Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2000-08-07
2003-06-10
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S366000, C435S325000
Reexamination Certificate
active
06576465
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of bone cell development. In particular, it concerns the identification of a class of accessory cells that are useful in stimulating bone cells and their precursors.
2. Description of Related Art
The bone:bone marrow interface represents a relatively unexplored area of the bone marrow microenvironment in which both hematopoietic and osteopoietic cells exist in close opposition to each other. Such physical proximity suggests that these two cellular lineages interact (i.e., regulate) one another. However, little information exists concerning the intimate relationship between bone and bone marrow.
The precise role of accessory cells in bone formation is poorly understood. Accessory cells, however, are an important source of growth regulators required for the controlled differentiation and proliferation of progenitor cells in other systems (Kaushansky and Lin, 1988; Fibbe et al., 1988; Lee el al., 1987). In addition, these cells also provide complex, functional extracellular matrix components that stimulate bone cell development. Accessory cells are a heterogeneous cellular composition consisting of macrophages, fibroblasts, adipocytes, endothelial cells (Dexter, 1982; Allen and Dexter, 1984; Dexter et al., 1977), and other poorly defined cells. Such diversity makes it extremely difficult to analyze the role of each cell type in bone cell development.
Established accessory cell lines provide a useful tool for the analysis of discrete stromal functions. While a number of spontaneously immortalized murine cell lines have been described (Hunt et al., 1987; Quesenberry et al., 1987; Collins and Dorshkind, 1987) attempts to establish corresponding human lines have failed (Lanotte et al., 1981). Human bone marrow cell lines are also described in Thalmeier et al. (1992).
Some of the problems associated with the establishment of human stromal cell lines have been solved by introducing SV40 encoding DNA into the cellular genome (Harigaya and Handa, 1985; Novotny et al., 1990; Singer et al., 1987; Aizawa et al., 1991; Ciuttini et al., 1992). These stromal cell lines have been used as model systems for analyzing stromal cell-progenitor cell interactions (Yang et al., 1988; Kohama et al., 1988; Nemunaitis et al., 1989; Slack et al., 1990). Nevertheless, the use of SV40 immortalized stromal cell lines as supportive feeder layers in LTBMCs still has two important drawbacks. First, SV40-immortalized cells grow very rapidly (Neufeld et al., 1987) and then enter a characteristic crisis leading to the death of the cells (Singer et al., 1987). Second, SV40-immortalized stromal cells are readily detached from culture flasks upon inhibition by irradiation or mitomycin C (Ciuttini et al., 1992).
Further, understanding the complex regulatory signals required to coordinate cellular development necessitates an understanding of progenitor cell responses to cytokines, to the surrounding extracellular matrix molecules, and to cell:cell interactions (Long, 1992; Long et al., 1992). Pragmatically, this requires that investigations into the control of a given cellular lineage utilize purified precursor cell populations, serum-free culture conditions, and purified (e.g., recombinant) growth factors.
The isolation and purification of human bone precursor cells utilizing immunology-based technology also has been extensively studied (Long et al., 1995; Long et al., 1992). These bone precursor cells are characterized as small-sized cells that express low amounts of bone proteins (osteocalcin, osteonectin, and alkaline phosphatase) and have a low degree of internal complexity (i.e., are immature). When stimulated to differentiate, these preosteoblast-like cells become osteoblast-like in their appearance, size, antigenic expression, internal structure, and mineralize extracellular matrix (Long el al., 1995; Long et al., 1990).
Recently, another antigenic determinant, STRO-1, was identified as a marker of human osteogenic cells (Gronthos et al., 1994). STRO-1 identifies clonogenic bone marrow stromal progenitor cells (CFU-F) in adult bone marrow (Simmons and Torok-Storb, 1991). When placed in mineralizing conditions, a portion of STRO-1-positive CFU-F becomes alkaline phosphatase positive, responds to 1,25-dihydroxy vitamin D
3
(1,25-OH D
3
) with increased osteocalcin production, and within four weeks undergoes mineralization (Gronthos et al., 1994). Thus, some STRO-1-positive bone marrow cells are clearly osteogenic in nature. Also, Riggs and colleagues have utilized negative immune selection to isolate a bipotential (i.e., osteoblast/adipocyte) precursor cell from human bone marrow (Rickard el al., 1996). All of the above investigations demonstrate that the use of partially purified bone cell populations allows the determination of phenotypic and functional data regarding the developmental characteristics of bone cells. Clearly, there are many studies that have attempted to isolate and purify populations of bone cells; however, to date, such studies have not addressed the necessity of microenvironmental accessory/stromal cells in regulating bone cell proliferation and/or differentiation.
Thus, there is a need to identify bone accessory cells that can proliferate at a high rate and can be used as accessory cells to support development of bone cells.
SUMMARY OF THE INVENTION
Therefore, the present invention provides compositions and methods relating to the stimulation of bone cells in vitro and in vivo.
The invention provides a method for isolating bone osteogenic accessory cells comprising the steps of (a) providing a starting cell population; (b) subjecting said population to density isolation to obtain a low density cell fraction; (c) subjecting said low density bone cell fraction to immune adherence based on TGF&bgr;II receptor expression; and (d) subjecting said immune adherent cells to positive selection based on cellular complexity. Also included in this method is plastic adherence and immunoaffinity purification. The immunoaffinity purification comprises selecting against osteonectin or osteocalcin expression or both. In addition, selection against expression of P-selectin, L-selectin, E-selectin, CD3, CD56, CD34, CD68 and vWF also is contemplated. The starting cell population may be any of a variety of cells but, in one embodiment, is bone marrow stromal cells.
Additional embodiments of the preceding method include culturing before or after any of steps (b)-(d), culturing said cells with an osteogenic cytokine, such as a member of the TGF&bgr; super family of cytokines, e.g., TGF&bgr;. The cells additionally may be characterized as between about 10 and about 70 &mgr;m in diameter.
In another embodiment, there is provided, a method of producing an osteogenic stimulatory factor comprising the steps of (a) culturing a cell population having the following characteristics: (i) having a buoyant density of between about 1.050 and about 1.090 g/cm
3
; (ii) absence of plastic adherence; (iii) presence of TGF&bgr;II receptor expression; (b) stimulating with TGF&bgr;, and (c) collecting the culture medium. The cells further may be characterized as being low complexity (SSC
lo
) cells.
In yet another embodiment, there is provided an isolated cell population having the following characteristics: (a) having a buoyant density of between about 1.050 and about 1.090 g/cm
3
; (b) absence of plastic adherence; and (c) presence of TGF&bgr;II receptor expression. The population also may be described as having the following characteristics: (a) presence of TGF&bgr;II receptor expression; (b) absence of expression of P-selectin, L-selectin, E-selectin, CD3, CD56, CD68, CD34 and vWF; and (c) low complexity as measured by flow cytometry.
In still yet another embodiment, there is provided a method for stimulating bone cell differentiation and/or maturation comprising the step of co-culturing a bone cell with an isolated cell population having the following characteristics: (a) having a buoyant density of between about 1.050 an
Afremova Vera
Fulbright & Jaworski L.L.P.
Saucier Sandra E.
The Regents of the University of Michigan
LandOfFree
Human bone accessory cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Human bone accessory cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Human bone accessory cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3151515