Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1994-11-01
2002-08-13
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S325000, C435S366000, C435S384000, C435S386000
Reexamination Certificate
active
06432711
ABSTRACT:
This invention relates to embryonic stem cells. More particularly, this invention relates to embryonic stem cells which are engineered with DNA and/or cultured in the presence of an agent, whereby such cells become capable of differentiating homogeneously into a desired primary cell line. Such homogeneous differentiation has not and cannot be achieved unless the methods described herein are applied.
Embryonic stem cells are pluripotent cells derived from the inner cell mass of pre-implantation embryos. (Evans et al.,
Nature
, Vol. 292, pgs. 154-156 (1981)). Embryonic stem cells can differentiate into any cell type in vivo (Bradley, et al.,
Nature
, Vol. 309, pgs. 255-256 (1984); Nagy, et al.,
Development
, Vol. 110, pgs. 815-821 (1990) and into a more limited variety of cells in vitro (Doetschman, et al.,
J. Embryol. Exp. Morph.
, Vol. 87, pgs. 27-45 (1985); Wobus, et al.,
Biomed. Biochim. Acta
, Vol. 47, pgs. 965-973 (1988); Robbins, et al.,
J. Biol. Chem.
, Vol. 265, pgs. 11905-11909 (1990); Schmitt, et al.,
Genes and Development
, Vol. 5, pgs. 728-740 (1991)). Embryonic stem cells, however, are more difficult to maintain in the laboratory and require the addition of a differentiation inhibitory factor (commonly referred to as leukemia inhibitory factor (or LIF) in the culture medium to prevent spontaneous differentiation (Williams, et al.,
Nature
, Vol. 336, pgs. 684-687 (1988); Smith, et al.,
Nature
, Vol. 336, pgs. 688-690 (1988); Gearing, et al,,
Biotechnology
, Vol. 7, pgs. 1157-1161 (1989); Pease, et al.,
Dev. Biol.
, Vol. 141, pgs. 344-352 (1990). LIF is a secreted protein and can be provided by maintaining embryonic stem cells on a feeder layer of cells that produce LIF (Evans, et al., 1981; Robertson,
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach
, Washington, D.C.: IRL Press (1987)) or by the addition of purified LIF (Williams, et al., 1988; Smith, et al., 1988; Gearing, et al., 1989; Pease, et al.,
Exp. Cell Res.
, Vol. 190, pgs. 209-211 (1990) to the medium in the absence of feeder layers. Differentiation of embryonic stem cells into a heterogeneous mixture of cells occurs spontaneously if LIF is removed, and can be induced further by manipulation of culture conditions (Doetschmann, et al., 1985; Wobus, et al., 1988; Robbins, et al., 1990; Schmitt, et al., 1991; Wiles, et al.,
Development
, Vol. 111, pgs. 254-267 (1991); Gutierrez-Ramos, et al.,
Proc. Nat. Acad. Sci.
, Vol. 89, pgs. 9111-9175 (1992)). Differentiation of stem cells into a homogeneous population, however, has not been achieved. Embryonic stem cell differentiation can be variable between different established embryonic stem cell lines and even between laboratories using the same embryonic stem cell lines.
It is an object of the present invention to provide embryonic stem cells which are capable of differentiating uniformly into a specific and homogeneous cell line, not achievable by previous methods.
In accordance with an aspect of the present invention, there is provided a method of producing a desired cell line from embryonic stem cells. The method comprises culturing embryonic stem cells under conditions which promote growth of the embryonic stem cells at an optimal growth rate. The embryonic stem cells then are cultured under conditions which promote the growth of the cells at a rate which is less than that of the optimal growth rate, and in the presence of an agent which promotes differentiation of the embryonic stem cells into the desired cell line.
