Biomedical subjects

S Gronthos

Publications and source records attributed to S Gronthos.

10 recordsLinked to original sources

PubMed1999

Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells.

Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO-1 antigen exclusively (STRO-1+/ALP-), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone-related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO-1-/ALP+ and STRO-1-/ALP- phenotypes appeared to represent fully differentiated osteoblasts, while the STRO-1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO-1/ALP NHBC subsets were also found to express the DNA-binding transcription factor CBFA-1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA-1 in each of the STRO-1/ALP subsets, with the exception of the proposed preosteoblastic STRO-1+/ALP- subpopulation. Furthermore, upon re-culture of the four different STRO-1/ALP subsets only the STRO-1+/ALP- subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO-1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.

Aged↗

PubMed1998

MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies.

Despite the importance of bone marrow stromal cells in hemopoiesis, the profile of surface molecule expression is relatively poorly understood. Mice were immunized with cultured human bone marrow stromal cells in order to raise monoclonal antibodies to novel cell surface molecules, which might be involved in interactions with hemopoietic cells. Three antibodies, WM85, CC9 and EB4 were produced, and were found to identify a 100-110 kDa antigen on bone marrow fibroblasts. Molecular cloning revealed the molecule to be MUC18 (CD146), a member of the immunoglobulin superfamily, previously described as a marker of metastatic melanoma. In addition to the expected expression on melanoma cell lines and endothelial cells, a number of human leukemic cell lines were found to express MUC18, including all six T leukemia lines tested, one of five B lineage lines and one of four myeloid lines. Analysis of bone marrow samples from patients revealed positivity in 20% of B lineage ALL (n = 20), one of three T-ALL, 15% of AML (n = 13) and 43% of various B lymphoproliferative disorders (n = 7). No apparent reactivity was observed with mononuclear cells from normal peripheral blood or bone marrow, including candidate hemopoietic stem cells characterized by their expression of the CD34 antigen. However, positive selection of bone marrow mononuclear cells labeled with MUC18 antibody revealed a rare subpopulation (<1%) containing more than 90% of the stromal precursors identified in fibroblast colony-forming assays. The structure and tissue distribution of MUC18 suggest a functional role in regulation of hemopoiesis.

Animals↗

PubMed1997

Integrin expression and function on human osteoblast-like cells.

The integrin family of cell adhesion molecules are a series of cell surface glycoproteins that recognize a range of cell surface and extracellular matrix (ECM)-associated ligands. To date, the precise role of individual integrin molecules in bone cell-ECM interactions remains unclear. Cell binding assays were performed to examine the ability of normal human bone cells (NHBCs) to adhere to different ECM proteins in vitro. NHBCs displayed preferential adhesion to fibronectin over collagen types I, IV, and vitronectin and showed low affinity binding to laminin and collagen type V. No binding was observed to collagen type III. The integrin heterodimers alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, and alpha v beta 5 were found to be constitutively expressed on the cell surface of NHBCs by flow cytometric analysis. The integrins alpha 4 beta 1 and alpha 6 beta 1 were not expressed by NHBCs. Subsequent binding studies showed that NHBC adhesion to collagen and laminin was mediated by multiple integrins where cell attachment was almost completely inhibited in the presence of a combination of function-blocking monoclonal antibodies (Mabs) to alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, and beta 1. In contrast, the adhesion of NHBCs to fibronectin was only partially inhibited (50%) in the presence of blocking Mabs to alpha 3 beta 1, alpha 5 beta 1, and beta 1. The attachment of NHBCs to collagen, laminin, fibronectin, and vitronectin was also found to be unaffected in the presence of a function-blocking Mab to alpha v beta 3. The results of this study indicate that beta 1 integrins appear to be the predominant adhesion receptor subfamily utilized by human osteoblast-like cells to adhere to collagen and laminin and in part to fibronectin.

Adult↗

PubMed1996

The biology and application of human bone marrow stromal cell precursors.

