Humoral Effect of a B-Cell Tumor on the Bone Marrow Multipotent Mesenchymal Stromal Cells.

The properties of bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) are altered in the patients with the diffuse large B cell lymphoma (DLBCL) without BM involvement. It was suggested that plasma from the patients contains soluble factors that affect MSCs. Plasma and BM-derived MSCs from the DLBCL patients at the onset of the disease and one month after the end of treatment were studied. Concentration of the plasma cytokines and gene expression in the MSCs were evaluated by the Bio-Plex Pro Human Cytokine Panel kit to measure 27 analytes and real-time PCR. Plasma and MSCs from the healthy donors were used as controls. Analysis of cytokines in the plasma from healthy donors and patients before and one month after the end of treatment revealed significant differences in the concentration of 14 out of 27 cytokines. Correlations between the levels of secreted cytokines were altered in the plasma from patients indicating that the immune response regulation was disturbed. Cultivation of the MSCs from the healthy donors in the medium supplemented with the plasma from patients led to the changes in the MSC properties, similar to those observed in the MSCs from patients. The BM-derived MSCs were shown to participate in the humoral changes occurring in the DLBCL patients. For the first time, it was shown that the precursors of the stromal microenvironment - multipotent mesenchymal stromal cells - are altered in the patients with DLBCL without bone marrow involvement due to the humoral effect of the tumor and the response of organism to it. Comprehensive analysis of the results shows that, when remission is achieved in the patients with DLBCL, composition of the plasma cytokines normalizes, but does not reach the level observed in the healthy donors. The discovery of a new aspect of the effect of the tumor B-cells on the organism could help to reveal general regularities of the humoral effect of various tumors on the bone marrow stromal cells.

The ability of multipotent mesenchymal stromal cells from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells.

BACKGROUND: The development of leukemia impairs normal hematopoiesis and marrow stromal microenvironment. The aim of the investigation was to study the ability of multipotent mesenchymal stromal cells (MSCs) derived from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells. METHODS: MSCs were obtained from the bone marrow of 14 patients with acute lymphoblastic (ALL), 25 with myeloid (AML), and 15 with chronic myeloid (CML) leukemia. As a control, MSCs from 22 healthy donors were used. The incidence of cobblestone area forming cells (CAFC 7-8 d) in the bone marrow of healthy donor cultivated on the supportive layer of patients MSCs was measured. RESULTS: The ability of MSCs from AML and ALL patients at the moment of diagnosis to maintain normal CAFC was significantly decreased when compared to donors. After chemotherapy, the restoration of ALL patients' MSCs functions was slower than that of AML. CML MSCs maintained CAFC better than donors' at the moment of diagnosis and this ability increased with treatment. CONCLUSIONS: The ability of patients' MSCs to maintain normal hematopoietic progenitor cells was shown to change in comparison with MSCs from healthy donors and depended on nosology. During treatment, the functional capacity of patients' MSCs had been partially restored.

Analysis of multipotent mesenchymal stromal cells used for acute graft-versus-host disease prophylaxis.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) are used for prophylaxis of acute graft-versus-host disease (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Not all samples of MSC are efficient for aGvHD prevention. The suitability of MSCs for aGvHD prophylaxis was studied. METHODS: MSCs were derived from the bone marrow (BM) of HCT donor and cultivated for no more than three passages. The characteristics of donor BM samples including colony-forming unit fibroblast (CFU-F) concentration, growth parameters of MSCs, and the relative expression levels (REL) of different genes were analyzed. MSCs were injected intravenously precisely at the moment of blood cell reconstitution. RESULTS: MSCs infusion induced a significant threefold decrease in aGvHD development and improved overall survival compared with the standard prophylaxis group. In ineffective MSC samples (9.4%), a significant decrease in total cell production and the REL of CSF1, FGFR1, and PDGFRB was observed. In all studied BM samples, the cumulative MSC production and CFU-F concentrations decreased with age. The expression levels of FGFR2, PPARG, and VEGF differed by age. CONCLUSIONS: A universal single indicator for the prediction of MSC eligibility for aGvHD prophylaxis was not identified. A multiparameter mathematical model for selecting MSC samples effective for the prevention of aGvHD was proposed.

Interleukin-1 beta enhances human multipotent mesenchymal stromal cell proliferative potential and their ability to maintain hematopoietic precursor cells.

Multipotent mesenchymal stromal cells (MMSCs) have been demonstrated to produce mature stromal cells and maintain hematopoietic progenitor cells (HPC). It was previously demonstrated that interleukin-1 beta (IL-1 beta) stimulates the growth of the stromal microenvironment in vivo. The aim of this study was to investigate the effect of IL-1 beta treatment of human MMSCs on their proliferative potential, gene expression, immunomodulating properties, and their ability to support HPCs in vitro. Human bone marrow-derived MMSCs were cultivated in standard conditions or with IL-1 beta. The cumulative cell production was assessed for five passages. After withdrawal of IL-1 beta, MMSC clonal efficiency was investigated, and the maintenance of HPCs on top of MMSCs layers was estimated using cobblestone area forming cell (CAFC) and long-term culture initiating cell (LTC-IC) assays. The effect of untreated MMSCs or MMSCs pretreated with IL-1 beta on lymphocyte proliferation was studied by CFSE staining. The relative expression level of various genes by MMSCs was analyzed using RT-qPCR. The administration of IL-1 beta elevated MMSCs clonal efficiency and total cell production but did not affect lymphocyte proliferation. MMSCs pretreatment with IL-1 beta enhanced their ability to maintain HPCs, as detected by CAFC assay, and it altered the expression levels of genes participating in HPC regulation by stromal cells, e.g., adhesion molecules (ICAM1) and growth factors (SDF1). This study revealed the ability of IL-1 beta to stimulate MMSCs proliferation and enhance their potential to maintain HPCs. MMSCs are considered a stromal niche component in vitro. The combined in vitro and previous in vivo data suggest that IL-1 beta is a systemic regulator of the stromal microenvironment.

Interleukin-1 beta is an irradiation-induced stromal growth factor.

Gamma irradiation of tissues and organs leads to many pathological consequences due to the formation of reactive oxygen species, DNA damage and the subsequent massive death of cells. The therapeutic use of gamma irradiation in the treatment of cancer is based on its penetrating power and damaging effects on tumor cells. Other effects from the irradiation are unnoticeable in comparison. Moreover, the long-term consequences of gamma irradiation are still poorly understood. When a donor bone marrow plug is implanted under the renal capsule of a syngeneic animal, а hematopoietic ectopic focus is formed. The size of the focus is increased in mice that received irradiation compared to non-irradiated ones, regardless of the amount of time between irradiation and bone marrow plug implantation. Long-term repetitive injections of blood serum from irradiated mice given to syngeneic non-irradiated recipients of bone marrow plugs also lead to the formation of enlarged foci. Hence, the blood of irradiated animals must contain an activity that induces the growth of a hematopoietic microenvironment. It was previously shown that the bones of irradiated animals secrete a growth factor required to create stromal microenvironments. The identity of this factor has, until now, been difficult to obtain. We demonstrated that interleukin 1 beta (IL-1) stimulates the growth of murine bone marrow stromal cells in vitro and in vivo. It was shown that the expression of the Il1b gene and the secretion of its product, IL-1, were activated in bone cells long after total body gamma irradiation. Hence, IL-1, or proteins regulated by this cytokine, appears to be the same stromal growth factor previously observed in the serum of irradiated animals. Our data demonstrate several non-canonical functions of IL-1. In addition, the presence of up-regulated levels of IL-1 long after irradiation points to an unknown mechanism governing its gene expression.

T Cell and Cytokine Dynamics in the Blood of Patients after Hematopoietic Stem Cell Transplantation and Multipotent Mesenchymal Stromal Cell Administration.

