Stem cells are best described in the context of normal human development which begins when a sperm fertilizes an egg and creates a single cell that has the potential to form an entire organism. The fertilized egg is"totipotent", i. e. its potential is total. Approximately four days after fertilization and after several cycles of cell division, these totipotent cells begin to specialize, forming an hollow sphere of cells, called a blastocyste. Inside the hollow sphere, there is a cluster of cells called the inner cell mass. These inner cell mass cells are"pluripotent"-they can give rise to many but not all types of cells necessary for fetal development. The pluripotent stem cells undergo further specialization into stem cells that are committed to give rise to cells with a particular function. These more specialized stem cells are described as"multipotent". The latter stem cells are still present in the adult human body, but their potential to develop is less than those of pluripotent embryonic stem cells. Examples of adult stem cells include certain bone marrow cells that give rise to all blood lineages but can turn also into liver cells or cardiac muscle cells. Neural stem cells give rise to neurons and glial cells but can turn also into heart, lung or liver cells.

A potential application of human pluripotent cells is the generation of cells and tissue that could be used for so-called"cell therapies". Many diseases and disorders result from disruption of cellular function or destruction of tissues of the body. Varied organisms like e. g. salamanders, starfish, polyps and zebrafish can regenerate new heads, limbs, internal organs or other body parts if the originals are lost or damaged. Regenerating organisms take one of two approaches to replace a lost body part. Some, such as flatworms and the polyp Hydra, retain populations of stem cells throughout their lives, which are mobilized when needed. Unlike adult stem cells in human tissues, which are thought to have a relatively restricted capacity for developing into different cell types, these cells retain the ability to re-grow many of the body's tissues. Other organisms including newts, segmented worms and zebrafish, convert differentiated adult cells-which have stopped dividing and

form part of the skin, muscle or another tissue-back into stem cells. This process is known as dedifferentiation.

In humans, ailing or destroyed tissue is often replaced by donated organs and tissues.

Unfortunately, the number of people suffering from these disorders far outstrips the number of organs available for transplantation. Pluripotent stem cells, stimulated to develop into specialized cells, offer the possibility of a renewable source of replacement cells and tissue to treat myriad of diseases, conditions, and disabilities including Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, skin alterations and diseases like e. g. burns or wrinkles, heart disease, diabetes, osteoarthritis and rheumatoid arthritis. However, if the full potential of donor embryo-derived cells lines is to be realized, the problem of donor-patient matching will need to be addressed. One approach, which avoids the problem of transplant rejection would be to generate stem cell lines from the patient's own cells through therapeutic cloning. The use of such so-called reprogrammed adult cells for cell therapies would reduce or even avoid the practice of using stem cells derived from human embryos of fetal tissue, sources that trouble many people on ethical grounds.

Several studies have shown that it is possible to re-program or dedifferentiate even mammalian somatic cells by taking the nucleus from an adult cell, transfer the nucleus to an oocyte devoid of maternal chromosomes, and achieve embryonic development directed by the transferred nucleus. The described technique named"nuclear transfer"was first performed with amphibians (Gurdon and Laskey, J. Embryol. Exp. Morphol. 1970 Sep ; 24 (2) : 227-48) but has more recently been established in mammals as well. For example, Willadsen (Nature 1986 Mar 6-12; 320 (6057): 63-5) described a procedure to investigate the development of sheep embryos in which whole blastomeres from 8-and 16- cell embryos were combined with enucleated or nucleated halves of unfertilized eggs. Robl et al performed nuclear transplantation in bovine embryos (J. Anim Sci. 1987 Feb; 64 (2): 642-7) and re-programming was successfully done in nuclear transplant rabbit embryos (Stice and Robl, Biol Reprod. 1988 Oct; 39 (3): 657-64).

These remarkable phenomena clearly indicate genetic programs that define cell differentiation are reversible and egg cytoplasm contains factors and activities to reprogram even terminally differentiated somatic nuclei so that they dedifferentiate and acquire pluripotency/totipotency required for normal development. However, the identities of these reprogramming factors are currently still to be defined.

The presently available methods for reprogramming of differentiated cells have distinct disadvantages. The transfer of somatic nuclei to embryos is technically very demanding in any animal and in humans it raises massive ethical objections. Cell-cell fusion on the other hand leads to aneuploid cells containing aberrant chromosome numbers, which are inherently different in character from normal cells and may be cancerous. Furthermore, they are immunologically non-compatible with the host, because one full set of chromosomes in the cell hybrid is of embryonic stem cell origin, typically derived from a foreign donor. Therefore it would be beneficial if other methods could be developed for converting differentiated cells to embryonic cell types, especially given their potential for producing different differentiated cell types for therapeutic use.

