Human Marrow Stromal Cell Line Research Articles

Bone Marrow Stroma–Secreted Cytokines Protect JAK2V617F-Mutated Cells from the Effects of a JAK2 Inhibitor

Signals emanating from the bone marrow microenvironment, such as stromal cells, are thought to support the survival and proliferation of the malignant cells in patients with myeloproliferative neoplasms (MPN). To examine this hypothesis, we established a coculture platform [cells cocultured directly (cell-on-cell) or indirectly (separated by micropore membrane)] designed to interrogate the interplay between Janus activated kinase 2-V617F (JAK2(V617F))-positive cells and the stromal cells. Treatment with atiprimod, a potent JAK2 inhibitor, caused marked growth inhibition and apoptosis of human (SET-2) and mouse (FDCP-EpoR) JAK2(V617F)-positive cells as well as primary blood or bone marrow mononuclear cells from patients with polycythemia vera; however, these effects were attenuated when any of these cell types were cocultured (cell-on-cell) with human marrow stromal cell lines (e.g., HS5, NK.tert, TM-R1). Coculture with stromal cells hampered the ability of atiprimod to inhibit phosphorylation of JAK2 and the downstream STAT3 and STAT5 pathways. This protective effect was maintained in noncontact coculture assays (JAK2(V617F)-positive cells separated by 0.4-μm-thick micropore membranes from stromal cells), indicating a paracrine effect. Cytokine profiling of supernatants from noncontact coculture assays detected distinctly high levels of interleukin 6 (IL-6), fibroblast growth factor (FGF), and chemokine C-X-C-motif ligand 10 (CXCL-10)/IFN-γ-inducible 10-kD protein (IP-10). Anti-IL-6, -FGF, or -CXCL-10/IP-10 neutralizing antibodies ablated the protective effect of stromal cells and restored atiprimod-induced apoptosis of JAK2(V617F)-positive cells. Therefore, our results indicate that humoral factors secreted by stromal cells protect MPN clones from JAK2 inhibitor therapy, thus underscoring the importance of targeting the marrow niche in MPN for therapeutic purposes.

Abstract 516: Marrow stromal cell rendered stability to chronic lymphocytic leukemia cells is partly mediated by Pim kinase

Abstract Chronic lymphocytic leukemia (CLL) was thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports reveal that at least 1% of CLL cells undergo replication in marrow and the accumulation is not merely due to intrinsic defect in apoptosis but in part due to extrinsic signals delivered by microenvironment. Although several entities of marrow stromal cell (MSC) microenvironment have been elucidated, the critical elements involved in CLL survival remain obscure. Pim proteins are constitutively active Ser/Thr kinases and to date Pim-1, -2, -3 have been identified. Their activity on target proteins influences a number of cellular processes such as transcription, translation, proliferation and survival. Previously, we elucidated that Pim kinases are highly expressed in CLL cells compared to normal lymphocytes [Blood 114:4150, 2009]. Based on this background, we hypothesized that Pim and their substrates may essentially contribute to MSC-driven CLL cell survival. To test this, we co-cultured CLL lymphocytes with NKTert stromal cells (a human MSC line) that mimic marrow microenvironment. Evaluation of Pim in CLL cells by immunoblotting (n=10) revealed that Pim-1 and -2 expression increased with supporting MSC, with more pronounced increase in Pim-2. A similar trend was observed in total and phospho cMyc (Ser62, n=8) suggesting that Pim is a co-activator of MYC and MYC is a Pim-driven transcription factor, and their levels largely correlate. This is consistent with the observed increase in the global RNA synthesis (uridine incorporation) in CLL cells co-cultured with MSC. Histone H3, a transcriptional activator and a Pim target, is important for MYC driven transcription. In CLL cells co-cultured with NKTert cells, phospho Histone H3 (Ser10) diminished significantly starting as early as 1 hr (n=10). The mechanism underlying the decrease and changes at the early time points are under investigation. In conjunction with transcription, translation is also governed by Pim kinases through phosphorylation of translation suppressor protein 4E-BP1. In this regard, our preliminary data shows that in CLL cells 4E-BP1 phosphorylation status was not affected by MSC. This is in concordance with the steady-state levels of total protein synthesis (leucine incorporation) in presence of stromal support. CLL cells remained replicationally quiescent on MSC (Ki67 staining) and hence cell cycle substrates of Pim (p21, p27, cdc25a and c) were not analyzed. In terms of survival (annexin/PI binding), the viability of CLL cells on MSC increased by 10-25% at day 4, 5, 6 of incubation (n=4, 6, 5, respectively). Consistent with this data, in CLL cells Bad phosphorylation (Ser112), a Pim substrate, was elevated with stromal support. Taken together, we demonstrate that MSC-rendered stability to CLL cells by Pim kinases is in part mediated by increased transcription and disrupted apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 516. doi:10.1158/1538-7445.AM2011-516

