Human Long-term Marrow Cultures Research Articles

Mechanisms That Regulate the Cell Cycle Status of Very Primitive Hematopoietic Cells in Long-Term Human Marrow Cultures. I. Stimulatory Role of a Variety of Mesenchymal Cell Activators and Inhibitory Role of TGF-β

Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive, high-proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum, which is absent from fetal calf serum. However, this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules, ie, platelet-derived growth factor (PDGF), interleukin-1 (IL-1), transforming growth factor alpha (TGF-alpha), and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays, although all have known actions on mesenchymal cells including, in some cases, the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly, a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-beta, a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1, blocked their stimulating activity. These results suggest a model in which indirect, local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo.

Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-beta

Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive, high- proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum, which is absent from fetal calf serum. However, this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules, ie, platelet- derived growth factor (PDGF), interleukin-1 (IL-1), transforming growth factor alpha (TGF-alpha), and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays, although all have known actions on mesenchymal cells including, in some cases, the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly, a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-beta, a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1, blocked their stimulating activity. These results suggest a model in which indirect, local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo.

High-efficiency gene transfer to human hematopoietic cells maintained in long-term marrow culture [see comments

We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony- stimulating factor and interleukin-1 beta or medium conditioned by a human marrow “stromal” cell line to medium conditioned by agar- stimulated human leukocytes both during the infection and the colony growth period. Similar increases were also noted in the proportion of neor BFU-E, although the efficiencies overall were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term cultures with marrow exposed to virus under the same growth factor-supplemented conditions but without any immediate selection step resulted in sustained production of a high proportion of neor CFU-GM and BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular analysis was used to confirm the presence of the neo gene after culture. These results demonstrate that stable, high-efficiency gene transfer can be accomplished to the most primitive class of human hematopoietic cells currently detectable that may also have in vivo reconstituting potential. Further use of this approach should provide new insights into human hematopoietic stem cell regulation and allow continued development and assessment of gene therapy procedures.

High-Efficiency Gene Transfer to Human Hematopoietic Cells Maintained in Long-Term Marrow Culture

We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony-stimulating factor and interleukin-1 beta or medium conditioned by a human marrow "stromal" cell line to medium conditioned by agar-stimulated human leukocytes both during the infection and the colony growth period. Similar increases were also noted in the proportion of neor BFU-E, although the efficiencies overall were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term cultures with marrow exposed to virus under the same growth factor-supplemented conditions but without any immediate selection step resulted in sustained production of a high proportion of neor CFU-GM and BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular analysis was used to confirm the presence of the neo gene after culture. These results demonstrate that stable, high-efficiency gene transfer can be accomplished to the most primitive class of human hematopoietic cells currently detectable that may also have in vivo reconstituting potential. Further use of this approach should provide new insights into human hematopoietic stem cell regulation and allow continued development and assessment of gene therapy procedures.

Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration- dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage- independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL- 6 may have indirect as well as direct influences on hematopoietic cell proliferation.

Human Marrow Stromal Cells: Response to Interleukin-6 (IL-6) and Control of IL-6 Expression

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration-dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage-independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL-6 may have indirect as well as direct influences on hematopoietic cell proliferation.

Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow.

Bisphosphonates inhibit bone resorption in vivo and in vitro by unknown mechanisms. The effect of bisphosphonates on the formation of osteoclasts from their mononuclear hematopoietic precursors was investigated using human long-term marrow cultures in which multinucleated cells form that express most of the known features of the osteoclast phenotype (e.g., bone resorption, tartrate-resistant acid phosphatase, calcitonin responsiveness, and reactivity with specific MAbs). The five bisphosphonates that were tested strongly inhibited 1,25-dihydroxyvitamin D3-stimulated formation of these cells with the same relative potencies as they inhibit bone resorption in vivo. Two representative compounds (3-amino-1-hydroxypropylidene-1,1-bisphosphonate and dichloromethylene bisphosphonate) failed to inhibit the proliferation of precursors of the osteoclast-like cells. However, these compounds decreased the proportion of mononuclear and multinucleated cells expressing an osteoclast antigen, thus suggesting a degree of specificity for cells of the osteoclast lineage. We conclude that bisphosphonates are potent inhibitors of osteoclast-like cell formation in long-term human marrow cultures, and that this may be related to their ability to inhibit bone resorption in vivo.