In general, a growth rate which is less than the optimal growth rate, is a growth rate from about 10% to about 80%, preferably from about 20% to about 50%, of the maximum growth rate for embryonic stem cells. The growth rates for embryonic stem cells can be determined from the doubling times of the embryonic stem cells. In general, the optimum doubling time for embryonic stem cells is from about 13 hours to about 18 hours, and more particularly, from about 15 hours to about 16 hours.
In one embodiment, when the embryonic cells are being cultured under conditions which promote growth of the cells at an optimal growth rate, the embryonic stem cells are cultured in the presence of a medium including leukemia inhibitory factor (LIF), and serum selected from the group consisting of: (i) horse serum at a concentration of from about 5% by volume to about 30% by volume; and (ii) fetal bovine serum at a concentration of from about 15% by volume to about 30% by volume. In one embodiment, the serum is horse serum at a concentration of about 10% by volume. In another embodiment, the serum is fetal bovine serum at a concentration of about 15% by volume.
In yet another embodiment, when the embryonic stem cells are cultured at an optimal growth rate, the embryonic stem cells are cultured in the absence of a feeder layer of cells.
In one embodiment, the agent which promotes differentiation of the embryonic stem cells is selected from the group consisting of retinoic acid and nerve growth factor, and the desired cell line is a neuronal cell line.
In one embodiment, in addition to culturing the cells in the presence of the stimulating agent selected from the group consisting of retinoic acid and nerve growth factor, the embryonic stem cells are grown in the presence of a cytokine. Cytokines which may be employed include, but are not limited to, Interleukin-1, Interleukin-3, Interleukin-4, Interleukin-6, colony stimulating factors such as M-CSF, GM-CSF, and CSF-1, steel factor, and erythropoietin.
In a further embodiment, the agent which promotes differentiation of the embryonic stem cells is selected from the group consisting of dimethylsulfoxide and hexamethylene bis-acrylamide, and the desired cell line is a muscle cell line, such as a smooth muscle cell line, or a skeletal muscle cell line, or a cardiac muscle cell line. In one embodiment, the agent is dimethylsulfoxide. In another embodiment, the agent is hexamethylene bis-acrylamide.
In one embodiment, in addition to culturing the embryonic stem cells in the presence of an agent which promotes differentiation of the embryonic stem cells into a muscle cell line, the embryonic stem cells also are grown in the presence of a cytokine, examples of which are hereinabove described.
In yet another embodiment, when the embryonic stem cells are cultured in the presence of the agent which promotes differentiation of the embryonic stem cells into a desired cell line, the embryonic stem cells also are cultured in the presence of fetal bovine serum at a concentration of about 10% by volume.
In a further embodiment, when the embryonic stem cells are cultured in the presence of the agent which promotes differentiation of the embryonic cells into a desired cell line, the embryonic stem cells also are cultured in a three-dimensional format.
Thus, Applicants have found that one may produce a homogenous desired cell line from embryonic stem cells by culturing the embryonic stem cells initially under conditions which favor the growth or proliferation of such embryonic stem cells at an optimal growth rate, and then culturing the cells under conditions which decrease the growth rate of the cells and promote differentiation of the cells to a desired cell type.
In a preferred embodiment, the embryonic stem cells cultured in a standard culture medium (such as, for example, Minimal Essential Medium), which may include supplements such as, for example, glutamine, and &bgr;-mercaptoethanol. The medium may also include leukemia inhibitory factor (LIF), or factors with LIF activity, such as, for example, CNTF or IL-6, and horse serum. LIF, and factors with LIF activity, prevents spontaneous differentiation of the embryonic stem cells, and is removed prior to the addition of the agent. Horse serum promotes differentiation of the embryonic stem cells into the specific cell type after the addition of the agent to the medium. After the cells have been cultured for a period of time sufficient to permit the cells to proliferate to a desired number, the cells are washed free of LIF, and then cultured under conditio
Dinsmore Jonathan H.
Ratliff Judson
Diacrin, Inc.
Olstein Elliot M.
Tate Christopher R.
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