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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies
  • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells

Definitions

• TECHNICAL FIELD This invention is in the field of molecular biology and medicine. More specifically, it relates to novel non-mouse embryonic stem (ES) cells and methods of obtaining these non-mouse ES cells.
• ES embryonic stem
• BACKGROUND Genetically modified laboratory animals are widely used in drug development and as model systems of human disease. Many of these transgenic animals, especially loss-of- function mutants, are mouse models that have been generated by using mouse embryonic stem (ES) cells. Briefly, gene targeting in ES cells uses the phenomenon of homologous recombination to disrupt or knock-out the function of a particular gene. (See, U.S. Patent Nos. 5,464,764; 5,487,992; 5,631,153 and 5,627,059 for techniques relating to mouse embryonic stem cells). There is a rapidly growing number of mouse mutants that have been created by inactivation of genes in ES cells.
• mice are produced to understand the function of known genes in vivo, to discover new genes .and to create animal models of human diseases, (see, e.g., Chisaka et al. (1992) 355:516-520; Joyner et al. (1992) in POSTIMPLANTATION DEVELOPMENT IN THE MOUSE (Chadwick and Marsh, eds., John Wiley & Sons, United Kingdom) pp:277-297; Dorin et al. (1992) Nature
• Gerhart et al. report isolation of human gonad-precursor cells from aborted fetuses. (See, Beardsley (1998) Scientific American, on line). When these cells are implanted into mice with no functioning immune system, they apparently give rise to tumors containing various cell types. However, it is not clear that these isolated human cells are pluripotent ES cells.
• Transgenic rats offer several advantages over mouse models.
• rats are larger, which makes surgical procedures possible, and they provide a larger amount of material for biochemical analysis.
• a second advantage of rat models is that a large existing database of information has been generated for rat models, especially in the areas of neurodegenerative disease, cardiovascular research and diabetes. No comparable database exists for mice.
• Rats are also the preferred animal model for drug development assays.
• Transgenic rats allow sophisticated physiological measurements that are not possible in transgenic mice. For example, because of their size, rats harboring human transgenes can offer primate-specific analyses in a non-primate experimental system.
• mice carrying the same transgene have no disease phenotype.
• a transgenic model of HLA-B27-associated human autoimmune disorders was first attempted in mice, but no pathology developed (Taurog et al. (1988) J. Immunol. 141:4020-30).
• Transgenic HLA-B27 rats however, have pathologies that closely resemble the human disease (Hammer et al. (1990) Cell 63:1099-1112).
• rat ES cells could be used to exploit all of the genetic manipulations now possible only in mice. If rat
• null mutations could be introduced into the rat homologues of human genes, and in combination with the human transgenes, would provide a "humanized” animal that could replace the primate for many studies.
• the present invention provides the first pluripotent rat ES cells.
• the invention provides a method which can be universally applied to the generation of ES cells from all species.
• the present invention includes an isolated population of non-mouse embryonic stem cells.
• the cells are rat ES cells.
• the invention provides a method of obtaining embryonic stem cells from a target species, the method comprising: (a) co-culturing cells obtained from an embryo of the target species with non-target embryonic stem cells under conditions which favor growth of embryonic stem (ES) cells from the target species; and (b) isolating the ES cells from the target species.
• the non-target embryonic stem cells used in the co-culture are from a species other than the target species, such as mouse.
• the target ES cells can be derived from inner cell masses (ICMs) or can be primordial germ cells (PGCs).
• the non-target embryonic stem cells of used in the co- culture lack a positive selection marker.
• the positive selection marker can be a gene encoding antibiotic resistance or a gene encoding HPRT resistance (HPRT).
• the embryonic stem cells used in the co-culture carry a negative selection marker, for example HPRT or herpes simplex virus thymidine kinase (HSV-tk).
• the embryo cells can be cultured on a feeder layer of cells.
• the feeder layer is SNL 76/7 cells.
• the non-target ES cells used in the co-culture methods are mitotically inactivated.
• the present invention includes genetically modified non- mouse ES cells.
• the genetic modification comprises insertion of a transgene.
• the genetic modification comprises disrupting the function of one or more genes.
• the invention includes a chimeric embryo containing the isolated population of ES cells or genetically modified ES cells.
• the chimeric embryo contains ES cells that have been genetically modified to include a transgene.
• the chimeric embryo contains ES cells that have genetically modified to disrupt the function of one or more genes.
• the invention includes an animal containing cells arising from an isolated population of target ES cells.
• the animal contains cells arising from rat ES cells that have been genetically modified to include a transgene.
• the animal contains cells arising from rat ES cells that have genetically modified to disrupt the function of one or more genes.
• Figure 1 A shows 400 x magnification, Nomarski optics of normal rat blastocysts from the Brown Norway strain
• Figure IB shows 400 x magnification, Nomarski optics of rat blastocysts from the Fischer 344 strain where implantation was delayed 10 days
• Figure 1C shows 200 x magnification, phase contrast of a group of delayed rat blastocysts from the Long Evans strain where implantation was delayed 8 days.
• Figure ID blastocysts attached after 3 days in culture on SNL 76/7 fibroblast feeder layer.
• the inner cell mass (ICM) is visible at the center of the cultures.
• FIGS. 1 and 2 are half-tone reproductions of photographs showing cultures derived from the ICMs of rat blastocysts.
• Figures 2 A -and 2B show colonies in secondary culture of FRDB-1 cell line. The morphology of the colonies resembles mouse ES cells at this stage of derivation.
