Characteristics of leukemia stem cells of murine myeloproliferative disease involving the liver.

We studied surface phenotype of tumor cells and characterized leukemia stem cells in various cell populations with phenotypes of stem and committed precursor cells in the hierarchy of hemopoietic stem cells. Transplantable murine leukemia was used as the model. Bone marrow and liver cells from mice in the terminal phase of the disease were stained with antibodies to various surface markers and analyzed on a flow cytofuorometer. The cells were sorted by various differentiation markers using a system of magnetic separation Miltenyi Biotec MACS and then transplanted to syngeneic recipients. In some cases, limiting dilutions were used for measuring the concentration of leukemia-initiating cells. All transplanted cell populations caused death of recipients: c-kit- CD45- over 23.9 days, c-kit+ over 22.2 days, c-kit- CD45+ over 15.4 days, Ter119+ over 18.2 days, and Ter119- over 17.7 days. The concentrations of leukemia cells determined by the method of limiting dilutions was 1 per 37,000 c-kit+ bone marrow cells and 1 per 45 unsorted liver cells from sick animals. Thus, leukemia stem cells retain hierarchic organization in the studied model and can differentiate at least into myeloid and erythroid cells without loosing self-maintenance capacity. This model can be used for the study of regulation of self-maintenance mechanisms in various hierarchic populations of leukemia stem cells.

Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2).

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1-155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.

Stromal clonogenic precursors of hemopoietic microenvironment and their rank in the hierarchy of mesenchymal stem cells.

The hierarchy of stromal precursor cells was studied. Changes in the number of fibroblast CFU in foci forming in irradiated recipients were analyzed. These precursor cells are most close known descendants of mesenchymal stem cells, while inducible precursors rank lower in the hierarchy and are the cells directly enlarging the hemopoietic area in irradiated recipients.

Characteristics of transplanted mouse myeloproliferative disease developed after repeated injections of granulocytic colony-stimulating factor.

Transplanted myeloproliferative disease developed in mice against the background of repeated injections of granulocytic CSF was characterized using morphological and molecular biological methods. It was demonstrated that transplanted myeloproliferative disease had a non-viral nature and is probably induced by repeated injections of granulocytic CSF. Tumor cells actively populate the liver of sick animals, which leads to their rapid death. Expression of Myc, Abl, G-CSF, and MPO genes is enhanced, which is typical of myeloid neoplastic transformation.

Infection of stromal and hemopoietic precursor cells with lentivirus vector in vivo and in vitro.

We developed a method for gene transfer into mesenchymal stromal cells. Lentivirus vector containing green fluorescent protein gene for labeling stromal and hemopoietic precursor cells was obtained using two plasmid sets from different sources. The vector was injected into the femur of mice in vivo and added into culture medium for in vitro infection of the stromal sublayer of long-term bone marrow culture. From 25 to 80% hemopoietic stem cells forming colonies in the spleen were infected with lentivirus vector in vivo and in vitro. Fibroblast colony-forming cells from the femoral bones of mice injected with the lentivirus vector carried no marker gene. The marker gene was detected in differentiated descendants from mesenchymal stem cells (bone cavity cells from the focus of ectopic hemopoiesis formed after implantation of the femoral bone marrow cylinder infected with lentivirus vector under the renal capsule of syngeneic recipient). In in vitro experiments, the marker gene was detected in sublayers of long-term bone marrow cultures infected after preliminary 28-week culturing, when hemopoiesis was completely exhausted. The efficiency of infection of stromal precursor cells depended on the source of lentivirus. The possibility of transfering the target gene into hemopoietic precursor cells in vivo is demonstrated. Stromal precursor cells can incorporate the provirus in vivo and in vitro, but conditions and infection system for effective infection should be thoroughly selected.

[Impaired expression of genes regulating homeostasis of stem hemopoietic cells in stromal cells of patients with aplastic anemia].