The importance of the stromal tissue of the bone marrow in regulating hemopoiesis is well documented. However, several features of marrow stromal cell biology remain poorly understood, in particular, the ontogeny and phylogeny of the various stromal elements that comprise the microenvironment of the bone marrow. In this article we review recent data concerning the immunophenotype and functional characteristics of precursor cells for marrow stromal tissue. The study of these stromal precursor cells (SPC) represents an exciting new field of research that will almost certainly expand in the future as we gain a greater understanding of the cellular and molecular events, environmental cues, and growth factors that physiologically regulate the commitment and subsequent development of SPC. Although the field of marrow SPC biology is in its infancy, we predict that future studies will result in several novel clinical applications for SPC. We, therefore, conclude this article by speculating on a number of these potential applications and, thus, view SPC and their progeny as likely vehicles for several novel and important cellular therapies, including gene therapy.

Antineoplastic Agents↗

PubMed1995

The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro.

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU-F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic-activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO-1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU-F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU-F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half-maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF-1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.

Adult↗

PubMed1994

The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors.

The monoclonal antibody STRO-1 identifies clonogenic bone marrow stromal cell progenitors (fibroblast colony-forming units [CFU-F]) in adult human bone marrow. These STRO-1+ CFU-F have previously been shown to give rise to cells with the phenotype of fibroblasts, adipocytes, and smooth muscle cells. In this study, the osteogenic potential of CFU-F derived from the STRO-1+ fraction of adult human bone marrow was determined. CFU-F were isolated from normal bone marrow aspirates by fluorescence activated cell sorting, based on their expression of the STRO-1 antigen. Osteogenic differentiation was assessed by the induction of alkaline phosphatase expression, by the formation of a mineralized matrix (hydroxyapatite), and by the production of the bone-specific protein osteocalcin. STRO-1+ cells were cultured in the presence of dexamethasone (DEX; 10(-8) mol/L), ascorbic acid 2-phosphate (ASC-2P; 100 mumol/L), and inorganic phosphate (PO4i; 2.9 mmol/L). After 2 weeks of culture, greater than 90% of the cells in each CFU-F colony stained positive for alkaline phosphatase using a monoclonal antibody specific for bone and liver alkaline phosphatase. Alkaline phosphatase activity was confirmed by histochemistry. A mineralized matrix developed in the CFU-F cultures, after 4 weeks of culture in the presence of DEX, ASC-2P, and PO4i. Mineralization was confirmed by both light and electron microscopy. The mineral was identified as hydroxyapatite by electron dispersive x-ray microanalysis and by x-ray diffraction analysis. In replicate cultures, osteocalcin release was shown after exposure of the cells to 1,25-dihydroxyvitamin D3 (10(-7) mol/L) both by radioimmunoassay and Northern blot analysis. This work provides direct evidence that adult human bone marrow-derived CFU-F are capable of differentiating into functional osteoblasts and that osteoprogenitors are present in the STRO-1+ population.

Adult↗

PubMed1994

Potential adhesion mechanisms for localisation of haemopoietic progenitors to bone marrow stroma.

Haemopoiesis occurs in intimate physical association with the stromal elements of the bone marrow. Current evidence supports the hypothesis that the restriction of primitive haemopoietic progenitor cells (HPC) to the bone marrow involves developmentally regulated adhesive interactions between HPC and the stromal cell microenvironment. This review examines the expression and function of cell adhesion molecules (CAM) on human HPC and marrow stromal cells. These data demonstrate that a broad range of CAMs representing at least three adhesion molecule superfamilies (integrins, selectins, immunoglobulin gene superfamily) participate in these adhesive interactions. We discuss the potential contribution of these various adhesion molecules to homing of HPC to the bone marrow, their retention within the extravascular haemopoietic compartment and their egress into the peripheral circulation. It is likely that each process is mediated not by a single binding event but requires the coordinated participation of multiple receptor-ligand pairs.

Bone Marrow Cells↗

PubMed1994

Isolation, characterization and functional activity of human marrow stromal progenitors in hemopoiesis.

In conclusion, monoclonal antibody STRO-1 has proven to be an extremely valuable reagent for the identification, isolation and functional characterization of human bone marrow stromal cell precursors. We were able to isolate CFU-F free of contaminating hemopoietic progenitors and are beginning to construct a detailed picture of their cell surface phenotype and have developed assays to examine the conditions required for their growth and differentiation. In the future, we hope to determine whether multipotential stromal stem cells exist in human bone marrow and to establish culture conditions that selectively promote self-renewal or development along any of a number of stromal cell lineages. Such cells would be an ideal target for gene therapy and may provide a means of treating disorders of the hemopoietic system with a suspected stromal etiology, such as aplastic anemia. We have recently succeeded in generating bone cells from STRO-1+ marrow cells (Gronthos et al, in preparation) and thus marrow stromal cell precursors may also find application in the treatment of other disorders such as those of the osteogenic system. For the present, these must remain tantalizing possibilities.