Recently it was shown that somatic cell extracts from primary human T cells or from a transformed T-cell line can be used to reprogram gene expression in differentiated nuclei such as 293T fibroblasts (Hakelien et al. Nat Biotechnol 2002,20 : 460-6). Although in these experiments the fibroblasts acquired a phenotype characteristic for T cells, not embryonic cells, these experiments raise the possibility that an analogous method could be feasible to achieve embryonic reprogramming. WO 01/00650 discloses a method for re- programming mammalian somatic cells by the introduction of cytoplasm from bovine oocytes. However, the disclosed methods bear the disadvantage that bovine cytoplasm required for successful re-programming is only available in minute amounts since the oocytes are relatively small and difficult to obtain in larger quantities, as would be necessary for therapeutic use. Furthermore, in the described art, the cytoplasm is introduced into the recipient cell by complicated and, thus, expensive techniques like e. g. microinjection.

It is an object of the present invention to provide a method for reprogramming cells which avoids the drawbacks of the methods known in the art. The process should be suitable for use on a large scale and allow for an easy and fast introduction of the cytoplasm.

Preferably, the method should comprise a simple contacting step of the cells to be reprogrammed with the cytoplasm possessing reprogramming activities.

Accordingly, in a first aspect of the present invention there is provided a method for reprogramming a recipient cell comprising the following steps: (i) contacting the recipient

cell with the cytoplasm of a donor cell from an amphibian or from a bony fish and (ii) detecting the expression of embryonic markers in the recipient cell.

Reprogramming of a recipient cell can be achieved by inducing the expression of embryonic markers which render a differentiated cell into an undifferentiated cell of an early developmental stage. Thus, an object of the present invention is also a method for inducing the expression of embryonic markers in a differentiated cell, the method comprising (i) contacting the recipient cell with the cytoplasm of a donor cell from an amphibian or from a bony fish and (ii) detecting the expression of embryonic markers in the recipient cell, wherein the donor cell is totipotent, pluripotent or multipotent and wherein the contacting of step (i) is performed with the entire recipient cell and not only with the nucleus.

As used herein, "contacting"means bringing the recipient cell and the cytoplasm of the donor cell in contact, namely under the time and temperature conditions being required for the reprogramming process. In this respect, contacting is often referred to "incubating".

It has to be noted furthermore, that"contacting the recipient cell"means contacting the entire recipient cell and not only parts of it such as single organelles like e. g. the nucleus, as it has been disclosed in WO-A 02/26959.

In the context of the present invention, the amphibian belongs to the orders of Anura, Urodela or Gymnophiona, namely, for example, to the families (a) Bufonidae, such as Atelopus varius, Buff alvarius, Bufo americanus, Bufo bufo, Bufo calamita, Bufo canorus, Bufo cognatus, Bufo fowleri, Bufo houstonensis, Bufo marinus, Bufo periglenes, Bufo quericus, Bufo terrestris, Bufo veraguensis, Bufo viridis, Bufo woodhousei; (b) Centrolenidae, such as Centrolenella fleishmannii ; (c) Dendrobatidae, such as Dendrobates auratus, Dendrobates azurus, Dendrobates histrionicus, Dendrobates lehmannii, Phyllobates bicolor ; (d) Discoglossidae, such as Bombina bombina, Bombina variegata, Alytes obstetricanus; (e) Hylidae, such as Acris crepitans Agalychnis callidryas, Corythomantis greeningi, Cyclorana platycephala, Gastrotheca ceratophrys, Hemophractus johnsoni, Hyla andersonii, Hyla arborea, Hyla avivoca, Hyla chrysoscelis, Hyla cinerea, Hyla gratiosa, Hyla regilla, Hyla squirella, Hyla versicolor, Litoria caerulea, Litoria chloris, Osteopilus septentrionalis, Pseudacris crucifer, Pseudacris regilla, Pseudacris triseriata; (f) the Hyperolidae family, such as Hyperolius viridiflavus ; (g) Leptodactylidae, such as Leptodactylus pentadactylus, Syrrhophus marnocki, Telmatobius sp.; (h)