Rapid assay and identification of human haemopoietic growth factors

In order to facilitate the identification of human haemopoietic growth factors (HGFs), in complex conditioned media from limited cell sources, we have developed a method for the rapid assay of HGFs using bone marrow stem cells (BMSCs) obtained by Percoll density gradient enrichment of non-adherent cells from long-term human bone marrow culture. These BMSCs provide the basis for a 72 h assay where proliferation is triggered by the presence of GM-CSF, G-CSF and IL-3. The assay is up to 1000 times more sensitive than the conventional colony assay in detecting the presence of HGFs. We also describe methods for the rapid separation and identification of HGFs using fast protein liquid chromatography (FPLC) with phenyl-Superose and Mono Q columns. This enables individual HGFs present in complex conditioned media to be rapidly identified from the elution profile of HGF activity determined in the BMSC assay.

Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclastlike cells in vitro.

Interleukin-1 (IL-1) alpha and beta and tumor necrosis factor (TNF) alpha and beta are potent stimulators of bone resorption in vitro and in vivo. However, the mechanisms underlying this increased bone resorption have not been clearly defined. Increased bone resorption can result from increased activity of individual osteoclasts, increased numbers of osteoclasts, or both. Therefore, we have used a long-term human marrow culture system that forms multinucleated cells (MNC) with the characteristics of osteoclasts to examine the effects of IL-1 and TNF on osteoclast formation. Human recombinant IL-1 alpha and IL-1 beta, and human recombinant TNF-alpha and TNF-beta stimulated MNC formation from 4- to 60-fold. IL-1 alpha, IL-1 beta, TNF-alpha, and TNF-beta significantly increased MNC formation at very low concentrations: 2.5 x 10(-13) M for IL-1 alpha and IL-1 beta, 10(-11) M for TNF-alpha, and 10(-10) M for TNF-beta In addition, these cytokines enhanced MNC formation in the presence of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], a potent osteotropic factor that stimulates MNC formation by stimulating fusion of mononuclear precursor cells. Simultaneous addition of IL-1 and TNF to the cultures resulted in a synergistic stimulation of MNC formation. These results suggest that: (1) IL-1 and TNF stimulate bone resorption in part by increasing osteoclast formation and (2) an extremely low concentration of these factors can synergistically increase osteoclast formation in the absence of other factors, such as 1,25-(OH)2D3. These data suggest that synergistic interactions among cytokines play an important role in maintaining bone cell activity in normal and pathologic states.

Infection of Hematopoietic and Stromal Ceils in Human Continuous Bone Marrow Cultures by a Retroviral Vector Containing the Neomycin Resistance Gene

Stability and expression of the bacterial neomycin resistance gene (neor) transferred to human continuous marrow cultures by a retroviral vector [pZIP-NeoSV(X)] was evaluated over 4 weeks. Following infection of long-term human marrow cultures with pZIP-NeoSV(X), 10-15% of the stromal cells demonstrated high replating efficiency in a dose of the neomycin analogue G418 that was toxic to stromal cells from uninfected cultures. In contrast, G418 resistance was detected in less than or equal to 1% of GM-CFUc and CFU-GEMM derived from the same virus-infected compared to control cultures. Infection of human CFU-GEMM enriched 100 X by monoclonal antibody selection with pZIP-NeoSV(X) did not increase the percentage of neor progenitors. Marrow cells from cultures infected with pZIP-NeoSV(X) and a replication competent amphotropic virus transferred the vector and G418 resistance to HeLa cells at a frequency of 1/10(5) for nonadherent and 1/10(4) for adherent cells. Two established human hematopoietic (HL60 and K562) and one stromal cell line (KM101) stably expressed the neor gene. Thus, a higher efficiency of infection and expression of a gene transferred by pZIP-NeoSV(X) to permanent human hematopoietic tumor cell lines and fresh marrow stromal cells contrasts with a lower level of expression in fresh CSF-dependent human hematopoietic stem cells.

Transforming growth factor beta inhibits formation of osteoclast-like cells in long-term human marrow cultures.

Transforming growth factor beta (TGF-beta), a polypeptide present in abundant amounts in bone matrix, was examined for its effects on osteoclast formation by using a human bone marrow culture system in which multinucleated cells (MNCs) with osteoclast characteristics form. TGF-beta strongly inhibited MNC formation at concentrations as low as 1 ng/ml. TGF-beta also completely suppressed the effects of osteotropic factors known to stimulate MNC formation. The inhibitory effect of TGF-beta on osteoclast-like cell formation was more pronounced during the first week of culture, which corresponds to the period of proliferation of mononuclear osteoclast precursors. To examine whether the inhibitory effects of TGF-beta on MNC formation could be due to inhibition of the granulocyte/macrophage progenitor cell [colony-forming unit granulocyte/macrophage (CFU-GM)], the probable precursor for MNC, we tested the effects of TGF-beta on CFU-GM formation in presence of a source of colony-stimulating factor. Unexpectedly, TGF-beta at concentrations (0.1-1 ng/ml) that were inhibitory for MNC formation enhanced day 7 CFU-GM colony formation. This increase in CFU-GM colony formation seen in cultures containing TGF-beta resulted from significantly more granulocytic colonies being formed in the cultures, suggesting that TGF-beta may induce CFU-GM to differentiate preferentially to cells of the granulocytic lineage. Differentiation of CFU-GM to granulocytes rather than osteoclast precursors in response to TGF-beta would result in inhibition of MNC formation by depleting the precursor pool for MNC. These data suggest that inhibition of osteoclast-like cell formation by TGF-beta may be an important mechanism of control of local bone resorption.