AIM: To evaluate expression of genes participating in regulation of hemopoietic stem cells (HSC) in the cells of stromal sublayer of bone marrow long-term cultures in patients with aplastic anemia (AA); to determine effects of parathyroid hormone (PTH) on stromal microenvironment and on its ability to maintain HSC homeostasis. MATERIAL AND METHODS: Gene expression in the sublayer of the adherent cells (SAC) was examined with RT-PCt. SAC was for a long time treated with PTH, then their ability to secure survival of early hemopoietic precursors was tested. Changes in the function of stromal cells and expression of some genes were compared in 9 AA patients and 14 donors. RESULTS: Stromal sublayer of AA patients is characterized by low expression of Ang-1 and VCAM-1 genes and high VEGF expression compared to mean level of healthy donors. PTH stimulates expression of different genes participating in HSC regulation in stromal cells of some patients and improves survival of early hemopoietic hemopoietic precursors on such sublayers. CONCLUSION: AA patients have severe defects in SAC interaction with stroma. In some cases the defects can be partially compensated with application of PTH.

Delayed effects of long-term administration of granulocyte colony-stimulating factor to mice.

We studied the effects of chronic administration of granulocyte colony-stimulating factor in nonmobilizing doses to mice. Over 18 months of the study, 55% animals of the treatment group died of unknown cause, blood diseases and tumors were found in 20% mice, and in 5% animals pathological changes were absent. Control mice had no diseases (normal values of total and differential leukocyte count). The diagnoses made over the first 7 months mainly included myeloproliferative diseases. Solid tumors were found at later terms. Suppurative inflammation at the site of injection was observed in all mice after 3-month treatment with granulocyte colony-stimulating factor. Our results indicate that chronic administration of granulocyte colony-stimulating factor in low doses leads to the development of etiologically different tumors and sharply reduced animal life span. The use of granulocyte colony-stimulating factor during allogeneic transplantation of hemopoietic stem cells can be hazardous for donors.

[Effect of parathyroid hormone (PTH 1-34) on hemopoiesis in long-term cultures of human bone marrow].

AIM: To study effects of parathyroid hormone (PTH) used in therapy of osteoporosis on hemopoiesis in long-term culture of human bone marrow (LCBM) in terms of its potential influence on stem hemopoietic and stromal cells. MATERIAL AND METHODS: For a long time LCBM was treated with PTH (1-34) and compared for cell production, concentration of late and early hemopoietic precursors. Maintenance of hemopoiesis and adhesion at early hemopoiesis precursors on the stromal sublayers treated with PTH (1-34) was used as a functional test. A relative level of expression of genes participating in regulating proliferation and self-support of stem hemopoietic cells was studied. RESULTS: PTH (1-34) in the above concentrations did not affect hemopoiesis in LCBM. Stromal sublayer treated with PTH (1-34) for a long time supports cell precursors better, their adhesion to such sublayers enhances. CONCLUSION: PTH (1-34) in pharmacological but small concentrations had no irreversible effects on hemopoiesis, i.e. contraindications for its use in the treatment of osteoporosis were not revealed.

Changed homing of hemopoietic precursor cells after long-term treatment with parathyroid hormone.

Changes in the capacity of hemopoietic stromal microenvironment to promote homing of hemopoietic stem cells from different hierarchical compartments were evaluated in mice treated with parathyroid hormone by determining their 24-h precipitation factor. This parameter did not change for splenic short-living hemopoietic stem cells (splenic CFU) and considerably decreased for the bone marrow of mice treated with parathyroid hormone. For earlier long-living hemopoietic stem cells (cells forming the cobblestone area on day 28) the precipitation factor after injections of parathyroid hormone did not change in the bone marrow and decreased in the spleen. These data suggest that parathyroid hormone decreases the efficiency of homing of short-living hemopoietic stem cells in the bone marrow.

Stromal regulation of hemopoietic stem cells in long-term human bone marrow tissue cultures under the effect of parathyroid hormone.

