FGFR3 as a marker for mesenchymal skeletal progenitor cells

Details FGFR3 as a marker for mesenchymal skeletal progenitor cells FGFR3 as a marker for mesenchymal skeletal progenitor cells

1998-06-12

2003-02-11

Caputa, Anthony C. (Department: 1642)

Drug, bio-affecting and body treating compositions

Extract, body fluid, or cellular material of undetermined...

Derived from musculoskeletal system, other than cardiac muscle

C424S093700, C530S300000

Reexamination Certificate

active

06517872

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns a method for identifying mesenchymal skeletal progenitor cells by identification of cells which feature on their surface fibroblast growth factor receptor 3 (FGFR3).
The present invention further concerns a method for obtaining mesenchymal skeletal progenitor cells by utilizing FGFR3-binding agents. The invention still further concerns a substantially pure culture of mesenchymal skeletal progenitor cells as well as pharmaceutical compositions and implants comprising said mesenchymal skeletal progenitor cells.
By another aspect the invention concerns a method for identification of cartilage-bony tumor and pharmaceutical compositions for the treatment of cartilage-bony tumor.
BACKGROUND OF THE INVENTION
Skeletal growth depends both on proper function of the tissue cellular elements—the chondrocytes, and their cell membrane receptors in the cartilaginous growth centers of the long bones, as well as on the normalcy and levels of circulating and local hormones and growth factors. Growth disorders are therefore classified into two distinct categories (a) failures in a circulating factor, and (b) failures in the target cartilaginous tissue.
The course of normal differentiation begins with mesenchymal stem cells which differentiate to skeletal progenitor cells which can either differentiate to precartilaginous stem cells, which eventually form the cartilage, or to preosteogenic stem cells which eventually form the bone.
In attempts to trace the mesenchymal stem cells supporting growth and their routes of migration in normals and in the family of growth disorders, there are difficulties, including the lack of proper markers for these specific mesenchymal stem cells. For example, spotty and incomplete information is available regarding the original location and the routes of migration of the growth plate stem cells, supporting the longitudinal and the transverse growth. A long lasting dispute of over a hundred years, which may be called “Ranvier versus La Cro2ix” is still perpetuating. In 1889 Ranvier stated “Cells forming the periosteal bone, originate from the cells of the growth plate”, while in 1951 La Croix declared “Appositional growth occurs from cells of the peri-chondral periphery”. Ranvier's theory gained support at the early seventies from Rigal, Hert, J. (Acta Anat (Bazel) 82:420-436 (1972)) and others, and in the nineties by Langenskiold et al. (Acta. Orthop. Scand., 64:683-687 (1993)), suggesting that cells from the germinal layer migrate to the borderline of the bone groove, serving as the source for both longitudinal and transverse bone growth.
A full understanding of the various types of cartilage cells and the factors that effect mesenchymal differentiation, however, has been hampered due to failure to locate the original location of the primary reservoir of these cells and thus the limitations of in vitro cell culture. One difficulty has been the lack of specific phenotypic markers to follow successive differentiation events. Type II collagen secretion is considered a major early marker of chondrocyte differentiation, while the synthesis of alkaline phosphatase is an early marker of osteoblast differentiation. Mature osteoblasts also produce osteopontin, osteonectin, and osteocalcin, three extracellular matrix proteins deposited together with type I collagen into mineralized bone matrix. Unfortunately, only a few differentiation markers have been identified, and several of these, such as alkaline phosphatase, osteopontin, and osteonectin, are not specific for osteogenic differentiation, while others, such as osteocalcin, are rarely expressed in vitro. In addition, mesenchymal cell lines and primary cultures of differentiating chondrocytes and osteoblasts display a variable phenotype and are often a mixture of cell types at different stages of differentiation (Eriebacher, A. et al,
Cell
80:371-378, (1995); Yamaguchi, T. P. and Rossant, J.,
Current Opinion in Genetics and Developmnent
5:485-491 (1995)).
Thus it would have been highly desirable to develop a marker capable of locating precisely the site and source of stem cells supporting and contributing to both longitudinal and transverse growth and for bone repair both for better understanding of the mechanism of mesenchymal development in normal and pathological conditions, as well as for the purpose of obtaining a substantially pure culture of mesenchymal skeletal progenitor cells for therapeutical purposes.
SUMMARY OF THE INVENTION
The present invention is based on the surprising finding that fibroblast growth factor receptor 3 (FGFR3) serves as a marker for mesenchymal skeletal progenitor cells. The present invention is further based on the surprising finding that the anatomical location of mesenchymal skeletal progenitor cells is in the perichondrium in the La Croix groove.
The term “niesenchiymal skeletal progenitor cells” will be used in the following to denote the following types of cells: (a) mesenchymal stem cells which are able to differentiate to skeletal progenitor cells, (b) skeletal progenitor cells, (c) precartilaginous stem cells, and (d) preosteogenic stem cells or a combination of two or more of the above cell types. The mesenchymal skeletal progenitor cells all share the property of contribution to the growth of bone and/or cartilage, show enhanced proliferation properties as compared to other types of cartilage and bone derived cells and also a tendency to migrate in the presence of suitable chemotactic agents such as fibroblast growth factor 9.
These mesenchymal skeletal progenitor cells, in early stages of embryonal and neonatal life, support the growth of both articular and physis growth-plate cartilages. However, quite early in life, a few months post-birth, the connection of these stem cells to the articular zone is abolished leading to the poor self-wound healing of articular cartilage. Such mesenchymal skeletal progenitor cells continue to maintain the cell source for the longitudinal and latitudinal (transverse) growth of long bones, until the closure of the physis (at the age of 18-22 years), and continue to provide the stem cell reservoir of the periosteum, involved in the callous of bone fractures all through life. In adult life, especially at advanced ages, a technique for tracing undifferentiated cell source with a potential to establish proliferating chondrocytes has previously failed due to the scarcity of such a cell source and the inadequatability of markers for such undifferentiated cells.
By using the discovery on which the present invention is based, namely that FGFR3 is a marker for mesenchymal skeletal progenitor cells, it was possible to develop a method for identification of mesenchymal skeletal progenitor cells by identifying those cells which feature FGFR3 on their surface. Such a method may be important for tracing mesenchymal skeletal progenitor cells for example for better understanding of pathological conditions of growth arrest involving FGFR3 receptors for example those leading to genetic dwarfism-achondroplasia or persistent expression in multiple hereditary exostosis and reexpression in primary osteoarthritic osteophytes.
Thus the present invention provides a method for identifying mesenchymal skeletal progenitor cells comprising:
(a) applying a fibroblast growth factor receptor 3 (FGFR3) binding agent to assayed cells or tissue under conditions allowing ligand-receptor binding;
(b) determining which cells bound said FGFR3 binding agent, said cells being mesenchymal skeletal progenitor cells.
The FGFR3 binding agent which may be an antibody or fibroblast growth factor 9 (FGF9) should be labeled and applied to the assayed tissue, for example to tissue of the joint. Those regions which are labeled serve as a source for mesenchymal skeletal progenitor cells.
Preferably, the source for the mesenchymal skeletal progenitor cells is the perichondrium at the region of La Croix, and the region which meets the synovial membrane and the periosteum.
The method of the present invention may be used to identify and l

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