JP2002536976A - Pluripotent cells-1 - Google Patents

Abstract

  (57) [Summary] The present invention relates to an isolated pluripotent cell comprising at least a portion of the cytoplasm from a teratocarcinoma cell and a somatic cell nucleus. The invention also relates to a method for producing such a cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides an isolated pluripotent cell comprising at least a portion of the cytoplasm from a teratocarcinoma cell and a somatic cell nucleus;
Methods for preparing such cells; therapeutic compositions of said cells; and uses thereof.

[0002] Embryonic development in animals is a highly regulated developmental process that combines cell proliferation and cell / tissue differentiation to produce a complete organism. The synergy of cell proliferation and differentiation has been the subject of intense research, and the information derived from this has contributed to our understanding of cell function and disease. Examples are, but not limited to: regulation of gene expression, cell differentiation, oncology, teratology.

[0003] The development of mammalian embryos is significantly conserved during the early stages. After fertilization, the early embryo completes four rounds of cutting to form a 16-cell morula. These cells complete several more rounds of division and develop into blastocysts, where they can divide into two distinct regions; the inner cell mass, which forms the embryo Yes,
And trophectoderm, which will form extra embryonic tissue (eg, placenta). Cells that form part of the embryo until blastocyst formation are said to be totipotent (eg,
Each cell has the developmental potential to form a complete embryo, and all cells need to support the growth and development of the embryo).

[0004] During blastocyst formation, cells comprising the inner cell mass (ICM) are said to be pluripotent (ie, each cell has a developmental potential to form various tissues) ). Embryonic stem cells are derived primarily from two embryonic sources. Pluripotent cells isolated from the inner cell mass are termed embryonic stem cells (ES cells). Another source of pluripotent cells is derived from primordial germ cells isolated from the septum or genital bulge of the embryo 8.5-12.5 days after sexual intercourse, which ultimately differentiate into germ cells. These pluripotent cells are called embryonic germ cells (EG cells). Each of these types of pluripotent cells has a similar developmental potential with respect to differentiation into another cell type.

[0005] It is important to note that a complete embryo can be produced from a single pluripotent cell (eg, ES or EG cells). Thus, when compared to totipotent cells, pluripotent cells have an increased commitment to terminal differentiation. Until very recently, in vitro culture of human ES cells was not possible. The first indication that conditions can be determined that allow the establishment of human EC cells in culture is WO 96
/ 22362. This application describes cell lines and growth conditions that allow for the continuous expansion of markers associated with primate ES cells that exhibit a range of properties or stem cells with pluripotent properties.

Examples are, but not limited to, the following: Specific surface marker SSEA-3
(+), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+) (Shevinsky et al.,
1982; Kannagi et al., 1983; Andrews et al., 1984a; Thomson et al., 1995) and expression of alkaline phosphatase (+). Furthermore, the established primate cell line disclosed in WO 96/22362 has a stable karyotype and continues to grow in continuous culture in an undifferentiated state. The primate ES cell line also
It retains the ability to form tissue from one embryonic germ layer (endoderm, mesoderm and ectoderm).

[0007] More recently, Thomson et al. (Science 282: 1145-1147, 1998) reported that human E
The conditions under which S cells can be established in culture were announced. Also, the above properties exhibited by primate ES cells were demonstrated by human ES cell lines. In addition, human cell lines exhibit high levels of telomerase activity, a property of cells that exhibit the ability to divide continuously in culture.

[0008] Another source of pluripotent embryonic stem cells is derived from primordial embryonic germ cells (EG cells) located within the septum or genital ridge of the embryo. WO 98/43679 describes the isolation of EG cells from the gonads or gonads of the human embryo. The EG cells described in WO 98/43679 exhibit characteristics common to primate and human ES cells (eg, expression of cell surface markers, continuous growth in culture in undifferentiated state, normal karyotype and Ability to differentiate into selected tissues under defined conditions).

[0009] The use of in vitro culture of pluripotent stem cells, especially human cells, has been studied for basic research (eg, as a model for studying genetic engineering and / or tissue differentiation) and transplantation of tissue damaged through injury or disease. It is clear that it has important consequences for both and / or substitution. The establishment of an in vitro culture of human ES and EG cells is a major step towards realizing the full potential of this technology; pluripotent natural ES cells and EG cells, under controlled conditions, This is because they can differentiate into various cell types and / or tissues and organs that can have a wide variety of uses.

Examples include, but are not limited to, replacement of damaged and / or diseased coronary arteries and / or major arteries; organs with damage and / or disease (eg, kidney disease (eg, Replacement of damaged neurons (eg, Alzheimer's disease, Parkinson's disease, spinal cord injury) or cancer. Also, as will be apparent to one of skill in the art, diseases such as AIDS can benefit from tissues derived from ES cells or EG cells. T cell depletion through virus-induced cell death is a major contributor to the immune-compromised state of AIDS patients.

