Factors required for bone marrow stromal fibroblast colony formation in vitro.

Marrow stromal fibroblasts (MSFs) are essential for the formation of the haemopoietic microenvironment and bone; however, regulation of MSF proliferation is poorly understood. MSF colony formation was studied in primary mouse and human marrow cell cultures. After a brief exposure to serum, MSF colony formation occurred in the absence of both serum and non-adherent marrow cells, if medium conditioned by marrow cells was present (serum-free conditioned medium, SF-CM). In mouse and human cultures stimulated to proliferate by SF-CM, neutralizing antibodies against PDGF, TGF-beta, bFGF and EGF specifically suppressed MSF colony formation. The degree of supression was species-dependent, with the most profound inhibition achieved in mouse cultures by anti-PDGF, anti-bFGF and anti-EGF, and in human cultures by anti-PDGF and anti-TGF-beta. Serum-free medium not conditioned by marrow cells (SFM) did not support MSF colony formation. In mouse cultures in SFM, human recombinant bFGF and bovine natural bFGF were able to partially substitute for the stimulating effect of SF-CM. Other growth factors, including TGF-beta1, TGF-beta2, PDGF, EGF, IL-6, IGF-I and IGF-II, showed no activity when tested alone. In human cultures in SFM, none of the growth factors, alone or in combination, stimulated MSF colony formation. Mouse and human MSFs grown in SF-CM formed bone and a haemopoietic microenvironment when transplantated into immunodeficient mice in vivo, and therefore were functionally equivalent to MSFs generated in the presence of serum. These data indicate that stimulation of the initial proliferation of an MSF precursor cell is complex, and requires participation of at least four growth factors: PDGF, bFGF, TGF-beta and EGF. In addition, mouse and human MSF precursor cells have different requirements for each of the growth factors.

Structure and molecular regulation of bone matrix proteins.

The organic matrix of bone contains several protein families, including collagens, proteoglycans, and glycoproteins, all of which may be extensively modified by posttranslational events, such as phosphorylation and sulfation. Many of the glycoproteins contain Arg-Gly-Asp (RGD), the integrin-binding sequence, within their structure, whereas other constituent proteins contain gamma-carboxyglutamic acid. The deposition of bone matrix by cells in the osteoblastic lineage is regulated by extrinsic factors, such as systemic and local growth factors and physical forces, and factors that are intrinsic to the cell, such as position in the cell cycle, maturational stage, and developmental age of the donor. Recent studies of several bone matrix gene promoters have identified cis- and trans-acting elements that are responsible for gene activity, although the precise sequence of regulatory events is not known. Development of in vitro assays, coupled with studies of the appearance of these proteins during development in vivo, provides insight into the functions of these proteins during the various stages of bone metabolism. Potential roles for these proteins include proliferation and maturation of stem cells, formation of matrix scaffolding elaborated by bone-forming cells, modeling, and remodeling. Changes in the functional properties of the extracellular matrix may be involved in a variety of disease processes, including osteoporosis and oral bone loss.

Bone marrow stromal colony formation requires stimulation by haemopoietic cells.

In mouse bone marrow cultures plated at low cell density, stromal colonies formed from colony-forming unit fibroblastic (CFUf) failed to develop unless the cultures were supplemented with irradiated feeder cells. Colony-stimulating activity was produced by irradiated bone marrow and spleen cells and by platelets, was dose dependent, not species specific and was maximal at high serum concentration. The efficiency of CFUf colony formation was 1.7 x 10(-4) for mechanically disaggregated and 14.6 x 10(-4) for trypsinised bone marrow cells. The colonies formed in the presence of feeder cells comprised hundreds of fibroblasts. In the absence of feeder cells, small fibroblast foci and single fibroblasts only were present in cultures. PDGF, IL-3 and EGF did not substitute for the colony-stimulating activity of feeder cells. These results suggest that CFUf colony formation requires growth factor(s) released by platelets and megakaryocytes which remain to be identified.

Stromal stem cells: marrow-derived osteogenic precursors.

