Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Tissue – other than lymphoid tissue
Reexamination Certificate
1997-07-14
2001-03-13
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Tissue, other than lymphoid tissue
C424S093700, C424S093710
Reexamination Certificate
active
06200606
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention generally relates to the isolation of precursor cells and their use in bone and cartilage regeneration procedures and, more particularly, is directed to a method for isolating bone/cartilage precursor cells from a variety of body tissue types utilizing cell surface antigen CD34, other precursor cell surface antigens on CD34+ cells, and other positive and negative cell selection techniques.
Osteogenesis and chondrogenesis are highly complex biological processes having considerable medical and clinical relevance. For example, more than 1,400,000 bone grafting procedures are performed in the developed world annually. Most of these procedures are administered following joint replacement surgeries, or during trauma surgical reconstructions. The success or failure of bone grafting procedures depends largely on the vitality of the site of grafting, graft processing, and in the case of allografts, on immunological compatibility between donor and host. Compatibility issues can largely be negated as an important consideration in the case of autologous grafting procedures, which involve taking bone tissue from one site of the patient for transplantation at another site. While autologous bone grafts are generally successful they do require additional surgery in order to harvest the graft material, and not uncommonly are accompanied by post-operative pain, hemorrhage and infection.
Cartilage regeneration and replacement procedures are perhaps even more problematic. Unlike osteogenesis, chondrogenesis does not typically occur to repair damaged cartilage tissue. Attempts to repair damaged cartilage in any clinically meaningful fashion have met with only limited success. In many cases, the most effective treatment for cartilage damage is prosthetic joint replacement.
These and other difficulties with presently available bone-grafting and cartilage regeneration procedures have prompted intensive investigations into the cellular and molecular bases of osteogenesis and chondrogenesis. Some promising research to date has been in the identification and isolation of bone and cartilage precursor cells from marrow and other tissues.
Early investigations into the complexity of bone marrow demonstrated that lethally irradiated animals could be rescued by marrow transplants, suggesting that bone marrow contained a restorative factor having the capacity to regenerate the entire hematopoietic system. More recent experiments have shown that marrow also has the capacity to regenerate bone and other mesenchymal tissue types when implanted in vivo in diffusion chambers. (See e.g. A. Friedenstein et al. “Osteogenesis in transplants of bone marrow cells.” J. Embryol. Exp. Morph. 16, 381-390,1960; M. Owen. “The osteogenic potential of marrow.” UCLA Symp. on Mol. and Cell. Biol. 46, 247-255, 1987) Results of this nature have led to the conclusion that bone marrow contains one or more populations of pluripotent cells, known as stem cells, having the capacity to differentiate into a wide variety of different cell types of the mesenchymal, hematopoietic, and stromal lineages.
The process of biological differentiation, which underlies the diversity of cell types exhibited by bone marrow, is the general process by which specialized, committed cell types arise from less specialized, primitive cell types. Differentiation may conveniently be thought of as a series of steps along a pathway, in which each step is occupied by a particular cell type potentially having unique genetic and phenotypic characteristics. In the typical course of differentiation a pluripotent stem cell proceeds through one or more intermediate stage cellular divisions, ending ultimately in the appearance of one or more specialized cell types, such as T lymphocytes and osteocytes. The uncommitted cell types which precede the fully differentiated forms, and which may or may not be true stem cells, are defined as precursor cells.
Although the precise signals that trigger differentiation down a particular path are not fully understood, it is clear that a variety of chemotactic, cellular, and other environmental signals come into play. Within the mesenchymal lineage, for example, mesenchymal stem cells (MSC) cultured in vitro can be induced to differentiate into bone or cartilage in vivo and in vitro, depending upon the tissue environment or the culture medium into which the cells are placed. (See e.g. S Wakitani et al. “Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage” J. Bone and Joint Surg, 76-A, 579-592 (1994); J Goshima, V M Goldberg, and Al Caplan, “The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks” Clin. Orthop. 262, 298-311 (1991); H Nakahara et al. “In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells” Exper. Cell Res. 195, 492-503 (1991)).
Studies of this type have conclusively shown that MSC are a population of cells having the capacity to differentiate into a variety of different cell types including cartilage, bone, tendon, ligament, and other connective tissue types. Remarkably, all distinct mesenchymal tissue types apparently derive from a common progenitor stem cell, viz. MSC. The MSC itself is intimately linked to a trilogy of distinctly differentiating cell types, which include hematopoietic, mesenchymal, and stromal cell lineages. Hematopoietic stem cells (HSC) have the capacity for self-regeneration and for generating all blood cell lineages while stromal stem cells (SSC) have the capacity for self-renewal and for producing the hematopoietic microenvironment.
It is a tantalizing though controversial prospect whether the complex subpopulations of cell types present in marrow (i.e. hematopoietic, mesenchymal, and stromal) are themselves progeny from a common ancestor. The search for ancestral linkages has been challenging for experimentalists. Identifying relatedness among precursor and stem cell populations requires the identification of common cell surface markers, termed “differentiation antigens,” many of which appear in a transitory and developmentally-related fashion during the course of differentiation. One group, for example, has reported an ancestral connection among MSC, HSC, and SSC, though later issued a partial retraction (S. Huang & L. Terstappen. “Formation of hematopoietic microenvironment and hematopoietic stem cells from single human bone marrow stem cells” Nature, 360, 745-749, 1992; L. Terstappen & S. Huang. “Analysis of bone marrow stem cell” Blood Cells, 20, 45-63, 1994; E K Waller et al. “The common stem cell hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors” Blood, 85, 2422-2435, 1995). However, studies by another group have demonstrated that murine osteoblasts possess differentiation antigens of the Ly-6 family. That finding is significant in the present context because the Ly-6 antigens are also expressed by cells of the murine hematopoietic lineage. (M. C. Horowitz et al. “Expression and regulation of Ly-6 differentiation antigens by murine osteoblasts” Endocrinology, 135, 1032-1043, 1994). Thus, there may indeed be a close lineal relationship between mesenchymal and hematopoietic cell types which has its origin in a common progenitor. A final answer on this question must await further study.
One of the most useful differentiation antigens for following the course of differentiation in human hematopoietic systems is the cell surface antigen known as CD34. CD34 is expressed by about 1% to 5% of normal human adult marrow cells in a developmentally, stage-specific manner (CI Civin et al., “Antigenic analysis of hematopoiesis. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells” J.Immunol., 133, 157-165, 1984). CD34+ cells are a mixture of immature blastic cells and a small percentage of mature, lineage-committed cells of the myeloid, erythroid and lymphoid series. Perhaps 1% of CD34&
Nousek-Goebl Nancy
Peterson Dale R.
Barnes & Thornburg
DePuy Orthopaedics, Inc.
Martin Alice O.
Witz Jean C.
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