[0011] However, there are practical and ethical difficulties associated with the use of materials derived from human embryos. Moreover, such allogenes, when implanted in other humans, elicit a severe immune response in the host and can thus be destroyed. It has been known for many years that amphibian somatic nuclei have the ability to give rise to whole organisms when transplanted into egg cells whose nuclei have been removed or inactivated (Gurdon 1974). ). Thus, the egg cytoplasm, which can actually reprogram somatic cells to a totipotent state, must control the determination of the pluripotency of these cells.

[0012] The nuclei of mammalian somatic cells have also been shown to retain this placidity and can be reprogrammed when transferred to enucleated oocytes (Campbell et al; Wakayama). other). Moreover, nucleated mouse ES cells were shown to be able to reprogram somatic nuclei, but in this case, reprogramming was performed because heterokaryons were produced that promote cytoplasm and nucleus from both types of cells. It is difficult to determine the actual mechanism of state activity.

In all these cases, these somatic cells are reprogrammed by cellular factors derived from oocytes or ES cells, and again in humans this raises practical and ethical issues. In addition, embryonal carcinoma cells (embryonal carcinoma) derived from teratoma (feranomo)
Can reprogram somatic nuclei to produce cells with pluripotent properties.

[0014] Teratomas, ie tumors that promote a wide range of somewhat organized tissue, have been known in humans for many years. Typically, they occur as both male and female gonad tumors, but also at other sites. The gonadal types of these tumors are generally thought to be derived from germ cells, and typically have the same area of histology, the extra gonadal types mismigrated during embryonic development.
It is widely believed to result from "mis-placed" germ cells. However, the derivation of non-germ cells from persistent embryonic stem cells can be considered in some cases, especially for teratomas that occur in neonates.

[0015] Teratoma is generally classified as a germ cell tumor (GCT) because of its presumed germ cell origin in many cases, and these also include a range of other histological types such as seminiferous epithelium It develops tumors (often called anaplastic germinomas in women), embryonal carcinoma (EC), yolk sac carcinoma and choriocarcinoma. Does the GCT contain teratoma factors,
Alternatively, it may contain any combination of these tissue types, not including. The combination of teratoma and EC is often further described as teratocarcinoma. Normally, human GC
T is pure seminomas (no other histological types in it), and nonseminoma GCTs (NSGCTs), which may or may not contain teratoma factors. Which can contain a combination of Confusingly, NS
GCT also contains seminomas tumor factors.

Ovarian GCTs are most commonly benign and contain only well-differentiated somatic tissue, which can include bones, muscles, and nerves. Often, well organized tissues are found, including teeth and hair. In contrast, human testicular GCTs are always malignant and contain any combination of the aforementioned tumor histologies. Although any teratoma factor present may be less organized than a benign human ovarian teratoma, somatic cell types such as nerves, muscle, bone and cartilage may be very recognizable. Testicle
GCT is rare but has a peak incidence after adolescence and is the most common type of cancer in young men aged 20 to 35 years.

Early histopathological studies of human GCT suggest that EC cells are somatic cells that resemble early embryonic cells and give rise to all other cell types in GCT, excluding seminomas. Led to. (In the now dominant view, seminomas are more closely resembling the primordial germ cells from which these tumors develop, and may represent an early stage in tumor development and progression). In a detailed experimental study of GCTs, it was discovered that male laboratory mice of line 129 develop spontaneous testicular teratocarcinoma.

Studies in these mice show that these tumors are derived from germ cell tumors, and in fact they migrate into the germ ridges of developing embryos in laboratory mice-on days 11-13 of gestation At times, it was confirmed that it originated from primordial germ cells (PGC). Also, EC
Evidence has been provided to support the hypothesis that cells are pluripotent stem cells capable of generating the full range of differentiated cells found in teratocarcinomas. Subsequently, it was found that similar teratocarcinomas and teratomas can be derived from embryos implanted at ectopic sites.

[0019] There is no animal model for this term because seminomas were not observed in laboratory mice due to obscurity. Spermatocyte seminomas do not occur in dogs, they also occur in older men, but appear to be very different tumor types, with different etiologies to GCTs.

[0020] EC cell lines that can be maintained in vitro have been derived from several murine teratocarcinomas. Some of these EC cell lines have been found to retain the pluripotent phenotype and are either transplanted back into syngeneic mice (in which case teratocarcinomas will form) or By performing in vitro manipulations, one could be induced to differentiate into a range of cell types. Some EC
The cell line spontaneously differentiated when grown to confluence. Other cell lines required a feeder layer of irradiated or mitomycin-treated cells if they should retain the EC phenotype, and spontaneously differentiated when removed from feeder cells.

