Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of co-culturing cells
Reexamination Certificate
1999-05-21
2001-05-01
Hauda, Karen M. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of co-culturing cells
C435S372000, C435S347000, C435S325000, C435S366000, C424S093200, C424S093700
Reexamination Certificate
active
06225119
ABSTRACT:
The present invention relates to the field of hematopoietic cell differentiation, and more particularly to the fields of megakaryocytopoiesis and thrombocytopoiesis. 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 megakaryocytes, the megakaryocytes are able to express the product of the transduced gene.
BACKGROUND OF THE INVENTION
The process of megakaryocytopoiesis is initiated with the terminal commitment of pluripotent hematopoietic stem cells (HSCs) into a differentiation pathway that results in the production of mature platelets into the circulation (Ellis, M. H. et al.
Blood,
9:1-6 (1995). The events involve megakaryocyte proliferation followed by megakaryocyte maturation into platelets, and these processes are regulated by a number of cytokines with specific megakaryocytic maturational activity and a variety of cell adhesive interactions.
Factors known to support and regulate megakaryocyte growth and development include megakaryocyte growth and development factor and cytokines (Bertolini, F. et al.
Blood,
89(8):2679-2688 (1997); Nagahisa, H. et al.
Blood,
87(4):1309-1316 (1996)); and bone marrow stromal cells (Guerriero, A. et al.
Blood,
90(9):3444-3455 (1997)). A process which would facilitate the development and maturation of megakaryocytes into platelets which does not require the use of exogenous factors would be invaluable since the time to platelet recovery following bone marrow or peripheral blood progenitor transplantation can be very protracted.
SUMMARY OF THE INVENTION
The inventors have discovered that human mesenchymal stem cells (hMSCs) are capable of driving the process of megakaryocytopoiesis in vitro to produce megakaryocytes and platelets. Moreover, such a result can be achieved with or without the addition of exogenous cytokines or megakaryocyte maturation factors such as thrombopoietin (TPO). Human mesenchymal stem cells can support the maturation of co-cultured megakaryocytic precursor cells into platelets.
Accordingly, the present invention is directed to a method of co-culturing human mesenchymal stem cells in vitro with hematopoietic stem cells. The mesenchymal stem cells support the growth of the hematopoietic stem cells, their differentiation into megakaryocytes, and their maturation to platelets.
In another aspect, the present invention involves a method for treating a patient in need of megakaryocytes. In accordance with one aspect of the invention, human mesenchymal stem cells are administered in an amount effective to enhance the in vivo production of megakaryocytes from CD34+ cells. In accordance with another aspect of the invention, CD34+ megakaryocyte precursor cells are administered in the presence of mesenchymal stem cells to a subject in need thereof.
In another embodiment, the invention provides a method for treating a patient in need of platelets comprising infusing mesenchymal stem cells with or without hematopoietic stem cells, and thereafter administering TPO or other growth factors to induce or upregulate mesenchymal stem cells to signal hematopoietic stem cells to differentiate into megakaryocytes and further into platelets.
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 differentiate into megakaryocytes that also carry the new genetic material. These transduced megakaryocytes cells are able to express the exogenous gene product. Thus, transduced megakaryocyte progenitor cells, the megakaryocytes differentiated therefrom, and resultant platelets can be used for applications where treatment using such modified megakaryocytes is beneficial. For example, these modified cells can be used as a delivery system for therapeutic proteins encoded by the exogenous gene for treatment of inherited and/or acquired disorders of blood coagulation and wound healing, as well as pathogen defense.
Accordingly, the present invention provides a method of obtaining genetically modified megakaryocytes, 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 megakaryocytes which contain the exogenous genetic material.
In one embodiment, the method of producing megakaryocytes comprises co-culturing transduced hematopoietic stem cells with mesenchymal stem cells such that after differentiation of the hematopoietic stem cells into megakaryocytes, the megakaryocytes also contain the exogenous genetic material.
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Ellis, et al.,Blood Reviews,vol. 9, pp. 1-6 (1995).
Handin et al. Blood Principles & Practice of Hematology, J.B. Lippincott Company, pp. 172, 175-176, 1951, 1399-1401, Oct. 1995.*
Stedman's Medical Dictionary, Williams & Wilkins, p. 1093, Aug. 1995.*
Nagahisa et al. Blood 87(4):1309-1316, Feb. 1996.*
Andrews et al. Journal of Experimental Medicine 172:355-358, Jul. 1990.*
Srour et al. Blood 82(11):3333-3342, Dec. 1993.*
Bertolini et al. Blood 89(8):2679-2688, Apr. 1997.*
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Frey et al. Blood 91(8):2781-2792, Apr. 1998.*
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Qasba Pankaj
Thiede Mark A.
Hauda Karen M.
Lillie Raymond J.
Olstein Elliot M.
Osiris Therapeutics, Inc.
Woitach Joseph T.
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