Role of pokeweed mitogen spleen conditioned medium in regulating haematopoiesis in long-term human marrow cultures.

The modulatory effect of pokeweed mitogen spleen conditioned medium (PWM-SCM) on the growth of myeloid progenitors (CFU-C) and on the composition of the stromal layer in human bone marrow long-term cultures was studied. In the presence of PWM-SCM there was enhanced CFU-C growth in the adherent layer throughout 6 weeks of incubation, with 2-fold, 2-fold and 3.4-fold increases during wk 2, 4 and 6 respectively (p less than 0.05). In the non-adherent layer of control cultures, however, there was a rapid decline in haematopoiesis which could not be offset by PWM-SCM. The differentiation pattern of CFU-C (entailing monocyte - macrophage progenitors CFU-M, granulocytic progenitors CFU-G or monocyte - granulocyte progenitors CFU-GM) was not altered by PWM-SCM. Fewer lipid-containing cells developed in the adherent layer of cultures treated with PWM-SCM than in control cultures, but the distribution pattern of monocytes - macrophages or granulocytes was not affected. These results suggest that in long-term cultures of human marrow cells there may be extensive haematopoiesis in the presence of PWM-SCM. Whether this is a direct effect on progenitor cells of stimulating factors, or rather of accessory cells in the adherent layer, remains to be clarified.

P16. Generation of osteoblast-like cells from long-term human marrow cultures in vitro

The progressive transformation of one organ system into another is a fundamental signature of fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of extraskeletal bone formation in humans. In all affected individuals, FOP is caused by heterozygous missense gain-of-function mutations in Activin receptor A type I (ACVR1), a bone morphogenetic protein (BMP) type I receptor. Loss of autoinhibition of the mutant receptor (mACVR1) results in dysregulated BMP pathway signaling, and is necessary for the myriad developmental features of FOP, but does not appear sufficient to induce the episodic flare-ups that lead to disabling post-natal heterotopic endochondral ossification (HEO) and that are a hallmark of the disease. Post-natal FOP flare-ups strongly implicate an underlying immunological trigger involving inflammation and the innate immune system. Recent studies implicate canonical and non-canonical TGFβ/BMP family ligands in the amplification of mACVR1 signaling leading to the formation of FOP lesions and resultant HEO. BMP and Activin ligands that stimulate mACVR1 signaling also have critical regulatory functions in the immune system. Cross-talk between the morphogenetic and immunological pathways that regulate tissue maintenance and wound healing identifies potential robust therapeutic targets for FOP. Here we review current evidence for an immunological trigger for flare-ups and HEO in FOP, propose a working schema for the pathophysiology of observed phenomena, and highlight outstanding questions under investigation.

Current status of bone marrow purging prior to autografting for Hematological malignancies—future directions

While the debate remains largely open concerning the importance of purging in malignant hemopathies, we are currently witness to the extraordinary development of complex and expensive technologies associating several methods (immunochemoseparation procedure, drug cocktails …). Such techniques may be justified for acute leukemias at high risk of relapse when the residual disease is almost perceptible. Current objectives are oriented in several directions: development of techniques for detecting residual disease, development of immunophysical procedure (magnetic and floating immunobeads) and long-term human marrow cultures for selection of normal hematopoietic stem cells. Future progress in ABMT depends not only on the effective ex-vivo purging of marrow but also on development of more effective conditioning regimens ( in vivo purging).

