsinki (1964). BM was aspirated from the patient during diagnostic punctures before the treatment and for patients with acute leukemia 37 and 100 days; for patients with CML on average at the 125th day and 220th day after the start of the treatment. The BM samples of 22 healthy donors were used as controls.
MSCs were derived from 5-10 ml of BM and cultivated at the density 3 х 106 cells in T25 culture flasks in aMEM with 10% FCS.
The ability of MSCs from patients to maintain normal hematopoietic precursor cells - coble stone area forming cells (CAFC) was performed by the limiting dilution method. One day prior to assay, MSCs from donors and patients were explanted with 1000 cells per well of 96-well plates. As a control, the cell line MS5 supporting growth of hematopoietic precursor cells was used. On the following day, with complete medium change, BM cells from healthy donors were implanted in four serial dilutions. The frequency of CAFC in the normal BM was performed using Poisson's equation and presented as the percent of control.
The total RNA was extracted from MSCs at passage 1 using the standard method. The relative gene expression levels (REL) were determined by normalizing the expression of each target gene to the levels of beta-actin and glyceraldehyde 3-phosphate dehydrogenase and calculated using the ΔΔCt method.
Results
Total cell production of MSCs from ALL (5.6±1.5) x 106 and AML (5.4±1.0) x 106 patients decreased at the moment of diagnosis, whereas the production of MSCs from CML (7.9±1.9) x 106 patients did not differ from the donors (7.1±1.04) x 106. Reduced cell production is likely associated with a significant decrease in the expression level of FGF2, VEGF, BGLAP and SOX9 genes (Table).
The ability of MSCs derived from the BM of AML patients at the onset of the disease to maintain normal CAFC (59.3±6.8) was significantly decreased (p = 0.02) when compared to donors (74.9±9.5). At the end of the first course of chemotherapy, the ability to maintain CAFC in patients' MSCs reached that of the donors(80.7±5.8); 2 months later, the CAFC frequency on MSCs from AML patients doubled (107.9±18.4) in comparison with the start of the disease (p = 0.04).
The ability of MSCs derived from ALL patients to support CAFC was lower than that of MSCs of AML patients both before treatment (57.8±12) and 37 days after the start (57.2±7.4). Three months after the initiation of treatment of these patients, the ability of MSCs to maintain CAFC recovered (73.7±13.3) and reached that of the donors' MSCs.
MSCs of CML patients (100.8±9.9) at the disease onset maintained CAFC better than donors' MSCs. This ability increased with treatment at 125th day (109.6±14.4) and at 220th day (169.6) after the start of the treatment. The expression levels of LIF, IGF1, IL6, CSF1 increased significantly in CML-derived as well as AML-derived MSCs, but changes were more pronounced in the case of CML.
Table. REL of different genes in MSCs derived from patients at the onset of the disease. Gene AML ALL CML Donors FGF2 2.8±0.2* 2.0 ±0.4* 4.2±0.5 6.0±0.9 VEGF 1.5±0.1* 1.5±0.2* 1.7±0.2 2.5±0.9 LIF 9.1±1.3* 7.1±2.3* 21.8±7.5 2.2±0.4 IL6 12.7±1.8* 11.3±4.4 22.5±4.8* 6.2±1.8 CSF1 3.6±0.9* 2.1±1.2 0.96±0.1* 0.7±0.1 IGF1 1.3±0.3* 1.49±0.7 2.6±0.5* 0.6±0.1 BGLAP 0.6±0.2* 0.8±0.4* 1.7±0.4 2.7±0.1 SOX9 1.2±0.1* 0.9±0.3* 1.2±0.2 1.7±0.2 (*significantly different from donors)
Conclusions
Functional changes in MSCs, which are the part of BM stromal microenvironment and, in particular, a niche for the HSCs was revealed. The ability of MSCs to support CAFC is dramatically changed in patients with hematological malignancies; the nature of the functional alterations of MSCs depends on the diagnosis. In cases of acute leukemia, MSCs' ability to maintain CAFC normalized with the treatment, whereas the strengthening of this capacity was revealed in cases of CML.

Disclosures
Turkina: Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis Pharma: Consultancy.