Microphylidae, such as Gastrophryne carolinensis, Gastrophryne olivacea; (i) the Myobatrachidae family, such as Mixophyes schevilli, Rheobatrachus silus; (j) Pelobatidae, such as Pelobates fuscus, Megophrys nasuta, Scaphiopus bombifrons, Scaphiopus couchii, Scaphiopus holbrooki, Scaphiopus intermontanus (k) Pipidae, such as Pipa pipa, Pipa sp., Xenopus laevis, Xenopus tropicalis ; (1) Pseudidae, such as Pseudis paradoxus; (m) Ranidae, such as Mantella aurantiaca, Rana arvalis, Rana aurora, Rana dalmatina, Rana esculenta, Rana ridibunda, Rana blairi, Rana cascadae, Rana catesbeiana, Rana clamitans, Rana grylio, Rana heckscheri, Rana okaloosae, Rana palustris, Rana pipiens, Rana septentrionalis, Rana sphenocephala, Rana sylvatica, Rana temporaria, Rana virgatipes; (n) Rhacophoridae, such as Rhacophorus schlegelii, Rhacophorus nigropalmatus; (o) Rhinophrynidae, such as Rhinophrynus dorsalis ; (p) Ambystomatidae, such as Ambystoma annulatum, Ambystoma californiense, Ambystoma gracile, Ambystoma jeffersonianum, Ambystoma laterale, Ambystoma maculatum, Ambystoma opacum, Ambystoma talpoideum, Ambystoma texanum, Ambystoma tigrinum, Ambystoma tremblai ; (q) Amphiumidae, such as Amphiuma tridactylum ; (r) Cryptobranchidae, such as Cryptobranchus alleganiensis ; (s) Hynobiidiae, such as Hynobius nigrescens; (t) Plethodontidae, such as Aneides lugubris, Desmognathus fuscus fuscus, Desmognathus ochrophaeus, Ensatina eschscholtzii, Eurycea bislineata, Eurycea junaluska, Eurycea longicauda, Eurycea lucifuga, Eurycea nana, Eurycea sosorum, Eurycea wilderae ; Gyrinophilus porphyriticus, Hemidactylium scutatum, Phaeognathus hubrichti, Plethodon cinereus, Plethodon cylindraceus, Plethodon glutinosus, Plethodon richmondi, Pseudotriton montanus, Pseudotriton ruber, Typhlomolge rathbuni; (u) Proteidae, such as Necturus maculosus; (v) Salamandridae, such as Cynops ensicauda, Chioglossa lusitanica, Cynops pyrrhogaster, Euproctes asper, Euproctes montanus, Notophthalmus viridescens, Salamandra salamandra, Taricha torosa, Triturus alpestris, Triturus vulgaris, Triturus cristatus, Triturus helveticus, Tylototriton shanjing; (w) Sirenidae, such as Pseudobranchus striatus, Siren intermedia, Siren lacertina ; (x) Caecilidae, such as Afrocaecilia taitana, Gymnophis multiplicata ; (y) Ichthyophiidae, such as Ichthyophis glutinosus; and (z) Typhlonectidae, such as Typhlonectes sp.

In a preferred embodiment of the present invention, the donor cell is isolated from an amphibian belonging to the Pipidae family. It is even more preferred if the donor cell providing the cytoplasm which is used for re-programming of somatic cells is isolated from Xenopus laevis.

Also included in the present invention are donor cells derived from the class of bony fishes (Osteichthyes), e. g. from the orders of Coelacanthidae, Ceratodiformes, Polypteriformes, Acipenseriformes Lepisosteiformes, Amiiformes, Elopiformes, Anguilliformes, Notacanthiformes, Clupeiformes, Osteoglossiformes, Mormyriformes, Salmoniformes, Stomiatiformes, Myctophiformes, Esociformes, Cetomimiformes, Ctenothrissiformes, Gonorynchiformes, Cypriniformes, Siluriformes, Percopsiformes, Batrachoidiformes, Gobiesociformes, Lophiiformes, Gadiformes, Atheriniformes, Beryciformes, Zeiformes, Lampridiformes, Gasterosteiformes, Channiformes, Synbranchiformes, Scorpaeniformes, Dactylopteriformes, Pegasiformes, Perciformes, Pleuronectiformes, Tetraodontiformes.

It is further preferred for the method of the present invention that the donor cell is an oocyte, e. g. an egg, or any other cell of an early developmental stage, including zygotes, embryonic stem cells, embryonic germ cells, primitive ectoderm-like cells or any other embryonic or adult stem cells being toti-, pluri-or multipotent. In a preferred embodiment of the present invention the donor cell is an oocyte.

In the context of the present invention, the recipient cell is terminally or partially differentiated and it can be of any suitable type and organism. The type and organism of the recipient cell can be different from type and organism of the donor cell. Preferably, the recipient cell is a somatic cell. Organisms possessing suitable recipient cells include but are not restricted to any vertebrate class like e. g. mammals, birds, amphibians, reptiles and fishes, with mammals being the preferred class. The mammal can for example be from the families of felidae, canidae, and/or other carnivora, muridae and/or other rodents, suidae, bovidae, equidae and/or other ungulates, pongidae and/or hominidae with recipient cells derived from humans being most preferred.

The recipient cell can be taken from any organ within any of said organisms, e. g. from heart, liver, lung, kidney, blood, brain, eye, pancreas, skin, stomach, intestine, bladder or bones. In addition, the term"recipient cell"refers to a variety of cell types, including epithelial and endothelial cells, fibroblasts, keratinocytes, mucosa cells and other skin cell types, leukocytes, erythrocytes, platelets, dendritic cells and other blood cells, astrocytes, oligodendrocytes, glial cells and other cells of the nerve system. In general, the method of the present invention can be applied to any recipient cell, whenever a source of cells in less differentiated state is desired, e. g. if the re-growth of a damaged tissue is favored.

The cytoplasm of the donor cell can be introduced into the recipient cell by any method known to a skilled artisan, e. g. by microinjection or by import via membrane-derived vesicles.

In a most preferred embodiment of the present invention the cytoplasm of the donor cell enters the recipient cell by itself without the provision of an external substance commonly used for the permeabilization of cells or without other manipulations like e. g, injection being necessary. The cytoplasm of the specific donor cells used in the present invention permits the penetration into the cell by itself. It is believed that this is due to the presence of certain compounds in the cytoplasm which permeabilize the plasma membrane of the recipient cell or which mediate the cellular uptake by endocytosis..

Furthermore, the cytoplasm of the donor cell can be introduced into the recipient cell by using a non-ionic detergent. This is also a preferred embodiment of the present invention.

The non-ionic detergent generally includes fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty amine ethoxylate, fatty acid ethoxylate, fatty acid ester ethoxylate, alkanolamides, glycosidic tensides and aminoxides. The recipient cell is contacted with the cytoplasm of the donor cell in the presence of the non-ionic detergent under suitable buffer conditions allowing for a permeabilization of the recipient cell. Preferably the non-ionic detergent is a alkylphenol ethoxylate or a glycosidic tenside. More preferred is the use of a glycosidic tenside. Particular suitable for the transfer of cytoplasma is the use of the glycosidic tenside digitonin or a derivative thereof.

A suitable buffer, as used herein, can contain substances with buffering capacity such as HERPES, Tris, CAPSO, DIPSO, MOPS, MES, HEPPSO or PIPES, with H-EPES being preferred, varied ionic salts such as potassium acetate or sodium acetate, cheating agents such as EGTA and diverse protease inhibitors.

The successful reprogramming of the recipient cell using the cytoplasmic extract of amphibian oocytes can be detected by methods known to the person skilled in the art, e. g. by determining the expression of suitable embryonic markers in the recipient cell. Suitable embryonic markers are known to the person skilled in the art ; they include, but are not restricted to SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, germ cell alkaline phosphatase and telomerase. Preferred embryonic markers of the present invention are OCT-4, germ cell alkaline phosphatase and telomerase.

Preferably, the expression of at least one embryonic marker, more preferably of at least two, even more preferably of at least three, of at least four, of at least five, of at least six embryonic or at least seven embryonic markers is detected. Preferred combinations of embryonic markers include SSEA-3 and SSEA-4; SSEA-3, SSEA-4, TRA-1-60 and/or TRA-1-81; SSEA-3, SSEA-4, T. RA-1-60, TRA-1-81 and/or OCT4; SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT4 and/or germ cell alkaline phosphatase; SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT4, germ cell alkaline phosphatase and/or telomerase as well as other combinations thereof.

The embryonic markers can be identified by methods well known to the person skilled in the art, including comparative PCR, RT-PCR, Southern Blotting, Northern blotting, subtractive hybridization, representational difference analysis (RDA), RNase protection assay, serial analysis of gene expression (SAGE) and mRNA differential display (DD) methods performing a scan for differentially expressed genes using cDNA microarray for hybridization studies. The preferred method to detect embryonic markers in the context of the present invention is RT-PCR.

In a further embodiment of the present invention oocyte cytoplasm is fractionated by, methods known to the person skilled in the art. Egg cytoplasm contains factors and activities to reprogram even terminally differentiated somatic nuclei so that they dedifferentiate and acquire pluripotency/totipotency required for normal development. The fractions of the cytoplasm can further contain factors that mediate the permeabilization of the recipient cell. Feasible fractionation methods include e. g. salt (ammonium sulfate) fractionation, equilibrium density centrifugations exploiting e. g. percoll or sucrose gradients, chromatographic methods such as gel permeation chromatography using e. g. sephadex or sephacryl resins, capillary gas chromatography (CGC) or high performance liquid chromatography (HPLC), spectroscopic detection such as Fourier transform infrared, atomic emission detection, inductively coupled plasma detection, mass spectrometric techniques, such as e. g. matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS).

One or more of the obtained fractions can be used for reprogramming of terminally or partially differentiated somatic cells, after having screened the fractions for their reprogramming capacity according to the requirements of the present invention.

Reprogramming and/or permeabilizing factors within the cytoplasm, or the fractions thereof, can be, among others, nucleic acids, in particular mRNA, proteins, proteoglycans, proteolipids and varied small molecules. It is possible to use the reprogramming and/or permeabilizing factors as such, after isolation and as mixtures thereof.

Nucleic acids bearing reprogramming or permeabilizing activity can be detected by comparative PCR, RT-PCR, Southern Blotting, Northern blotting, subtractive hybridization, representational difference analysis (RDA), RNase protection assay, serial analysis of gene expression (SAGE) and mRNA differential display (DD) methods using cellular extracts or fractions that do not possess reprogramming or permeabilizing activities for comparison. Methods to determine the amount and presence of proteins bearing reprogramming or permeabilizing activity comprise, among others, 2D-gel electrophoresis, Western blotting, immunoprecipitation, ELISA, and RIA using cellular extracts or fractions that do not possess reprogramming or permeabilizing activities for comparison.

The invention further contemplates a screening method for the purpose of identifying reprogramming and/or permeabilizing factors comprising the following steps: (i) contacting the recipient cell with the cytoplasm or a fraction thereof of a donor cell from an amphibian or from a bony fish and (ii) detecting the expression of embryonic markers in the recipient cell.

More detailed, the screening method comprises the following steps: (i) contacting a first portion of recipient cells with the cytoplasm of amphibian or bony fish oocytes, (ii) contacting a second portion of recipient cells with a control cytoplasm, (iii) comparing the expression level of embryonic markers in the recipient cells of (i) with the expression level of embryonic markers in the recipient cells of (ii), (iv) comparing the expression pattern of the cytoplasm of amphibian or bony fish oocytes with the expression pattern of the control cytoplasm, and (v) isolating at least one factor being present in the cytoplasm which induced the expression of embryonic markers.

For comparing of the expression patterns and isolating of the factor, well-known methods which are described supra are available to the skilled artisan.

A further object of the present invention are pharmaceutical preparations which comprise an effective dose of the cytoplasm, a fraction thereof and/or at least one identified

reprogramming factor, fragment or derivative thereof, and a pharmaceutically acceptable carrier, i. e. one or more pharmaceutically acceptable carrier substances and/or additives.

The cytoplasm, a fraction thereof or a reprogramming factor thereof, respectively the medicaments containing them, can be used for the treatment of diseases which require the replacement or renewal of certain cell types. Examples of such diseases comprise Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, skin alterations and diseases like e. g. burns or wrinkles, heart disease, diabetes, osteoarthritis and rheumatoid arthritis.

In another aspect of the present invention, the nucleic acids which are identified of having reprogramming activity can be isolated by methods known to a skilled artisan and used to recombinantly express proteins encoded by said nucleic acids. The nucleic acids can thus be used for transfecting differentiated cells which are destined to be reprogrammed into a less differentiated for reasons obvious to the person skilled in the art.

Therefore, the present invention is also directed to a recombinant DNA and an expression vector which includes a nucleic acid displaying reprogramming activity operably linked to regulatory control nucleic acid which effects expression of the nucleic acid in a host cell.

The present invention is further directed to a host cell which contains such a recombinant DNA or such an expression vector.

For the purpose of overexpression in differentiated cells, the nucleic acid can be placed in expression vectors capable of expressing an encoded protein, polypeptide or peptide. Nucleic acids encoding a desired protein or polypeptide are derived from cDNA or genomic clones using conventional restriction digestion or by synthetic methods, and are ligated into vectors from which they may be expressed. Vectors may, if desired, contain nucleic acids encoding portions of other proteins, thereby providing a fusion protein.

Expression vectors include plasmids designed for the expression of proteins or polypeptides fused to or within bacterial phage coat proteins. The DNA encoding the desired protein or polypeptide, whether in a fusion, premature or mature form, may be ligated into expression vectors suitable for any host. The DNA encoding the desired polypeptide may also contain a signal sequence to permit secretion from the intended host. Both prokaryotic and eukaryotic host systems are contemplated. Preferably the host cells are of mammalian, most preferably they are of human origin.

The host cells being transformed by the methods described as well as the recombinant DNA and/or the vector can be components of a kit which is used for treatment of disorders requiring the regrowth or the renewal of cells. Examples of such disorders include diseases requiring replacement or renewal of cells, preferably Parkinson's disease, Alzheimer's disease, spinal cord injury, stroke, skin alterations and diseases like e. g. burns, or wrinkles, heart disease, diabetes, osteoarthritis and rheumatoid arthritis.

The present invention includes furthermore embryonic stem cells possessing toti-or pluropotency which have been obtained by reprogramming differentiated cells, preferably human cells, by applying the methods of the present invention. Such cells can be used for therapeutic purposes, i. e. they can be used as a source to give rise to any differentiated cell type, which can be used for cell transplantation therapies.

Furthermore, the present invention refers to the use of a cytoplasm of amphibian or bony fish oocytes for in vitro reprogramming a mammalian somatic cell. Preferably, the mammalian somatic cell is selected from the group consisting of epithelial cells, endothelial cells, fibroblasts, keratinocytes, mucosa cells, leukoyctes, erythrocytes, platelets, dendritic cells, astrocytes and oligodendrocytes. More preferably, the mammalian somatic cell is a fibroblast or a leukocyte DESCRIPTION OF THE FIGURES: Figure 2a Expression of Oct-4, Germ Cell Alkaline Phosphatase (GCAP), Natriuretic Peptide Receptor Type A (NPR-A), ß-actill mRNA in human 293T cell spheres Panel 1 shows amplification for Oc/-4 (38 cycles), panel 2 for Germ Cell Alkaline Phosphatase (GCAP, 44 cycles), panel 3 for Natri1/retic Peptide Receptor Type A (NPR-A, 41 cycles). Samples were normalized for theirfl-actit ? expression as shown in panel 4 (24 cycles). Lane 1 = 293T spheres treated with Digitonin (+) and egg extract (+); lane 2 = Digitonin +, egg extract- ; lane 3 = Digitonin-, egg extract +; lane 4 = Digitonin-, egg extract-; lane 5 = untreated control cells. Lanes 6-10 correspond to lanes 1-5, but without Superscript 11 Reverse Transcriptase in the cDNA synthesis. Lane 11 = egg extract as template, lane 12 = water control. 10y1 samples each were loaded onto 1.8% agarose gels with 1xTBE.