We studied the interaction between different categories of hemopoietic precursors with parathyroid hormone-activated stromal microenvironment. Improved survival of early precursors capable long-term hemopoiesis maintenance and increased number of later short-term repopulating precursors was demonstrated on the model of co-culturing of human bone marrow cells on a layer of adherent cells of long-term bone marrow cultures treated with parathyroid hormone. These changes correlate with increased expression of genes involved in the maintenance of the hemopoietic stem cells in the sublayer activated by parathyroid hormone. Simultaneously, the expression of some stromal differentiation genes, adhesion molecules for hemopoietic stem cells, and growth factors increased in adherent cell layers treated with parathyroid hormone. These findings attest to activating effect of parathyroid hormone on cells forming the niches for both early and later hemopoietic precursors, and hence parathyroid hormone can be used as a potential agent promoting expansion of early hemopoietic stem cells ex vivo.

Ex vivo expansion of hemopoietic precursor cells on a sublayer treated with parathyroid hormone.

The kinetics of hemopoietic precursor cells was studied in cultures treated with parathyroid hormone in a concentration of 10(-7) M. Long-term culturing of bone marrow with parathyroid hormone did not change the number of mature cells, while the number of precursors forming colonies in semisolid media increased 7-fold and the number of cells forming cobblestone areas on day 28 increased 9-10-fold. After 24 h culturing of bone marrow cells on an irradiated sublayer pretreated with parathyroid hormone for 8 and 12 weeks, the number of early hemopoietic precursor cells forming cobblestone areas on day 28 of culturing increased 2-and 5.5-fold, respectively. The expression of Bmi-1 gene responsible for self-maintenance of stem hemopoietic cells increased in cultures treated with parathyroid hormone. It seems that parathyroid hormone can be used for expansion of hemopoietic stem cells ex vivo, which is essential for their transplantation to patients.

Effect of TNF expression by stromal cells on hemopoietic and stromal precursor cells in long-term bone marrow cultures derived from TNF-deficient mice.

The effect of TNF expression by wild type stromal sublayer on hemopoiesis was studied in cultures after second inoculation of TNF-deficient bone marrow cells. Long-term maintenance of hemopoiesis was determined solely by the absence of autocrine expression of TNF by hemopoietic cells. The production of hemopoietic precursors of all studied types in TNF-deficient cultures on a wild type sublayer was significantly higher than in cultures without TNF production. Presumably, initial expression of TNF in the sublayer promotes the survival of hemopoietic precursors with high proliferative potential in the culture. It is also obvious that TNF is required for normal functioning of stromal precursor cells.

Stromal cell strains derived from suspension fraction of bone marrow cultures of tumor necrosis factor-deficient mice.

Culturing of suspension fraction of a long-term bone marrow culture derived from tumor necrosis factor (TNF)-deficient mice for 75 days produced cells forming adherent cell strains. The cells of all strains expressed RNA of various stromal differentiating markers in various combinations. The cells of many strains simultaneously expressed genes encoding products characteristic of different differentiation lineages. The derived strains maintained hemopoiesis for 10 weeks. RNA-analysis of gene expression by cells of these strains showed that they express a set of various growth factors and cytokines. It was hypothesized that suspension fraction of long-term bone marrow culture derived from TNF-deficient mice includes immature stromal precursor cells, which were never detected in long-term bone marrow culture derived from wild-type mice.

Regeneration of the spleen in intact animals and radiation chimeras.

Regeneration of the splenic tissue after partial splenectomy is incomplete in adult non-irradiated mice and lethally irradiated animals reconstituted with donor syngeneic bone marrow. Transplantation of the splenic tissue to intact adult animals after partial splenectomy resulted in virtually complete regeneration of the spleen. In chimeras recovery of the splenic tissue was decreased; autotransplantation of the whole spleen or its part did not lead to appreciable changes in the weight and cellularity of this organ. No more than 30% splenic tissue is restored after complete splenectomy and transplantation of the splenic tissue in intact and chimeric mice.

Effect of parathyroid hormone PTH (1-34) on hemopoietic and stromal stem cells.

