Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
1999-06-08
2001-07-03
Brusca, John S. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S347000, C435S455000, C435S373000, C424S093210
Reexamination Certificate
active
06255112
ABSTRACT:
The present invention relates to hematopoietic stem cells and more particularly to a process and composition for differentiating human hematopoietic stem cells into a committed osteoclast lineage in the absence of exogenous osteoclastogenesis-inducing factors. The present invention further relates to genetically modified hematopoietic stem cells in the presence of human mesenchymal stem cells, such that when the hematopoietic stem cells differentiate into osteoclasts, the osteoclasts are able to express the product of the transduced gene.
BACKGROUND OF THE INVENTION
Osteoclasts are terminally differentiated hematopoietic cells responsible for physiological bone resorption. Dysfunction in osteoclast differentiation and/or activity is the cause of a variety of human metabolic bone diseases including osteoporosis (for review see Teitelbaum et al., 1995). Osteoporosis, or progressive bone loss, is attributed to a shift in the critical balance of osteoclast and osteoblast activities responsible for proper skeletal integrity. Osteoblasts, which build bone, are one terminally differentiated lineage descending from mesenchymal stem cells.
Hematopoietic progenitors in bone marrow are driven into the osteoclast cell lineage under the influence of local regulatory factors. Diverse cell types including stromal cells, which arise from mesenchymal stem cells (MSCs), are present in bone marrow. Stromal cells have been shown to produce factors that regulate osteoclast physiology (for review see Mundy et al., 1995). Histological pictures of bone show close vicinity between marrow stromal cells and hematopoietic cells. Further, the relationship between the differentiated status of bone marrow stromal cells and their potential to regulate neighboring osteoclast progenitors is poorly documented. The importance of heterotypic cell-to-cell interactions mediated by cadherin-6 between stromal cells and osteoclast progenitors using a mouse model of osteoclastogenesis has been reported (Mbalaviele et al., 1998). Several studies demonstrated that osteoclast differentiation required the presence of stromal/osteoblast cells (Takahashi et al., 1988; Mbalaviele et al., 1995). Recent studies using cells derived from rodent marrow have demonstrated that osteoprotogerin ligand (OPGL) expressed on the membrane of stromal cells is a key factor of osteoclastogenesis (Lacey et al. 1998; Kong et al. 1999). However, it has also been reported that human osteoclast cells differentiated from progenitor cells in the presence of cocktails of cytokines and that stromal cells were not necessary (Matayoshi et al., 1996). The formation of human osteoclast cells (Ocl) displaying bone-resorbing activity has been reported (Takahashi et al., 1995; James et al., 1996; Sarma et al., 1996; Quinn et al., 1997), however, critical events which take place during osteoclastogenesis are unclear.
Bone is a composite matrix consisting of both inorganic and organic elements. The organic phase contains proteins, while the inorganic component contains calcium salts. It is known that during osteoclastic bone resorption, dissolution of the inorganic phase precedes that of the organic phase and that high concentrations of calcium (>40mM) are released locally from bone matrix. In turn, increased calcium concentrations have a negative feedback on osteoclast fliction, a regulatory mechanism to prevent excessive bone resorption. Calcium is known to regulate the expression of several genes. Recently, a calcium response element that enhances human keratin-l gene expression after exposure of keratinocytes to extracellular calcium (0.6 mM) has been characterized.
Osteoclasts are not easily isolated due to their low cell number in vivo, their fragility and tendency to adhere to other bone cells. Thus, there is a need for a method of efficiently producing quantities of osteoclasts to facilitate, for example, the design of effective therapeutics aimed at preventing abnormal osteolysis.
Accordingly, it is an object of the present invention to provide an efficient method of producing osteoclasts in vitro.
It is a further object of the invention to provide a method to produce and maintain osteoclasts in vitro without the addition of exogenous growth factors in numbers sufficient to provide an ongoing source of osteoclasts, for example, for administering osteoclasts to an individual in need thereof.
Accordingly, it is an object of the present invention to regulate hematopoietic stem cell differentiation towards osteoclast cells in the absence of exogenous growth factors, cytokines and hormones.
It is a still further object of the present invention to provide a method of producing osteoclasts from genetically modified hematopoietic cells such that when the hematopoietic cells differentiate into osteoclasts, the osteoclasts express the gene product of interest.
SUMMARY OF THE INVENTION
The present inventors have discovered that mesenchymal stem cells support osteoclast cell differentiation of hematopoietic stem cells.
Thus in one aspect of the present invention, there is provided a method for inducing the differentiation of hematopoietic progenitor cells in vitro into osteoclast cells, comprising co-culturing the hematopoietic cells with human mesenchymal stem cells.
When hematopoietic progenitor cells were co-cultured with mesenchymal stem cells, the hematopoietic progenitor cells differentiated towards osteoclast cells which were identified by the expression of markers characteristic of osteoclasts including multinuclearity, calcitonin and vitronectin receptors, and more importantly, bone resorbing activity.
It has further been discovered that the differentiation of the hematopoietic stem cells occurred in the absence of exogenous (added) growth factors, cytokines and hormones known to be required for inducing differentiation of hematopoietic stem cells.
Accordingly, one aspect of the present invention provides a method of producing osteoclasts in vitro comprising co-culturing hematopoietic stem cells with human mesenchymal stem cells. In a preferred embodiment, the cells are co-cultured in the same culture vessel such that physical cell-to-cell interactions occur between the hematopoietic progenitor cells and the mesenchymal stem cells.
It has also been discovered that when hematopoietic stem cells, which have been modified to carry exogenous genetic material of interest, are co-cultured with mesenchymal stem cells, the transduced hematopoietic stem cells differentiated into osteoclasts that also carried the new genetic material. These transduced osteoclast cells are able to express the exogenous gene product. Transduced osteoclast progenitors and the osteoclasts differentiated therefrom can be used for applications where treatment using such modified osteoclasts is beneficial, for example, in the alleviation of the effects of osteoporosis.
Accordingly, the present invention provides a method of obtaining genetically modified osteoclasts, comprising transducing hematopoietic progenitor cells with exogenous genetic material and placing the transduced hematopoietic cells under conditions suitable for differentiation of the hematopoietic stem cells into osteoclast cells which contain the exogenous genetic material.
In one embodiment, the method of producing osteoclasts comprises co-culturing transduced hematopoietic stem cells with mesenchymal stem cells such that after differentiation of the hematopoietic stem cells into osteoclasts, the osteoclasts also contain the exogenous genetic material.
The invention further relates to hematopoietic stem cells that are transduced with a polynucleotide that encodes a product that downregulates osteoclast differentiation and/or activity, and the use thereof.
In a particularly preferred embodiment, the expressed gene product downregulates osteoclast differentiation and/or activity. Hematopoietic stem cells are transduced with a retroviral vector containing a tandem of a calcium response element, tartrate resistant acid phosphatase (TRAP) promoter (specifically expressed in osteoclasts) and a nucleic acid sequence encodi
Mbalaviele Gabriel
Thiede Mark A.
Brusca John S.
Lillie Raymond J.
Olstein Elliot M.
Osiris Therapeutics, Inc.
Ousley Andrea
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