Molecular Changes in Immunological Characteristics of Bone Marrow Multipotent Mesenchymal Stromal Cells in Lymphoid Neoplasia.

Immune system and bone marrow stromal cells play an important role in maintaining normal hematopoiesis. Lymphoid neoplasia disturbs not only development of immune cells, but other immune response mechanisms as well. Multipotent mesenchymal stromal cells (MSCs) of the bone marrow are involved in immune response regulation through both intercellular interactions and secretion of various cytokines. In hematological malignancies, the bone marrow stromal microenvironment, including MSCs, is altered. Aim of this study was to describe the differences of MSCs' immunological function in the patients with acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). In ALL, malignant cells arise from the early precursor cells localized in bone marrow, while in DLBCL they arise from more differentiated B-cells. In this study, only the DLBCL patients without bone marrow involvement were included. Growth parameters, surface marker expression, genes of interest expression, and secretion pattern of bone marrow MSCs from the patients with ALL and DLBCL at the onset of the disease and in remission were studied. MSCs from the healthy donors of corresponding ages were used as controls. It has been shown that concentration of MSCs in the bone marrow of the patients with ALL is reduced at the onset of the disease and is restored upon reaching remission; in the patients with DLBCL this parameter does not change. Proliferative capacity of MSCs did not change in the patients with ALL; however, the cells of the DLBCL patients both at the onset and in remission proliferated significantly faster than those from the donors. Expression of the membrane surface markers and expression of the genes important for differentiation, immunological status maintenance, and cytokine secretion differed significantly in the MSCs of the patients from those of the healthy donors and depended on nosology of the disease. Secretomes of the MSCs varied greatly; a number of proteins associated with immune response regulation, differentiation, and maintenance of hematopoietic stem cells were depleted in the secretomes of the cells from the patients. Lymphoid neoplasia leads to dramatic changes in the functional immunological status of MSCs.

Acute Myeloid Leukemia Causes Serious and Partially Irreversible Changes in Secretomes of Bone Marrow Multipotent Mesenchymal Stromal Cells.

In patients with acute myeloid leukemia (AML), malignant cells modify the properties of multipotent mesenchymal stromal cells (MSCs), reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the role of MSCs in supporting leukemia cells and the restoration of normal hematopoiesis by analyzing ex vivo MSC secretomes at the onset of AML and in remission. The study included MSCs obtained from the bone marrow of 13 AML patients and 21 healthy donors. The analysis of proteins contained in the MSCs-conditioned medium demonstrated that secretomes of patient MSCs differed little between the onset of AML and remission; pronounced differences were observed between MSC secretomes of AML patients and healthy donors. The onset of AML was accompanied by a decrease in the secretion of proteins related to ossification, transport, and immune response. In remission, but not at the onset, secretion of proteins responsible for cell adhesion, immune response, and complement was reduced compared to donors. We conclude that AML causes crucial and, to a large extent, irreversible changes in the secretome of bone marrow MSCs ex vivo. In remission, functions of MSCs remain impaired despite the absence of tumor cells and the formation of benign hematopoietic cells.

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.

Hematopoiesis during Ontogenesis, Adult Life, and Aging.

In the bone marrow of vertebrates, two types of stem cells coexist-hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Hematopoiesis only occurs when these two stem cell types and their descendants interact. The descendants of HSCs supply the body with all the mature blood cells, while MSCs give rise to stromal cells that form a niche for HSCs and regulate the process of hematopoiesis. The studies of hematopoiesis were initially based on morphological observations, later extended by the use of physiological methods, and were subsequently augmented by massive application of sophisticated molecular techniques. The combination of these methods produced a wealth of new data on the organization and functional features of hematopoiesis in the ontogenesis of mammals and humans. This review summarizes the current views on hematopoiesis in mice and humans, discusses the development of blood elements and hematopoiesis in the embryo, and describes how the hematopoietic system works in the adult organism and how it changes during aging.

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.

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.

Multipotent Mesenchymal Stromal Cells from Porcine Bone Marrow, Implanted under the Kidney Capsule, form an Ectopic Focus Containing Bone, Hematopoietic Stromal Microenvironment, and Muscles.

Multipotent mesenchymal stromal cells (MSCs) are an object of intense investigation due to their therapeutic potential. MSCs have been well studied in vitro, while their fate after implantation in vivo has been poorly analyzed. We studied the properties of MSCs from the bone marrow (BM-MSC) before and after implantation under the renal capsule using a mini pig model. Autologous BM-MSCs were implanted under the kidney capsule. After 2.5 months, ectopic foci containing bones, foci of ectopic hematopoiesis, bone marrow stromal cells and muscle cells formed. Small pieces of the implant were cultivated as a whole. The cells that migrated out from these implants were cultured, cloned, analyzed and were proven to meet the most of criteria for MSCs, therefore, they are designated as MSCs from the implant-IM-MSCs. The IM-MSC population demonstrated high proliferative potential, similar to BM-MSCs. IM-MSC clones did not respond to adipogenic differentiation inductors: 33% of clones did not differentiate, and 67% differentiated toward an osteogenic lineage. The BM-MSCs revealed functional heterogeneity after implantation under the renal capsule. The BM-MSC population consists of mesenchymal precursor cells of various degrees of differentiation, including stem cells. These newly discovered properties of mini pig BM-MSCs reveal new possibilities in terms of their manipulation.

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.

Recovery of Donor Hematopoiesis after Graft Failure and Second Hematopoietic Stem Cell Transplantation with Intraosseous Administration of Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cells (MSCs) participate in the formation of bone marrow niches for hematopoietic stem cells. Donor MSCs can serve as a source of recovery for niches in patients with graft failure (GF) after allogeneic bone marrow (BM) transplantation. Since only few MSCs reach the BM after intravenous injection, MSCs were implanted into the iliac spine. For 8 patients with GF after allo-BMT, another hematopoietic stem cell transplantation with simultaneous implantation of MSCs from their respective donors into cancellous bone was performed. BM was aspirated from the iliac crest of these patients at 1-2, 4-5, and 9 months after the intraosseous injection of donor MSCs. Patients' MSCs were cultivated, and chimerism was determined. In 6 out of 8 patients, donor hematopoiesis was restored. Donor cells (9.4 ± 3.3%) were detected among MSCs. Thus, implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. These results suggest that MSCs could participate in the restoration of niches for donor hematopoietic cells or have an immunomodulatory effect, preventing repeated rejection of the graft. Perhaps, intraosseous implantation of MSCs contributes to the success of the second transplantation of hematopoietic stem cells and patient survival.

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.

What do we know about the participation of hematopoietic stem cells in hematopoiesis?

The demonstrated presence in adult tissues of cells with sustained tissue regenerative potential has given rise to the concept of tissue stem cells. Assays to detect and measure such cells indicate that they have enormous proliferative potential and usually an ability to produce all or many of the mature cell types that define the specialized functionality of the tissue. In the hematopoietic system, one or only a few cells can restore lifelong hematopoiesis of the whole organism. To what extent is the maintenance of hematopoietic stem cells required during normal hematopoiesis? How does the constant maintenance of hematopoiesis occur and what is the behavior of the hematopoietic stem cells in the normal organism? How many of the hematopoietic stem cells are created during the development of the organism? How many hematopoietic stem cells are generating more mature progeny at any given moment? What happens to the population of hematopoietic stem cells in aging? This review will attempt to describe the results of recent research which contradict some of the ideas established over the past 30 years about how hematopoiesis is regulated.