Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1998-07-01
2001-07-17
Clark, Deborah J. R. (Department: 1633)
Drug, bio-affecting and body treating compositions
Lymphokine
C514S002600, C514S012200, C930S140000
Reexamination Certificate
active
06261549
ABSTRACT:
BACKGROUND OF THE INVENTION
Mesenchymal stem cells (MSCs) are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into more than one specific type of mesenchymal or connective tissues (i.e. the tissues of the body that support the specialized elements; e.g. adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines. Human mesenchymal stem cells (hMSCs) are reactive with certain monoclonal antibodies, known as SH2, SH3 and SH4. (See U.S. Pat. No. 5,486,359).
Hematopoietic stem cells (HSCs) are the formative pluripotential blast cells found inter alia in bone marrow and peripheral blood that are capable of differentiating into any of the specific types of hematopoietic or blood cells, such as erythrocytes, lymphocytes, macrophages and megakaryocytes. After mobilization of HSCs from bone marrow by administration of certain factors such as G-CSF and GM-CSF and subsequent recovery from peripheral blood, HSCs have also come to be referred to as peripheral blood progenitor cells (PBPCs). Human hematopoietic stem cells (hHSCs) and PBPCs are reactive with certain monoclonal antibodies which are now recognized as being specific for hematopoietic cells, for example, CD34.
Thus, hMSCs and hHSCs are readily distinguishable by their immunospecific profiles and, for the sake of clarity herein, will be referred to, for example, herein as SH2
+
-CD14
−
hMSCs or SH2
−
-CD14
+
hHSCs as needed.
Human hematopoietic stem cell (hHSC), or peripheral blood progenitor cell (PBPC), transplantation has become an accepted method for dose-intensification in the treatment of several neoplastic diseases.
1,2
Various procedures have been used for HHSC mobilization and removal from the circulation by apheresis. At present, most methods exploit the rebound in circulating progenitors that occurs after cytotoxic chemotherapy.
3
Together, the short-term administration of either GM-CSF or G-CSF, enhances the yield of hHSC, as measured by the number of CD34
+
cells
4,5,6
which, when used for autografting at ≧2.5×10
6
CD34
+
hHSC/kg recipient, ensures fast hematopoietic recovery.
4,5,6
Several mechanisms seem to be involved in the growth factor-mediated release of marrow progenitor cells.
7
Among them, it appears that after exposure to G-CSF or GM-CSF, adhesion molecules are shed from the surface of marrow resident primitive multilineage cells, therefore allowing them to enter the circulation
5
. This concept, has been strengthened by several observations showing that growth factors (like G-CSF, GM-CSF, IL-3 and SCF) modulate the expression or function of several cytoadhesive molecules on the surface of hematopoietic progenitor cells.
8,9
In addition, reports cite that cytokines produce profound morphological and immunohistochemical changes in marrow stroma and in the contiguous extracellular matrix.
10,11
SUMMARY OF THE INVENTION
Accordingly, the invention provides a method for obtaining human mesenchymal stem cells wherein the human mesenchymal stem cells are recovered from peripheral blood obtained from an individual. More particularly, the invention provides a method for recovering peripheral blood containing a population of cells enhanced in human mesenchymal stem cells from an individual which method comprises (i) administering to said individual at least one growth factor and, thereafter, (ii) recovering hMSCs from the peripheral blood from said individual. Growth factors which can be used are those, for example, which are known to mobilize hematopoietic stem cells. The growth factors preferably include G-CSF, GM-CSF and combinations thereof.
