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.

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.

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.

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.

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.

Alterations in hematopoietic microenvironment in patients with aplastic anemia.

Mechanisms of hematopoietic failure in patients with aplastic anemia (AA) are obscure. We investigate alterations in the hematopoietic microenvironment in AA patients. We present the results of studying mesenchymal stromal cells (MSC), fibroblastic colony-forming units (CFU-F), and adherent cell layers (ACL) of long-term bone marrow cultures (LTBMC) from bone marrow (BM) samples of AA patients. MSC of AA patients proliferated longer than those of donors. In half of the patients' MSC cultures, adipogenesis was impaired. Osteogenic differentiation was not achieved in 36% of AA MSC. CFU-F formed enlarged colonies, and their concentration in the BM of AA patients was significantly increased. Our data suggest that the physiological activation of the stromal microenvironment is characteristic of AA. We detected a decrease in the expression of the angiopoetin-1 (ANG-1) and vascular cell adhesion molecule-1 (VCAM-1) genes, together with an increase in the expression of vascular endothelial growth factor (VEGF) in ACL of AA patients. This indicates abnormal regulatory patterns in both osteoblastic and vascular contexts. Addition of AA patients' serum to donors' LTBMC for 3 weeks induced similar gene expression alterations. The addition of parathyroid hormone (PTH) resulted in the expression levels of analyzed genes returning to normal, in both AA LTBMC and donor cultures treated with AA serum. The physiologic status of the BM stromal microenvironment (MSC, CFU-F, and ACL of LTBMC) of AA patients was altered.