Oct-4 and GCAP are overexpressed in egg extract treated spheres compared to spheres not treated with egg extract and control cells. Complementary to this pattern, NI'R A is overexpressed in spheres not treated with egg extract and in control cells compared to egg extract treated spheres.

Figure 2b Expression of Oct-4, ß-actin m. RNA in mouse NAH 3t3 cell spheres The right panel shows amplification for Oct-d (47 cycles). Samples were normalized for their ßactin expression as shown in the left panel (30 cycles). For both panels: lane 1 = NIH 3T3 spheres treated without Digitonin (-) and with egg extract (+); lane 2 = Digitonin -, egg extract- ; lane 3 = untreated control cells. 10yl samples each were loaded onto 1.8% agarose gels with IxTBE.

Oct-4 is overexpressed in the egg extract treated sphere compared to the sphere not treated with egg extract and control cells.

Figure 2c Expression of Oct-4, Germ Cell Alkaline Phosphatase (GCA. P) and ß-actin mRNA in human leukocyte spheres Panel 1 shows amplification for Oc-4 (38 cycles) and panel 2 for Germ Cell Alkaline Pho. sphatase (GCAP, 44 cycles). Samples were normalized for their ßactin expression as shown in panel 3 (24 cycles). Lane 1 = leucocyte spheres treated with Digitonin (+) and egg extract (+) ; lane 2 = Digitonin +, egg extract- ; lane 3 = Digitonin-, egg extract +; lane 4 = Digitonin-, egg extract- ; lane 5 = untreated control cells. Lanes 6-10 correspond to lanes 1-5, but without Superscript II Reverse Transcriptase in the cDNA synthesis. Lane 11 = egg extract as template, lane 12 = water control. 10y1 samples each were loaded onto 1. 8% agarose gels with 1xTBE.

Oct-4 and GCAP are overexpressed in egg extract treated spheres compared to spheres not treated with egg extract and control cells.

Figure 3 Expression of SSEA-1 and SSEA-4 Figure 3 shows the expression of the Stage Specific Embryonic Antigens 4 and 1 (SSEA 4, 1) in human Leukocyte clusters under various treatments with Egg Extract and Digitonin as well as the bright field view of the same cells. Clusters were fixed with 90% Acetone (SSEA 4) or 100% Ethanol (SSEA 1) and IgG-Cy3 (SSEA 4) or IgM-Cy3 (SSEA 1) was used as a secondary antibody. As a control (last line), the first antibody was replaced by an equal amount of unspecific human Ig's. The clusters were cultivated on Mitomycin-treated STO feeder cells in DMEM + 10% FCS and transferred to a new dish every 2-3 days.

EXAMPLES : A. Production os Xenopus laevis egg extract The production of Xeeloplls laevis egg extract essentially followed a protocol by Leno, 1998. Frogs were injected with 600IU hCG at 21 : 00h the day before the preparation. The following morning eggs were squeezed in glass petri dishes, one petri dish per frog. The eggs were dejellied with 2% cysteine HCI, freshly prepared with NaOH to pH 7.8, and 10min rocking at room temperature. Afterwards eggs were washed three times with Ix Barth solution in plastic beakers, misshape eggs removed and eggs transferred to 50ml Falcon tubes on ice. Eggs were then washed two times with ice cold Extraction Buffer (50mM HEPES/KOH, pH 7.4, 50mM KC1, 5mM MgCl2, 2mM 2-mercaptoethanol, 5mM EGTA, second wash with 10µg/ml each Cytochalasin B, Leupeptin, Aprotinin, Pepstatin A). Eggs were spun at 1000rpm for I min, excessive buffer was carefully removed and the eggs transferred to 2ml Eppendorf tubes. Eggs were crushed by centrifugation at 10. 000g for 15min at 4°C, the middle layer sucked out with a pipette tip and transferred to a new tube. This centrifugation step was repeated with 16. 000g to clean the extract. Afterwards 2% glycerol was added, 20pll samples snap-frozen in liquid nitrogen and stored at-80°C.

B. Treatment of 293T, N ! R 3T3 ceHs and human tenkocytes with digitonin and Xenopus egg extract 293T or NIH 3T3 cells were grown to sub-confluency in DMEM + 10% FCS +1% Glutamine and 10% C02, cells washed out of the culture flask by PBS and transferred to 15ml Falcon tubes. Cells were then centrifuged at 500rpm for 5min (same conditions for

