Stromal Precursor Research Articles

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.

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 In Vitro

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.

Effect of Serum from Mice Immunized with Group A Streptococcus Antigens on the Efficiency of In Vitro Cloning of Stromal Precursor Cells

The efficiency of cloning of stromal precursor cell in mouse bone marrow culture increases significantly (2-3-fold) in the presence of serum from mice immunized with type 5 group A streptococcus antigens (5-20 microl serum/ml culture medium) in comparison with intact animal serum. The levels of TNF-alpha and IFN-gamma are significantly reduced (2.7 times and more than 6-fold, respectively) in the sera of immunized mice in comparison with normal serum. Serum levels of IL-2, -4, -5, -10, and -12 were about the same in both groups; no granulocyte-macrophage CSF was detected. These data attest to appreciable effect of immunization with streptococcal antigens on the bone marrow stromal tissue; this effect is presumably mediated through serum cytokines.

Nonadhesive populations in cultures of mesenchymal stromal cells from hematopoietic organs in mouse and rat

The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells.

Systemic Endocrine Instigation of Indolent Tumor Growth Requires Osteopontin

The effects of primary tumors on the host systemic environment and resulting contributions of the host to tumor growth are poorly understood. Here, we find that human breast carcinomas instigate the growth of otherwise-indolent tumor cells, micrometastases, and human tumor surgical specimens located at distant anatomical sites. This systemic instigation is accompanied by incorporation of bone-marrow cells (BMCs) into the stroma of the distant, once-indolent tumors. We find that BMCs of hosts bearing instigating tumors are functionally activated prior to their mobilization; hence, when coinjected with indolent cells, these activated BMCs mimic the systemic effects imparted by instigating tumors. Secretion of osteopontin by instigating tumors is necessary for BMC activation and the subsequent outgrowth of the distant otherwise-indolent tumors. These results reveal that outgrowth of indolent tumors can be governed on a systemic level by endocrine factors released by certain instigating tumors, and hold important experimental and therapeutic implications.

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.

Sensitivity of Mesenchymal Stem Cells and Their Progeny to Medicines Used for the Treatment of Hematoproliferative Diseases

Background/Aims: The influence of cytostatic medicines on mesenchymal stem cells (MSC) and their progeny, fibroblastic colony-forming units (CFU-F), was investigated. Methods: Mice were treated with busulfan, cyclophosphamide, cytarabine, methotrexate and bortezomib, as used in clinical practice. MSC and CFU-F were analyzed 3 days and 6 weeks after the treatment termination. To estimate MSC numbers, the ectopic foci formation method was used. Briefly, a donor bone marrow plug was transplanted under the renal capsule of a syngeneic animal, leading to ectopic foci formation. The systemic response of the hematopoietic microenvironment to these drugs was studied using the same method applied to recipients pretreated with the medicines. Results: CFU-F concentration was halved in the bone marrow of mice treated with busulfan, methotrexate and cyclophosphamide, and was not restored for the next 6 weeks. Proliferative potential and differentiation abilities of MSC were not affected by these medicines. The enlargement of foci size in mice treated with cytostatic agents was not conditioned by MSC, but by more mature stromal precursor cells. Conclusions: Cytostatic medicines affect stromal precursors in 2 ways: they decrease CFU-F concentration in the ‘steady-state’ bone marrow, while stimulating growth of the stromal microenvironment during its de novo formation. MSC are not sensitive to the cytostatic agents used.

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.

Morphofunctional status and osteogenic differentiation potential of human mesenchymal stromal precursor cells during in vitro modeling of microgravity effects

We studied the effects of long-term (20-day) simulated microgravity (clinostatic exposure) and osteogenic differentiation stimuli on cultured mesenchymal stromal precursor cells isolated from human bone marrow. Clinostatic exposure significantly reduced proliferative activity of mesenchymal stem cells in comparison with the static and dynamic control, increased the number of large flat cells in the culture, and stimulated migration activity of cells. Phenotypic studies of surface antigens (CD90, CD54, CD106, CD105, CD34, CD45, class 1 HLA) during clinostatic exposure of mesenchymal stem cell cultures showed differences in their expression between experimental and control groups. Studies of osteogenesis of precursor cell showed that cell differentiation potential can be directed towards osteogenesis by a combination of clinostatic exposure and differentiation stimuli. The results confirm gravity sensitivity of human bone marrow precursor cells and open new vistas for understanding of the mechanisms of bone tissue loss in humans under conditions of space mission.

