Method of culturing embryonic stem cells with the use of amniotic membrane-origin factor

#The present invention relates to a method of culturing embryonic stem cells using an amnion-derived factor, a culture agent/culture kit of embryonic stem cells comprising an amnion-derived factor, a screening method for an amnion-derived factor useful for culturing embryonic stem cells, a method of preparing amniocytes useful for culturing embryonic stem cells and the like.

#Embryonic stem cells are promising candidates for a source of cells for cell transplantation for Parkinson's disease and diabetes mellitus. However, even embryonic stem cells derived from mice, humans and other primates require the coexistence with mouse-derived feeder cells (stromal cells) in their culture and differentiation induction, and this represents a major barrier against their clinical applications.

#Recently, the present inventors developed a method of inducing the differentiation of mouse and monkey embryonic stem cells into neurons at high efficiency (SDIA method) (see pamphlet for International Patent Publication No. WO01/088100; pamphlet for International Patent Publication No. WO03/042384; Kawasaki et al., Neuron, vol, 28, p. 31-40 (2000); Kawasaki et al., Proceedings of the National Academy of Sciences of the USA, vol, 99, p. 1580-1585 (2002); Mizuseki et al., Proceedings of the National Academy of Sciences of the United States of America, vol, 10, p. 5828-5833 (2003); Ooto et al., Invest. Ophthalmol. Vis. Sci., vol, 44, p. 2689-2693 (2003)). Using this method, the present inventors succeeded in producing in vitro dopamine-secreting neurons, which are expected to be applied to transplantation therapy for Parkinson's disease, and motor neurons, which are speculated to be applied to treatment for amyotrophic lateral sclerosis, from mouse and monkey embryonic stem cells (see pamphlet for International Patent Publication No. WO01/088100; pamphlet for International Patent Publication No. WO03/042384; Kawasaki et al., Neuron, vol, 28, p. 31-40 (2000); Kawasaki et al., Proceedings of the National Academy of Sciences of the USA, vol, 99, p. 1580-1585 (2002); Mizuseki et al., Proceedings of the National Academy of Sciences of the United States of America, vol, 100, p. 5828-5833 (2003); Ooto et al., Invest. Ophthalmol. Vis. Sci., vol, 44, p. 2689-2693 (2003)).

#The present invention is hereinafter described in more detail by means of the following examples, which, however, merely show illustration and are not to be construed as limiting scope of the present invention.

#After consent in writing with regard to the use of her placenta was obtained from a pregnant woman with no systemic complications who was scheduled to undergo caesarian section and determined to be negative for infections by serological testing, the amnion was aseptically collected from the placenta extirpated in caesarian section. The amnion collected was washed with antibiotic-containing physiological saline to remove blood components and chorion, after which the amnion was cut into pieces of appropriate size and rapidly frozen in antibiotic-supplemented 50% glycerol/DMEM in a deep freezer, after which they were preserved in a dedicated refrigerator at −80° C.

#After the amnion cryopreserved (7 months) was thawed, it was reacted in 0.02% EDTA at 37° C. for 2 hours, and the cells were removed from the amnion to prepare cell-free amnion tissue. Next, a porous membrane culture insert was inserted into a 6-well plate (diameter of each well: 3.5 cm), and the cell-free amnion tissue was mounted on the porous membrane culture insert with the luminal side thereof up. Next, to promote ES cell adhesion, the cell-free amnion tissue was pre-coated with gelatin or fibronectin.