Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers

Abstract. Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies. When transplanted in diffusion chambers after 20–30 cell doublings in vitro , the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation. The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC. Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells.     

Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers

Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies. When transplanted in diffusion chambers after 20-30 cell doublings in vitro, the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation. The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC. Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells.     

Enhancement of human muscle growth in diffusion chambers by bone marrow cells

Samples of minced human muscle were cultured in millipore diffusion chambers incubated in the peritoneal cavities of mice. In about half the chambers the minced muscle samples were mixed with autogenous bone marrow cells which lead to improved myogenic growth. A similar but less marked effect was produced by mononuclear cells from the patients' blood. No growth enhancement occurred when the muscle and marrow cells were separated by a filter in double chambers. In addition to accelerated myogenesis, the chambers with added bone marrow cells had a much lower incidence of infection. This work may have practical clinical implications for the treatment of muscle injuries. Local implantation of autogenous marrow cells (+/- minced muscle) may prove useful in improving myogenic regeneration.     

A count of osteogenic precursor cells in the bone marrow and their multiplication in cultures

The progeny of clonogenic stromal medullary fibroblasts from rabbits was cultivated by repeated passage. As a result of several passages the number of cells under consideration could be raised hundreds of thousand times as compared with the initial cell quantity. The strains of stromal medullary fibroblasts were found to have osteogenic properties; during reverse transplantation to the body they formed osseous tissue, creating the medullary organs. It was shown that during cultivation, the amount of osteogenic units in a cell culture dramatically rose, i. e. the osteogenic precursor cells multiplied and were self-maintained. According to all these signs the clonogenic stromal cells of the bone marrow can be regarded as stem osteogenic cells.     

Human bone marrow cells synthesize collagen, in diffusion chambers, implanted into the normal rat

Endosteal bone marrow cell populations were harvested, from freshly removed human femoral trabecular bone fragments, by enzymatic digestion. Diffusion chambers inoculated with fractions of this cell population were then implanted in the peritoneal cavities of three Albino Wistar rats for six weeks. The human cells were found both to survive within the chambers and to produce a collagen-containing extracellular matrix.     

Human bone marrow colony formation in diffusion chambers. Normal values.

In situ hybridization techniques have previously employed a series of manipulations to effect denaturation of chromosomal DNA and reannealing of DNA-RNA hybrids. This report presents a new protocol which combines the denaturation and reannealing processes. DNA is heated in a solution of 50% formamide, 50% 4 × SSC containing the RNA to be hybridized. After l h at 70 °C the preparation is slowly cooled to 37 °C over a period of 6 h and incubated at 37 °C for an additional 10 h. This technique eliminates the possibility of premature reannealing of the DNA while employing hybridization conditions which, in vitro, lead to accurate base pairing.     

Heterogeneity of stromal precursor cells isolated from rat bone marrow

Bone marrow stroma contains mesenchymal stem cells (MSC) which are precursor for at least mesenchyma-derived cells. Recent investigations revealed a lot of questions concerning MSC biology that should be further refined. The aim of this study was the comparative analysis of rat bone marrow stroma cells cultures. Mesenchymal precursor cells isolated from rat bone marrow were passed up to 50 times. Comparative morphological and immunophenotypical analysis of these cultures was carried out as well as their ability to osteogenic differentiation was studied. The isolated cultures contained morphologically different types of cells and thus showed a high heterogenity level. Morphology of these cell types was described. The heterogeneity level was reported to decrease over time. It was found out that subcultures isolated from different rats shared the same immunophenotype characteristics (CD90+, CD44+, CD54+, CD 106+, CD45-, CD11b-), but differed in their morphology as well as in ability to osteogenic differentiation. Thus MSC identification requires more specific marker and functional tests to be used.     

Ability of locally irradiated bone marrow to form erythroid colonies in diffusion chambers

It was shown that mouse bone marrow locally exposed to ionizing radiation during the period of T-lymphocytes accumulation can form, int the diffusion chambers in the absence of exogenous erythropoietin, a large number of giant and small erythroid colonies.     

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.     

Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats

Summary Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40–43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20–30 days. Cells were cultured in either -minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 g/ml ascorbic acid, or the above medium supplemented with either 10 mM Na--glycerophosphate, 10^-8 M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Glaprotein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both -glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Glaprotein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both -glycerophosphate and, particularly, dexamethasone.     

Molecular-genetic and immunophenotypic analysis of antigen profile and osteogenic and adipogenic potentials of mesenchymal stromal cells from fetal liver and adult bone marrow in rats

Comparative characteristics of mesenchymal stromal cells (MSCs) from adult bone marrow and fetal liver are of great interest due to the similar functions performed by these organs on the organization of a hemopoietic microenvironment at various developmental periods. It is known that MSCs play a pivotal role in the formation of niches for hemopoietic stem cells. The histogenetic relation of MSCs from these two hemopoietic organs cannot be ruled out. An analysis of antigen profile using immunocytochemistry and RT-PCR has confirmed that the studied cell populations fit the MSC criteria and have no contaminations of hemopoietic, lymphoid, and endothelial cells beginning at the second passage. Comparative analysis of osteogenic and adipogenic marker expression revealed MSC from fetal liver to have a weaker potential for adipogenesis and the extremely low capability for terminal osteogenic differentiation, in contrast to pronounced osteo- and adipogenic potentials of adult bone marrow MSC. The similar cell phenotype but different differentiation potentials under identical conditions of cultivation in vitro seem to be due to different developmental programs of the pre- and postnatal histogenesis of these MSC.     

Electronic sorting of granulocyte precursor cells from stimulated bone marrow.

A commerical cell sorter was used to obtain preparations of cells in various stages of granulocyte development from rabbit marrows stimulated by inflammatory response. Marrow cells were fractionated on density gradients of Ficoll/Hypaque and each fraction sorted using light scatter. Trial and error selection of appropriate gradient fractions and light scatter windows allowed sorting of early (blast cells, promyelocytes), intermediate (myelocytes, metamyelocytes) and late stage (band cells, polys) granulocytes with enhanced purity.     

Endothelial cells influence the osteogenic potential of bone marrow stromal cells

Background  

Improved understanding of the interactions between bone cells and endothelial cells involved in osteogenesis should aid the development of new strategies for bone tissue engineering. The aim of the present study was to determine whether direct communication between bone marrow stromal cells (MSC) and human umbilical vein endothelial cells (EC) could influence the osteogenic potential of MSC in osteogenic factor-free medium.     

A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue

To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.     

Purmorphamine enhances osteogenic activity of human osteoblasts derived from bone marrow mesenchymal cells

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells, but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1, 2 or 3 microM purmorphamine, or vehicle. At 7, 14 and 21 days, cell proliferation, viability, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability, but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine.