Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse

Primary cultures were initiated from marrow, spleen, and bone explants of an adult H-2K^b-tsA58 transgenic mouse (immortomouse). All cultures were initiated in immortalizing conditions, and an additional marrow culture was first incubated for 1 week in standard conditions and then switched to immortalizing conditions. Marrow cells immediately immortalized were designated the marrow immediate population (MIP); those immortalized after 1 week were termed the marrow delayed population (MDP). MIP and MDP cells both contained a mixture of fibroblastic or flattened cells, and the MIP cells contained an additional subpopulation of adipocytic (Oil Red-O positive) cells. Alkaline phosphatase expression was induced by dexamethasone (10⁻⁷ M) in MDP cells while MIP, spleen, and bone explant cells had only a low level of expression. MDP and MIP cells differentiated into bone when combined with porous calcium phosphate ceramics and implanted subcutaneously into nude mice while bone- and spleen-derived cells did not. Clones were isolated from the MDP and MIP cell populations and tested for differentiated phenotypes. Some MIP-derived clones exhibited adipocytic characteristics while MDP-derived subclones were negative. Histologic examination of porous ceramic implanted clones showed that all of the clones had osteogenic potential. Clones exposed to either dexamethasone, human recombinant bone morphogenetic protein-2, or horse serum plus hydrocortisone showed differences in expression of adipocytic or osteogenic markers. These immortalized cultures have retained both adipocytic and osteogenic potential even after 1 year of continuous culture, and provide a model system for clonal analysis of the developmental potential of marrow-derived mesenchymal precursor cells. © 1996 Wiley-Liss, Inc.     

Clonogenic stromal cell count in the bone marrow of mice

The number of fibroblast colonies in bone marrow cultures depends on FCFC concentration in explanted cells and FCFC cloning efficiency. For mouse bone marrow the efficiency of fibroblast colony formation increases in the presence of the feeder (irradiated bone marrow of spleen cells). Colony-stimulating feeder activity does not depend on the presence of phagocytic and stromal cells in the feeder cell population. Trypsinization of the bone marrow leads to the release of additional FCFC and the increase of their concentration in bone marrow cell suspensions.     

Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell-cell interactions in osteoblast differentiation

Glucocorticoids, notably dexamethasone (Dex), have been reported to be a requirement for osteoprogenitor cell differentiation in young adult rat bone marrow stromal cell populations. We have reinvestigated the requirement for Dex and analyzed the frequency of osteoprogenitor cells present. Stromal cells were grown as primary or first subcultures in the presence or absence of Dex and their expression of osteogenic markers (alkaline phosphatase activity, hormone responsiveness, and matrix molecules, including type I collagen, osteopontin, bone sialoprotein, and osteocalcin), as well as their functional capacity to differentiate to form a mineralized bone nodule, were assessed. Dex increased, but was not an absolute requirement for, the expression of osteogenic markers. Bone nodule formation was plating cell density dependent and occurred under all combinations of treatment with or without Dex but was maximal when Dex was present in both the primary and secondary cultures. Dex increased CFU-F by approximately 2-fold, but increased CFU-O (osteoprogenitor cells; bone nodule forming cells) by 5- to 50-fold depending on the cell density and duration of treatment. Neither CFU-F nor CFU-O expression followed a linear relationship in limiting dilution analysis until very high cell densities were reached, suggesting cooperativity of cell types within the population and a multitarget phenomenon leading to osteoprogenitor differentiation. When a large number of nonadherent bone marrow cells or their conditioned medium was added to the stromal cells, osteoprogenitors comprised approximately 1/100 of plated adherent cells and their expression followed a linear, single-hit relationship. By contrast, rat skin fibroblasts or their conditioned medium totally inhibited bone nodule formation. These data support the hypothesis that in marrow stroma, as in other bone cell populations such as those from calvaria, there are at least two classes of osteoprogenitor cells: those differentiating in the absence of added glucocorticoid and those requiring glucocorticoid to differentiate, that more than one cell type is limiting for stromal osteoprogenitor differentiation suggesting a role for heterotypic cell-cell interactions in osteogenesis in this tissue, and that Dex may be acting directly and/or indirectly through accessory cells in the bone marrow to alter osteoprogenitor cell expression.     

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2.Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.     