Multipotent mesenchymal stromal cells (MSCs) are currently under intensive investigation for the treatment and prevention of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), owing to their substantial immunomodulatory properties. The responses of recipients to MSC infusion following allo-HSCT are not yet well understood. T cells are central to the adaptive immune system, protecting the organism from infection and malignant cells. Memory T cells with different phenotypes, gene expression profiles, and functional properties are critical for immune processes regulation. The aim of this study was to study the dynamics of memory T cell subpopulations and cytokines in the blood of allo-HSCT recipients after MSC administration. In clinical trial NCT01941394, patients after allo-HSCT were randomized into 2 groups, one receiving standard GVHD prophylaxis and the other also receiving MSC infusion on the day of leukocyte recovery to 1000 cells/µL (engraftment, day E0). Blood samples of patients from both groups were analyzed on days E0, E+3, and E+30. T cell subpopulations were studied by flow cytometry, and cytokine concentrations were evaluated by the Bio-Plex Pro Human Cytokine Panel. Administration of MSCs to patients on day E0 did not affect the overall dynamics of restoration of absolute numbers and proportions of T and B lymphocytes after 3 and 30 days. At 3 days after MSC injection, only the numbers of CD8+ effector cells (CD8+TE, CD8+TM, and CD8+EM) were found to increase significantly. A significant increase in the number of CD4+ cells after 30 days compared to day E0 was observed only in patients who received MSCs, indicating faster recovery of the CD4+ cell population following MSC injection. An increase in CD8+ cell number by day E+30 was significant regardless of MSC administration. To characterize the immune status of patients following allo-HSCT in more detail, changes in the cytokine concentration in the peripheral blood of patients on days E0, E+3, and E+30 after MSC administration were investigated. On day E+30, significant increases in the numbers of CD4+CM and activated CD4+CD25+ cells were observed. The concentrations of proinflammatory and anti-inflammatory cytokines IL-6, IL-8, IL-17, TNF-α, and IFN-γ were increased significantly in patients injected with MSCs. Analysis of growth factor levels showed that in the group of patients who received MSCs, the concentrations of G-CSF, GM-CSF, PDGFbb, FGFb, and IL-5 increased by day E+30. Among the cytokines involved in regulation of the immune response, concentrations of IL-9, eotaxin, IP-10, MCP-1, and MIP-1a were increased after 30 days irrespective of MSC administration. The administration of MSCs exerts a positive effect on the restoration of T cell subpopulations and immune system recovery in patients after allo-HSCT.

Functional Characteristics of the Mouse Il1b Promoter in Various Tissues Before and After Irradiation.

Interleukin-1 beta (IL1B) is a key inducer of inflammation and an important factor in the regulation of hematopoietic stem cells and mesenchymal stromal progenitors. Irradiation of mice with ionizing radiation has been shown to induce a lasting increase in IL1B concentration in peripheral blood. One of the possible mechanisms may be demethylation of CpG cytosines in the Il1b promoter, which has not been characterized in detail for the mouse. In this study, the methylation level of CpGs located in a region between -3562 and -208 bp upstream of the start of transcription is studied in muscles, bones, liver, thymus, spleen, bone marrow, lymph nodes, lungs, and brain. The methylation level is compared to Il1b expression. Tissue-specific features of CpG methylation are established. It is demonstrated that the region between -2420 and -2406 bp is likely a part of the mouse Il1b promoter/enhancer and may determine the base level of Il1b expression in various tissues. Irradiation at a dose of 6 Gy does not change the methylation profile of most studied CpGs, and therefore, the cause of the stably increased IL1B level after irradiation is unlikely to be a change in the methylation of the studied CpGs in investigated tissues.

