Counts of Stromal Precursor Cells in the Bone Marrow and Heterotopic Bone Marrow Transplants from Mice Immunized with Group A Streptococcus Antigens during Different Periods after Immunization

The counts of stromal precursor cells in bone marrow transplants obtained from animals 2 months after their immunization with killed type 5 group A streptococcus vaccine drop almost 3-fold in comparison with transplants from normal donors. Six months after donor immunization, the count of stromal precursor cells in the transplants reaches the normal level. The count of stromal precursor cells in bone marrow transplants from normal mice transplanted to recipients 6 months after their immunization with killed streptococcus vaccine also virtually did not change in comparison with the counts in bone marrow transplants from normal donors transplanted to normal recipients. The weight and size of bone capsules of 6.5-month bone marrow transplants in intact recipients after transplantation from donors immunized 2 months before with killed type 5 group A streptococcus vaccine was 3-fold lower than in bone marrow transplants collected from intact donors. The content of stromal precursor cells in the femoral bone marrow of animals immunized with killed streptococcus vaccine was 2.5 time higher in comparison with the parameter in the femoral bone marrow of normal mice even 8 months after immunization. The results indicate a significant long-acting effect of streptococcal antigens on the bone marrow stromal tissue, specifically, on its osteogenesis potential. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 1, pp. 78-81, January, 2009     

Effects of immunization with group a streptococcal antigens on the transplantability of mouse bone marrow stromal stem cells, counts of stromal precursor cells, and their osteogenic characteristics

Immunization of CBA mice with killed group A streptococcus (type 5) vaccine changed the counts of stromal precursor cells (CFC-F) in bone marrow transplants at different donorrecipient combinations (normal, N, or immune, I). CFC-F counts in bone marrow transplants from normal mice transplanted to immunized animals decreased 4-6-fold depending on the transplant age in comparison with similar transplants in normal recipients. The percentage of CFC-F colonies with alkaline phosphatase (osteogenesis marker) activity decreased more than 2-fold. Similarly, the count of CFC-F in the transplants was 2-fold lower during delayed (7 months) period after bone marrow transplantation from immunized donors (8–12 days after the end of immunization) to intact recipients, while 2 months after transplantation it was 3-fold lower. The mean optical density of the bone capsule in preparations stained for glycogen and alkaline phosphatase was 1.5-3 times lower in the N → I and I → N experiments in comparison with the control (N → N). On the other hand, CFC-F count in the femoral bone marrow of immunized animals was significantly (3.5-2.5 times) higher during the period from 8 days to 8 months after the end of immunization compared to CFC-F count in the femoral bone marrow of intact mice. These results attest to a significant prolonged effect of streptococcal antigens on the bone marrow stromal tissue. These data also indicate that not all CFC-F, the counts of which increased in response to antigens, are responsible for transplantability of the stromal tissue in heterotopic transplantation. Immunization by streptococcal antigens seemed to suppress transplantability and osteogenic activity of stromal stem cells. The efficiency of CFC-F cloning in mouse bone marrow cultures increased significantly (2-3-fold) in the presence of sera from immune mice. The levels of TNF-α and IFN-γ were low in this serum (2.7 and 6 times lower, respectively) in comparison with normal serum. Presumably, the effects of streptococcal antigens on stromal tissue were mediated through serum cytokines.     

Counts of Stromal Precursor Cells in Heterotopic Bone Marrow Transplants in Mice Immunized with Group A Streptococcus Antigens

The count of stromal precursor cells in bone marrow transplants from CBA mice, transplanted to animals immunized with killed type 5 group A streptococcus vaccine, decreased 4.5–6.5 times (depending on the transplant age) in comparison with the grafts transplanted to normal recipients. The counts of stromal precursor cells in 1.5–3-month bone marrow transplants from animals immunized with killed streptococcal vaccine transplanted to normal mice were virtually the same, while in 7-month transplants they decreased 2-fold in comparison with their counts in bone marrow transplants from normal CBA mice transplanted to normal animals. The content of stromal precursor cells in the femoral bone marrow of animals immunized with killed streptococcal vaccine was appreciably (3.5 times) higher than in the bone marrow of normal mice. The results attest to an appreciable effect of streptococcal antigens on the bone marrow stromal tissue and suggest that not all stromal precursor cells, whose count increases after injection of antigens, are responsible for transplantability of the stromal tissue in case of its heterotopic transplantation. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 7, pp. 77–80, July, 2005     