• Figure 2C shows a third passage of cell line BNRB- 1.
• the colonies have a more epithelial morphology, typical of endoderm.
• Figures 2A-C are 200 x magnification, phase contrast microscopy.
• FIG 3 panels A through E are half-tone reproductions of 200 x magnification of cultured blastocysts.
• Figures 3 A and 3C show alkaline phosphatase (AP) staining of inner cell mass cultured rat and mouse blastocysts, respectively.
• Figure 3B shows rat blastocyst- derived cells (line BNRB-1) stained with AP;
• Figure 3D shows mouse ES cells stained with AP.
• Figure 3E shows AP positive cells within a colony derived from 9 day Sprague- Dawley embryo. These cells may be derived from primordial germ cells.
• Figure 4A shows a phase contrast view of Long Evans rat blastocyst inner cell mass (ICM) cells cultured for 3 days before staining;
• Figure 4B shows fluorescence microscopy of the 3 day cultured ICM cells stained with anti SSEA-1 antibody.
• Figure 4C shows mouse ES cells stained with anti SSEA-1.
• SSEA-1 is heterogeneously expressed.
• Figure 4D shows colonies from the BNRB-1 cell line stained with SSEA-1. The rat line is also heterogeneous for SSEA-1 labeling.
• Figure 5A shows a cystic structure, similar to mouse ES cell-derived simple embryoid body, formed by BRNB-1 cell line after culture in suspension.
• Figure 5B shows mo ⁇ hological changes in the BNRB-1 cell line grown on a rat embryonic fibroblast feeder layer
• Figure 5C shows morphological changes in mouse ES cells grown on a rat embryonic fibroblast feeder layer.
• Figure 6, panels A through D are half-tone reproductions of lOx magnification, phase contrast photographs showing the mo ⁇ hology of cell co-cultures of rat blastocysts •and mouse ES cells.
• Figure 6A depicts ICM from a delayed Long Evans rat blastocyst after three days of culture on mouse AB-1 ES cell feeder layer. The ICM cells look like mouse ES cells, and no differentiation is apparent.
• Figure 6B shows second passage of LE rat ICM by mechanical dissociation.
• FIG. 6C shows LE rat ICM after 7 passages.
• the ICM explant looks like mouse ES cells, but spherical cells are much more abundant. These fast- growing spherical cells are believed to be endoderm. After several more passages of the cells having ES morphology, there were very few alkaline phosphatase (AP)-positive cells remaining.
• Figure 6D shows AP staining of an early passage rat ICM. The spherical endoderm-like cells are negative.
• Figure 7, panels A through C are half-tone reproductions of photographs of rat primordial germ cells (PGC) in culture.
• Figure 7 A shows a lOx magnification phase contrast photograph of PGC cultures derived by dissociating the hindgut tissue and allantois from a 10.5 day LE rat embryo and co-culturing with mouse ES cells for two passages. After two passages (mouse ES cell confluence), the mouse ES cells were removed by negative selection. The remaining rat cells were passaged once more and the culture examined at 17 days. Surviving rat cells are apparent.
• Figure 7B shows a 20x magnification the cells in Figure 7A.
• Figure 7C shows the same colony as shown in Figures 7 A and 7B stained for alkaline phosphatase (AP) after 21 days in culture. The morphology and staining indicates that these cells have properties of ES cells.
• AP alkaline phosphatase
• the present invention provides pluripotent embryonic stem (ES) cells.
• the ES cells are typically not mouse and, although isolated rat ES cells are exemplified herein, the claimed methods can be applied to any species.
• the invention provides substantially purified populations of non-mouse, for example rat, ES cells.
• the methods of producing ES cells involve co-culturing embryo cells from a target species (e.g., inner cell masses (ICMs) from blastocysts or primordial germ cells (PGCs)) with non-target ES cells.
• the non-target ES cells can be from any species, for instance mouse.
• the non- target ES cells contain a negative selection marker.
• embryonic stem cell or "ES cell” are used to refer to cells of the early embryo that can give rise to all differentiated cells, including germ line cells. Although not yet isolated from every species, all animals are believed to have ES cells.
• Mouse embryonic stem cells (the most well-characterized ES cell) are derived from the pluripotent inner cell mass of blastocysts, and their pluripotence can be maintained by an appropriate culture environment.
• Mouse ES cells lines have been established in culture using feeder cells such as irradiated fibroblasts or cultured in medium conditioned by established teratocarcinoma stem cell lines. Some mouse ES cells can also be propagated without a feeder cell layer in the presence of differentiation inhibiting activity (DI A) or leukemia inhibitory factor (LIF) which prevent spontaneous differentiation of cells in culture.
• DI A differentiation inhibiting activity
• LIF leukemia inhibitory factor
• non-mouse and target ES cells refer to cells derived from any animal other than mouse.
• Preferred non-mouse or target cells are rat.
• non-target ES cells refers to cells derived from any animal other than the target species.
• Preferred non- target ES cells are mouse. When they .are tr.anspl.anted to host blastocysts, ES cells contribute to formation of chimeric animals, and if the germ cells of a chimera are ES cell-derived, the offspring of the chimera carry the genome of the ES cells ("germ-line transmission").
