Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of detaching cells – digesting tissue or establishing...
Reexamination Certificate
2000-02-21
2003-08-05
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of detaching cells, digesting tissue or establishing...
C435S001100, C435S325000, C435S363000, C435S375000
Reexamination Certificate
active
06602711
ABSTRACT:
CROSS REFERENCES TO RELATED APPLICATIONS
Not applicable.
BACKGROUND OF THE INVENTION
Undifferentiated primate embryonic stem (“ES”) cells can be cultured indefinitely and yet maintain the potential to form differentiated cells of the body. See U.S. Pat. No. 5,843,780; J. Thomson, et al., 282 Science 1145-1147 (1998); and J. Thomson, et al., 38 Biology 133-165 (1998). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
Primate ES cells thus provide an exciting new model for understanding the differentiation and function of human tissue, and offer new strategies for drug discovery and testing. They also promise new therapies based on the transplantation of ES cell-derived tissues. For example, human and rhesus monkey ES cells injected into immunocompromised mice form benign teratomas with advanced differentiated derivatives representing all three embryonic germ layers. Easily identified differentiated cells in human ES cell teratomas include smooth muscle, striated muscle, bone, cartilage, gut and respiratory epithelium, keratinizing squamous epithelium, hair, neural epithelium, and ganglia.
Human and non-human primate ES cell lines provide a particularly powerful new model for understanding normal human development and thus also for understanding abnormal human development. Because of the potential risk to the resulting child, experimental manipulation of the post-implantation human embryo is ethically unacceptable and as a result functional studies on human embryos are lacking. Consequently, what is known about human development in the early post-implantation period is based almost entirely on static histological sections of a few human embryos and on analogy to experimental embryology studies of the mouse.
However, early mouse and primate development differ significantly. For example, human and mouse embryos differ in the timing of embryonic genome expression, in the formation, structure, and function of the fetal membranes and placenta and in the formation of an embryonic disc instead of an egg cylinder. The earliest events of human development are critically involved in human infertility, pregnancy loss, and birth defects. Primate ES cells offer a new window for understanding these early human developmental events and for understanding the pathogenesis of developmental failures.
Primate ES cells also provide a potentially unlimited source of differentiated, euploid, non-transformed cells for investigators interested in the normal function and pathology of specific differentiated primate cells. Such purified populations of specific ES cell-derived cells will also likely be useful for drug discovery, toxicity screens, and will provide a source of cells for transplantation.
For tissues such as the heart that completely lack a tissue-specific stem cell, primate ES cells will prove even more valuable. Primate ES cells also offer the promise of new transplantation therapies. When disease results from the destruction or dysfunction of a limited number of cell types, such as in Parkinson's disease (dopaminergic neurons), or juvenile onset diabetes mellitus (pancreatic &bgr;-islet cells), the replacement of those specific cell types by ES cell derivatives could offer potentially life long treatment.
To accomplish these goals, it is desirable to more efficiently differentiate ES cells to specific lineages. Considerable progress in causing non-primate ES cell differentiation to neural, hematopoietic, and cardiac tissue has been made. See e.g. T. Doetschman, et al., 87 J. Embry. And Exper. Morph. 27-45 (1985); G. Keller, 7 Current Op. In Cell Biol. 862-869 (1995); U.S. Pat. No. 5,914,268. In each of these examples, ES cells were first formed into “embryoid bodies”, three-dimensional ES cell aggregates that facilitate subsequent differentiation.
However, analogous experiments on primate ES cells demonstrated that embryoid body formation by conventional murine protocols fail. In such conventional protocols ES cells are dispersed to single cells, and either allowed to aggregate into embryoid bodies under conditions that prevent cell attachment to the substrate, or the ES cells are allowed to grow into embryoid bodies from single cells or clusters suspended in methylcellulose. We have learned that primate ES cells die rapidly when dispersed to single cells if attachment is prevented, so they do not successfully aggregate, and they therefore do not grow out from clones in methylcellulose.
It can therefore be seen that a need exists for improved methods for producing primate embryoid bodies, and differentiated cells derived therefrom.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method for producing primate embryoid bodies from colonies of primate embryonic stem cells that are adhering to a substrate. One removes the adhering colonies of the embryonic stem cells from the substrate in clumps. One then incubates the clumps in a container under conditions in which the clumps are essentially inhibited from attaching to the container and coalesce into embryoid bodies. For purposes of this application, a clump is a grouping of two or more stem cells, preferably a clump large enough to be visible to the naked eye.
In one preferred form the removal step is in the presence of an agent that promotes disassociation of the clumps from the substrate as clumps. A purely chemical agent such as VERSENE® calcium disodium EDTA chelating agent can be used. However, more preferred is a proteinase that preferentially acts on the extra cellular matrix such as dispase, collagenase, catalase, neuraminidase, pancreatin, pancreatic elastase or trypsin. If trypsin is used the removal step must be conducted quickly and at relatively low concentrations in order to prevent the trypsin from also destroying the clumps. Enzyme EDTA mixes can also be used to advantage.
In another form the removal step involves mechanically scraping the clumps from the substrate as clumps.
In another aspect the incubation step can be conducted by agitating the container (e.g. by gently rocking, shaking, or vibrating it), the container for the incubation step can be a non-attaching bacterial grade culture plastic, and/or the incubation step can be in the presence of a serum-free medium which lacks serum attachment factors.
In another aspect the invention provides primate embryoid bodies that have been derived (directly or indirectly) using the above methods.
In still another aspect the invention provides differentiated cells derived (directly or indirectly) from the embryoid bodies.
In accordance with the present invention, primate ES cells that have been cultured under standard conditions (see e.g. U.S. Pat. No. 5,843,780) are permitted to overgrow, pile up and/or otherwise closely associate in clumps on a substrate (e.g. a plastic tissue culture plate with standard feeder layer). They are then removed as clumps from the substrate (e.g. by incubating the colonies with an enzyme which attacks the ES cell colony's attachment to the substrate more strongly than ES cell attachments with ES cells). In such a case the enzyme could be dispase at a concentration of about 10 mg/ml.
Alternatively, the clumps could be removed as clumps by mechanically scraping with a micropipette, cell scraper, or the like.
The essentially intact colonies are then incubated under non-attaching conditions (preferably continuous rocking of the culture dish, culture on non-attaching bacterial grade culture plastic, and/or continuous culture in the presence of serum-free medium which lacks serum attachment factors). The colonies can then quickly coalesce into compact embryoid bodies, which can thereafter be allowed to differentiate either in continuous suspension, or after re-attachment to a substrate. Such embryoid bodies can be used to derive differentiated derivatives of endoderm, mesoderm, and ectoderm, and for obtaining other desired lineages.
It is an advantage of the present invention that it provides effective methods of forming primate embryoi
Marshall Vivienne S.
Swiergiel Jennifer J.
Thomson James A
Crouch Deborah
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
Woitach Joseph
LandOfFree
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