Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow

Abstract

In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.

Isolation and Characterization of Human Clonogenic Osteoblast Progenitors Immunoselected from Fetal Bone Marrow Stroma Using STRO-1 Monoclonal Antibody

“…Several purification strategies for the isolation of early osteoblast precursors from BM stroma have been described. (22,31,42,43) However, in these studies the cells were maintained in short-term cultures and no clonal cell lines were generated. In this report, we describe for the first time the isolation and establishment of immortalized clonogenic human osteoprogenitor cells immunoselected from fetal human BM stroma.…”

“…In the presence of WGA or other lectins, 80% of BM cells agglutinate and form clusters that sediment, and previous studies have shown that majority of stromal progenitors (WGA + ) reside in this agglutinated fraction, (23,33) whereas hematopoietic lineage cells and their progenitors have no lectin-binding sites on their cell surface. (22,35,36) Osteoprogenitors can be sorted from mouse BM based on their binding to WGA and Sca-1 expression. (31) Sca-1 antigen is coexpressed by cells of both stromal and hematopoietic lineage (CD34 + ) cells.…”

“…(8,(16)(17)(18)(19)(20) Functional characteristics of human BM cells also differ from those of other species. For example, dexamethasone (Dex) diminishes the osteogenic capabilities of mouse BM stromal precursors (21,22) and induces differentiation in human BM stromal cells. (16)(17)(18)(19)(20) Moreover, although Dex has no effect on the growth of rat marrow stromal cells, (23) it stimulates proliferation of human marrow stromal cells.…”

“…The initial step was based on biophysical properties of BM stromal cells, i.e., density, nonadherence, and lectin-binding. (22,30,31) The second stage was, by analogy with the purification of hematopoietic progenitors from BM, a positive selection procedure based on magnetic activated cell sorting (MACS) and utilizing a murine monoclonal antibody (MAb), STRO-1, (7) which recognizes all the clonogenic stromal precursors in human marrow, including all assayable osteoprogenitors. (7,32,33) With the aim of generating clonogenic STRO-1 + cells, the immunoselected cell population was immortalized with an origindefective mutant of the SV-40 large tumor antigen (T-Ag) (34) and cloned.…”

IL-1α, IL-1β, IL-6, and TNF-α Steady-State mRNA Levels Analyzed by Reverse Transcription-Competitive PCR in Bone Marrow of Gonadectomized Mice

“…In this experimental set-up, cells are obtained from the central bone marrow compartment, whereas cells at the bone surface do not detach and are not included. (29) Bone marrow cells were resuspended in 0.9 ml of 4 M guanidinium isothiocyanate lysis buffer and stored at Ϫ20°C until RNA isolation. Bone marrow RNA was isolated according to the method described by Chomczynski and Sacchi.…”