Jon N. Beresford

Vascular Pericytes Express Osteogenic Potential In Vitro and In Vivo

At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three‐dimensional nodule‐like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage‐specific expression of markers of primitive clonogenic marrow stromal cells (STRO‐1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.

Further Characterization of Cells Expressing STRO‐1 in Cultures of Adult Human Bone Marrow Stromal Cells

Primitive cells of the osteoblast lineage are not well characterized but are known to be present within the STRO‐1+ fraction of adult human bone and marrow. A survey of human osteosarcoma cell lines revealed that STRO‐1 is expressed by MG‐63 but not SaOS‐2. Among murine cell lines tested, expression of STRO‐1 was detected in the bipotential (adipocyte/osteoblast) line BMS‐2 but not the committed osteoblast precursor MC3T3‐E1. A proportion of cultured adult human bone marrow stromal cells (BMSCs) consistently expressed the STRO‐1 antigen. The expression of a range of cell surface antigens was studied in relation to STRO‐1 by flow cytometry and several, including the bone/liver/kidney isoform of alkaline phosphatase (ALP), were found to subtype the STRO‐1+ population of BMSCs. Further, BMSCs dual‐labeled with antibodies recognizing STRO‐1 and ALP could be assigned to one of four fractions: STRO‐1−/ALP−, STRO‐1+/ALP−, STRO‐1+/ALP+, and STRO‐1−/ALP+. Cells from each fraction could be isolated in high purity and, when recultured, remained viable and exhibited a limited degree of phenotypic stability. Using reverse transcriptase‐polymerase chain reaction, cells in the four fractions were found to express different levels of transcripts for the parathyroid hormone receptor (PTHr) and bone sialoprotein (BSP). The expression of transcripts for the nuclear transcription factor core‐binding factor alpha 1/osteoblast‐specific factor‐2 (CBFA1/OSF2) was restricted to those fractions expressing STRO‐1 and/or ALP. Treatment with 10 nM dexamethasone consistently increased the proportion of cells present in those fractions which expressed the highest levels of transcripts for PTHr and BSP (STRO‐1+/ALP+ and STRO‐1−/ALP+) while simultaneously decreasing the proportion present in the STRO‐1+/ALP− fraction. In conclusion, the expression of STRO‐1 in vitro remains a characteristic of less well differentiated cells of the osteoblast lineage; in cultures of BMSCs and in established human osteosarcoma cell lines, there is an inverse association between the expression of STRO‐1 and ALP; dual labeling of BMSCs with monoclonal antibodies recognizing STRO‐1 and ALP permits the identification and isolation of cells of the osteoblast lineage at different stages of differentiation.

Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1–4

Autologous marrow stromal cells have been proposed as an adjuvant in the treatment of bone defects and diseases. This will require the development of culture conditions that permit their rapid expansion ex vivo while retaining their potential for further differentiation. Fibroblast growth factor (FGF)-2 has been proposed as a candidate for the ex vivo expansion of cells with enhanced osteogenic potential, and we have explored this possibility further using cells obtained from a large cohort of adult human donors. Treatment with FGF-2 (0.001-2.5 ng/mL) had no detectable effect on colony formation, but markedly increased their proliferative potential and that of their immediate progeny, as shown by the increases in colony size and cell number. Based on the observed increase in the expression of the developmental markers STRO-1 and alkaline phosphatase (AP), a major target for the actions of FGF-2 appears to be the more primitive cells of the osteoblast lineage, and that, when added in combination with the synthetic glucocorticoid dexamethasone (Dx), it interacts positively to promote further cell maturation. The maintenance of adequate levels of ascorbate was shown to be a critical component in determining the nature of the effect of FGF-2 on AP expression. Variation in the response (predominantly in the magnitude and/or sensitivity) of the cultured cell populations to treatment with FGF-2 was apparent, but a preliminary analysis indicated that this was not due to differences in the age or gender of the donors used. The cultured cell populations were found to express multiple FGF receptors (FGFRs; 1-4) and the observed changes in the spectrum and abundance of FGFRs expressed in relation to that of STRO-1 and AP are consistent with their expression being developmentally regulated during the process of osteogenic differentiation. These results provide novel insights into the mechanism of action of FGF-2 on human cells of the osteoblast lineage and support the use of this factor, alone or in combination with Dx, for the rapid, ex vivo expansion of cell populations with enhanced osteogenic potential.

Osteoblasts and osteoclasts in adult human osteophyte tissue express the mRNAs for insulin-like growth factors I and II and the type 1 IGF receptor

Insulin-like growth factors (IGFs) are among the most abundant growth factors present in bone. In vitro, bone-derived cells both produce and respond to IGFs I and II, suggesting that these growth factors play an autocrine role in the regulation of bone turnover. In vivo, however, particularly in adult bone, their sites of expression have not been well documented. We have used, therefore, the technique of in situ hybridization to study the expression of the mRNAs for IGFs I and II and the type 1 IGF receptor in adult human osteophyte tissue. Throughout the developing osteophyte there was a strong association between osteogenesis and the expression of all three mRNA transcripts. The highest levels of expression were observed in active osteoblasts. Hybridization signals were weak or absent in flat cells lining quiescent surfaces and in cells of the bone marrow, including those that expressed alkaline phosphatase activity. Osteocytes and cells of the periosteum were negative. At sites of endochondral bone formation newly differentiated and hypertrophic chondrocytes expressed the mRNAs for IGFs and IGF receptor whereas cells of the perichondrium were negative. A striking finding of this investigation was that osteoclasts at sites of bone and calcified cartilage resorption expressed high levels of all three mRNA transcripts. These results support the hypothesis that locally produced IGFs are important regulators of bone formation. The differential expression of all three transcripts among cells of the osteoblast lineage suggests that IGFs may be involved in the maintenance of the mature osteoblast phenotype rather than in inducing the differentiation of marrow precursors or controlling the osteoblast-osteocyte transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and culture of bone-forming cells (osteoblasts) from human bone.

The most conspicuous function of the osteoblast is the formation of bone. During phases of active bone formation, osteoblasts synthesize bone matrix and prime it for subsequent mineralization. Active osteoblasts are plump, cuboidal cells rich in organelles involved in the synthesis and secretion of matrix proteins. Unlike fibroblasts, they are obviously polarized, secreting matrix onto the underlying bony substratum which consequently grows by apposition. Some osteoblasts are engulfed in matrix during bone formation and are entombed in lacunae. These cells are described as osteocytes and remain in the bone matrix in a state of low metabolic activity. At the completion of a phase of bone formation, those osteoblasts which avoided entombment in lacunae lose their prominent synthetic function and become inactive osteoblasts, otherwise known as bone-lining cells. In mature bone, lining cells cover most of the bone surfaces. Osteocytes and bone-lining cells should not be considered as inactive cells since they play a major role in the regulation of bone modeling and remodeling and in calcium homeostasis (1).

IGF-I does not affect the proliferation or early osteogenic differentiation of human marrow stromal cells

The ability of insulin-like growth factor-I (IGF-I) to regulate the proliferation and differentiation of primitive osteogenic precursors (CFU-F) has been investigated in cultures of bone marrow stromal cells (BMSC) derived from a large cohort of adult human donors. Treatment with IGF-I (0.1-20 ng/mL, days 0-28) had no consistent effect on the number or size of colonies that formed or the proportion of colonies that expressed the developmental marker alkaline phosphatase (AP). At the end of primary culture, similar numbers of cells were harvested from the control and IGF-I-treated groups and there was no detectable difference in the expression of AP (activity or percentage of positive cells) or the developmental marker STRO-1. This was found to be the case whether IGF-I was added alone or in combination with 10 nM dexamethasone (Dx), a known inducer of osteogenic differentiation in this cell culture system. In contrast, cells derived from the same cohort of donors responded to treatment with fibroblast growth factor-2 (FGF-2) with an increase in the number and size of the colonies that formed, in proliferation and in the number of cells recovered in STRO-1(+)/AP(+) (osteoprogenitor) fraction. Further analysis revealed that the majority of BMSC expressed the alpha and beta subunits of the type 1 receptor for IGF-I (IGF-IR), in the expected 1:1 ratio. Treatment with Dx did not affect the expression of these receptor subunits (percentage of positive cells or number of sites per cell) but did increase the proportion of cells present in the IGF-I(+)/AP(+) fraction. The results of this investigation suggest that the beneficial effects of IGF-I on the skeleton are not mediated primarily via an effect on osteoprogenitor fraction and are thus consistent with the hypothesis that the effects of IGF-I are differentiation dependent and restricted largely to the more mature cells of the osteoblast lineage.