Immunophenotypic characteristics of multipotent mesenchymal stromal cells that affect the efficacy of their use in the prevention of acute graft vs host disease.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) are widely used in the clinic due to their unique properties, namely, their ability to differentiate in all mesenchymal directions and their immunomodulatory activity. Healthy donor MSCs were used to prevent the development of acute graft vs host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). The administration of MSCs to patients was not always effective. The MSCs obtained from different donors have individual characteristics. The differences between MSC samples may affect their clinical efficacy. AIM: To study the differences between effective and ineffective MSCs. METHODS: MSCs derived from the bone marrow of a hematopoietic stem cells donor were injected intravenously into allo-BMT recipients for GVHD prophylaxis at the moment of blood cell reconstitution. Aliquots of 52 MSC samples that were administered to patients were examined, and the same cells were cultured in the presence of peripheral blood mononuclear cells (PBMCs) from a third-party donor or treated with the pro-inflammatory cytokines IL-1ß, IFN and TNF. Flow cytometry revealed the immunophenotype of the nontreated MSCs, the MSCs cocultured with PBMCs for 4 d and the MSCs exposed to cytokines. The proportions of CD25-, CD146-, CD69-, HLA-DR- and PD-1-positive CD4+ and CD8+ cells and the distribution of various effector and memory cell subpopulations in the PBMCs cocultured with the MSCs were also determined. RESULTS: Differences in the immunophenotypes of effective and ineffective MSCs were observed. In the effective samples, the mean fluorescence intensity (MFI) of HLA-ABC, HLA-DR, CD105, and CD146 was significantly higher. After MSCs were treated with IFN or cocultured with PBMCs, the HLA-ABC, HLA-DR, CD90 and CD54 MFI showed a stronger increase in the effective MSCs, which indicated an increase in the immunomodulatory activity of these cells. When PBMCs were cocultured with effective MSCs, the proportions of CD4+ and CD8+central memory cells significantly decreased, and the proportion of CD8+CD146+ lymphocytes increased more than in the subpopulations of lymphocytes cocultured with MSC samples that were ineffective in the prevention of GVHD; in addition, the proportion of CD8+effector memory lymphocytes decreased in the PBMCs cocultured with the effective MSC samples but increased in the PBMCs cocultured with the ineffective MSC samples. The proportion of CD4+CD146+ lymphocytes increased only when cocultured with the inefficient samples. CONCLUSION: For the first time, differences were observed between MSC samples that were effective for GVHD prophylaxis and those that were ineffective. Thus, it was shown that the immunomodulatory activity of MSCs depends on the individual characteristics of the MSC population.

Alterations in multipotent mesenchymal stromal cells from the bone marrow of acute myeloid leukemia patients at diagnosis and during treatment.

We analyzed multipotent mesenchymal stromal cells (MMSCs) from the bone marrow (BM) of 33 acute myeloid leukemia (AML) patients at diagnosis, after the first course of chemotherapy (day 37), and at days 100 and 180 after diagnosis. All patients were treated according to the AML 01.10 protocol. Cumulative production of MMSCs from AML patients at diagnosis was normal but increased during treatment. Most of the studied genes were upregulated at AML diagnosis, some (IL6, IL1B, LIF) remained upregulated during treatment, and others were downregulated (FGFR1, ICAM1) or normalized. A few genes were normal at diagnosis but decreased during treatment (FGF2, FGFR2, VEGF, SDF1, SOX9, TGFB1). The upregulation of proinflammatory genes both at diagnosis and during remission reflects ongoing inflammation. PDGFRB expression was upregulated in MMSCs from patients in relapse versus those in remission. The AML 01.10 protocol downregulates the expression of genes related to proliferation, differentiation and niche formation.

Alterations of the bone marrow stromal microenvironment in adult patients with acute myeloid and lymphoblastic leukemias before and after allogeneic hematopoietic stem cell transplantation.

Bone marrow (BM) derived adult multipotent mesenchymal stromal cells (MMSCs) and fibroblast colony-forming units (CFU-Fs) of 20 patients with acute myeloid leukemia (AML) and 15 patients with acute lymphoblastic leukemia (ALL) before and during 1 year after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) were studied. The growth characteristics of MMSCs of all patients before allo-HSCT were not altered; however, relative expression level (REL) of some genes in MMSCs, but not in CFU-Fs, from AML and ALL patients significantly changed. After allo-HSCT, CFU-F concentration and MMSC production were significantly decreased for 1 year; REL of several genes in MMSCs and CFU-F-derived colonies were also significantly downregulated. Thus, chemotherapy that was used for induction of remission did not impair the function of stromal precursors, but gene expression levels were altered. Allo-HSCT conditioning regimens significantly damaged MMSCs and CFU-Fs, and the effect lasted for at least 1 year.