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Although adult bone-marrow-derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non-dental stem-cell-mediated odontogenesis remain an open question. STRO-1(+) (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic-activated cell-sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2-day-old rat incisors. Co-cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up-regulated alkaline phosphatase activity, cell-cycle modification, and the expression of tooth-specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth-shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin-pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin-like structures and fibrous connective tissues respectively. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non-dental stem cell populations.

Mechanism of mobilization of mesenchymal stem cell under the effect of granulocyte colony-stimulating factor

We studied the role of cell components and extracellular matrix of the microenvironment in the mechanisms of mobilization of mesenchymal bone marrow stem cells under the effect of granulocyte colony-stimulating factor in vitro and in vivo on CBA/CaLac mice. It was found that the mechanisms underlying the dissociation effect of granulocyte colony-stimulating factor do not include its direct effect on the interaction of precursor cells with fibronectin. The major role in this process is played by changes in functional state of stromal precursors and cell component of the microenvironment induced by granulocytic colony-stimulating factor and mediated by regulatory systems of the microorganism.

Origination of stromal precursor cells replenishing the population of these cells in heterotopic mouse bone marrow transplants after curettage in recipients

Curettage of bone marrow cavities of two bones (femoral and crural) in recipient mice causes a drastic (more than 7-fold) increase in the count of stromal precursor cells in heterotopic bone marrow transplants. Stromal colonies in cell cultures from these transplants consist of fibroblasts with an appreciable admixture of macrophages. All y chromosome-typed colonies from cultures of female donor bone marrow transplants in recipient males (intact and subjected to curettage) contained cells carrying and not carrying y chromosome. Quantitative results of Y chromosome typing of cells from colonies corresponded to the fibroblast/macrophage ratio in colonies and the predominant localization of the label corresponded to predominant localization of macrophages (at the periphery of colonies). The results indicate that the pool of bone marrow stromal precursor cells under conditions of increased demands originates from local sources, which confirms ample data on inability of these cells to migration.

Surface patterning: Tool to modulate stem cell differentiation in an adipose system

There are several issues that need to be better understood before breast tissue-engineering becomes viable clinically. One of the key issues is the interaction between cells and the microtopography of the implant surface. The aim of this study was to evaluate the efficacy of D1 cells, multipotent mouse bone marrow stromal precursors, in differentiating to fat and to characterize their metabolic activity (lactic acid released and glucose consumed) and lipid production when cultured on patterned poly-L-lactide (PLLA) films. It was determined that, with appropriate stimulation, the D1 cells displayed morphological characteristics of adipocytes and produced lipid. The results show that the patterned surfaces did affect the rate of lipid production. Polynomial models were proposed to predict the metabolic activity of the cells over a period of time.

Effect of hypoxia on stromal precursors from rat bone marrow at the early stage of culturing

We studied the effect of hypoxia on stromal precursors from rat bone marrow. Under hypoxic conditions the cultures were characterized by low heterogeneity, the percent of large spread morphologically different cells decreased. Hypoxia increased proliferative activity of cells, had no adverse effect, and decreased the percent of cells expressing hemopoietic markers.

Role of hyaluronidase in the regulation of functions of mesenchymal precursor cells

The possibility of using hyaluronidase for the regulation of the state of different pools of mesenchymal precursors in vivo was demonstrated. The reaction of mesenchymal precursor cells depended on the dose of the enzyme. Administration of hyaluronidase in a dose of 20 U/mouse increased the content of stromal precursors and mesenchymal stem cells in the bone marrow and promotes mobilization of precursor cells induced by granulocyte CSF. Administration of high dose of hyaluronidase (100 U/mouse) reduced the content of mesenchymal precursors in hemopoietic tissue and abolished granulocyte CSF-induced mobilization of mesenchymal precursor cells of different maturity.