Adult↗

PubMed1992

A differential sensitivity to recombinant human interferon-alpha 2a between normal and chronic myeloid leukaemic peripheral blood granulocyte-macrophage colony-forming units.

The sensitivity to recombinant human interferon-alpha 2a (IFN) of peripheral blood granulocyte-macrophage colony-forming units (PB CFU-GM) from patients with chronic myeloid leukaemia (CML) was studied in a semi-solid clonogenic assay, and compared with normal PB CFU-GM. Like normal PB CFU-GM, the growth of CML PB CFU-GM in vitro was found to be dependent on the plating concentration used. The optimal CFU-GM growth occurred when CML PB mononuclear cells (MNC) were plated at low concentrations in the range of 0.01-0.1 x 10(5)/ml, compared to the range of 0.3-3.0 x 10(5)/ml optimal for CFU-GM growth in normal subjects. The optimal plating concentration for CML PB CFU-GM was similar to that observed in PB collected from patients with ovarian carcinoma during haematological recovery following chemotherapy-induced myelosuppression (recovery phase). The recovery phase PB was used as a source of non-leukaemic cells with a higher incidence of CFU-GM similar to that of CML. IFN produced a dose-related inhibition of CFU-GM growth in normal, recovery phase ovarian carcinoma and CML, PB MNC. The IFN concentration required to inhibit 50% of the CFU-GM in culture (LD50) was found to be significantly influenced by the plating concentration. When cells were cultured at 1.0 x 10(5) MNC/ml the mean LD50 for 7 CML patients was similar to that in normal (n = 5) or recovery phase (n = 5) peripheral blood, 273 i.u./ml, 1047 i.u./ml and 795 i.u./ml, respectively. In contrast when CML cells were cultured at 0.03 x 10(5) MNC/ml the concentration for optimal CML CFU-GM growth, the mean LD50 was significantly lower than that in normal PB and recovery phase PB, 4 i.u./ml, 251 i.u./ml and 78 i.u./ml, respectively (p less than 0.05). This is the first report of a differential sensitivity to IFN between CML and non-CML progenitors using an optimized PB CFU-GM assay system and proposes that further study of the in vitro culture of CML progenitors may increase our understanding of the clinical effects of IFN.

Cells, Cultured↗

PubMed1992

The detection of Philadelphia chromosome negative metaphases in long-term bone marrow cultures of the peripheral blood from patients with chronic myeloid leukemia predicts response to interferon-alpha 2a.

The cytogenetic response of 10 patients with chronic myeloid leukaemia (CML) to human recombinant interferon-alpha 2a (rhIFN alpha 2a) was compared to the Philadelphia chromosome (Ph) status of the pre-treatment peripheral blood cells after in vitro culture under long-term bone marrow culture (LTBMC) conditions. Pre-treatment light density peripheral blood cells were cultured in LTBMC on sex-mismatched irradiated allogeneic stromal layers with weekly cytogenic examination of metaphases in the non-adherent cell fraction. This was correlated with the patients' response to rhIFN alpha. Two groups of patients, five showing a cytogenetic response (responsive) and five who failed to achieve a cytogenetic response (nonresponsive) were studied. At the initiation of the LTBMCs the Ph' was found to be present in 100% of the cells analysed for nine patients and 97% for one patient. Pretreatment peripheral blood from four responsive patients demonstrated a decline in the proportion of Ph'-positive cells (Ph+) after 1 to 2 weeks in LTBMC. In contrast, peripheral blood from all the non-responsive subjects showed persistence of the Ph+ clone in 100% of the cells analysed out to a maximum of 3 to 5 weeks in LTBMC. A significant difference was observed (Fisher exact test, p = 0.023) between the two patient groups in respect to the appearance of normal clones in the nonadherent population. The presence of Ph- metaphases in LTBMC of peripheral blood cells of CML patients may predict their cytogenetic response to rhIFN alpha 2a.

Bone Marrow↗