Wednesday, July 19, 2006

More on the re-exam request of WARF's stem cell patents

Further to the re-exam request on WARF patents of July 18, 2006, one notes that there is an assertion that claims of both US 5,843,780 and US 6,200,806 are anticipated by US 5,166,065.

The abstract of the '065 patent states:

The present invention relates generally to the use of leukaemia inhibitory factor (LIF) in the maintenance and derivation of embryonic stem (ES) cells in culture. The ES cells are maintained and/or derived from animal embryos by culturing said cells or embryos in a culture medium containing an effective amount of LIF for a time and under conditions sufficient to maintain and/or derive said ES cells. The ES cells may be passaged in LIF and used to make chimaeric animals.

Text within the '065 states:

However, it is known that 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. (1975) Proc. Natl. Acad. Sci. USA 72:1441-1445) or when cultured in medium conditioned by certain cells (e.g. Koopman, P. and Cotton, R. G. H. (1984) Exp. Cell Res. 154:233-242; Smith, A. G. and Hooper, M. L. (1987) Devel.Biol. 121:1-91). In the absence of feeder cells or conditioned medium, the ES cells spontaneously differentiate into a wide variety of cell types, resembling those found during embryogenesis and in the adult animal. The factors responsible for maintaining the pluripotency of ES cells have, however, remained poorly characterised.


Accordingly, a first aspect of the present invention relates to a method for the isolation of embryonic stem (ES) cells from animal embryos in vitro which method comprises deriving ES cells from said embryos in culture medium, said culture medium containing an effective amount of leukaemia inhibitory factor (LIF), for a time and under conditions sufficient for the development of said ES cells. The embryos used may be isolated from animals including, but not limited to, humans and a number of other animal species such as birds (e.g. chickens), mice, sheep, pigs, cattle, goats and fish.

A second aspect of the present invention, contemplates a process for maintaining animal embryonic stem (ES) cells in vitro while retaining their pluripotential phenotype, which process comprises culturing said cells in a culture medium containing an effective amount of leukaemia inhibitory factor (LIF) under conditions sufficient to maintain said cells. The ES cells in accordance with this aspect of the invention include cells from humans, mice, birds (e.g. chickens), sheep, pigs, cattle, goats and fish.

An example is given of a stem cell line from a mouse.

The first claim of the '065 states:

A method for the isolation of embryonic stem (ES) cells from mammalian embryos in vitro which method comprises deriving and maintaining said embryos in culture medium containing an effective amount of recombinant leukaemia inhibitory factor (LIF) for a time and under conditions sufficient for the development of said ES cells.

A paper in the journal Stem Cells (Vol 22, p. 770) in the year 2004 had reported:

Anecdotal and contradictory accounts exist for the action of LIF in the culture of human embryonic stem cells, and the nature of LIF signaling and whether the LIF-STAT3 pathway is conserved in human embryonic stem cells (hESCs) has not been systematically explored.

Separately, mouse stem cells can be cultured indefinitely in the presence of leukemia inhibitory factor (LIF), a glycoprotein cytokine.

Human embryonic stem cells are in many respects similar to mouse embryonic stem cells, but they do not require LIF for their maintenance.

The re-exam request makes much of the fact that the '780 and '806 patents of Thomson (WARF) did NOT cite the '065 patent (Williams). HOWEVER, one notes that the '780 patent of Thomson DOES CITE the work of Williams:

Mouse ES cells maintain an undifferentiated state through serial passages when cultured in the presence of fibroblast feeder layers in the presence of Leukemia Inhibitory Factor (LIF) (Williams, et al., Nature 336: 684-687, 1988). If LIF is removed, mouse ES cells differentiate.

The '780 patent does mention LIF:

FIG. 2 is a set of phase-contrast photomicrographs demonstrating the morphology of undifferentiated rhesus ES (R278.5) cells and of cells differentiated from R278.5 in vitro (bar=100 .mu.). Photograph A demonstrates the distinct cell borders, high nucleus to cytoplasm ratio, and prominent nucleoli of undifferentiated rhesus ES cells. Photographs B-D shows differentiated cells eight days after plating R278.5 cells on gel treated tissue culture plastic (with 10.sup.3 units/ml added human LIF) . Cells of these three distinct morphologies are consistently present when R278.5 cells are allowed to differentiate at low density without fibroblasts either in the presence or absence of soluble human LIF.

More importantly, the '780 patent mentions (and the re-exam request does not mention) a previous failed attempt to isolate hESC using LIF:

No other primate (human or non-human) ES cell line is known to exist. The only published permanent, euploid, embryo-derived cell lines that have been convincingly demonstrated to differentiate into derivatives of all three germ layers have been derived from rodents (the mouse, rat, and hamster), and possibly from rabbit. The published reports of embryo-derived cell lines from domestic species have failed to convincingly demonstrate differentiation of derivatives of all three embryonic germ layers or have not been permanent cell lines. Research groups in Britain and Singapore are informally reported, later than the work described here, to have attempted to derive human ES cell lines from surplus in vitro fertilization-produced human embryos, although they have not yet reported success in demonstrating pluripotency of their cells and have failed to isolate permanent cell lines. In the only published report on attempts to isolate human ES cells, conditions were used (LIF in the absence of fibroblast feeder layers) that the results below will indicate will not result in primate ES cells which can remain in an undifferentiated state. It is not surprising, then that the cells grown out of human ICMs failed to continue to proliferate after 1 or 2 subcultures, Bongso et al. Hum. Reprod. 9: 2100-2117 (1994).


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