The invention further provides a method for recovering an isolated, culture-expanded population of human mesenchymal stem cells from the mesenchymal stem cell-enriched peripheral blood of an individual by (i) administering to said individual at least one growth factor; (ii) recovering mesenchymal stem cell-enriched peripheral blood from said individual; (iii) culturing the mesenchymal stem cell-enriched peripheral blood or a fraction thereof; and (iv) isolating a culture-expanded population of human mesenchymal stem cells from other cells in said mesenchymal stem cell-enriched peripheral blood. The steps of culturing and isolating can also be in the inverse order. That is, the mesenchymal stem cells can be isolated from the peripheral blood and then be culture-expanded. Approaches to such isolation include leucopheresis, density gradient fractionation, immunoselection and differential adhesion separation. The culture media can be chemically defined serum free media or can be a “complete medium”, such as DMEM or DMEM-1 g containing serum. Suitable chemically defined serum free media are described in U.S. Ser. No. 08/464,599, filed Jun. 5, 1995, and “complete media” are described in U.S. Pat. No. 5,486,359, issued Jan. 23, 1996.
The invention further provides a method for preserving ex vivo an isolated, culture-expanded population of human mesenchymal stem cells from the mesenchymal stem cell-enriched peripheral blood of an individual by (i) administering to said individual at least one growth factor; (ii) recovering mesenchymal stem cell-enriched peripheral blood from said individual; (iii) culturing the mesenchymal stem cell-enriched peripheral blood; (iv) isolating a culture-expanded population of human mesenchymal stem cells from other cells in said mesenchymal stem cell-enriched peripheral blood; and (v) preserving the isolated, culture-expanded population of human mesenchymal stem cells. Preferably, the preservation is by cryopreservation.
The invention further provides a method for treating an individual in need of treatment with an isolated, culture-expanded population of human mesenchymal stem cells by (i) administering to said individual at least one growth factor; (ii) recovering mesenchymal stem cell-enriched peripheral blood from said individual; (iii) culturing the mesenchymal stem cell-enriched peripheral blood; (iv) isolating a culture-expanded population of human mesenchymal stem cells from other cells in said mesenchymal stem cell-enriched peripheral blood; and (v) administering said isolated, culture-expanded population of human mesenchymal stem cells to said individual. The cells can be administered by, for example, systemic infusion or local implantation into a site where de novo tissue generation is desired, such as by an open or arthroscopic procedure. The cells can be preserved prior to readministration.
REFERENCES:
patent: 5199942 (1993-04-01), Gillis
patent: 5486359 (1996-01-01), Caplan et al.
patent: 5612211 (1997-03-01), Wilson et al.
Paul, in “Fundamental Immunology”, Third Edition, Raven Press, New York, pp. 773, 774, and 788, 1993.*
Roitt et al., in Immunology, Third Edition, Mosby-Year Book Eurpoe Limited, pp. 8.14-8.15, 1993.*
Science, 284:143-147, 1999.*
Bensinger et al. Blood, 85:1655-1658, 1995.*
Fernandez et al. Bone Marrow Transplantation, 20:265-271, 1997.*
Brandt, J., et al., “Cytokine-dependent Long-Term Culture of Highly Enriched Precursors of Hematopoietic Progenitor Cells from Human Bone Marrow,”J. Clin. Invest.,86:932-941 (1990).
Chao, N.J., et al., “Granulocyte Colony-Stimulating Factor ‘Mobilized’ Peripheral Blood Progenitor Cells Accelerate Granulocyte and Platelet Recovery After High-Dose Chemotherapy,”Blood,81(8):2031-2035 (1993).
Dedhar, S., et al., “Human granulocyte-macrophage colony-stimulation factor active on a variety of cell types of nonhemopoietic origin,”Proc. Natl. Acad. Sci. USA,85:9253-9257 (1988).
Fernández, M. and Minguell, J.J., “Adhesive Interactions in the Hematopoietic System: Regulation by Cytokines,”Proc. Soc. Exp. Biol. Med.,313-323 (1996).
Goldman, J., “Peripheral Blood Stem Cells for Allografting,”Blood,85(6):1413-1415 (1995).
Gronthos, S., et al., “The STRO-1+ Fraction of Adult Human Bone Marrow Contains the Osteogenic Precursors,”Blood,84(12):4164-4173 (199
Fernández Mireya
Minguell José J.
Clark Deborah J. R.
Kerr Janet M
Lillie Raymond J.
Olstein Elliot M.
Osiris Therapeutics, Inc.
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