Simian virus 40-transformed adherent cells from human long-term marrow cultures: cloned cell lines produce cells with stromal and hematopoietic characteristics

Adherent cells from long-term marrow cultures from 23 individuals were transformed with wild-type simian virus 40 (SV40). After transformation, cloned cell lines were developed that even after rigorous subcloning invariably produced both stromal cells and round cells. The stromal cells expressed cytoskeletal filaments similar to those of long-term marrow culture adherent cells and produced interstitial and basal lamina collagen types. The round cells had the electron microscopic appearance of primitive hematopoietic cells and when examined with cytochemical stains and monoclonal antibodies to hematopoietic differentiation antigens had reaction patterns suggestive of cells from several lineages. Most round cells expressed the pan- hematopoietic T-200 determinant, and lesser percentages expressed the early T cell antigens CD-1 and CD-3, HLA-DR determinants, the monocytic antigen recognized by Leu M3, and the myeloid antigens detected by monoclonal antibodies 1G10 and 12.8. In addition, when plated in semisolid medium in the presence of a source of colony-stimulating activity, up to 11% of the cells formed colonies consisting of blastlike cells that also expressed hematopoietic cell surface determinants. The data suggest that adherent cells in long-term marrow cultures contain a cell that after transformation by SV40 obligately produces cells with hematopoietic as well as stromalike features.

Phenotype and functional characterisation of multi-nucleated cells (MNC) derived from long-term cultures of human adult and fetal bone marrow

Phenotype and functional characterisation of multi-nucleated cells (MNC) derived from long-term cultures of human adult and fetal bone marrow

Recombinant human interferon-gamma inhibits formation of human osteoclast-like cells.

Interferon-gamma (IFN-gamma) inhibits osteoclastic bone resorption in vitro, but the mechanism responsible for this inhibition is unknown. We have used a long-term human marrow culture system that forms multinucleated cells (MNC) with osteoclast characteristics to test the effect of recombinant human IFN-gamma on MNC formation. The addition of 1,25-dihydroxy-vitamin D3 (1,25D3) at 10(-8) M to these cultures significantly increased both MNC formation and the number of nuclei per MNC. IFN-gamma at 100 U/ml strongly inhibited both of these effects of 1,25D3 in this system. IFN-gamma significantly inhibited MNC formation at very low concentrations (4 U/ml), with 10 U/ml inhibiting 1,25D3-stimulated MNC formation by 50%. In contrast, 100 U/ml of IFN-gamma were required to inhibit the growth of granulocyte-macrophage colony-forming cells, the probable progenitor for MNC, by 50%. Treatment of cultures with IFN-gamma for only the first or last week of culture significantly inhibited MNC formation stimulated by 1,25D3. Autoradiographic studies with [3H]thymidine showed that IFN-gamma did not inhibit proliferation of precursors for MNC. Additionally, IFN-gamma inhibited MNC formation stimulated by parathyroid hormone or interleukin 1. These results suggest that IFN-gamma inhibits MNC formation, and that IFN-gamma inhibits bone resorption in part by inhibiting osteoclast formation.

Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures.

Transforming growth factor-alpha (TGF-alpha) is synthesized by a variety of tumor cell lines and stimulates osteoclastic bone resorption in vitro. The mechanism by which TGF-alpha increases osteoclast activity is unknown. We used a human marrow culture system that forms osteoclast-like multinucleated cells (MNCs) to determine the effects of recombinant human TGF-alpha on MNC formation. Addition of 0.01 ng/ml TGF-alpha for the 1st week followed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for the subsequent 2 wk significantly increased MNCs. Treatment of these cultures with TGF-alpha without later addition of 1,25(OH)2D3 did not increase MNC formation. Autoradiographic studies revealed that TGF-alpha stimulated proliferation of precursors for MNCs, and 1,25(OH)2D3 increased their rate of fusion into MNCs. Addition of murine epidermal growth factor (EGF) (0.1 ng/ml) followed by 1,25(OH)2D3 also significantly stimulated MNC formation. These data suggest that TGF-alpha and EGF may stimulate bone resorption by increasing the proliferation of osteoclast precursors, which leads to increased numbers of osteoclasts.

Formation of osteoclast-like multinucleated cells is human long-term marrow cultures in controlled by osteotropic hormones

Formation of osteoclast-like multinucleated cells is human long-term marrow cultures in controlled by osteotropic hormones

CGA-7 and HHF, two monoclonal antibodies that recognize muscle actin and react with adherent cells in human long-term bone marrow cultures

The CGA-7, a monoclonal antibody that reacts with smooth muscle cell actin but not with endothelial cell or fibroblast actin, and HHF, a monoclonal antibody that reacts with smooth muscle, skeletal muscle, and cardiac muscle actin, both recognize microfilaments present within adherent cells from actively hematopoietic human long-term marrow cultures. Macrophages, monocytes, and cultured marrow fibroblasts do not react with either antibody. These data suggest that the anti-actin antibodies may serve as useful markers for in vitro microenvironmental cells and lend support to the hypothesis that stromal cells from long- term marrow cultures are different from marrow fibroblasts and may constitute a unique cell lineage.