Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-08-29
2004-10-05
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S001100, C424S093100, C424S093210
Reexamination Certificate
active
06800480
ABSTRACT:
1 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates to the field of stem cell culture media and to methods for culturing such cells. More particularly, the present invention provides methods and materials for culturing primate-derived primordial stem cells in a substantially undifferentiated state with and without a feeder layer. The present invention has applications in the areas of cell culture, tissue transplantation, drug discovery, and gene therapy.
1.2 The Related Art
Stem cells are cells capable of differentiation into other cell types having a particular, specialized function (“fully differentiated” cells) or other types of stem cells which are capable of differentiation into a more narrow range of cell types (“pluripotent” cells). Stem cells having the ability to differentiate into any type of cell, i.e., pluripotent or fully differentiated, are called “totipotent”. Such cells are also referred to as “primordial stem cells”. There has been great interest in isolating and growing primordial stem cells from primates, especially from humans, as such primordial stem cells could provide a supply of readily available cells and tissues of all types for use in transplantation, drug discovery, and gene therapy in humans.
Methods for isolating and growing primordial stem cells from primates have been described. Procedures for isolating and growing human primordial stem cells are described in co-pending U.S. patent application Ser. No. 08/829,372. Procedures for obtaining Rhesus monkey and other non-human primate primordial stem cells are described in co-pending U.S. patent applications Ser. Nos. 08/376,327; 08/591,246; 08/665,217; and WO 96/22362. Each of these patent applications is incorporated herein by reference in its entirety and for all purposes. In addition, methods for isolating Rhesus monkey primordial stem cells are described in Thomson et al. (1995
Proc. Natl. Acad. Sci. USA
92:7811-7848) also incorporated herein by reference in its entirety and for all purposes.
Unfortunately, current methods for growing primordial stem cells derived from primates in culture have not been as clearly defined as, and are relatively inefficient compared with, methods for culturing primordial stem cells for other species such as mouse. For example, current methods of culturing primate-derived primordial stem cells require a feeder layer that complicates and slows the process of cell cultivation. In addition, the formulation of an optimal culture media for propagating undifferentiated totipotent primate-derived primordial stem cells remains to be determined.
In particular, it is desirable to maintain cultures of totipotent primordial stem cells for extended periods or indefinitely. The ability to maintain cultures of undifferentiated, totipotent, primate-derived primordial stem cells for long periods facilitates the use of such cells for therapeutic purposes. Moreover, it would be desirable to grow cultures of substantially undifferentiated primate-derived primordial stem cells for periods sufficient to allow the production of primate-derived primordial stem cells having multiple genetic modifications for the production of tissues and for gene therapy.
2 SUMMARY OF THE INVENTION
The present invention provides methods and reagents for culturing primate-derived primordial stem cells in a substantially undifferentiated state. The methods and materials described herein provide improved culturing conditions that allow the preparation of primate-derived primordial stem cells having single or multiple genetic modifications. Such modified cells have important applications in gene therapy and tissue transplantation/implantation therapies.
In one aspect, the present invention provides a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state. In one embodiment, the cell culture medium of the invention comprises a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. This basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from the group of feeder cells, such as mouse embryo fibroblast cells and STO cells, and an extracellular matrix derived from the feeder cells. The medium further includes non-essential amino acids, an anti-oxidant (for example, &bgr;-mercaptoethanol), and, optionally, a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.
In more specific embodiments, the basic medium of the cell culture medium has an osmotic pressure of less than about 300 mOsm/kg. Still more specific embodiments are those for which the basic medium has an osmotic pressure of about 280 mOsm/kg. Yet other embodiments of the cell culture medium of the present invention include those for which the basic medium has an endotoxicity of less than about 0.1 endotoxin units per ml. More specific embodiments for which the endotoxicity of the basic medium is less than about 0.1 endotoxin units per ml are those embodiments for which the endotoxicity of the base medium is about 0.03 endotoxin units per ml.
In other embodiments the cell culture medium of the invention further includes a second growth factor. In a preferred embodiment, the second growth factor is selected from the group consisting of: Anti-IL-8, Anti-TGF-&bgr;5, Anti-BDNF, Anti-TNF-&bgr;, Anti-VEGF, Anti-TGF-&bgr;, IL-11, IL-6, IL-6+soluble IL-6 receptor, IL-1&agr;, IL-1&bgr;, LIF, Anti-HB-EGF, IL-17, TFG-&bgr;-1 LAP, MCP-1, bFGF, FGF-4, PDGF Soluble Receptor A, dexamethasone and Forskolin.
Suitable growth factors for use in the present invention can be determined using a method for screening for growth factors that is provided in another aspect of the present invention. According to one embodiment of this aspect of the invention, primate-derived primordial stem cells are grown using a cell culture medium of the present invention in the presence of a putative growth factor. A determination is made as to whether the putative growth factor enhances the growth of undifferentiated primate-derived primordial stem cells. Substances that enhance the growth of primate-derived primordial stem cells are classified as growth factors.
In another aspect, the present invention provides a culture of primate primordial cells, comprising at least one primate-derived primordial stem cell in fluid contact with the cell culture medium of the invention. Such cells can be human- or Rhesus-derived primordial stem cells, for example.
In still another aspect, the present invention provides methods for producing primate cell lines having one or more genetic modifications. According to one embodiment of this aspect of the present invention, primordial stem cells are grown using a cell culture medium of the invention. A first gene or nucleic acid is introduced into, or a first gene is modified in, these cells and a first clone population is derived. In a further embodiment, a second gene or nucleic acid is introduced into, or a second gene is modified in, the cells of the first clone population and a second clone population is derived. In some embodiments, the primordial stem cells are derived from human embryonic cells. In other embodiments the primordial stem cells are PSC43 cells, an aneuploid variant of Rhesus embryonic stem cells that is capable of growing in a feeder-free cell culture as described hereinbelow. This cell line has been found effective for screening for growth factors for cell culture media.
These and other aspects and advantages will become apparent when the Description below is read in conjunction with the accompanying Examples.
3 DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION
The present invention provides methods and materials for culturing primate-derived primordial stem cells in a substantially undifferentiated state and for identifying and quantifying undifferentiated primate-derived primordial stem cells. In addition, the present invention also provides s
Bodnar Andrea G.
Chiu Choy-Pik
Gold Joseph D.
Inokuma Margaret
Murai James T.
Crouch Deborah
Earp David J.
Geron Corporation
Schiff J. Michael
Woitach Joseph
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