• ES cells are those which have been shown to be pluripotent as determined by assays and methods known in the art and described herein. Pluripotent cell lines are not limited to blastocyst-derived lines; recently, a cell line that possesses at least the in vitro pluripotence of ES cells was derived from mouse primordial germ cells (see, Matsui et al. (1992) Cell 70:841-847; Matsui and Hum (1997) Cell 10(l):63-8; Buehr, M. (1997) Exp. Cell Res. 232:194-207). ES cells can be easily manipulated. They are useful as models for studies of cellule differentiation, presumably because the factors they produce and secrete are important for the control of early embryonic development in vivo.
• ES cells harboring foreign DNA can be used to generate lines of true-breeding transgenic animals. Methods of genetic manipulation are known to those skilled in the art.
• An "isolated” or “purified” population of cells is substantially free of cells and materials with which it is associated in nature. By substantially free or substantially purified is meant at least 50% of the population are ES cells, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of non-pluripotent ES cells with which they are associated in nature.
• a "cell line” or “cell culture” denotes higher euk-aryotic cells grown or maintained in vitro. It is understood that the descendants of a cell may not be completely identical in mo ⁇ hology, genotype or phenotype to the parent cell.
• embryo refers to tissue obtained from any stage of an animal's development prior to birth. In the course of mammalian development, for instance, the fertilized egg cleaves to form a mulberry-shaped cluster of cells called the "morula.”
• the morula transforms into a "blastocyst" - a nearly spherical, fluid-filled structure.
• the outer cells of the blastocyst are “trophectoderm” cells and give rise to the placenta and other extraembryonic structures.
• the embryo itself is derived from the “inner cell mass” or “ICM,” an accumulation of cells at one pole of the blastocyst.
• Other embryonic tissue sources include delayed blastocysts and primordial germ cells.
• the term "target animal” or “target species” refers to the species from which the isolated ES cells of the present invention are derived.
• Suitable species include, but are not limited to, rat, human, bovine and sheep.
• selectable marker refers to a gene whose expression allows one to identify cells that have been transformed or transfected with a vector containing the marker gene. Selectable markers can be "positive” or “negative” and dominant or recessive.
• a “positive selection marker” refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced antibiotic resistant genes.
• suitable antibiotic resistant genes are neomycin resistance (Neo 1 ) which confers resist-ance to the compound G418, hygromycin resistance and puromycin resistance.
• HPRT hypoxanthine phosphoribosyl transferase
• negative selection marker refers to a gene encoding a product that can be induced to selectively kill the cells that carry the gene.
• negative selection markers include he ⁇ es simplex virus thymidine kinase (HSV-tk) and HPRT. Cells carrying the HSV-tk gene are killed when gancyclovir or FIAU (1(1,2- deoxy-2-fluoro- ⁇ -D-rabinofuranosyl)-5-iodouracil) is added and cells carrying mammalian tk are killed using 5-bromodeoxyuridine (5BdU). Cells carrying the HPRT marker can be selectively killed with 6-thioguanine (6TG).
• Other examples of selectable markers both positive and negative will be known to those in the .art.
• “Positive-negative selection” refers to the process of selecting cells that carry a DNA insert integrated at a specific targeted location (positive selection) and also selecting against cells that carry a DNA insert integrated at a non-targeted chromosomal site (negative selection).
• transgenic animal refers to a genetically engineered animal or offspring of genetically engineered animals.
• a “chimeric embryo” is an embryo that has populations of cells with different genotypes.
• a transgenic animal or chimeric embryo usually contains material from at least one unrelated organism, such as from a virus, plant, or other animal.
• the term "transgene” refers to a polynucleotide from one source that has been inco ⁇ orated into genome of another organism. The transgene can be obtained from any source, for instance, isolated from a different organism, species or synthetically produced.
• the transgene can be a gene, gene fragment or multiple genes. Suitable sizes of transgenes can be determined by methods known in the art.
• the present invention provides the first isolated population of rat embryonic stem cells.
• the invention also provides novel methods of deriving ES cells from non-mouse species. By employing novel culture conditions, the present inventor has derived isolated populations of rat ES cells from embryonic tissue. These novel methods are equally applicable to target species other than rats. Previous groups have unsuccessfully attempted to culture rat ES cells by amount and/or kind of growth factors and feeder cells added to the culture medium. No matter what the medium, previous rat cells cannot be maintained in culture without differentiating. These cultured rat cells are not transmitted into the germ lines of chimeric rats. It appears as though rat embryonic cells reach a critical stage in culture.
• the present invention overcomes this problem by co-culturing the rat embryonic cells with cells of a non-target ES cell line. Contact with the ES cell line appears to support the isolated embryonic cells through this crisis in culture. Once past this critical point, the rat ES cells will proliferate on their own and maintain their undifferentiated, pluripotent phenotype.
• ES cells can be isolated from any species. Examples described herein include ES cells derived from the inbred line of Long Evans
• LE rats available from Simonsen (inbred for 16 generations) or from Sprague-Dawley rat strains. LE rats have appropriate coat color (black, hooded) for identifying chimeras when the LE ES cell candidates are injected into an albino strain. Large numbers of embryos have been obtained from this strain and these cells adapt well to tissue culture conditions. LE animals can also be time-mated by the supplier, reducing the size of the animal colony that must be maintained and the time involved in raising animals to breeding age. Unlike the mouse, strains of rat vary greatly in timing of embryonic implantation and each reacts differently to superovulation and delayed implantation procedures. To the extent that these variations affect the present invention, they can readily be determined by methods known in the art.
• Non-mouse, target ES cells can be derived from any suitable cell.