Ultrasound mimics the effect of mechanical loading on bone formation in vivo on rat ulnae.

While the effect of ultrasound as an extreme example of low-magnitude high-frequency stimulation has been explored in the response of bone to injury, little is known about its effect on normal bone. This experiment was designed to test the hypothesis that ultrasound exerts a similar influence on bone as mechanical stimulation at a physiological level. Three groups of female Wistar rats were anaesthetised (6 per group). In one group, the left ulna was loaded cyclically in vivo 40 times, repeated on a further 5 occasions on alternate days. In a second group, transcutaneous low-intensity pulsed ultrasound stimulation was applied to the left ulnae for the same duration as the period of loading. In a third group, loading and ultrasound stimulation were applied concurrently. The right ulna served as non-loaded control in each animal. At the end of the experiment after 14 days, both ulnae were removed. Induced bone formation was assessed by measuring the proportion of medial periosteal bone surface with double label (dLS/BS, %) and by calculation of mineral apposition rate (MAR) from the inter-label distance. All three treatments induced a significant periosteal response, increasing dLS/BS values from <10% in control limbs to >80% in treated limbs. Increases in MAR of experimental ulnae versus contralateral control ulnae were 2.9 (+/-0.9), 8.6 (+/-2.4) and 8.7 microm (+/-3.2) for the ultrasound only, ultrasound and load, and load only groups, respectively. The effects of loading plus ultrasound were not significantly different from ultrasound alone. These data suggest that ultrasound is able to induce changes in bone that share at least some features with mechanical loading.

Variations in genome-wide gene expression in identical twins – a study of primary osteoblast-like culture from female twins discordant for osteoporosis

BackgroundMonozygotic twin pairs who are genetically identical would be potentially useful in gene expression study for specific traits as cases and controls, because there would be much less gene expression variation within pairs compared to two unrelated individuals. However the twin pair has to be discordant for the particular trait or phenotype excluding those resulting from known confounders. Such discordant monozygotic twin pairs are rare and very few studies have explored the potential usefulness of this approach.ResultsWe studied genome-wide gene expression in primary osteoblast-like culture from marrow aspirates obtained from three pairs of monozygotic twins. We used the latest Affymetrix microchip contains probe sets for more than 20,000 genes. Two pairs were discordant for bone mineral density at the hip by more than one standard deviation, and the third pair was unrelated concordant and used as control. Only 1.5% on average of genes showed variation in expression within pairs as compared to 5% between pairs or over 15% from the literature. Importantly we identified several groups of genes showing variations within the discordant pairs and not within the concordant pair such as chondroitin beta 1,4 N-acetylgalactosaminyltransferase, inhibin beta A, interleukin 1 beta and colony stimulating factor 1 macrophage. These genes are known to have potential roles in bone physiology relating to bone density, osteoporosis and osteoarthritis.ConclusionUsing the example of osteoblast-like cells in our monozygotic discordant twins for osteoporosis, we identified genes showing differential expression. Although without further experiment, we cannot confirm or conclude these are genes definitely related to bone physiology, we believe we have shown the potential and cost-effectiveness of further gene expression studies in discordant monozygotic twin pairs. A replication study for confirmation is essential.

The expression of mRNA for insulin-like growth factors and their receptor in giant cell tumors of human bone.

Insulin-like growth factors I and II are among the most abundant growth factors found in bone, and their expression also has been reported in a variety of neoplastic tissues. Using the technique of in situ hybridization, the authors have studied the expression of the messenger ribonucleic acid (mRNA) for insulin-like growth factors I and II and the Type 1 insulin-like growth factor receptor in giant cell tumors of human bone (n = 8). The expression of the mRNA for insulin-like growth factors I and II and the Type 1 insulin-like growth factor receptor was observed in giant cells and in the mononuclear stromal cell component but not in cells of the fibrous connective tissue at the growing edge of the tumor. The multinucleated cells expressed tartrate resistant acid phosphatase and showed reactivity with osteoclast selective monoclonal antibodies, confirming the close relationship between these giant cells and osteoclasts. Thus, these results are consistent with the possibility that locally produced insulin-like growth factors have an important regulatory role in the growth and development of giant cell tumors of bone. In addition, they suggest that these growth factors may function as autocrine or paracrine regulators of the proliferation and differentiation of cells of the osteoclast lineage.