all further centrifugation steps) and resuspended in 1 ml ice cold Transport Buffer (20mM HEPES, pH 7.3, 110mM KAc, 5mM NaAc, 2mM MgAc, 1mM EGTA, 2mM DTT, 1µg/ml each Aprotinin, Leupeptin, Pepstatin A). 500mi human blood samples, obtained from HIV, HBV, HCV negative donors, were ca. 5 times enriched for leukocytes by removing plasma and erythrocytes after centrifugation in donor bags. The leukocyte- enriched blood sample was diluted 1 : 1 with PBS and 25m1 laid over 16ml Ficoll in a 50ml Falcon tube. After centrifugation for 10min at 2000rpm with deactivated breaks, ca. 1 ml of the purified buffy coat layer with leukocytes was removed by pipetting and transferred into a 15ml Falcon tube. The tube was filled up with PBS, centrifuged for 10min at 2000rpm, the supernatant discarded and 0. 5ml ice cold Transport Buffer added to the pellet. Cells were centrifuged again, resuspended in lml Transport Buffer + 35p, g/ml digitonin (from 20mg/ml stock solution in DMSO) and incubated on ice for 5min. Cells were centrifuged, resuspended in lml Transport Buffer, centrifuged and resuspended in 40pal Incubation Mix (2µl 20mg/ml BSA, 2µl 20mM ATP, 2yl 100mM Phosphocreatine, 2yl 400U/ml Creatiner Kinase, 2µl RNAsin, 10y1 Transport Buffer, 20ptl egg extract) for 30min at 37°C Incubation was terminated by addition of lml ice cold Transport Buffer, cells centrifuged,. resuspended in DMEM + 10% FCS +1% Glutamin, plated out in 4 cm petri dishes and kept at 10% CO2 The appearance of spheres was assessed at day 5 (day 1 = experiment) ; and documented by photography. Spheres were transferred to agarose coated petri dishes. later to prevent attachment at the bottom. Control experiments were performed without digitonin (incubation in Transport Buffer only), without egg extract (incubation in Incubation Mix with Transport Buffer replacing egg extract) or without digitonin and egg extract.

For cultivation on growth-arrested feeder cells, 300-350µl STO cells (mouse embryonic fibroblasts stably transfected with human Leukemia Inhibitory Factor (LIF), American Type Culture Collection, Manassas, Virginia, USA) were seeded in 3ml MEMa Medium into a 3cm 0 Petri dish and grown overnight. The next day, Iml MEMa + 25yg/ml fresh Mitomycin (Roche Diagnostics, Mannheim, Germany) was added and incubated for 2h at 37°C in an incubator with 10% CO2. Cells were then washed 3 times with MEMa and 3ml fresh MEMa finally added. STO cells were allowed to recover for 2h before the MEMa Medium was exchanged to the desired Medium to grow clusters or spheres, four of which were seeded onto one plate. Each 2-3 days the clusters or spheres were carefully picked by a lOut pipette tip to avoid too much carry-over of STO cells and transferred onto new dishes.

C. RT-PCR for embryonic markers Oct-4, Germ Cell Alkaline Phosphatase, Telomerase RNA prearation RNA preparation of spheres and clusters with or without digitonin treatment, with or without egg extract or untreated control cells essentially followed a protocol developed by Rappolee at al., 1989. Spheres, cell clusters and untreated control cells were harvested at day 5-18 by glass needle aspiration and transferred to 100µl Denaturing Solution (4M GnSCN, 0.5% Sacrosinate, 1% ß-mercaptoethanol, 20µg rRNA), laid over 1001l1 5.7M CsCI and spun for 3. 5h at 70. 000rpm in an Optima TL Tabletop Ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) in a TL 100.2 rotor. The supernatant was discarded in several steps with new pipette tips each time to avoid DNA contamination. The pellet was then washed in 70% EtOH, dried, resuspended in 201. t1 H20 DEPC, transferred to an Eppendorf tube and 6µl 2M NaAc pH 4.0 and 60p11 100% ice cold EtOH added. The samples were precipitated at 4°C overnight and the pellet washed with 70% EtOH.

DNAase digestion The dried pellet was resuspended in 1. PLI H20 DEPC and 0. 18y1 lOx Reaction Buffer (all reagents Invitrogen Life Technologies, Carlsbad, CA, USA) and 0. 3µl DNAse 1 were added and incubated for 15min at room temperature. The DNA. se digestion was stopped by addition of 0.18µl EDTA and heat inactivation at 65°C for 15min.

RT reactiorr Four microliter 50ng/pt1 random hexamers (all reagents Invitrogen) were added to the DNAse treated : RNA, incubated at 70°C for 10min and immediately placed on ice. Then l). d lOx PCR buffer, 1µl 25mM MgCl2, 0.5µl 10mM dNTP, 1µl 0.1M DTT, 0. 1 PLI RNAsin (40U/µl) were added and incubated at 25°C for 5min. 2pll of the sample were transferred to a new tube for RT negative control reactions; to the rest 0. 4µl Superscript 11 RT was added, incubated at 25°C for 10mil1 and at 42°C for 50min. The reaction was terminated by heating to 95°C for 5min.