• Non-limiting examples are blastocysts (non-delayed), blastocysts whose implantation has been experimentally delayed (delayed blastocysts), and primordial germ cells.
• PGCs can be isolated from various stages of embryonic development, for instance stage 13 embryos. For humans, cells obtained from spontaneous and elective abortions can be employed. Cells can also be obtained from embryos produced by in vitro fertilization techniques.
• Blastocysts can be isolated by any method known in the art. For example, timed- mated females can be sacrificed on about day 4.5 after mating (day 0.5 is the morning after mating), and blastocysts are collected from the uteri by the method described for mice, for example in A. Bradley in TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH (E.J. Robertson, ed., 1987).
• timed-mated females are ovariectomized when embryos are in the oviducts, approximately day 3.5 after mating.
• animals receive daily injections of progesterone (5 mg/animal/day, subcutaneous injection), as described in
• blastocysts that have not implanted are collected from the uterus using any method described in the art (e.g., A. Bradley, supra). Delayed blastocysts are usually larger than normal blastocysts, and lack the zona pellucida layer. Blastocysts and delayed blastocysts may be cultured as described herein, preferably on feeder cells in individual wells of a 24- well plate.
• Embryos produced by in vitro fertilization can be cultured to the blastocyst stage for the isolation of ICMs.
• Primordial germ cells can be also be isolated from embryos. Although the stage of the embryo tissue is not believed to be critical, in an embodiment directed to production of rat ES cells, timed mated females are sacrificed on approximately day 9.5 of pregnancy and embryos dissected from extraembryonic tissues. At this age, rat embryos are at stage 13, equivalent to the mouse on day 8.5. (stage is determined by somite number). The caudal region of the embryos, preferably from the last somite to the allantois, is dissociated into a single cell suspension with trypsin (0.5%) and gentle trituration with a micropipette. Cells are plated, for instance into in 24-well dishes with or without feeder cells as described below. At this stage, there are approximately several hundred PGCs in each rat embryo.
• the present invention employs culture conditions which promote ES cell derivation.
• the primary blastocysts from which the ES cells are derived are grown in any appropriate medium under any conditions which allow for growth and proliferation of the ES cells.
• one suitable medium is mouse ES medium (DMEM with glutamine and high glucose (Gibco) supplemented with 15% fetal bovine serum (FBS: HyClone), 1 X non-essential amino acids, 0.1 mM 2-mercaptoethanol, and antibiotics).
• Primary primordial germ cells are preferably cultured in mouse ES medium containing exogenous growth factors, for example LIF, SCF and bFGF.
• exogenous growth factors for example LIF, SCF and bFGF.
• Other growth factors which may be used will be known to those skilled in the art.
• concentrations of the growth factors can be readily determined by those skilled in the art. As described herein, 2000 U/mL LIF (Gibco); mouse SCF, 60 ng/mL; human bFGF, 20 ng/mL (Genzyme) have been shown to be effective.
• the cells can be cultured in ES cells prepared from other species, for example ES cells previously prepared as described herein. Secondary .and subsequent PGC cultures can be cultured in the same medium or a medium lacking exogenous growth factors.
• the inner cell mass In normal and delayed blastocyst culture, the inner cell mass (ICM) is visible after about three days of culture.
• the ICMs are removed under conditions that minimize contamination with other cell types, for example, after about 3 to 5 days in culture.
• the ICM is removed using a micropipette and then dissociated with 0.25% trypsin.
• the ICM is dispersed either to a single cell suspension or more preferably, gently to produce small groups of cells.
• ICM cultures are cultured in 6 well dishes and colonies arising from the dispersed ICMs will be selected by mo ⁇ hological criteria.
• Exogenous growth factors for example, bFGF, LIF, and SCF
• bFGF for example, bFGF, LIF, and SCF
• the ICM cultures are cultured on a feeder layer, for instance mitotically inactivated SNL 76/7 cells, a feeder line that produces leukemia inhibitory factor (LIF) and stem cell factor (SCF).
• LIF leukemia inhibitory factor
• SCF stem cell factor
• Feeder cells that induce differentiation should not be used.
• a critical step in deriving embryonic stem cells is culturing the embryo tissue in the presence of pluripotent embryonic stem cells. Without being bound by one theory, it seems that the co-culture method is effective because of cell contact between ES cells and because of self-conditioning of the culture medium by ES cells.
• the present inventor noted that purified growth factors are not sufficient to provide optimal maintenance of pluripotence .and proliferation of undifferentiated ES cells. For mouse ES cells to have a high probability of germ line transmission, mouse ES cells must be cultured on feeder layers, with serum. Most importantly, the inventor also noted that mouse ES cells differentiate much more easily and often when they were cultured at low density rather than high density. These observations led the present inventor to the claimed co-culturing methods.
• the embryo tissue can come from any source, preferably a non- mouse donor.
• the embryo cells are ICMs from non-delayed or delayed blastocysts.
• they are primordial germ cells.
• they are delayed blastocysts.
• At least one embryo tissue cell is used although between 1 -and 50, preferably between about 5 and 10 single cells can also be combined into a single culture.
• the selected embryo tissue cell(s) can be isolated and immediately co-cultured with the non-target embryonic stem cells.
• the embryo tissue cells are cultured in vitro for a short time before adding the non-target embryonic stem cells to the culture, for instance for approximately 3 days.
• the co-culture is established before the embryo cells begin to differentiate.
