Compositions and methods of making embryonic stem cells

Details Compositions and methods of making embryonic stem cells Compositions and methods of making embryonic stem cells

1997-02-28

2001-06-26

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of culturing cells in suspension

C435S385000, C435S386000, C435S387000

Reexamination Certificate

active

06251671

ABSTRACT:

FIELD OF THE INVENTION
The field of the invention is human and nonhuman mammalian spermatogenesis.
BACKGROUND OF THE INVENTION
The bone morphogenetic proteins (BMPs) are members of a large, highly conserved, family of extracellular polypeptide signaling molecules related to transforming growth factor-&bgr; (TGF-&bgr;). There is now considerable evidence from expression studies, and from the in vivo effects of misexpression and mutations, that Bmp genes play key roles at many different stages of embryonic development, in both invertebrates and vertebrates (Kingsley et al., 1994, Dev. Biol. 166:112-122; Massague et al., 1994, Trends Cell Biol. 4:172-178; Hogan, 1995, Sem. Dev. Biol. 6:257-265). In the mouse, both spontaneous and induced mutations in a number of Bmp genes have shed light on their function in vivo. The first example to be described was a series of short ear mutations, which result from alterations in the Bmp5 gene (Green, 1968, J. Exp. Zool. 167:129-150; Kingsley et al., 1992, Cell 71:399-410; King et al., 1994, Dev. Biol. 166:112-122). Null mutants are viable, but have defects in cartilage development in specific parts of the skeletal system, as well as abnormalities in the lung, kidney and ureter in some genetic backgrounds.
Mutations in other Bmp genes have been generated by homologous recombination in embryonic stem cells. For example, Bmp7 homozygous null mutant mice die shortly after birth with major defects in eye, kidney and limb development (Dudley et al., 1995, Genes Dev. 9:2795-2807; Luo et al., 1995, Genes Dev. 9:2808-2820). Most Bmp4 homozygous mutant embryos die around the time of gastrulation and many exhibit a deficiency in extraembryonic and posterior/ventral mesoderm (Winnier et al., 1995, Genes Dev. 9:2105-2116), a finding consistent with the effect of BMP4 on mesoderm patterning in Xenopus embryos (Jones et al., 1992, Development 115:639-647; Graff et al., 1994, Cell 79:169-179; Harland, 1994, Proc. Natl. Acad. Sci. USA 91:10243-10246). Mutations have also been described in other members of the BMP superfamily, including mouse nodal, and Gdf5 (brachypodism) (Zhou et al., 1993, Nature 361:543-547; Conlon et al., 1994, Development 120:1919-1928; Storm et al., 1994, Nature 368:639-643).
Spermatogenesis takes places within the confines of the seminiferous tubules in the testis. A typical tubule is ensheathed by an outer basal lamina. Inside the lamina and attached to it is a layer of spermatogonial cells which continue to divide very slowly from puberty to late adult life. The self-renewing spermatogonial stem cell (known as an A
O
/A
S
cell) is very rare and it gives rise to other less primitive spermatogonial cells. These give rise to non-dividing spermatocytes which leave the basal layer and move towards the center of the tubule. These spermatocytes undergo meiosis and eventually give rise to mature sperm. The spermatogonia and differentiated derivatives are in intimate contact with the somatic Sertoli cells. Little is known about the growth factors/cytokines which regulate the proliferation of the spermatogonial stem cells, their differentiation into spermatocytes, the entry of the spermatocytes into meiosis, their differentiation into sperm and the way in which this whole complex process is co-ordinated in vivo. It has been difficult to obtain populations of cells having high concentrations of the most primitive spermatogonial stem cells in part because they form a very small proportion of the total spermatogenic cells of the testis. Additionally, cultures of spermatogonia generally only remain viable for short periods of time, that is, about 24-48 hours.
SUMMARY OF THE INVENTION
The invention relates to a method of proliferating mammalian spermatogonial stem cells, comprising culturing spermatogonial stem cells in the presence of BMP8, or a biologically active fragment or an agonist thereof, to effect proliferation of the cells.
Also included in the invention is a method of differentiating mammalian spermatogonial stem cells, comprising culturing spermatogonial stem cells in the presence of BMP, or a biologically active fragment or an agonist thereof, to effect differentiation of the cells.
In addition, the invention relates to a method of extending viability of a mammalian spermatogonial cell population, comprising culturing the spermatogonial cell population in the presence of BMP8, or a biologically active fragment or an agonist thereof, thereby extending the viability of the cultured spermatogonial cell population.
In another aspect of the invention, there is provided a method of inducing apoptosis of spermatocytes in culture comprising incubating the spermatocytes in the substantial absence of BMP8, or a biologically active fragment or an agonist thereof, thereby inducing apoptosis of spermatocytes.
Also included in the invention is a method of inhibiting proliferation of spermatogonial stem cells in culture, comprising incubating a population of spermatogenic cells in the substantial absence of BMP8 to effect inhibition of proliferation of the cells.
Further provided is a method of effecting the proliferation of mammalian spermatogonial stem cells in vivo in a mammal, comprising administering to the mammal BMP8, or a biologically active fragment or an agonist thereof, suspended in a pharmaceutically acceptable carrier, to effect proliferation of the cells in the mammal.
The invention also includes a method of effecting differentiation of mammalian spermatogonial stem cells in vivo in a mammal, comprising administering to the mammal BMP8, or a biologically active fragment or an agonist thereof, suspended in a pharmaceutically acceptable carrier, to effect differentiation of the cells in the mammal.
A method of extending viability of a mammalian spermatogonial cell population in vivo in a mammal is also provided. The method comprises administering to the mammal BMP8, or a biologically active fragment or an agonist thereof, suspended in a pharmaceutically acceptable carrier, thereby extending the viability of the mammalian spermatogonial cell population in the mammal.
The invention also relates to a method of inducing apoptosis of spermatocytes in vivo in a mammal comprising administering to the mammal BMP8, or a biologically active fragment or an agonist thereof, suspended in a pharmaceutically acceptable carrier, thereby inducing apoptosis of spermatocytes in the mammal.
A method of inhibiting proliferation of spermatogonial stem cells in vivo in a mammal is further provided. The method comprises administering to the mammal an antagonist of BMP8 suspended in a pharmaceutically acceptable carrier, to effect inhibition of proliferation of the cells in the mammal.
In yet another aspect, the invention includes a method of selectively obtaining a proliferating population of spermatogonial stem cells in culture, comprising adding BMP8 to the population of cells, thereby selectively obtaining a proliferating population of spermatogonial stem cells.
There is also included a method of treating infertility in a male mammal, comprising administering to the mammal BMP8, or a biologically active fragment or an agonist thereof, suspended in a pharmaceutically acceptable carrier.
Ina preferred embodiment, the BMP8 is administered to the testes of the mammal.
The invention includes a mammalian male contraceptive comprising a BMP8 antagonist.
The invention also includes a method of identifying an antagonist of BMP8, comprising adding a test compound to a culture of spermatogonial cells in the presence or absence of BMP8 and measuring the level of proliferation or differentiation of the cells, wherein a lower level of proliferation or differentiation of the cells in the presence of the test compound, compared with the level of proliferation or differentiation of the cells in the absence of the test compound, is an indication that the test compound is a BMP8 antagonist.
Further included is a method of identifying an agonist of BMP8, comprising adding a test compound to a culture of spermatogonial cells in the presence or absence of BMP8 and measuring the

Affiliated with

Also associated with

Rating

Compositions and methods of making embryonic stem cells does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Compositions and methods of making embryonic stem cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compositions and methods of making embryonic stem cells will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2454975