Differences in the Differentiation Potential and Relative Levels of Gene Expression in the Bone Marrow-Derived Fibroblast Colony-Forming Units in Patients during the Onset of Aplastic Anemia Depending on the Disease Severity.

The differentiation potential of individual clones of fibroblast CFU (CFU-F) was studied and the relative expression level of genes was analyzed in the culture of CFU-F from the bone marrow in patients with non-severe and severe forms of aplastic anemia at the onset of the disease. The differentiation potential of CFU-F clones was determined by the relative expression of marker genes using quantitative PCR. In aplastic anemia, the ratio of CFU-F clones with different differentiation potential changes, but the molecular mechanisms of this phenomenon are different in non-severe and severe aplastic anemia. In the culture of CFU-F in non-severe and severe aplastic anemia, the relative expression level of genes associated with the maintenance of the hematopoietic stem cell in the bone marrow niche changes, but the decrease in the expression of immunoregulatory genes occurs in severe form only, which may reflect differences in the pathogenesis of non-severe and severe aplastic anemia.

Bone Marrow Multipotent Mesenchymal Stromal Cells from Patients with Aplastic Anemia Retain Their Ability to Support Hematopoietic Precursors despite Pronounced Changes in Gene Expression.

The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are studied insufficiently. The aim of this work was to test the ability of MSC from patients with aplastic anemia to maintain hematopoietic precursors and to analyze the expression of genes associated with hematopoiesis and immune response. The ability of MSC to maintain hematopoietic precursors was determined by counting cobblestone area-forming cells; gene expression was analyzed by quantitative PCR. It was shown that MSC of patients with aplastic anemia preserve their ability to maintain hematopoietic precursors. Pronounced changes in the expression of the VEGFA and ANGPT1 genes were found. MSC from aplastic anemia patients with PNH clone significantly differ from those from aplastic anemia patients without PNH clone in terms of the expression of the SDF1, IL1R, and VEGFA genes. Changes in gene expression can be associated with the pathogenesis of the disease.

Stable Lentiviral Vector Transfer into Mesenchymal Stem Cells In Vivo.

Green fluorescent protein (eGFP) gene was transferred into mouse mesenchymal stem cells in vivo using a lentiviral vector. In 2 months after injection of the lentivirus into the cavity of the femoral bone, up to 30% fibroblast CFU in the bone marrow of infected mice contained the alien gene. The transferred gene was found in more than 50% of adherent layers of longterm bone marrow cultures formed by mesenchymal stem cells from the infected mice bone marrow; 4% fibroblast CFU obtained from these layers were labeled. Ectopic hemopoiesis foci developed after transplantation of the bone marrow from infected mice under the renal capsule of syngeneic recipients contained bone tissue labeled with the alien gene in 57% cases and labeled fibroblast CFU in 11%. The data confirm the possibility of gene transfer with the lentiviral vectors into the mesenchymal stem cells in vivo.

Peculiarities of Gene Transfer into Mesenchymal Stem Cells.

Murine mesenchymal stem cells in long-term bone marrow culture were genetically labeled using lentiviral vector carrying enhanced green fluorescent protein (eGFP) reporter gene under SFFV promoter or without it. We studied the developmental fate of labeled mesenchymal stem cells in stromal cell layers of long-term bone marrow culture and in ectopic hemopoietic foci formed by these stromal layers under the renal capsule of syngeneic mice. The frequency of labeled polypotent stromal precursors (fibroblast CFU) was analyzed in adherent cell layers of long-term culture and ectopic foci formed from them. The proportion of labeled fibroblast CFU in ectopic foci increased by 10 times in case of implantation of adherent cell layers infected with lentivirus containing eGFP reporter gene without promoter. eGFP expression leads to rejection of labeled stromal cells. Labeling with eGFP-carrying vector without promoter makes possible long-term tracking of mesenchymal stromal cells.

[Characteristics of stromal cell precursors in patients after allogeneic bone marrow transplantation].

UNLABELLED: AIM. To study the elements of the mesenchymal stromal cell compartment (multipotent mesenchymal stromal cells (MMSCs)) and their more mature progenies of fibroblast colony-forming units (CFU-F) in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). SUBJECTS AND METHODS. The total production of MMSCs after 5 passages, the time of their growth, and the concentration of CFU-F in the bone marrow from patients were determined using the control sections before transplantation and over time for 2 years after allo-HSCT. What is more, the genetic affiliation of the MMSCs from the patients after allo-HSCT and their immunophenotype were studied. RESULTS: The MMSCs from the patients after allo-HSCT belong to a recipient and have the immunophenotype that meets the international standard for these cells. The total production of MMSCs in the cultures obtained from the bone marrow of the patients with hematologic diseases was decreased. Not all the samples from the patients after allo-HSCT are able to undergo 5 passages. In addition, the time of growth substantially increases and the total production of cells decreases in all the analyzed cultures. These indicators are gradually restored; however, they never achieve the mean values in donors. The concentration of CFU-F in the bone marrow from the patients are reduced as compared to that in the donors prior to transplantation and decreased still further after allo-HSCT. These cell precursors are not restored for at least 2 years following allo-HSCT. CONCLUSION. Both examined categories of the cell precursors of the stromal environment suffer from both the disease itself and allo-HSCT in the patients with hematologic diseases.