• the primary embryo tissue cultures can be cultured on a feeder layer of cells, for instance STO or SNL 76/7cells. Any known pluripotent embryonic stem cells can be used in the co-culture condition. Although potentially as few as one ES cell may be required in co-culture, more may be required, for example between 10 and 100.
• the non-target ES cells are mouse ES cells. Procedures for the isolation of mouse ES cells have been described (see, e.g., Martin, 1981; Ledermann et al., 1991 and Matsui et al, 1992).
• the non-target embryonic stem cells are mouse ES cells having a negative selectable marker gene. Accordingly, the mouse ES cells can be selectively removed from the co-cultures.
• suitable selectable markers including, for example HPRT and thymidine kinase.
• Other negative selection marker genes will be known to those skilled in the art.
• the non-target mouse ES cells are a cell line that lacks HPRT, either by knock-out or by natural mutation. Suitable ES cell lines are incapable of reverting to HPRT-positive (HAT-resistant) phenotype.
• the co-cultured cells from the target species are HPRT-positive and survive HAT treatment. As described in the
• non-target ES cells have been mitotically inactivated by gamma-irradiation.
• Another means to identify the ES cells from the target species is to use, as the source of the co-culture, blastocysts from a cross between wild-type female rats and a male transgenic, wherein the transgene is a reporter gene.
• female Sprague-Dawley rats can be crossed with a homozygous male carrying a randomly inserted, stably integrated LacZ reporter gene, under the control of a metallothionine promoter.
• the LacZ protein expression is thus inducible by metal ions such as zinc or cadmium.
• the blastocysts isolated from these crosses are then co-cultured with selectable non-target ES cells. After selectively killing the added non-target ES cells, the remaining cells can be induced to express LacZ in culture.
• the claimed methods result in rat populations that are blue (i.e. express LacZ).
• ES cells obtained using the methods claimed herein can also be assayed for ES cell phenotype.
• Typical cell surface markers expressed by ES cells include alkaline phosphatase and anti-SSEA-1.
• In vitro assays for differentiation using embryoid body formation followed by culture to produce differentiated cell types, .and retinoic acid induction of differentiation.
• the pluripotency of putative ES cells can also be demonstrated by showing the ability of subclones derived from isolated single cells to differentiate into a wide variety of cell types and by the formation of teratocarcinomas when injected into a whole animal.
• the cells can be assayed at any stage of the process.
• an "antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta. epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• Monoclonal or polyclonal antibodies can be used to detect ES cell markers.
• the anti-SSEA-1 recognizes a glycolipid on the surface of undifferentiated mouse stem cells.
• Alkaline phosphatase (AP) activity is characteristic of primordial germ cells, blastocyst, inner cell mass and ES cells.
• Figures 6 and 7 show that rat ICM and PGCs co-cultured with mouse ES cells are AP positive (see Figures 6D and 7C). Both AP assays and SSEA- 1 antibodies are commercially available.
• Pluripotent ES cells can differentiate in culture into embryoid bodies containing multiple cell types.
• ES cells are multi-layered and the embryoid bodies which result from these multi-layered cells contain endodermal, mesodermal .and ectodermal tissues and structures.
• undifferentiated cells have one epithelial layer and develop embryoid bodies composed of a single cell type, epithelial cells.
• inducing agents such as retinoic acid or 3-methoxybenzamide
• One easily observed indicator of pluripotence is formation from an embryoid body of cardiac tissue which spontaneously contracts in the culture dish.
• the ES cells described herein form embryoid bodies having a variety of cell types, including spontaneously contracting cardiac tissue. See, for example, Example 4, section B.2.
• any strain of a particular species could be used as a host blastocyst for injection of ES cells of that species.
• experiments with mouse ES cells have shown that some combinations do not work as well as others, and the choice of host can be critical to obtaining germ-line transmissions.
• a preferred host is the Fischer 344 strain, since this albino strain would allow assessment of ES cell contribution from LE ES cells by observing pigmentation.
• ES cells derived from albino rat strains e.g., Fischer 344
• the Fischer 344 albino animal lacks coat color markers to detect contribution of ES cells in a chimera when injected into an albino host blastocyst, so promising cell lines will be injected into blastocysts of a pigmented strain, for example, Long Evans or Dark Agouti.
• the coat color markers are a convenience rather th.an a necessity, since ES cell-derived cells could be detected by other means.
• a marked cell line can be created by introducing ⁇ -galactosidase or tyrosinase plasmids; ⁇ -galactosidase is detectable by staining, and has the advantage of allowing examination of chimeras as embryos.
• the tyrosinase gene would convert the albino cells to cells capable of producing pigment, allowing direct visualization of coat color chimerism.
• Methods for ES cell transfection are described herein and are known in the art, ⁇ -galactosidase and tyrosinase plasmids are commercially available. The advantage of this approach is that any animal strain could be used for the blastocyst host, including the strain from which the ES cells originated.
• ES cell technology The strength of ES cell technology is the ability to genetically modify cells in culture, analyze the cells for the correct modification, then produce a new line of animals from the cells.
• methods for introducing genetic modifications and for analyzing the ES cells have developed rapidly in the last several years.
• Gene targeting strategies allow inactivation of specific genes by homologous recombination. Selection techniques have been developed to improve identification of rare targeted events and to introduce subtle mutations, (see, e.g., Hasty et al. (1991) Nature 350:243-246; U.S. Patent No. 5,629,159).
