Changes in Bone Marrow Stromal Progenitor Cells in Patients with Hematoblastosis at the Onset of the Disease.

Multipotent mesenchymal stromal cells (MSC) are the key regulators of hematopoiesis. We studied changes in MSC characteristics in patients with myeloid leukemia and patients with lymphoproliferative diseases. MSC were obtained from the bone marrow of patients at the time of diagnostic puncture using a standard technique. Their proliferative potential and expression of genes associated with differentiation and regulation of hematopoiesis were studied. The total cell production of MSC in patients with leukemia at the onset of the disease did not differ from that in the group of healthy donors. The relative expression of the IL6, TGFb1 and TGFb2, PPARG genes was similar in all patients. The relative expression of the JAG1, LIF, IGF1, CSF1, IL1b, and IL1bR1 genes in MSC of patients with leukemia was enhanced and the relative expression of SDF1 was unchanged in comparison with MSC from donors. MSC from patients with leukemia were characterized by enhanced relative expression of PDGFRA and PDGFRB, and reduced expression of SOX9. Changes functions of the stromal microenvironment in patients with hemoblastoses attested to the role of stromal cells in the maintenance and spread of tumor cells.

Clonal Composition of Human Multipotent Mesenchymal Stromal Cells: Application of Genetic Barcodes in Research.

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

Investigation of the Mesenchymal Stem Cell Compartment by Means of a Lentiviral Barcode Library.

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.

Analysis of results of acute graft-versus-host disease prophylaxis with donor multipotent mesenchymal stromal cells in patients with hemoblastoses after allogeneic bone marrow transplantation.

Allogeneic bone marrow transplantation (allo-BMT) is currently the only way to cure many hematoproliferative disorders. However, allo-BMT use is limited by severe complications, the foremost being graft-versus-host disease (GVHD). Due to the lack of efficiency of the existing methods of GVHD prophylaxis, new methods are being actively explored, including the use of donors' multipotent mesenchymal stromal cells (MMSC). In this work, we analyzed the results of acute GVHD (aGVHD) prophylaxis by means of MMSC injections after allo-BMT in patients with hematological malignancies. The study included 77 patients. They were randomized into two groups - those receiving standard prophylaxis of aGVHD and those who were additionally infused with MMSC derived from the bone marrow of hematopoietic stem cell donors. We found that the infusion of MMSC halves the incidence of aGVHD and increases the overall survival of patients. Four of 39 MMSC samples were ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index) and the MMSC properties of these donors (growth parameters, level of expression of 30 genes involved in proliferation, differentiation, and immunomodulation) revealed no significant difference between the MMSC that were effective or ineffective for preventing aGVHD. We used multiple logistic regression to establish a combination of features that characterize the most suitable MMSC samples for the prevention of aGVHD. A model predicting MMSC sample success for aGVHD prophylaxis was constructed. Significant model parameters were increased relative expression of the FGFR1 gene in combination with reduced expression levels of the PPARG and IGF1 genes. Depending on the chosen margin for probability of successful application of MMSC, this model correctly predicts the outcome of the use of MMSC in 82-94% of cases. The proposed model of prospective evaluation of the effectiveness of MMSC samples will enable prevention of the development of aGVHD in the maximal number of patients.

Proliferative potential of multipotent mesenchymal stromal cells from human bone marrow.

We studied the capacity of multipotent mesenchymal stromal cells isolated from human bone marrow (BM) to long-term passaging, cloning, and re-cloning. Initial multipotent mesenchymal stromal cells and cells after gene labeling were studied. Multipotent mesenchymal stromal cells were obtained from donors (13-59 years) and cultured for 7 passages. Third generation lentivector was used for delivery of green fluorescent protein marker gene. The procedure of infection revealed reduced proliferative potential of multipotent mesenchymal stromal cells from elder donors. Hierarchy of precursor cells differing by their proliferative potential was demonstrated in the culture of multipotent mesenchymal stromal cells. Three categories of multipotent mesenchymal stromal cells were identified: mature cells incapable of proliferation (75.7±2.4% population) and cells with low and high proliferative potential (17.6±2.1 and 6.7±0.3%, respectively). The relative content of these cells insignificantly differed from passage to passage. The efficiency of cloning also remains stable, but re-cloning capacity sharply decreased after passage 3 and completely disappeared in multipotent mesenchymal stromal cells after cryopreservation. Thus, cultured multipotent mesenchymal stromal cells represent a heterogeneous and hierarchically organized population and the characteristics of this population depend of the duration of culturing and age of BM donor. This should be taken into account when using multipotent mesenchymal stromal cells in clinical practice.

Characteristics of mesenchymal stromal precursor cells labeled with lentiviral vector in long-term bone marrow culture.

Mouse mesenchymal stromal precursor cells were labeled with lentiviral vector in long-term bone marrow culture. We studied the fate of labeled cells in the stromal sublayer of the long-term bone marrow culture and in ectopic hemopoiesis foci formed from the labeled cultures. The incidence of labeled polypotent fibroblast CFU in sublayers of long-term bone marrow culture and in ectopic hemopoiesis foci formed from these sublayers under the renal capsule of syngeneic mice was also analyzed. It was shown that the marker gene was present in about 40% cells of the stromal sublayer and 30% fibroblast CFU and that effective gene transfer did not affect the total production of hemopoietic cells. The size of ectopic hemopoietic foci formed after implantation of labeled sublayers of the long-term bone marrow culture under the renal capsule did not differ from the control. Differentiated cells of the osseous shell in these foci carried the marker gene in 40% cases. Analysis of fibroblast CFU in these foci showed that despite the total concentration of fibroblast CFU was comparable to that in the bone marrow, the concentration of labeled fibroblast CFU was about 6%, which suggests that one more class of precursors probably exists in the hierarchy of stromal cells presumably between mesenchymal stem cells and fibroblast CFU. Our findings demonstrate the capacities of mesenchymal stem cells to self-maintenance and differentiation without losing the marker gene integrated into the genome.

Reactive oxygen species produced in mitochondria are involved in age-dependent changes of hematopoietic and mesenchymal progenitor cells in mice. A study with the novel mitochondria-targeted antioxidant SkQ1.

Lifelong treatment of mice with the effective mitochondria-targeted antioxidant SkQ1 [10-(6'-plastoquinonyl) decyltriphenylphosphonium] does not affect hematopoietic stem cells (HSC) and more differentiated hematopoietic progenitors but significantly decelerates age-dependent changes in peripheral blood. During the first 13 months, SkQ1 (0.9 or 28.8 nmol/kg day) prevents age-dependent myeloid shift (increase in the proportion of granulocytes and decrease in the proportion of lymphocytes). During the next year of treatment the effect disappears, and the hemogram of 2-year-old treated mice does not differ from the control. The number of mesenchymal stem cells (MSC) in the bone marrow does not change during 2 years of treatment with SkQ1, but the concentration of MSC progeny fibroblast colony-forming units (CFU-F) increases with dose of SkQ1. The concentration of CFU-F after 1 and 2 years treatment with SkQ1 is twice higher than in young mice. Our data indicate that the stromal environment of hematopoietic cells could be the primary target of age-dependent changes mediated by reactive oxygen species produced in mitochondria. The anti-aging effects of SkQ1 described here are in perfect agreement with the inhibitory effects of this antioxidant on aging observed in the other models.