Possibilities of physical-chemical regulation of stem cells' pool

Modulating influence of electric impulses, visible light, some microelements is studied in parameters which are near to physio-logic ones on life activity of progenitor and mature cells of blood system in vitro and in situ. Content of granulocyte and fibroblastoid colony-forming units, concentration of colony-stimulating and colony-inhibiting activities, their concentration were determined , myelograms and morphologic signs of myelocariocytes apoptosis and necrosis were scored, saturation by chrom ions of peripheral blood erythrocytes was studied in mice bone marrow. The results showed that cell effects of physical-chemical influ-ences are small specific. Nevertheless, one may perform both positive and negative regulating influence on life activity of stromal precursors in dependence of energy density and stimulus concentration . The effect is due not only by direct but mediated reactions regulating direction and intensity of metabolic intracellular processes. It is suggested that one of target cells capable of change in-crease program of hemopoietic and stromal cells-progenitors of bone marrow in dependence of physical stimulus strength are lym-phocytes.

Heterogenecity of stromal cell precursers isolated from rat bone marrow

Bone marrow stroma contains mesenchymal stem cells (MSC) which are progenitor cells, at least for tissues arising from mesechyma. The study of MSC biology yields controversial data. Therefore further experiments are needed to characterize these cells. The aim of our research was to compare primary cultures and subcultures of stromal precursor cells isolated from rat bone marrow. Long-term cultures of these cells isolated from 5 animals have been obtained. Morphological, immunophenotypic, and functional (capacity to osteogenic differentiation) characteristics of the cells have been investigated. We show that the cell morphology in the cultures is highly heterogenic. Morphological cell types are described. Heterogeneity of stromal cells declines on late passages. Cell cultures isolated from different animals have the same immunophenotypic markers (CD90, CD44, CD54, CD106, CD45, CD11b) but different morphological characteristics and a different capacity to osteogenic differentiation during long-term cultivation. The data show that more specific markers and functional tests should be applied to identify MSC.

Effect of α-fetoprotein on the count of bone marrow and splenic stromal precursor cells and proliferation of their cultural descendants

The efficiency of cloning of stromal precursor cell increased more than 2-fold in splenic cultures and more than 3-fold in bone marrow cultures 24 h after injection of Profetal preparation to mice in vivo. The number of nucleated cells did not change in the bone marrow and slightly increased in the spleen. Addition of Profetal in vitro 2-fold decreased the efficiency of stromal precursor cells colony formation in mouse splenic cultures and dose-dependently decreased this process in bone marrow cultures derived from these animals, the maximum (5-fold) inhibitory effect was observed in a dose of 50 microg/ml. Addition of Profetal to cultured human bone marrow fibroblasts did not change the content of stromal fibroblasts in cultures. These data indicate the possibility of indirect effect of alpha-fetoprotein on the number of stromal precursor cell in hemopoietic and lymphoid organs.

State of stem cell pools in experimental diabetes mellitus

We studied the state of pools of mesenchymal precursor cells of different maturity in the bone marrow and peripheral blood and the dynamics of the content of regional parenchymal stem cells and stromal precursors in the pancreas during experimental diabetes mellitus. Reduced content of organ-specific stem cells and increased content of stromal precursors in the pancreas in the absence of compensatory reaction of deep reserve mechanisms, mesenchymal bone marrow cells, were revealed.

Participation of mesenchymal precursor cells in wound healing on skin flap model

We studied the state of different pools of mesenchymal precursor cells in the bone marrow, peripheral blood, and wound surface after modeling of tissue damage by skin flap removal. The participation of regional and circulating stromal precursors in the healing of skin defect and the absence of compensatory reaction of the regeneratory process deep reserve, mesenchymal stem cells of the bone marrow, was demonstrated.