• Efficient methods for ES cell analysis, using microtiter plates and rapid DNA preparation techniques allow screening of thousands of clones for extremely rare events.
• ES cell technology has recently generated an important breakthrough in transgenic animal research.
• ES cells were used for producing transgenic mice which express large genomic DNA fragments cloned in yeast artificial chromosomes (YACs).
• YACs yeast artificial chromosomes
• A. Identification of genes and genetic pathways involved in AD Human and rat ES cells can be made into neuroblasts that differentiate to neurons in culture, by manipulation of the culture conditions (e.g., adding retinoic acid, removing serum from the medium, changing culture substrata, adding specific growth or growth- inhibition factors, or ligands for cell surface receptors).
• the cells can be made into embryoid bodies, which enhances certain kinds of differentiation, then treated with other factors that enhance neuronal differentiation.
• neuroblasts or neurons can be selected from other cell types by first transfecting the ES cells with a transgene in which a neuron- or neuroblast-specific promoter drives expression of a selectable marker, a novel cell surface macromolecule, or a reporter gene (such as Green Fluorescent Protein or LacZ).
• a selectable marker such as Green Fluorescent Protein or LacZ
• a reporter gene such as Green Fluorescent Protein or LacZ
• novel cell surface macromolecule In the case of cells trnasfected with a novel cell surface macromolecule gene, expression of the novel cell surface macromolecule would serve to allow selection of neurons by a number of means including, but not limited to, antibody binding, adhesion to substrata and by providing a fluorescent tag.
• the cells expressing the reporter gene can be collected by FACS. Neurons or neuroblasts can also be separated from other cells by density gradient centrifugation or by using another intrinsic property that distinguishes them from other cells.
• a gene expression profile of the ES-derived cells can be made by mRNA detection methods including, but limited to, Northern blot analysis, RNAse protection, reverse- tr.anscription PCR and cDNA expression arrays or microarrays of expressed sequence tags
• ESTs oligonucleotides or cDNA.
• the mRNA can be first amplified, preferably by a linear amplication method. Genes that are expressed in neurons but not in their ES cell precursors are candidates for neuron-specific drug targets. Further analysis of candidate genes is done by performing genetic manipulations of the precursor ES cells. For example, genes known to be involved in AD, such as the preselinins, apolipoprotein E, amyloid precursor protein (APP), can be "knocked out" by gene t-argeting through homologous recombination. Since the ES cells are diploid, both copies of the gene can be modified by selecting for gene conversion events or by targeting both alleles. In another example, the same genes are modified in situ by "knock-in” methods such as the cre-lox procedure and the hit and run procedure. Modification of protein domains and
• genes are added, as cDNA constructs or as large genomic transgenes (e.g., BACs and YACs from genomic libraries).
• Human genes are added to the rat cells, as well as to the human cells.
• the genes to be modified are not limited to AD-related genes, but could include any gene that is of interest.
• a protein expression profile of the cells can be made using, for example, immunoblot analysis, ELISA, two-dimentional polyacrylamide gel electrophoretic analysis and histochemical .analysis.
• Specific proteins that are of immediate interest with regard to AD are fragments of APP such as soluble APP and A-beta amyloid.
• Cells expressing these genes, for example, and other genes associated with AD can be used to identify drug candidates and to test hypotheses about the specific interactions of genes, signaling factors, .and proteins in neurons and in AD. Such cells can provide a picture of gene expression and its control in neurons.
• Drugs that are designed to directly affect neurons can be tested in human and rat ES cell-derived neurons. The results have predictive value both for preclinical animal studies and for clinical trials. Drugs that work through other cell types can be tested by co- culturing rat or human ES cell derived neurons with glial cells, such as astroglia, microglia, and oligodendroglia. The cell types for co-culture are obtained, for example, as primary cultures, cell lines and by deriving them from the human ES cells. Toxicity of drugs can also determined by using similar cultures. The effects of drugs are assessed by a variety of assays including, but not limited to, biochemical, immunochemical, or gene expression assays.
• ES cells can be modified genetically to examine the effects of single nucleotide polymo ⁇ hisms (SNPS) or larger genetic differences on drug efficacy and direct toxicity.
• SNPS single nucleotide polymo ⁇ hisms
• the differentiated cells are included in models of the blood brain barrier (BBB), to test the effectiveness of drugs that must cross the BBB.
• BBB blood brain barrier
• neurons and neuron-associated cells derived from human ES cells are repair of tissues damaged by neurodegenerative disease and injury.
• striatal grafts of dopaminergic neurons for Parkinson's disease for Parkinson's disease (PD), repair of brain regions damaged by ischemic stroke, and to bridge spinal cord lesions.
• Additional uses include transplantation of neurons into regions of the brain or body affected by: AD, peripheral neuropathy, ALS (amyotrophic lateral sclerosis), and other neurodegenerative diseases such as ataxias (trinucleotide-repeat diseases).
• the cells to be transplanted include neurons and neuron-supportive cells, such as glia or fibroblasts, or combinations of cells that support each other in a transplant.
• Such cells for implant are genetically unchanged, or are modified to produce specific neurotransmitters, such as dopamine for PD, or specific growth factors to support themselves or other cells.
• specific neurotransmitters such as dopamine for PD, or specific growth factors to support themselves or other cells.
• the ES cells can be genetically modified to express different HLA types, to express molecules that mask the cells from the host immune system and/or by knocking out antigenic macromolecules.
