Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of culturing cells in suspension
Reexamination Certificate
1998-03-31
2001-06-12
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of culturing cells in suspension
C435S383000, C435S366000
Reexamination Certificate
active
06245566
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of in vitro culture of undifferentiated cells and methods of producing such cells. More specifically, the invention relates to methods and compositions for producing human embryonic germ (EG) cells and methods of using such cells. The invention has applications in the areas of cell culture, tissue transplantation, drug discovery, and gene therapy.
2. Description of Related Art
Pluripotent embryonic stem cells have traditionally been derived principally from two embryonic sources. One type of mouse pluripotent cell can be isolated in culture from cells of the inner cell mass of a pre-implantation embryo and are termed embryonic stem (ES) cells (Evans & Kaufman,
Nature
292: 154-156, 1981). A second type of mouse pluripotent stem cell can be isolated from primordial germ cells (PGCs) located in the mesenteric or genital ridges of days 8.5-12.5 post coitum mouse embryos and has been termed embryonic germ cell (EG) (Matsui et al.,
Nature
353:750-751, 1991; Resnick et al.,
Nature
359:550-551, 1992; Hogan, U.S. Pat. No. 5,453,357). Both types of cells are pluripotent and demonstrate germline genetic transmission in the mouse.
ES and EG cells propagated in vitro can contribute efficiently to the formation of chimeras, including germline chimeras. Importantly, both of these cell types can be genetically manipulated in vitro without losing their capacity to generate germline chimeras.
Thus, ES and EG cells are useful in methods for the generation of transgenic animals. Such methods have a number of advantages as compared with more conventional techniques for introducing new genetic material into such animals, such as zygote injection and viral infection. First, the gene of interest can be introduced and its integration and expression characterized in vitro. Second, the effect of the introduced gene on the ES or EG growth can be studied in vitro. Third, the characterized ES or EG cells having the novel genes can be efficiently introduced into embryos by blastocyst injection or embryo aggregation, and the consequences of the introduced gene on the development of the resulting transgenic chimeras monitored during prenatal or postnatal life. Fourth, the site in the ES or EG genome at which the introduced gene integrates can be specified, permitting subsequent gene targeting and gene replacement (Thomas & Capecci,
Cell
51:503-512, 1987).
However, the EG or ES cell lines studied to-date only retain the stem cell phenotype in vitro when cultured under special conditions. The conditions include culturing the cells on a feeder layer of fibroblasts (such as murine STO cells, e.g., Martin & Evans,
Proc. Natl. Acad. Sci USA
72:1441-1445, 1975) when cultured in medium conditioned by certain cells (e.g. Koopman & Cotton,
Exp. Cell
154:233-242, 1984; Smith & Hooper,
Devel Biol.
121:1-91, 1987), or by the exogenous addition of leukemia inhibitory factor (LIF). Such cells can be grown relatively indefinitely using the appropriate culture conditions. However, the factors responsible for maintaining the pluripotency of ES and EG cells remain poorly characterized and are often dependent upon the species from which the cells have been harvested.
In the absence of feeder cells, exogenous leukemia inhibitory factor (LIF), or conditioned medium, ES or EG cells spontaneously differentiate into a wide variety of cell types, including cells found in each of the endoderm, mesoderm, and ectoderm germ layers. With the appropriate combinations of growth and differentiation factors, however, cell differentiation can be controlled. For example, mouse ES and EG cells can generate cells of the hematopoietic lineage in vitro (Keller et al.,
Mol. Cell. Biol.
13:473-486, 1993; Palacios et al.,
Proc. Natl. Acad. Sci USA
92:7530-7534, 1995; Rich,
Blood
86:463-472, 1995). Additionally, mouse ES cells have been used to generate in vitro cultures of neurons (Bain et al.,
Developmental Biology
168:342-357, 1995; Fraichard et al.,
J. Cell Science
108:3161-3188, 1995), cardiomyocytes (heart muscle cells) (Klug et al.,
Am. J. Physiol.
269:H1913-H1921, 1995), skeletal muscle cells (Rohwedel et al.,
Dev. Biol.
164:87-101, 1994), and vascular cells (Wang et al.,
Development
114:303-316, 1992).
Subsequent to the work with mouse embryos, several groups have attempted to develop similar embryonic stem cell lines from sheep, pig, and cow. A cell line with embryonic stem cell-like appearance has reportedly been cultured from porcine embryos using culture conditions similar to mouse (Evans et al., PCT Application WO90/03432; Notarianni et al.,
J. Reprod. Fert., Suppl.
41:51, 1990; Piedrahita et al.,
Theriogenology
34:879, 1990; Notarianni et al.,
Proceedings of the
4th World Congress on Genetics Applied to Livestock Productions, 58, Edinburgh, July 1990). Other groups have developed avian stem cell lines from chickens (Pain et al.,
Development,
122:1996). However, human ES and EG cell lines have not been reported.
Any method that would allow production of human ES and EG is desirable, since human ES or EG cell lines would permit easier study of early human development, and the use of such human EG cell lines would enable the development of cell cultures for transplantation, manufacture of bio-pharmaceutical products, and development of biological-based sensors. Importantly, the ability to produce large quantities of human cells has important working applications for the production of substances, such as insulin or factor VIII which currently must be obtained from non-human sources or donors; implantation to treat disease, such as Parkinson's disease; tissue for grafting; and screens for drugs and toxins.
SUMMARY OF THE INVENTION
The present invention provides a human embryonic pluripotent germ cell (EG) line and a method of producing such cells. The invention also provides methods of using EG cells.
EG cells are derived from primordial germ cells (PGCs) cells isolated, according to one embodiment, from gonadal tissues, genital ridges, mesenteries or embryonic yolk sacs of human embryos. The PGCs are cultured under conditions that allow derivation of EG cells. The present invention also provides cell culture media for long term cell culture (more than 30 days) of the resulting EG cells.
The invention provides a cell line having the characteristics of a human embryonic germ cell. In one aspect, the cells are cultured in a medium including a growth factor, e.g., basic fibroblast growth factor (bFGF). In another embodiment, the cells are cultured in the presence of culture media containing an effective amount of either a ligand that binds to a receptor which can associate with glycoprotein 130 (gp130) or an antibody that binds to and stimulates gp130; and a growth factor, which in one embodiment is bFGF. In another embodiment, the EG cell culture medium of the invention can include forskolin or other cAMP elevating compound. In another aspect, the EG cells are alkaline phosphatase positive. In yet another aspect, the EG cells express cell surface antigens SSEA-1 and SSEA-4. In still another aspect, the EG cells express cell surface antigens that bind with antibodies having the binding specificity of monoclonal antibodies TRA-1-60 (ATCC HB-4783) and TRA-1-81 (ATCC HB-4784). The cells can also express cell surface antigen SSEA-3.
In one aspect, the invention provides a method for screening to identify compounds that affect EG cell function. In one embodiment, the method includes incubating components comprising the compound and at least one EG cell under conditions sufficient to allow the compound and cell to interact; and determining the effect of the compound on an EG cell function before and after incubating in the presence of the compound. A cell function that may be modulated (e.g., inhibited or stimulated) by the compound includes, but is not limited to, differentiation, gene expression, production of growth factors, response to growth factors and modulation of cell membrane permeability. In a
Gearhart John D.
Shamblott Michael Joseph
Fish & Richardson P.C.
The Johns Hopkins University School of Medicine
Weber Jon P.
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