Evidence is discussed for the hypothesis that there are stromal stem cells present in the soft connective tissues associated with marrow and bone surfaces that are able to give rise to a number of different cell lines including the osteogenic line. Fibroblastic colonies, each derived from a single colony-forming unit fibroblastic (CFU-F), are formed when marrow cells are cultured in vitro. In vivo assays of CFU-F have demonstrated that some CFU-F have a high ability for self renewal and multipotentiality whereas some have more limited potential. In vitro studies also support the hypothesis and have shown that CFU-F are a heterogeneous population of stem and progenitor cells and that their differentiation in vitro can be modified at the colony level. Factors added to the medium can activate osteogenesis in a range of multipotential and more committed precursors. Different stromal cell lines can be promoted under different culture conditions. The number and hierarchy of cell lines belonging to the stromal fibroblastic system are not yet fully elucidated and more specific markers for the different lines are required before a better understanding can be achieved.

Bone formation in organ cultures of bone marrow.

Bone formation in organ cultures of intact marrow fragments from mouse is described. Marrow explants were cultured on the top surface of a millipore filter at a gas-liquid interface. Observations with both light- and electron microscopes demonstrated the formation of a well-organised trabecular matrix lined with osteoblast-like cells. The tissue and cells were positive for alkaline-phosphatase activity. Large amounts of thick, well-banded collagen fibrils and matrix vesicles typical of those found in bone were present. The tissue became mineralised in the presence of 10 mM Na-beta-glycerophosphate; in its absence a similar trabecular matrix developed but mineralisation did not take place.

Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers.

Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies. When transplanted in diffusion chambers after 20-30 cell doublings in vitro, the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation. The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC. Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells.

Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges.

Cells for study were obtained from mouse bone marrow by three methods: 1) Mechanical dissociation; 2) Trypsin release; and 3) Passage through a syringe and needle. The ratio of the fibroblast colony-forming cells in these suspensions was, respectively, 1:13:1. Of the colonies formed by cells obtained by trypsin release, 70% were alkaline phosphatase positive fibroblasts. Freshly isolated marrow cells and cells harvested, by trypsin treatment, from cultures established from the differently obtained bone marrow cell suspensions, were absorbed into porous sponges for transplantation under the renal capsule of mice. Ossicles containing bone marrow formed after transplantation of either freshly isolated cells, or cultured cells obtained from marrow by trypsin treatment, but not after the transplantation of cells cultured from marrow obtained by mechanical dissociation. The size of the bone marrow "organs" that formed was determined by both the number of cells grafted and the size of the sponge in which they had been absorbed for grafting.

Radiosensitivity and postirradiation changes of bone marrow clonogenic stromal mechanocytes.

The in vitro colony assay was used to determine radiosensitivity and post-irradiation changes of guinea pig clonogenic bone marrow stromal fibroblasts (CFU-F). D0 of the CFU-F survival curve proved to be about 200 rad, while n is 1.4. Immediately after total-body exposure to 400 rad and to local exposure of tibias to 400 rad, the number of CFU-F diminishes in accordance with D0; it sharply increases after 6 hours and then decreases again. In the period between the eighth and twelfth day following local irradiation, the number of CFU-F doubled in the course of 30-35 hours but later the rate of recovery slowed down. Practically no CFU-F were traced during 3 months after local exposure to 2 krad.

Origin of bone marrow stromal mechanocytes in radiochimeras and heterotopic transplants.

Strain histocompatibility antigens (detected by indirect immunofluorescence reaction) and sex chromosomes were used as markers of donor and recipient cells in monolayer cultures of bone marrow from radiochimeras and from heterotopic transplants. In contrast to macrophages and hemopoietic cells all bone marrow fibroblasts precursors in radiochimeras were of recipient origin; in heterotopic transplants they were of donor origin. These data point to the decisive role of stromal mechanocytes, and not macrophages or hemopoietic cells, in creating the bone marrow hematopoietic microenvironment.

Fibroblast precursors in normal and irradiated mouse hematopoietic organs.

Using the in vitro colony assay, clonogenic fibroblast precursor cells (CFU-F) were detected in the bone marrow, spleen and thymus from adult mice. The survival curve for CFU-F of mouse bone marrow irradiated in vitro has a D0 of 220 r. Regeneration of bone marrow CFU-F after whole-body irradiation with 150 r is characterized by a marked secondary loss and post-irradiation lag and dip, lasting 6 days, followed by return to normal values by about the 25th day. This pattern of post-radiation recovery of CFU-F is similar to that of the CFU-s. In addition, during the first 6 hours following irradiation the number of CFU-F increased approximately twofold.