Long-term administration of parathyroid hormone causing activation and proliferation of osteoblasts to mice increases the concentration of primitive hemopoietic precursor cells (cobblestone area-forming cells) in long-living bone marrow culture after 28-35 days. The concentrations of later precursors forming colonies in the spleen and the concentration of cobblestone-area forming cells in long-living bone marrow culture after 7 days decrease, while the concentration of more differentiated cells forming colonies in the culture does not change. Transplantation of the bone marrow from mice treated with parathyroid hormone under the renal capsule of syngeneic recipients results in the formation of a focus of ectopic hemopoiesis not differing by size from the control. Injection of parathyroid hormone to mice during the growth of the ectopic focus did not modulate its size. These foci tolerate retransplantation procedure similarly as controls. Hence, parathyroid hormone has no effect on mesenchymal stem cells responsible for transfer of the stromal microenvironment. Therefore, the number of stem hemopoietic cells in the body is regulated by not stromal stem cells, but their better differentiated descendants.

Dynamics of precursor cell composition in bone marrow culture derived from mice deficient by tumor necrosis factor.

Hemopoiesis in a long-term bone marrow culture derived from mice deficient for tumor necrosis factor was maintained for more than 130 weeks, which was 4 times longer than in cultures from wild type mice. The dynamics of hemopoietic precursor cells of different maturity was studied in the suspension fraction of the culture. The incidence of granulocyt-macrophage precursor cells and cell forming cobblestone areas was studied by the method of limiting dilutions in culture. In contrast to cultures derived from wild type mice, in long-term bone marrow culture derived from tumor necrosis factor deficient mice, first, the incidence of early precursor cells gradually increased and then the incidence of all precursor cells sharply increased.

Lentivirus vector can integrate in the genome and exist and replicate in the cell as an episome.

Lethally irradiated mice were reconstituted with few purified primitive hemopoietic stem cells containing sequences of a gene encoding green fluorescent protein. The gene was transferred using a lentivirus vector. The presence of the marker gene in splenocyte colonies derived from the bone marrow of reconstituted mice and in cells of other hemopoietic and non-hemopoietic organs was studied during the life. It was shown that the lentivirus vector can persist for a long time and replicate in hemopoietic cells as an episome.

Long-term self-maintenance of hemopoietic precursors in bone marrow culture derived from tumor necrosis factor-deficient mice is not a result of neoplastic transformation.

In long-term bone marrow cultures derived from tumor necrosis factor-deficient mice the total cell production and the total duration of hemopoiesis are increased (the latter is comparable with mouse life span). Telomerase activity in cells of nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice increases with time and peaks after 1-year culturing. Karyotyping of nonadherent and adherent cells of long-term bone marrow cultures revealed instability of nonadherent cells and hyperploidy of the stromal sublayer cells, which attested to the presence of a neoplastic transformation. However, cell differentiation is not blocked in long-term bone marrow cultures. The nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be cultured without exogenous growth factors; in the presence of growth factors the cells proliferate, but cannot be passaged; stromal sublayer cells cannot be passaged as well. Intraperitoneal and intravenous injections of nonadherent cells to recipients with normal and radiation-attenuated immunity induced no tumor growth. Hence, peculiar dynamics of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be explained by neoplastic transformation.

Abnormalities in long-term bone culture from mice deficient in tumor necrosis factor: induction of apoptosis and cell proliferation.

The total duration of hemopoiesis and the total cell production in long-term bone marrow cultures from mice deficient by tumor necrosis factor are increased, while proliferation of granulocyte-macrophage precursor cells, the main cell populations in long-term bone marrow cultures, did not differ from that in wild-type mice. In bone marrow cultures from knockout mice the intensity of apoptosis remained low during 40-week culturing and was similar to that in early wild-type cultures (10 weeks). Then, the intensity of apoptosis in bone marrow cultures from knockout mice did not differ or even surpassed that in wild-type cultures. However, we observed no inhibition of hemopoiesis in bone marrow cultures from knockout mice. The absence of autocrine expression of tumor necrosis factor did not affect proliferation of precursor cells, but modulate the intensity of apoptosis. It remains unclear whether changes in apoptosis are related to intensive cell production.