[Treatment of acute host-versus-graft reaction in patients after allogeneic hematopoietic cell transplantation with multipotent mesenchymal stromal cells from a bone marrow donor].

AIM: To evaluate the efficiency of administration of multipotent mesenchymal stromal cells obtained from a bone marrow donor for the treatment of an acute host-versus-graft reaction (HVGR) resistant to therapy with glucocorticosteroids (GCS). SUBJECTS AND METHODS: The experience in treating 6 patients with GCS-resistant acute HVGR following allogeneic hematopoietic stem cell transplantation is given. The patients were intravenously injected cultured multipotent mesenchymal stromal cells (MMSC) in a dose of 10(6) per kg body weight. RESULTS: Four weeks after MMSC administration, a complete or partial response was obtained in 3 cases; clinical improvement was noted in 2; one patient showed no response. MMSC therapy proved to be effective in 5 of the 6 cases with acute HVGR resistant to GCS therapy. CONCLUSION: MMSC therapy turned out to be effective in case of acute HVGR resistant to GCS therapy.

Analysis of expression of genes involved in immune response modulation in silent multipotent mesenchymal stromal cells.

The expression of some genes modulating the immune response was studied in multipotent mesenchymal stromal cells (MMSC) from the bone marrow of a healthy donor. Non-activated MMSC expressed IL-6 and IL-10, complement H factor, macrophage growth factor, prostaglandin E2 synthase, and indoleamine-2,3-dioxygenase. The expression of all these genes was higher in female MMSC. A close inverse relationship between IL-6 expression in MMSC and male donor age, close relationship between body weight index and fibroblast CFU concentration in female donor bone marrow and between indoleamine-2,3-dioxygenase and macrophage growth factor in MMSC from these donors were detected. The expression of the analyzed genes was higher in MMSC of donors who had no antibodies to cytomegalovirus, herpes simplex virus, and Epstein-Barr virus in the blood. The results demonstrate the MMSC regulation of immune reactions by MMSC at the cell and organism levels.

[The basic properties of mesenchymal stromal cells from the donor bone marrow: superficial markers].

AIM: To characterize a superficial phenotype and to make a cytogenetic analysis of bone marrow (BM) mesenchymal stromal cells (MSC) from donors. MATERIALS AND METHODS: The study analyzed BM samples from 11 healthy donors. The phenotype of obtained MSC was analyzed using cytofluorometry. Chromosomal analysis was carried out at the first-second passage. RESULTS: The superficial phenotype of MSC was steady-state during 8 passages and conformed to the worldwide standard for this cell population. The marker NGFR+ was detectable only during the first 2 passages and the count of CD146+ cells was decreased to 50% as consecutive passages were carried out, which confirms that MSCs have lost their neural and endothelial differentiation capacity. MSCs are stably able to differentiate only into the mesenchymal lineage. The detection of chromosomal rearrangements in MSCs at different stages of cultivation revealed no clonal rearrangements in any case. However, chromosomal aberrations were found 3-10% of metaphases at the first and second passages, which may be associated with chromosome instability in primary cultures. CONCLUSION: The pooled data suggest that the analyzed MSCs meet the conventional worldwide standards.

[The study of parameters of mesenchymal stromal cells differentiation in donors and patients with aplastic anemia].

AIM: To examine ability of mesenchymal stromal cells (MSC) of the bone marrow (BM) for differentiation in adipogenic and osteogenic differentiation in donors and patients with aplastic anemia (AA). MATERIAL AND METHODS: We obtained MSC cultures from BM cells of donors and AA patients and induced differentiation of mesenchymal cells with use of relevant reagents. Morphological changes in MSC were studied with light microscopy. A relative level of expression of differentiation marker genes in MSC cultures before and after induction of differentiation was analysed with reverse transcription-polymerase chain reaction. RESULTS: By morphological characteristics, MSC cultures in AA patients before and after differentiation induction do not differ from donor cultures, but relative expression of the genes of differentiation markers demonstrated that expression was different in male and female donors; MSC before and after induction of differentiation differ in donors and AA patients. CONCLUSION: Further studies are needed for detection of functional changes in precursors of stromal microenvironment and understanding of the disease pathogenesis.