• the transgenic ES cells described herein can be used for in vitro screening or testing of compounds.
• the ES cells expressing genes involved in drug metabolism, such as the p450 gene can be used in determining the effects of compounds on development and/or differentiation.
• the ES cells express the human p450 gene.
• the genetically modified ES cells described herein can be also used to create animal models of disease, useful in in vivo screening of potential therapeutics. Such animal models can include "humanized" rats (or other commonly used laboratory species). These "humanized” animals are created from ES cells which have been genetically modified to carry human genes associated with disease states. Such a generated model animal is useful for the post-discovery, pre-clinical phase of drug development.
• BN Brown Norway rats of breeding age was established. Approximately 40 BN rats were housed in microisolator cages and replaced as animals were used in experiments. For development of the primordial germ cell procedures (see below), timed-mated Sprague Dawley rats (SD; Simonsen) were used. Timed-mated animals (F344 and Long-Evans) from a local supplier (Simonsen) were also used. The Long-Evans strain from Simonsen is a hooded rat that is about 50% black and 50% white. Although traditionally an outbred strain, Simonsen animals are effectively inbred, having been derived through 16 generations of brother-sister matings. For blastocyst injection, techniques were developed for producing large numbers of blastocysts from the Fischer 344 strain.
• the F344 strain and the Simonsen LE rat produced large numbers of blastocysts and worked well for experimental procedures.
• the LE animals were not homogeneously pigmented, but provided the coat color markers required for assessment of chimeras.
• the AGUS strain and AB2 rats were chosen since they are nonhooded albino strains.
• Adult animals of these strains were not commercially available in sufficient numbers to use for the short term project. Instead, the methods for blastocyst injection were developed using F344 animals, for which an abundant source of adult pathogen-free animals (Simonsen) were available.
• host blastocysts from the F344 strain should allow easy visualization of ES cell contribution in chimeras.
• the culture medium used in most experiments was based on mouse ES cell medium, .and contained high glucose DMEM (Gibco) supplemented with 15% fetal bovine serum (Hyclone), IX nonessential amino acids (Gibco), and 2-mercaptoethanol (0.1 mM).
• Conditioned medium was prepared from Buffalo Rat Liver cells (BRL: ATCC), mouse
• AB-1 ES cells (provided by A. Bradley), and a rat blastocyst-derived cell line we derived (BNRB-1).
• Medium was conditioned by 2 days of culture with the cell lines, then filtered and frozen for future use. For experiments, the conditioned media were used at 50% mixed with fresh medium.
• Exogenous growth factors used were leukemia inhibitory factor (LIF: 1000-2000 U/mL; Gibco/BRL), basic fibroblast growth factor (BFGF: 20ng/mL;
• Genzyme stem cell factor
• SCF stem cell factor
• Fig 1 Normal and delayed blastocysts (shown in Fig 1) were placed on fibroblast feeder layers made from LIF-producing mouse fibroblasts (SNL76/7) or embryonic rat fibroblasts and cultured for 3 to 7 days. All of the blastocysts attached and in almost all the inner cell mass (ICM) was visible (Fig Id).
• Culture medium LIF; LIF and SCF, or LIF, SCF, and bFGF
• the center mass of cells was usually removed from each culture 3-5 days after blastocyst culture, dissociated and subcultured on the same feeder type in the same medium.
• the secondary cultures always contained colonies of various mo ⁇ hology, including some that resemble the compact colony mo ⁇ hology of mouse ES cells (Fig 2). After about a week in secondary culture, colonies that resembled ES cells were dissociated and subcultured. The subcultured cells were passed once and then frozen. Cells lines were derived from a non- delayed Brown Norway blastocyst (BNRB-1), a Fischer 344 delayed blastocyst (FRDB-1) .and a Long-Evans strain. Two types of feeder layers were used for these experiments. SNL76/7 is a mouse fibroblast line used as feeder layers for mouse AB-1 ES cells (Soriano et al, 1991).
• Rat embryo fibroblasts were prepared from rat embryos by a method used for mouse embryos and described, for ex-ample, in Doetschman et al. (1985) J. Embryol Exp. Morph. 87:27-45.
• the feeder cells were mitotically inactivated by treatment with mitomycin C. Mitomycin C was toxic to the REF cells at the 10 ⁇ g/ml concentration used for SNL cells, so the concentration was reduced to 4 ⁇ g/ml, and the cells were treated for a shorter time.
• Cells were cultured on feeder layers in 6-well or 24-well tissue culture plates, glass coverslips, or glass culture slides (LabTek). Alternatively, feeder layers can be mitotically inactivated with gamma-irradiation (see, Robertson, supra).
• Primordial germ cells were cultured from tissues obtained from dissections of 9.5 day embryonic rats from Sprague-Dawley and Long-Evans (LE) strains using the methods reported for mouse, and stained the cultures for AP as described below. Several of the cultures contained colonies of cells that stained with AP (Fig 3e), and are therefore likely to be PGCs.
• Pluripotent ES cells were derived by co-culturing the rat cell lines described above with mouse ES cells.
• Rat ICMs for non-delayed blastocysts were cultured for 3 days on STO feeder layers. After 3 days, approximately 20-50 cells were present in the ICMs.
• the rat ICMs were mixed with a mouse ES cell line called Del 19.2. This line, which has a "knock-out" of 19.2 kb in the single (X-linked) copy of the HPRT gene, was obtained from Dr. Allan Bradley. Del 19.2 cells are HPRT- do not survive in HAT medium and are incapable of reverting to a HPRT+ phenotype.
