Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-12-12
2001-12-18
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S375000, C435S377000
Reexamination Certificate
active
06331406
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This 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 production of human pluripotent embryonic germ cells or (hEG) cell lines.
2. Description of Related Art
Pluripotent embryonic stem cells are derived principally from two embryonic sources. In the mouse, one type of pluripotent stem cell can be isolated from cells of the inner cell mass of a pre-implantation embryo and are termed embryonic stem (ES) cells (Evans and 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 genital ridges of day 8.5 post coitum mouse embryos and has been termed an 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.
The extent of pluripotency in pluripotent cell cultures is generally determined experimentally. For example, one method utilizes measuring the high intracellular levels of the enzyme alkaline phosphatase found in ES, PGCs, and EGs. Demonstration of intracellular alkaline phosphatase by histological staining was historically used to define and locate PGCs (Chiquoine,
Anat.Rec.
118: 135-146, 1954). Such staining remains one of the criteria for the definition of new pluripotent cell cultures.
ES and EGs propagated in vitro can contribute efficiently to the formation of chimeras, including germline chimeras, but in addition, both of these cell types can be genetically manipulated in vitro without losing their capacity to generate germ-line chimeras.
ES and EGs 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 EGs having a novel introduced gene 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, K. R. and Capecci, M. R.
Cell
51: 503-512, 1987).
However, it is known that EGs or ES cells and certain EC (embryonal carcinoma) cell lines will only retain the stem cell phenotype in vitro when cultured on a feeder layer of fibroblasts (such as murine STO cells, e.g., Martin, G. R. and Evans, M. J.
Proc. Natl. Acad. Sci. USA
72: 1441-1445, 1975) when cultured in medium conditioned by certain cells (e.g. Koopman, P. And Cotton, R. G. H.
Exp. Cell
154: 233-242, 1984; Smith, A. G. and Hooper, M. L.
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. They can be induced to differentiate in vitro using retinoic acid or spontaneously by removal of the feeder layer conditioned media or exogenous LIF. In addition, these cells can be injected into a mouse blastocyst to form a somatic and germ line chimera. This latter property has allowed mouse ES cells to be used for the production of transgenic mice with specific changes to the genome.
See M. Evans et al.,
Nature
292: 154 (1981); G. Martin,
Proc. Natl. Acad Sci. USA
78: 7638 (1981); A. Smith et al.,
Developmental Biology
121: 1 (1987); T. Doetschman et al.,
Developmental Biology
127: 224 (1988); A. Handyside et al.,
Roux's Arch Dev. Biol.
198: 48 (1989).
In the absence of feeder cells or conditioned medium, ES or EGs spontaneously differentiate into a wide variety of cell types, resembling those found during embryogenesis and in the adult animal. With the appropriate combinations of growth and differentiation factors, mouse ES and EGs generate cells of the hematopoietic lineage in vitro (Keller, G., et al.,
Mol. Cell. Biol.
13: 473-486, 1993; Palacios, R., E. Golunski, and J. Samaridis,
Proc. Natl. Acad. Sci. USA
92: 7530-7534, 1995; Rich, T.,
Blood
86: 463-472, 1995). Additionally, mouse ES cells have been used to generate in vitro cultures of neurons (Bain, G., et al.,
Developmental Biology
168: 342-357, 1995; Fraichard, A., et al.,
J. Cell Science
108: 3161-3188, 1995), cardiomyocytes (heart muscle cells) (Klug, M., M. Soonpaa, and L. Field,
Am. J. Physiol.
269: H1913-H1921, 1995), skeletal muscle cells (Rohwedel, J., et al.,
Dev. Biol.
164: 87-101, 1994), and vascular cells (Wang, R., R. Clark and V. Bautch,
Development
114: 303-316, 1992). The factors responsible for maintaining the pluripotency of ES and EGs remain poorly characterized and are often dependent upon the species from which the cells have been harvested.
Subsequent to the work with mouse embryos, several groups have attempted to develop 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 (M. Evans et al., PCT Application WO90/03432; E. Notarianni et al.,
J. Reprod. Fert., Suppl.
41: 51, 1990; J. Piedrahita et al.,
Theriogenology
34: 879, 1990; E. 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.,
Dev.
122:1996).
To date, there have been no reports for the establishment of human EG cells or cell lines. Any method which would allow production of human ES and EG would be desirable since, human 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.
SUMMARY OF THE INVENTION
The present invention provides a human pluripotent embryonic germ cell (hEG) line and a method of producing cells exhibiting an hEG phenotype. hEGs are derived from EGs isolated from gonadal tissues, genital ridges, mesenteries or embryonic yolk sacs of human embryos and cultured under conditions which allow long term cell culture (more than 30 days). The resulting hEG cells resemble ES cells in morphology, biochemical histotype and in pluripotency. These cells can be passaged and maintained for several months in culture and can survive cryopreservation.
One object of the invention is to provide a method for producing human cell lines which exhibit an ES cell-like phenotype. Another object of the invention is to provide human pluripotential embryonic germ cell (hEG) lines in general, as well as differentiated cell lines derived from hEGs. Another object is to provide human transgenic cells, cell lines, or tissues using the hEGs of the invention. Another object is to provide hEG cells or hEG derived stem cells of restricted developmental lineage for transplantation.
In one aspect, the invention provides a method for screening, compounds, including small molecules, that affect hEG cell function. The method includes incubating components comprising the compound and at least one hEG cell under conditions sufficient to allow the components to interact; and determining the effect of the compound on an hEG 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 differentiation, gene expression, production o
Gearhart John D.
Shamblott Michael Joseph
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
The John Hopkins University School of Medicine
Weber Jon P.
LandOfFree
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