Content of stromal precursor cells in heterotopic transplants of bone marrow in CBA mice of various ages

Efficiency of colony formation of stromal precursor cells in cultured bone marrow transplants from old (24 month) CBA mice implanted to young (2-month-old) mice almost 3-fold surpassed that in cultured transplants implanted to old recipients. The content of nucleated cells in bone marrow transplants from senescence accelerated mice SAMP increased more than 2-fold, if SAMR mice with normal aging rate were used as the recipients instead of SAMP mice. Bone marrow taken from old and young CBA mice endured the same number of transplantations if the recipient mice were of the same age (5 month). It was concluded that stromal tissue considerably changes with age and is under strict control of the body.     

Infection of stromal and hemopoietic precursor cells with lentivirus vector <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

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. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 1, pp. 25–28, January, 2007     

Candida albicans can stimulate stromal cells resulting in enhanced granulopoiesis

Previously, we have reported that although unperturbed granulocyte colony-stimulating factor (GCSF)-deficient (G-CSF-/-) mice are neutropenic, when challenged with Candida albicans, they develop a profound neutrophilia. In an attempt to understand the basis of Candida-induced neutrophilia in G-CSF-deficient mice, we have modified the Dexter bone marrow culture system to produce an in vitro model that mimics emergency granulopoiesis in vivo. In this model, stromal cultures are overlaid with bone marrow cells in the presence or absence of heat-inactivated (HI) Candida. Irrespective of the genotype of mice used as a source of bone marrow-derived stromal cells, stimulation of these cultures with HI Candida led to a significantly greater recovery of cells compared to unstimulated stromal cultures. In addition, there was a marked increase in the number of colony-forming units granulocyte-macrophage (CFU-GM), as well as in the percentage of granulocytes in the population of nonadherent cells recovered from HI Candida-stimulated cultures. The conditioned medium generated from stromal cultures derived from either wild-type or G-CSF-/- mice exposed to HI Candida, when applied to bone marrow cells in a soft agar clonogenic assay stimulated M-, GM-, and G- type colonies. Interleukin-3 (IL-3) and GM-CSF could not be detected in the conditioned medium from either HI Candida stimulated or unstimulated stromal cultures. However, IL-6 was detected in the conditioned media from both wild-type and G-CSF-/- stromal cultures. Addition of anti-IL-6 antibody significantly impaired granulopoiesis in unstimulated and HI Candida-stimulated, wild type, and G-CSF-/- stromal cultures. Conditioned medium generated from G-CSF/IL-6-deficient stromal cells had the capacity to stimulate bone marrow cells to form colonies comprised of granulocytes and macrophages in soft agar clonogenic assay. This study demonstrates that stromal cells can be stimulated with HI Candida and gives an insight into Candida mediated granulopoiesis.     

Functional activity of hemopoietic and stromal cells in various types of myelodysplastic syndrome

Using clonal methods for assessment of hemopoietic and stromal cells and long-term bone marrow cell cultures, we have demonstrated heterogeneity of myelodysplastic syndrome. Low content of stromal precursor cells in native bone marrow, peculiarities in the formation of the stromal layer and its hemopoiesis-stimulating capacity in long-term cultures, and altered properties of stromal precursor cells in long-term cultures indicate defect in the stromal microenvironment in myelodysplastic syndrome. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 14–18, January, 1999     

Evaluation of anti-inflammatory effects of Trombovazim in the model of liver ischemia-reperfusion

The study was carried out on CBA mice using the method of heterotopic transplantation. A fragment of the femoral bone marrow (¹/₂) or spleen (¹/₅ of the organ) was transplanted under the renal capsule of a recipient. The following donor-recipient cross-transplantation variants were studied: young → young (Y → Y), young → old (Y → O), old → old (O → O), and old → young (O → Y). Cell suspensions were prepared from 2-month transplants inoculated in monolayer cultures and the cloning effi ciency (ECF-F) of stromal precursor cells (CFC-F) was evaluated. The bone marrow transplant ECF-F and the count of CFC-F in the O → O group were 8-fold lower than in the Y → Y group. In the O → Y group, ECF-F was 3-fold higher than in the O → O group, but by 2.5 times lower than in the Y → Y group. ECF-F in Y → O group was 2-fold lower than in Y → Y group. The ECF-F and CFC-F count in spleen transplants in the O → O group were 4- and 6-fold lower, respectively, than in Y → Y group. However, in O → Y group ECF-F was 7-fold higher than in O → O group and higher than even in Y → Y group. The weight of induced ectopic bone tissue after transplantation of the osteoinductor (fragments of the allogenic urinary bladder mucosa) was 2-fold lower in the O → O vs. Y → Y group. However, comparison of the ectopic bone tissue weights in different experimental groups showed that osteoinductor activity of the bladder epithelium did not decrease, but increased 3-fold with age (O → Y:Y → Y). A 5-fold reduction of this proportion in groups where the osteoinductor was transplanted from old donors to old and young recipients (O → Y:O → O) could be attributed to age-specifi c reduction of the count of inducible osteogenic precursor cells (IOPC). The data in general suggest that age-specifi c reduction of the stromal precursor count and functional activity could be caused by the true reduction (exhaustion) of cell pool (bone marrow CFC-F; presumably, IOPC) and by the regulatory effects of the organism (bone marrow and splenic CFC-F, IOPC). These data seem to be signifi cant for understanding of the role of osteogenic stromal precursor cells in the development of age-associated bone tissue defects, for example, senile osteoporosis.     

Functional disturbances in hemopoietic microenvironment in various forms of myelodysplastic syndrome

We studied the ability of stromal sublayer of long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome to maintain the growth of normal granulocyte-macrophage colony-forming units in mixed cultures. There were changes in the hemopoietic microenvironment in these patients: decreased cellularity of the bone marrow and impaired formation of sublayers in long-term bone marrow cultures, production of growth factors, maintaining the growth of normal granulocyte-macrophage precursors by stromal cells. Dysfunction of macrophages in the stromal microenvironment was probably related to the presence of pathological macrophages. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 9, pp. 255–258, September, 2000     

Functional disturbances in hemopoietic microenvironment in various forms of myelodysplastic syndrome

We studied the ability of stromal sublayer of long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome to maintain the growth of normal granulocyte-macrophage colony-forming units in mixed cultures. There were changes in the hemopoietic microenvironment in these patients: decreased cellularity of the bone marrow and impaired formation of sublayers in long-term bone marrow cultures, production of growth factors, maintaining the growth of normal granulocyte-macrophage precursors by stromal cells. Dysfunction of macrophages in the stromal microenvironment was probably related to the presence of pathological macrophages. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 9, pp. 255–258, September, 2000     

Proliferative activity of clonogenic bone marrow stromal precursor cells

The proliferative activity of precursors of bone marrow stromal cells forming colonies (clones of fibroblasts) in monolayer cultures was studied by the thymidine-suicide method in vitro. Clonogenic cells of native bone marrow were shown not to be inhibited by thymidine-H³ with high specific activity, i.e., they virtually do not proliferate in vivo. Having started to proliferate 24 h after explantation, they then give rise to colonies of fibroblasts. In 6–14-day bone marrow cultures 39±4% of clonogenic cells die under the influence of thymidine-H³. Hence it follows that, unlike precursor cells in native bone marrow, clonogenic cells in primary cultures proliferate actively.N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 55–57, January, 1976.     

Expression of Genes of Glutathione Transferase Isoforms GSTP1-1, GSTA4-4, and GSTK1-1 in Tumor Cells during the Formation of Drug Resistance to Cisplatin

Injection of polyvinylpyrrolidone (synthetic type 2 T-independent antigen) stimulated the efficiency of clone-forming efficiency and the content of stromal precursor cells in CBA mice in the femoral bone marrow (almost 3-fold) and in the spleen (by 1.7 times) with the peak within 24 h and normalization by day 3 after immunization. The expression of IL-6, IL-8, and TNF-α genes in bone marrow and spleen cultures from immunized animals appeared on day 1 and disappeared on day 3. Hence, stimulation of stromal tissue in response to polyvinylpyrrolidone immunization was significantly less pronounced in comparison with immunization with S. typhimurium antigens. The counts of stromal precursor cells in these organs did not increase in CBA/N mice not responding to polyvinylpyrrolidone because they had no xidmutation in Brutton’s tyrosine kinase (Btk) gene, and the proinflammatory cytokine genes expression in primary cultures derived from these animals did not increase either. These data indicated that the degree of stromal tissue stimulation in immunized mice correlated with the immune response intensity. This indicated a close relationship between the stromal tissue and immune system. Stromal tissue seemed to be stimulated not only and not so much through the stromal cell Toll-like receptors, but mainly through interactions of immunocompetent and stromal cells, the former presumably playing the leading role in this process.     

Study of Genetic Stability of Human Bone Marrow Multipotent Mesenchymal Stromal Cells

Immunophenotype, proliferation rate, and genetic stability parameters of bone marrow multipotent mesenchymal stromal cells were studied. Despite the reduction of proliferative activity by passages 11–12, the cells retained the characteristic immunophenotype. The incidence of spontaneous aneuploidy for autosomes 6, 8, 11 and sex chromosomes was evaluated. Two cultures of mesenchymal stromal cells carrying aneuploid cell clones were detected: with chromosome 8 trisomy and X chromosome monosomy. The results indicate the possibility of genetic transformation and selection of mesenchymal stromal cells with abnormal karyotype during in vitro culturing.     

雌性小鼠骨髓移植给雄性小鼠后内源性骨髓细胞残存状态的研究

 目的:以雌性小鼠骨髓移植给雄性小鼠的方法,通过检测雄性小鼠血细胞的Y染色体来明确内源性骨髓细胞的残存状态。方法:将雌性或雄性C57BL/6小鼠作为受体,实验组用［137Cs］照射,6 h后每只经尾静脉注射供体小鼠骨髓细胞1×107。统计骨髓移植后14 d动物的存活率,并通过眶静脉采血观察外周血白细胞数量的变化,检测受体雄性小鼠体内Y染色体基因水平的变化以明确骨髓移植的效果。结果:分别用1 000、950和900 rad的照射剂量对受体小鼠进行照射后将供体小鼠的骨髓移植到受体小鼠体内,1 000和950 rad剂量时雌性受体小鼠可迅速恢复造血功能,而雄性受体小鼠则仅有48%的存活率。900 rad照射剂量骨髓移植后,雄性受体小鼠迅速恢复了造血功能,13 d后外周血白细胞计数基本恢复正常。移植后的雄性受体小鼠在5周内已检测不到外周血细胞Y染色体基因,表明雄性受体小鼠的骨髓被完全破坏,雌性供体小鼠的骨髓可完全替代受体雄性小鼠的骨髓并且发挥造血功能。结论: 在照射剂量900 rad照射后,雄性小鼠可以作为骨髓移植受体,为将来应用雄性小鼠作为骨髓移植受体动物开展有关心血管疾病的研究奠定了实验基础。     

Agar cultures of human clonogenic hemopoietic precursor cells for early diagnosis of some myeloproliferative diseases

Growth characteristics of human hemopoietic cells in erythremia and chronic myeloid leukemia were studied using agar cultures with and without hemopoietic growth factors. Agar cultures, similarly to cultures on other semisolid media (plasma clot, methylcellulose) can be used for early differential diagnosis of polycythemia vera (erythremia) and secondary erythrocytosis: erythremia, but not erythrocytosis, is characterized by spontaneous (erythropoietin-independent) formation of colonies from erythrocyte precursor cells. Spontaneous colony formation from granulocyte-macrophage precursor cells can serve as an important test for early diagnosis of chronic myeloid leukemia. The study of colony formation from granulocyte-macrophage precursors and of the capacity of bone marrow cells to form colonies from hemopoietic stromal precursor cells revealed new characteristics of the studied myeloproliferative diseases. Presumably, spontaneous colony formation from erythrocytic and myeloid precursors should be regarded as a sign of tumor transformation of the studied hemopoietic cells.     