• the rat ICM:mouse ES cell co-cultures were passed when confluent at least two times, with trypsin. After between about 5 days and 2 weeks, HAT medium was added to kill the mouse Del 19.2 cells. The rat cells survive HAT treatment because they are HPRT-positive. Hundreds of surviving colonies were obtained. As described in detail below, these colonies maintained markers of pluripotent ES cells.
• Ex-ample 4 Characterization of rat cells before and after co-culture Rat cells were characterized by various methods both before and after co-culture with ES cells. One cell type dominated in the first cell line derived (BNRB-1) in the absence of co-culture. These cells were round, retractile, and had a loose attachment to the substratum (Fig 2C).
• Alkaline phosphatase (AP) activity is characteristic of primordial germ cells, blastocyst inner cell mass, and ES cells of mouse.
• the inner cell mass rat blastocysts has
• AP activity was consistently observed in cultures of BNRB-1 cells (Fig 3B), but only in a small proportion of the cells.
• the staining was inhibited by levamisole (Vector Labs), which also inhibited AP activity in the control mouse ES cells.
• a quantitative assay was developed to provide a clear measure of the proportion of AP-stained cells. As shown in Table 2, after multiple passages the proportion of AP- positive cells in the BNRB-1 line was only 5%. The FRDB-1 line was approximately 50% positive. Mouse ES cell cultures (AB-1) were more th-an 90% positive. Rat cells were analyzed after co-culture by staining the colonies in the culture dish. The proportion of AP positive cells appeared to be almost 100% (see Figures 6 and 7).
• the BNRB-1 cells prior to co-culture showed an ability to differentiate in vitro, but they did not show the extent of pluripotence of mouse ES cells.
• the rat cells formed aggregates that developed into cyst-like structures (Fig 5A). These structures resemble the simple embryoid bodies bounded by endoderm that form initially by mouse ES cells; unlike the mouse cells, however, the rat embryoid cysts did not continue to differentiate into more complex structures. In most cases the cysts appeared to be bounded by a single epithelial layer, but some developed multiple layers.
• mouse embryoid bodies differentiate into a wide variety of cell types; the rat embryoid cysts failed to differentiate further when replated. This limited differentiation is consistent with idea that the dominant cell type in the population is differentiated endodermal cells.
• BNRB-1 cell colonies underwent mo ⁇ hological differentiation.
• mouse ES cells lost their AP stain and differentiated into a mo ⁇ hologically distinct cell type that resembled epithelial cells (Fig 5C).
• Fig 5B some of the rat cells on rat embryo fibroblast feeder layers showed less staining for AP and formed colonies that resembled epithelial cells (Fig 5B). Similar results were seen when the rat cells were cultured in retinoic acid, which induces differentiation in mouse ES lines (not shown).
• the isolated and resuspended rat ES cells differentiated into a number of mo ⁇ hologically different cell types, including a "beating" mass of heart tissue.
• the ability to form beating heart and other embryoid bodies is indicative of pluripotent ES cells.
• the cells are then implanted into an immune deficient host animal (e.g., nude rats or nude mice) to determine whether they formed teratomas.
• an immune deficient host animal e.g., nude rats or nude mice
• the cells are subcloned and karyotyped -and are injected into host blastocysts.
• blastocysts were obtained from PVG rats at day 5 after mating and plated into a single organ culture dish in 1 ml of ES cell medium (same as described earlier but with 20%) fetal bovine serum) containing 2000U of mouse LIF.
• the dish contained a mitotically inactivated STO cell feeder layer. Blastocysts were cultured for 3 days, when clusters of ICM cells were evident. The ICM cells were removed from the dish with a glass pipette, .and incubated for 30 min. in a solution of calcium and magnesium-free PBS containing ImM EGTA.
• the ICMs which dissociated into small clumps of cells, were placed in a 6-well culture dish on a feeder layer in ES medium (without LIF). To the same well were added 1.5 x 10 5 mouse ES cells (HPRT- Del 19.2 cells). A control well contained an equal number of mouse ES cells but no rat cells. After 3 days, the cells in both wells were dissociated with trypsin (0.25% in 1 mM
• the media changes were made often because the products of dying cells can often damage surviving cells in the same dish.
• the cultures were examined carefully with a microscope.
• the control culture contained STO feeder cells, but no visible embryonic stem cells, indicating that all of the mouse HPRT- cells had been killed by the HAT medium.
• the experimental culture originally containing rat ICMs, contained numerous colonies of cells.
• One of the 6-wells (“A") contained 38 colonies containing an estimated 100-500 cells; of these, three appeared to consist entirely of the differentiated cell type that has been previously described as "endoderm”, six colonies consisted of mixtures of cellular mo ⁇ hologies, and the remaining 29 consisted solely of cells that looked indistinguishable from mouse ES cells at the microscopic level.
• Well “B” contained 30 colonies; 7 were "endodermal”, three were mixed, and 20 consisted of ES-like cells. The colonies were stained for AP, and all colonies containing ES-like cells were positive for
• Rat ES cells isolated as described are tested for pluripotence in vivo by blastocyst injection.
• Long Evans rat ES cell are injected into host blastocysts from the albino Lewis strain. The blastocysts are allowed to develop to term in a surrogate mother and the pup examined. The pup has patches of brown coat and eye color, indicating contribution from the Long Evans ES cells.

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