Interleukin 10 inhibits transforming growth factor-beta (TGF-beta) synthesis required for osteogenic commitment of mouse bone marrow cells

Interleukin 10 (IL-10) suppressed TGF-beta synthesis in mouse bone marrow cultures. Coincidingly, IL-10 down-regulated the production of bone proteins including alkaline phosphatase (ALP), collagen and osteocalcin, and the formation of mineralized extracellular matrix. The mAb 1D11.16 which neutralizes TGF-beta 1 and TGF-beta 2, induced suppressive effects comparable to IL-10 when administered before the increase of cell proliferation in the culture. It appears that mainly TGF-beta 1 plays a role in this system since (a) TGF-beta 2 levels were undetectable in supernatants from osteogenic cultures, (b) no effect was observed when the anti-TGF-beta 2 neutralizing mAb 4C7.11 was added and (c) the suppressive effect of IL-10 could be reversed by adding exogenous TGF-beta 1. It is unlikely that TGF-beta 1 modulates osteogenic differentiation by changing the proliferative potential of marrow cells since 1D11.16 did not affect [3H]thymidine ([3H]TdR) incorporation or the number of fibroblast colony forming cells (CFU-F) which harbor the osteoprogenitor cell population. Furthermore, 1D11.16 did not alter [3H]TdR uptake by the cloned osteoprogenitor cell lines MN7 and MC3T3. Light and scanning electron microscopy showed that IL-10 and 1D11.16 induced comparable morphological changes in the marrow cultures. Control cultures contained flat adherent cells embedded in a mineralized matrix. In contrast, IL-10 and 1D11.16 treated cultures were characterized by round non-adherent cells and the absence of a mineralized matrix. In this study, the mechanism by which IL-10 suppresses the osteogenic differentiation of mouse bone marrow was identified as inhibition of TGF-beta 1 production which is essential for osteogenic commitment of bone marrow cells.     

Bone marrow-derived stromal cell line expressing osteoblastic phenotype in vitro and osteogenic capacity in vivo

Marrow stroma has been shown to have osteogenic potential. Here we report the characterization of a unique stromal cell line derived from mouse bone marrow (MBA-15), which expresses osteoblastic phenotype in vitro and forms bone in vivo. More than 70% of cells in culture were histochemically positive for alkaline phosphatase. The enzyme levels were enhanced threefold when cultures were treated with dexamethasone. Gel electrophoresis of [3H]-proline-labeled cultures showed that MBA-15 cells produced only type I collagen. These cells were responsive to PTH, as indicated by a 50-fold increase in intracellular cAMP. Prostaglandin E2, but not calcitonin, stimulated cAMP up to 70-fold. When cultures were grown to confluence and fed daily with ascorbic acid and beta-glycerophosphate, the cells formed a Von Kossa positive, thick extracellular matrix, shown to contain hydroxyapatite crystals. MBA-15 cells produced mineralized bone when implanted in diffusion chambers. These results indicate that the MBA-15 cell line possesses osteoblastic features in vitro and osteogenic capacity in vivo.     

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.     

Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ～50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ～80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ～3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ～50% decrease was seen in the expression of terminal osteogenic gene expression and a ～50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ～2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.     

Fibroblastic regulation of osteoblast function by prostaglandins

The effects of osteogenic inhibitory factors secreted by human periodontal ligament fibroblasts were studied in rat bone marrow stromal cell cultures. Serum-free conditioned medium from cultures of fibroblasts strongly depressed formation of mineralized tissue by bone marrow cell cultures. The inhibitory activity was reduced by treatment of fibroblast cultures with indomethacin or by pretreatment of conditioned medium with specific antibodies to prostaglandins (PGs) E₂ and F₂. Passage of conditioned medium over octadecyl columns enriched PGs four-fold and significantly increased inhibitory activity. Inhibition of mineralization was replicated by treatment of bone-cell cultures with PGs B₂, D₂, E₂, and I₂ at concentrations of 350 ng/ml to 350 pg/ml. All combinations of these agents were inhibitory but PGE₂ and PGF₂ exhibited the greatest inhibition at low concentrations (350 pg/ml). These experiments indicate that fibroblasts secrete PGs which can inhibit bone formation, and this may be one mechanism whereby fibroblasts can modulate osteogenesis at the interfaces of soft and mineralizing connective tissues.     

小鼠胎肝基质细胞造血刺激因子体外生物活性研究

本实验对基质细胞造血刺激因子－１（ＳＨＦ－１）的体外生物活性进行了研究。结果表明,ＳＨＦ－１可刺激小鼠骨髓ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－ＧＭ、ＣＦＵ－Ｍｉｘ集落的形成,它产生的这些广泛造血刺激作用是其自身所具活性的直接影响。正常小鼠骨髓细胞与ＳＨＦ－１在体外孵育４ｈ,其中ＣＦＵ－Ｓ的自杀率可提高约１０％,显示它对造血干细胞也有诱导增殖作用。     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

Osteogenic stem cells of the bone marrow

This paper presents literature and author's own data demonstrating that bone marrow contains determined osteogenic precursor cells with high potential to differentiation. They are stem cells of the bone and belong to the stromal cell line of the bone marrow which is histogenetically independent of hemopoietic cells. The paper presents detailed analysis of bone marrow stromal cells (CFUf) as well as of their osteogenic properties and requirements in growth factors. In conclusion mutual growth-stimulating interactions in the system of hemopoietic stromal cells are reviewed.     

Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro

The role of basic fibroblast growth factor (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.     

Population Dynamics of Clonogenic Stromal Precursor Cells in Hemopoietic and Lymphoid Organs during Bone Marrow Regeneration

This paper describes our study on the regeneration of hemopoietic and stromal components of bone marrow after mechanically emptying the medullar cavity of the guinea pig tibia. The intensity of hemopoiesis was determined from the number of hemopoietic cells, while the concentration and total number of stromal precursor cells were used to estimate the ability of the bone marrow to produce stromal structures, including its ability to restore a specific microenvironment. We found that there was no direct correlation between the recovery characteristics of hemopoietic and stromal cells. An increase in the population size of stromal precursor cells takes place early after curettage, and stromal fibroblasts become phosphatase-positive according to Gomori, which is characteristic of osteogenic tissue. We have also demonstrated that curettage of 3–5 tubular bones results in the growth of this cell population in the bone marrow of nonoperated bones and even in the spleen, which in guinea pigs participates only in lymphopoiesis.     

Effect of opioid peptides on proliferation and differentiation of skeletogenic cells of rabbit bone marrow in vitro

The influence of opioid peptides DSLET and DAGO in doses 10(-5), 10(-7) or 10(-10) mg per 1 ml of the medium on colony formation in the culture of stromal bone marrow fibroblast precursors was investigated 5. 10(-6) bone marrow cells were placed in plastic containers (Costar). 12 day old cell cultures were fixed with ethanol and stained with hematoxyline-eosin. Effectiveness of fibroblast colony formation (EFFC) was detected. Grown fibroblast colonies were stained after Gomory for alkaline phosphatase. Opioid peptides DSLET and DAGO in the used doses exerted no influence on EFFC and percentage phosphatase-positive colonies, which casts doubt on a presumable direct action of opioid peptides on stromal bone marrow cell-precursors. But it does not seem unlikely that opioid peptides may affect stromal bone marrow precursors of fibroblasts through the cell environment, particularly, via macrophages.     

Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro.

Rat bone marrow stromal cells were cultured in vitro. At days 14-15 of culture, dense clusters of polygonal cells were formed, and they mineralized 2-3 days later. The cells resembling osteoblasts or young osteocytes were histologically observed to be embedded in mineralized or unmineralized extracellular matrices of the nodules. Next, these mineralized nodules were electron-microscopically examined. The osteoblastic cells associated with the nodules had a well-developed rough endoplasmic reticulum, an evident Golgi apparatus and some mitochondria as their intracellular organellae. Some lysosomes and microfilaments were also visible in the cytoplasms. Moreover, some cells protruded cell processes toward the neighboring cells through the extracellular matrix. The extracellular matrix consisted of numerous collagen fibrils which were striated with 60-70 nm axial periodicity and which was similar to bone tissue collagen. A large number of matrix vesicles were scattered among the collagen fibrils in the unmineralized area of the nodules. In contrast, in the mineralized area, numerous matrix vesicles at different stages of maturation and many calcified spherules were observed. That is the mineralization in this culture system was considered to be initiated in association with the matrix vesicles and to progress along the collagen fibrils. From these findings, it was confirmed by the present study that the mineralized nodules formed in this bone marrow stromal cell culture were ultrastructurally similar to bone and that the mineralization also proceeded by going through the normal calcification process. This culture system is considered to be available to study osteogenic differentiation and calcification mechanisms.     

Osteogenic properties of adhesive cells in Dexter culture of the mouse bone marrow

Disaggregated cell suspensions obtained by mouse bone marrow fermentative digestion as well as stromal tissue obtained by marrow mild mechanical destruction were explanted. Both methods yield the cultures in which the hematopoiesis duration is comparable with dexter cultures. Adhesive cells from all of these three culture types were resuspended and in the porous gelatin sponges heterotopically transplanted under the kidney capsule of syngenic recipients. In the transplantation site there develops the hemopoietic organ containing reticular stroma, hemopoietic cells, and in most cases the well developed bone tissue. Thus, the adherent layers of mouse bone marrow dexter and similar cultures contain for a long period (not less than 2-3.5 months) the stromal fibroblast population which maintains its osteogenic and hemopoietic microenvironment transfer capacities.