PCR

PCR reactions were performed using 0. 6pl undiluted-0. 3tl 1 : 100 diluted cDNA template as determined by normalization for 3-actin expression at 24 or 30 cycles (see Figure 2a, b, c). To the cDNA in a 0. 2m1 Eppendorf tube 500nM primers (0. 5pt1 each) for Oct-4 (modified after Abdel-Rahman et al., 1995; for human cells : Inner 5' : GACAACAATGAAAATCTTCAGGAGA, Inner 3' : TTCTGGCGCCGGTTACAGAACCA ; for mouse cells : mOct4a o5' GTCTTTCCACCAGG and Inner 3'), Germ Cell Alkaline Phosphatase (Hung et al., 2001 ; GCAP 1 : CCATATCCTGAGGTGGATCAG, GCAP 2 : CAGGGAGGGAAG GTAATGAGT), Nalrils7etic Pelvtide Receptor Type A (Ortego and Coca-Prados, 1999 ; NPR-A 5' : CTTGGGGAGAGGGGGAGTA GCAC ; NPR-A 3' : GGGGGTCGGGGGAGCAGGTATTGT) and P-actin (5' : CGTGGGGCGCCCCAGGCACCA ; 3' : TTGGCCTTGGGGTTCAGGGGGG) and 47su1 of a mastermix containing Ix PCR buffer (all reagents Roche Diagnostics, Mannheim, Germany, except dNTP : MBI Fermentas, St. Leon-Rot, Germany) 2mM MgC12, 20ßM dNTP and 37. 9µl H2O were added. The samples were heated to 94°C, lpl Taq Polymerase added and 30-47 cycles performed with 94°C for 15sec, 62°C for 30sec and 72°C for 30sec followed by 72°C for lOmin and 4°C for 5min (see Figure 2a, b, c) in a PTC-200 thermal cycler (MJ Research, Watertown, MA, USA). Control reactions included RT negative samples, H20 samples and egg extract.

D. Immunohistochemistry for Stage Specific Embryonic Antigens (SSEA) 1,3, 4 and Tumor Rejection Antigens 1-60, 1-81 Next to the expression of totipotency-associated markers by RT-PCR, other markers were also assessed by immunofluorescence. These markers include Stage Specific Embryonic Antigens 1 (SSEA 1, Solter and Knowles, 1978) and SSEA 4 (Andrews et al. , 1982; Kannagi et al., 1983) as well as Tumor Rejection Antigens (TRA) 1-60 and 1-81 (Andrews et al. , 1984). While SSEA 4, TRA 1-60 and TRA 1-81 are all markers for undifferentiated cells in humans, SSEA 1 is a marker for differentiated cells.

Spheres, clusters and untreated control cells were washed in PBS, pipetted onto a SuperFrost Plus slide (Menzel Gläser, Braunschweig, Germany), and dissociated into several pieces by the edge of a second slide when necessary. After drying cells were fixed in 100% ice cold Methanol for 5min, 90% ice cold Acetone for 20sec or 100% ice cold Ethanol for 5min and slides allowed to dry on air. Slides were stored in plastic containers at-20°C or directly used for staining. Antibody reactions were performed using a dilution of the primary antibody (anti Stage Specific Embryonic Antigens (SSEA1 (1 : 150),-') (1: 5), 4 (1: 5); Developmental Studies Hybridoma Bank, University of lowa, USA) and Tumor

Rejection Antigens (TRA 1-60 (1: 5), 1-81 (1: 5); Prof. P. Andrews, University of Sheffield, UK) in dilution buffer (stock 500ml PBS, 5% FBS, 100Kl1 2% NaAzide) for 45min at room temperature, 5ll1 total volume. As a control, an equal amount of unspecific human Ig's (0. 6ng/pl) was used to replace the first antibody. After three washes with Dilution Buffer, the second antibody (anti mouse IgM Cy3 (1: 1500, Chemicon, Hofheim, Germany) or anti mouse IgG Cy3 (1: 500, Jackson Immuno Research Laboratories, West Grove, PA, USA) was applied for 2h at room temperature in 5ll1 total volume. The slides were washed again three times with Dilution Buffer and fixed with 100% ice cold Ethanol for 2-3min. After drying the slides on air, they were mounted with Fluoromount G and assessed under a fluorescent microscope. Photographs were taken with fluorescent light as well as with bright field.

Figure 3 shows the expression of SSEA 4 and 1 in human Leukocyte clusters under various treatments with Egg Extract and Digitonin as well as the bright field view of the same cells. The clusters were cultivated on growth-arrested mouse embryonic fibroblasts as feeder cells (STO cells), which are stably transfected with human Leukemia Inhibitory Factor (LIF).

SSEA 4, which is a marker for undifferentiated cells, is only detectable in Egg Extract- treated clusters. Clusters which were not treated with Egg Extract are negative for SSEA 4 as well as the negative control. Digitonin-treatment strongly enhances the rate of SSEA 4 positive cells.

SSEA 1, which is a marker for differentiated cells, is only detectable in clusters which were not treated with Egg Extract. Clusters which were treated with Egg Extract are negative for SSEA 1 as well as the negative control. Digitonin treatment does not alter the rate of SSEA 1 positive cells.

TRA 1-60 and 1-81 are only detectable in some Egg Extract-treated clusters (data not shown). Clusters which were not treated with Egg Extract are negative for TRA 1-60 and 1-81 as well as the negative control.