Clonal composition of human multipotent mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MMSCs) are a heterogeneous population consisting of cells with a distinct proliferative potential. The aim of this study was to define clonal composition in MMSCs and trace the dynamics of individual clones in MMSC subpopulations with different proliferative potentials during the process of cultivation. The investigation was performed at single-cell level using genetically marked cells. Specifically, human bone marrow MMSCs were infected with a lentiviral vector-bearing marker gene. Integration site analysis was performed for clones at each passage by ligation-mediated polymerase chain reaction and Southern blot hybridization. Sibling connections between clones and clonal composition of MMSC culture at each passage were revealed. The MMSC population contained multiple, different, mainly small, clones. It was found that large long-living clones with a high, but limited proliferative potential could be detected rarely in MMSCs population. These data suggest that the human MMSC population does not fit the "stem cell" criteria, however, MMSCs may contain a subpopulation of large clones with a high proliferative potential.

Multipotent Mesenchymal Stromal Cells for the Prophylaxis of Acute Graft-versus-Host Disease-A Phase II Study.

The efficacy and the safety of the administration of multipotent mesenchymal stromal cells (MMSCs) for acute graft-versus-host disease (aGVHD) prophylaxis following allogeneic hematopoietic cell transplantation (HSCT) were studied. This prospective clinical trial was based on the random patient allocation to the following two groups receiving (1) standard GVHD prophylaxis and (2) standard GVHD prophylaxis combined with MMSCs infusion. Bone marrow MMSCs from hematopoietic stem cell donors were cultured and administered to the recipients at doses of 0.9-1.3 × 10(6)/kg when the blood counts indicated recovery. aGVHD of stage II-IV developed in 38.9% and 5.3% of patients in group 1 and group 2, respectively, (P = 0.002). There were no differences in the graft rejection rates, chronic GVHD development, or infectious complications. Overall mortality was 16.7% for patients in group 1 and 5.3% for patients in group 2. The efficacy and the safety of MMSC administration for aGVHD prophylaxis were demonstrated in this study.

Leukemia cells invading the liver express liver chemokine receptors and possess characteristics of leukemia stem cells in mice with MPD-like myeloid leukemia.

OBJECTIVE: Massive liver infiltration by leukemic cells is an indicator of poor prognosis in some hemoblastoses. The aim of this study was to determine the mechanism of liver invasion by leukemic cells using the mouse model of transplantable myeloproliferative disease-like myeloid leukemia characterized by liver invasion. MATERIALS AND METHODS: CD45+ cells from the liver of mice transplanted with leukemic cells were sorted by magnetic separation. Gene expression alterations in CD45+ cells invading the liver were examined by polymerase chain reaction arrays and quantitative real-time polymerase chain reaction (including polymerase chain reaction arrays) analysis of selected genes. RESULTS: Liver chemokine receptors (Ccr1, Ccr2, Ccr5, and others) were expressed in cells invading the liver. The expression level of Ccr1 was increased 149-fold in comparison with CD45+ cells derived from the livers of healthy mice. Expression levels of several genes responsible for proliferation and self-renewal were elevated dramatically, which is in accordance with a high concentration of leukemia stem cells in the livers of moribund animals. The nuclear factor-κB signaling pathway and several oncogenes are also activated in these leukemia cells. CONCLUSIONS: Overexpression of liver-specific cytokine receptors allowed the leukemic cells to invade the liver. The high concentration of leukemia stem cells in the liver suggests the cells of this leukemia are able to adapt to new extramedullar niches. The model for the investigation and development of preventative strategies against massive liver invasion are described here.