High Throughput Sequencing Following Cross-Linked Immune Precipitation (HITS-CLIP) of Argonaute (AGO) Identifies Mir-193a as a Regulator of Jagged1 In Marrow Stromal Cells.

AbstractAbstract 3847MicroRNAs (miRNAs) are small non-coding RNAs with important roles in the post-transcriptional regulation of up to 30% of all vertebrate genes. Traditional methods to determine miRNA-mRNA interactions have included transcriptional profiling of miRNAs, bio-informatic prediction of miRNA-mRNA binding, analysis of 3' untranslated region (3'UTR) binding of miRNAs and over-expression of miRNAs in relevant cell types. These studies however fall short of demonstrating direct interaction between a miRNA and its target mRNAs. We applied a recently described biochemical technique of high throughput sequencing following cross-linked immune precipitation (HITS-CLIP) to dissecting the miRNA-mRNA interactions in two functionally distinct human marrow stromal cell lines. HITS-CLIP relies on the ability of ultraviolet (UV) radiation to cross-link RNA to proteins they are bound to, followed by immune-precipitation of the RNA-protein complex to isolate the cross-linked RNA and sequencing by high throughput techniques. As miRNA-mRNA interactions occur in close proximity to the argonaute proteins (AGO), an anti-argonaute monoclonal antibody was used to isolate the Ago-miRNA-mRNA complexes.The two stromal cell lines analyzed by HITS-CLIP (designated HS5 and HS27a) were isolated from a normal marrow primary long term culture (LTC), immortalized and extensively characterized for both function and expression profiles (mRNA and miRNA). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (CXCL12, Angiopoietin-1, Jag1 etc). In keeping with this, HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. Sequence reads from the HITS-CLIP analysis from each of the cell lines were aligned to the human genome using the UCSC genome browser to identify Ago-mRNA and Ago-miRNA binding sites in both the cell lines. Interestingly, corresponding datasets from HS5 and HS27a were similar for the majority of mRNAs and miRNAs, but distinct for those mRNAs (such as Jag1, CXCL12, IL6 and GCSF) and miRNAs (such as miR-886-3p, miR-221, miR-181a and miR-193a) known to be differentially expressed between the two cell lines. We then validated the use of the HITS-CLIP strategy in stromal cells by analyzing one such Ago-mRNA binding site for Jagged1 (Jag1). Jag1 is a ligand for Notch1 and is expressed in those cells that support the hematopoietic stem cell (HSC) niche. The Notch pathway is a highly conserved signaling system critical in regulating several tissue systems including hematopoietic cells. This binding site, 1749 bp downstream of the transcriptional start-site for Jag1 was significantly more enriched in HS5 compared to HS27a. The site was also a predicted binding site for miR-193a, a miRNA over-expressed in HS5 compared to HS27a cells. Over-expression of miR-193a in HS27a cells resulted in the down-regulation of Jag1 protein (as measured by Western blotting). To confirm the direct interaction between Jag1 and miR-193a, we cloned this purported binding site downstream of the luciferase gene and co-transfected the plasmid with miR-193a. Luciferase activity was down-regulated greater than 50% when compared to control transfections suggesting a direct effect of miR-193a on Jag1 transcript. In summary, our data suggest that HITS-CLIP methodology can be used to define in vivo spatial interactions between miRNA and mRNAs in the marrow microenvironment (ME). It can also be used to define miRNA-based regulation of specific genes such as Jag1, which are critical to defining functional niches in the ME. Disclosures:No relevant conflicts of interest to declare.