Effects of bone marrow conditioned media on proliferation of stromal clonogenic cellsin vitro

Fibroblast growth factors aFGF, bFGF, IGF-I, IGF-II, TGF-β, EGF, and PDGF did not stimulate the formation of stromal fibroblast colonies (CFC-F-colonies) in cultured murine adhesive bone marrow cells. It means that colony-stimulating activity of the bone marrow feeder cells with respect to the formation of stromal fibroblast colonies does not depend on the known growth factors. A scheme and conditions of culturing are developed for preparing conditioned media from bone marrow cell cultures which can replace the CFC-F colonystimulating activity of the bone marrow feeder cellsin vitro. Primary separation of conditioned media by ultrafiltration reveals that only the fraction with molecular weight of more than 65 kD exhibits CFC-F colony-stimulating activity. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 2, pp. 218–220, February, 1999     

Effect of Macrophage Migration Inhibition Factor on the Content of Stromal Precursor Cells in Mouse Bone Marrow and Efficiency of Bone Marrow Precursor Cell Cloning <Emphasis Type="Italic">In Vitro</Emphasis>

The content of stromal precursor cells in the bone marrow of mice decreased 2–5.7 times 24 h after injection of macrophage migration inhibition factor in doses of 0.1–50 ng/kg, this reduction depending on the dose of inhibition factor. The content of precursor cells in the bone marrow of mice increased 2-fold 24 h after injection of S. typhimurium bacterial mass. One day after injection of S. typhimurium bacterial mass, the count of precursor cells in mouse spleen was 7-fold higher than 24 h after injection of macrophage migration inhibition factor. The efficiency of cloning of mouse bone marrow stromal precursor cells in vitro was suppressed 1.7–2.8 times in the presence of macrophage migration inhibition factor in doses of 0.1 to 50 ng/ml culture medium. The effect of cloning inhibition was preserved, if macrophage migration inhibition factor was added to the culture medium after 2 days of bone marrow cell culturing. In general, macrophage migration inhibition factor inhibits stromal precursor cells in vivo and in vitro. The data also indicate that macrophage migration inhibition factor is not responsible for rapid and sharp increase in the count of stromal precursor cells after immunization of animals. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 667–670, December, 2008     

Origin of stromal mechanocytes in bone marrow cultures

The strain of origin of fibroblasts growing in monolayer bone marrow cultures of semisyngeneic heterotopic grafts and radiochimeras was investigated by the indirect immunofluorescence method using an isoantiserum. All fibroblasts from bone marrow of complete radiochimeras were shown to be of recipient origin whereas histocyte—macrophages in the same cultures were of donor origin. All fibroblasts in bone marrow cultures of heterotopic grafts belonged to the original donor, whereas 80% of the histiocytes were of recipient origin. This proves directly that the stromal mechanocytes and hematopoietic cells, and also the macrophages, belonged to different cell lines.Laboratory of Immunomorphology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Barovan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 10, pp. 1270–1271, October, 1976.     

Lentivirus transduction of bone marrow hemopoietic precursor cells with Lin-c-kit<Superscript>+</Superscript> phenotype <Emphasis Type="Italic">Ex Vivo</Emphasis> using a genetic construct containing green fluorescent protein gene

We developed a technology of labeling bone marrow precursor cells with the Lin^—c-kit⁺ phenotype in culture by green fluorescent protein gene using a lentivirus vector. The proposed system provides effective transduction of bone marrow precursor cells and high transgene expression level in vitro (27%). The integration of the transgene into the transduced cell genome in vivo was verified by the method of splenic colonies. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 667–671, June, 2007     

Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day-14 colony-forming unit-spleen (CFU-S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU-S formation nor the hemopoietic colonies in the recipient liver were observed. These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.