Direct actions of leptin on bone remodeling.

From the time of its original description almost a decade ago [1] leptin was regarded as a hormone originating in the adipocyte whose primary target was within the central nervous system. There, it regulates appetite and reproductive function. It has now become clear that leptin receptors are much more widely distributed than just the hypothalamus, and the skeleton has emerged as an important site of action of leptin. This is suggested by the demonstration of the signalling form of the leptin receptor in human osteoblasts [2–4], rat osteoblasts [5, 6], human chondrocytes [6], porcine chondrocytes [2], human mesenchymal stem cells undergoing osteogenic differentiation [7], and in osteoblastic and chondrocytic cell lines [5, 8]. Consistent with this receptor data, there is now abundant evidence indicating that leptin directly promotes the differentiation and growth of cells of the osteoblast lineage. Thomas et al. [9] found that leptin promoted differentiation of a human marrow stromal cell line into an osteoblast phenotype, and that it increased synthesis of bone matrix proteins, such as type I collagen and osteocalcin, in these cells. Similar findings have been reported by Gordeladze et al. [10] in studies of primary human osteoblasts, in which they also found that leptin reduced apoptosis and increased formation of mineralized bone nodules. Iwaniec et al. [11] have reported increased numbers of mineralized bone nodules in primary osteoblast cultures, and Reseland et al. [12] demonstrated this with both primary human osteoblast cultures and in osteosarcoma cell lines. We have found that low concentrations of leptin (>10 M) increase proliferation of isolated fetal rat osteoblasts to an extent comparable to that of IGF1 [6]. Thus, there is agreement across a range of in vitro models that leptin is anabolic to osteoblasts. There is also evidence that leptin directly regulates osteoclast development. Holloway et al. [13] have shown that leptin inhibits osteoclast generation from human peripheral blood mononuclear cells by reducing production of RANK in mononuclear cells. These findings are complemented by the report of Burguera et al. [14] that leptin increases osteoprotegerin levels and decreases RANK-ligand levels in human marrow stromal cells. Leptin (‡10 M) also inhibits osteoclastogenesis in mouse bone marrow cultures [6]. The effects of leptin on osteoclasts could be contributed to by its stimulation of the secretion of interleukin-1 receptor antagonist from monocytic and lymphocytic cells, which will reduce osteoclast recruitment [15, 16]. These in vitro findings of leptin effects on osteoclasts explain the clinical observations of an inverse relationship between serum leptin concentrations and bone resorption markers in human fetuses [17] and in postmenopausal women [18]. Thus, there is a consistent body of evidence suggesting that leptin will impact positively on bone density as a result of its dual effects on bone formation and resorption. Leptin may also impact on skeletal size since it acts on chondrocytes. We have demonstrated the presence of the leptin receptor in chondrocytes from human articular cartilage, and that proliferation of ovine and canine chondrocytes is promoted by leptin [6]. Systemic administration of leptin to adult mice increases the thickness of their growth plates [6]. Maor et al. [19] have shown specific binding sites for leptin in the chondrocyte cell population of skeletal growth centers in the mouse mandibular condyle. Leptin stimulated cell proliferation and increased the width of the chondroprogenitor zone in this model. These findings are reflected in the reduced femoral length of both the leptin-deficient mouse (Fig. 1) [2] and the leptin-resistant rat [20]. The positive effect of leptin on skeletal size suggests that this hormone may be a contributor to the increases in stature of successive generations of humans that has been observed over the last century or more [21]. As nutrition has improved, leptin concentrations may have increased with a consequent increase in growth and adult height. The final integration of the direct effects of leptin on bone cells seen in vitro occurs in at least three different ways. One is in human studies of the relationships between leptin and bone density. Circulating leptin conCorrespondence to: I. R. Reid; E-mail: i.reid@auckland.ac.nz Calcif Tissue Int (2004) 74:313–316 DOI: 10.1007/s00223-002-0015-z Calcified Tissue International

Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein

DNA spotted microarrays were used to compare gene expression profiles from 2 functionally distinct human marrow stromal cell lines: HS-27a, which supports cobblestone area formation by early hematopoietic progenitors, and HS-5, which secretes multiple cytokines that support the proliferation of committed progenitors. One unexpected result was the high level of interleukin-7 receptor (IL-7R) gene expression in HS-27a stromal cells. Northern blot analysis confirmed the IL-7R RNA expression, and Western blots for the IL-7R protein detected both a full-length (90-kd) IL-7R and a smaller 30-kd fragment in both HS-27a cells and primary stromal cell cultures, whereas only the 90-kd receptor protein was detected in peripheral blood mononuclear cells. Biotinylated IL-7 was shown to bind to HS-27a cells under physiologic conditions, and this binding was inhibited by blocking anti-IL-7 antibodies. Tyrosine phosphorylation of several proteins (55 kd, 30 kd, and 24 kd) in HS-27a cells was rapidly increased after incubation with recombinant IL-7. One of the phosphorylated proteins proved to be the 30-kd IL-7R fragment. Exposure of HS-27a cells to IL-7 resulted in a 10-fold increase in secretion of IL-6 into culture supernatants but no increase in the cytokines stromal cell-derived factor 1, macrophage inflammatory protein 1 alpha, or IL-1 beta. The up-regulation of IL-6 secretion is associated with a rapid but transient increase in detectable levels of IL-6 messenger RNA. These data suggest that IL-7 may function to regulate the milieu of the microenvironment by modulating IL-6 secretion by the IL-7R-expressing stromal elements.

Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.

Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34(+) cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34(+) cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34(+) cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 +/-52.1)-fold, total progenitor cells (CFC) by (74.5 +/-5.2)-fold and CD34(+) cells by 15.7-fold. Maximal expansions of CFC and CD34(+) cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 +/- 5.5)-fold and (57.0 +/- 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34(+) cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34(+) cells can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines.

Response of bipotential human marrow stromal cells to insulin-like growth factors: effect on binding protein production, proliferation, and commitment to osteoblasts and adipocytes.

Insulin-like growth factors (IGFs) are important regulators of the activity of mature osteoblasts, but their effects on osteoprogenitor cells in human bone marrow stroma are unclear. In this study, we assessed the effects of IGFs on a conditionally immortalized human marrow stromal cell line, hMS(3-4), which has the ability to differentiate to either mature osteoblasts or adipocytes. hMS(3-4) cells expressed functional receptors for IGFs as well as specific IGF-binding proteins (IGFBP-3, -4, -5, and -6). IGF treatment of hMS(3-4) cells did not alter IGFBP expression, but resulted in distinct posttranslational modifications of secreted IGFBP-3 and IGFBP-4 proteins. IGF-I, IGF-II, and their receptor-activating analogs significantly increased by 2-fold the proliferation rate of the hMS(3-4) cells, but had a more complex effect on hMS(3-4) cell differentiation. Treatment with IGFs did not affect gene expression of Cbfa1 or peroxisome proliferator-activated receptor gamma2 (transcription factors involved in commitment to osteoblast and adipocyte pathways, respectively), alkaline phosphatase, type I collagen, and osteocalcin (markers of the osteoblast lineage), or lipoprotein lipase and adipsin (markers of the adipocyte lineage) and did not change alkaline phosphatase activity or type I collagen and osteocalcin protein relative to total protein production. In contrast, IGFs significantly increased type I collagen expression in differentiated hMS(3-4) cells as well as mature osteoblasts and promoted lipid accumulation in differentiated adipocytes. In summary, hMS(3-4) cells express essential components of the IGF system and respond to IGF treatment with increased proliferation. There was no evidence for IGFs directly modulating the commitment of hMS(3-4) cells to either osteoblast or adipocyte pathways, and their effects on differentiation within these lineages were dependent on the stage of cell maturation.

Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation.

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2-6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2-6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2-stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor gamma2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.

Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes.

Both bone mass and serum leptin levels are increased in obesity. Because osteoblasts and adipocytes arise from a common precursor in bone marrow, we assessed the effects of human recombinant leptin on a conditionally immortalized human marrow stromal cell line, hMS2-12, with the potential to differentiate to either the osteoblast or adipocyte phenotypes. By RT-PCR and Western immunoblot analysis, the hMS2-12 cells expressed messenger RNA (mRNA) and protein for the leptin receptor. Leptin did not affect hMS2-12 cell proliferation, but resulted in dose- and time-dependent increases in mRNA and protein levels of alkaline phosphatase, type I collagen, and osteocalcin, and in a 59% increase in mineralized matrix. Leptin increased mRNA levels of lipoprotein lipase at 3 days, but decreased mRNA levels of adipsin and leptin at 9 days and decreased lipid droplet formation by 50%. Leptin did not affect the expression of Cbfa1 or peroxisome proliferator-activated receptor-gamma2, transcription factors involved in commitment to the osteoblast and adipocyte pathways, respectively. Thus, leptin acts on human marrow stromal cells to enhance osteoblast differentiation and to inhibit adipocyte differentiation. Our data support the hypothesis that leptin is a previously unrecognized, physiological regulator of these two differentiation pathways, acting primarily on maturation of stromal cells into both lineages.

IL-6 mediates the effects of IL-1 or TNF, but not PTHrP or 1,25(OH)2D3, on osteoclast-like cell formation in normal human bone marrow cultures.

A potent interleukin-6 (IL-6) antagonist (Sant 5), which binds tightly to the IL-6alpha receptor but has impaired gp130 heterodimerization, has been developed recently by site-directed mutagenesis of human IL-6. We report here that Sant 5 inhibits IL-6-stimulated osteoclast-like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL-1 or tumor necrosis factor alpha (TNF-alpha in these cultures. We further show that a neutralizing antibody to IL-6 also inhibits the stimulatory effects of IL-1 or TNF-alpha in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25-dihydroxyvitamin D3 stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL-6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast-like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL-6 mediates the effects of IL-1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL-6 antagonist and inhibit osteoclast formation in vitro.

CD14+ Cells in Granulocyte Colony-Stimulating Factor (G-CSF)-Mobilized Peripheral Blood Mononuclear Cells Induce Secretion of Interleukin-6 and G-CSF by Marrow Stroma

The ability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMCs) to induce secretion of cytokines in primary long-term marrow cultures (LTC) or in the human marrow stromal cell line HS23 was compared with that of marrow mononuclear cells. Equal numbers of G-PBMCs or marrow mononuclear cells were added to stromal cultures, supernatants were harvested at day 4 and levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, G-CSF, and tumor necrosis factor alpha (TNF alpha) were determined. G-PBMCs induced 21.4-fold higher levels of IL-6 and 12.5-fold higher levels of G-CSF in LTC cocultures compared with marrow mononuclear cells and induced 20.6-fold more IL-6 and 6.3-fold more G-CSF when added to HS23 cells. Experiments using sorted populations of CD20+, CD3+, and CD14+ cells showed that CD14+ cells within G-PBMCs were responsible for triggering the production of IL-6 and G-CSF. The effect did not require cell-cell contact and was inhibited when neutralizing antibodies to IL-1 alpha and IL-1 beta were used in combination. In these experiments, the greater stimulating ability of G-PBMCs is most likely attributable to the greater number of CD14+ cells in G-PBMCs (26.1+% +/- 2.3%) compared with marrow (2.5% +/- 0.8%), because equal numbers of CD14+ cells sorted from marrow and G-PBMCs showed comparable ability to induce IL-6 and G-CSF when placed directly on stromal cells.

Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation.

We established a human bone marrow stromal cell line (Saka) by infecting marrow adherent cells from semisolid marrow cultures with a recombinant simian virus-40 (SV40) virus. The cells expressed SV40 large tumor antigen, had a fibroblast-like shape, and expressed fibronectin and vimentin. They did not contain detectable alkaline phosphatase activity; express myeloid, lymphoid, or factor VIII-associated antigens; or develop adipocyte-like characteristics with dexamethasone treatment. Polymerase chain reaction analysis of Saka cell RNA detected expression of messenger RNAs for interleukin-6 (IL-6), IL-1 beta, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, and the 1,25-dihydroxyvitamin D3 receptor. Coculture of Saka cells with human marrow mononuclear cells enhanced formation of osteoclast-like multinucleated cells (MNC) in long term human bone marrow cultures. These MNC expressed calcitonin receptors and formed resorption lacunae on dentine. In contrast, coculture of marrow mononuclear cells with other SV40-transformed human marrow stromal cell lines did not increase MNC formation. Conditioned medium from Saka cells or coculture of bone marrow and Saka cells separated by a Millipore membrane did not enhance MNC formation. Addition of a neutralizing antibody to IL-6 or IL-1 beta blocked the effects of Saka cells on MNC formation. These results suggest that marrow stromal cells enhance osteoclast formation in part through direct cell to cell contact and production of IL-6 and/or IL-1 beta.

Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation

Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation

Functionally Distinct Human Marrow Stromal Cell Lines Immortalized by Transduction With the Human Papilloma Virus E6/E7 Genes

A replication-defective recombinant retrovirus containing the human papilloma virus E6/E7 genes (LXSN-16 E6E7) was used to immortalize stromal cells from human marrow. The E6/E7 gene products interfere with the function of tumor-suppressor proteins p53 and Rb, respectively, thereby preventing cell cycle arrest without causing significant transformation. Twenty-seven immortalized clones designated HS-1 to HS-27 were isolated, four of which are characterized in this report. Two cell lines, HS-5 and HS-21, appear to be fibroblastoid and secrete significant levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF), Kit ligand (KL), macrophage-inhibitory protein-1 alpha, interleukin-6 (IL-6), IL-8, and IL-11. However, only HS-5 supports proliferation of hematopoietic progenitor cells when cocultured in serum-deprived media with no exogenous factors. Conditioned media (CM) from HS-5 promotes growth of myeloid colonies to significantly greater extent than a cocktail of recombinant factors containing 10 ng/mL of IL-1, IL-3, IL-6, G-CSF, GM-CSF, and KL and 3 U of erythropoietin (Epo). Two additional clones, HS-23 and HS-27, resemble "blanket" cells, with an epithelioid morphology, and are much larger, broader, and flatter when compared with HS-5 and HS-21. These lines secrete low levels of growth factors and do not support proliferation of isolated progenitor cells in cocultures. CM from HS-23 and HS-27 also fail to support growth of myeloid colonies. Both HS-23 and HS-27 express relatively high levels of VCAM-1, yet HS-27 is the only line that supports the formation of "cobblestone" areas by isolated CD34+38lo cells. We hypothesize that HS-5, HS-21, HS-23, and HS-27 represent functionally distinct components of the marrow microenvironment.