In many cases, it has been discovered that when EC cells are maintained in suspension culture for several days, they form floating cell populations, which become vesicles and begin to differentiate. These floating populations are known as embryo-like objects; if they are subsequently attached to tissue culture surfaces, a wide range of differentiated cell types, including nerves and muscles, will grow from them. There will be. Also, a number of chemical factors, most notably retinoic acid, have also been found to induce the differentiation of a number of murine EC cells into a range of cell types. The exact cell type formed in response to either of these treatments was dependent on the particular EC cell line.

Access to EC cell lines has allowed detailed characterization of their properties and patterns of marker expression. Several surface antigens, notably the "F9 antigen" and stage-specific embryonic antigen-1 (SSEA-1), have been identified as being characteristically expressed by mouse EC cells. Furthermore, these cells were found not to express the major histocompatibility (MHC) antigen of class 1 -H-2 in mice. These features, as well as their morphology and their ability to differentiate, suggest that mouse EC cells resemble cells of the inner cell mass or primitive ectoderm of early mouse embryos.

[0023] Some EC cells are transferred to blastocysts of early mouse embryos, then re-implanted into pseudopregnant mothers and differentiated when developed to term for normal embryonic development. The finding of participation confirmed the similarity. In some cases, the chimeras that develop from such an EC cell-> embryo combination are normal with a wide contribution from the EC cell components; in some cases, the chimeras subsequently develop teratocarcinomas and have This suggests that the tumor phenotype was not completely suppressed. Only in very small numbers was germ cell chimerism reported.

EC cells have been shown to have a number of characteristics that characterize ES cells and / or EG cells. EC cells are relatively easy to establish in culture, express cell surface markers associated with ES cells and / or EG cells, can be maintained in a undifferentiated state in continuous culture, and It has the potential to differentiate into selected tissues both in vitro. However, EC cells also contain one or more mutations in the gene (oncogene), which mutations give rise to the additional undesired feature that the cells retain the potential to form tumors. Is evident.

It has been known for several years that selected chemical treatments of cells in culture can result in cells protruding nuclei that form separate nuclei and cytoplasms, termed nucleosomes and cytoplasts, respectively. Have been. As is well known in the art, separated parts of a cell can be reconstituted via cell fusion.

Examples include, but are not limited to: producing a cytoplast from one cell and fusing the cytoplast to a selected cell to domain a cytoplasmic hybrid, or As described above, a cybrid can be formed.
Furthermore, the nucleus can be fused to a selected cell to form a nuclear hybrid. The nucleus fuses during mitosis after disruption of the nuclear envelope and reconstitutes after cytokinesis to form a polyploid or aneuploid nucleus. These techniques are well known in the art and will not be described in extensive detail at this stage.

We prepared cytoplasts derived from EC cells and fused the selected somatic cells with the cytoplast to form cybrids. The goal of this approach is to reprogram differentiated somatic nuclei through contact with factors located within the ECC terminus, thus allowing the cyclids to dedifferentiate and take on the characteristic features of pluripotent cells. is there. This in turn provides the basis for establishing pluripotent cell lines, which, when exposed to various differentiation factors, include, among other things, selected differentiated tissues for use in transplantation therapy. Can be produced.

Advantageously, it is not necessary to use the collected embryo cells to induce cytoplasts. Thus, any ethical organization is avoided in using the embryo in this way. Furthermore, establishing EC cells in culture is a relatively inexpensive task when compared to the problem of establishing human somatic cell cultures in vitro. Finally, when the EC nuclei are removed from the donating EC cells, the genetic material with the potential oncogene does not transfer to the so-formed hybrid cells.

Thus, it is an object of the present invention to provide pluripotent cells that are not derived from embryonic tissue from a primary source. It is another object of the present invention to provide a method for combining at least a portion of the cytoplasm of an EC cell with a somatic cell nucleus. Yet another object of the present invention is to provide a pluripotent cell capable of differentiating into a selected tissue when exposed to a suitable factor. According to a first aspect of the present invention there is provided a cell comprising at least a portion of the cytoplasm derived from at least one embryonal carcinoma cell in combination with at least the nucleus of at least one somatic cell.

In a preferred embodiment of the invention, said cells, ideally cybrids, are characterized by having at least one pluripotent property. We believe that the acquisition of this pluripotent property is the result of the reprogramming of the somatic nucleus. As will be apparent to one of skill in the art, the cells of the invention can be most preferably induced by the development of cybrids; however, another option is to use somatic cells and rc
c. Obviously, this latter option is not preferred due to the potentially oncogene genome of the donating EC cells. Preferably, the further steps of removing EC nuclei are described below.

[0031] Ideally, said pluripotent properties include the ability to differentiate into at least one selected tissue type, preferably when exposed to at least one differentiation factor. Alternatively or additionally, said pluripotent property comprises the ability of said cells to grow in a undifferentiated state in culture. In yet another preferred embodiment of the present invention, said cells are capable of growing when continuously cultured in an undifferentiated state for at least 6 months, ideally 12 months. Alternatively or additionally, said pluripotent property comprises expression of at least one selected marker.

As is well known in the art, pluripotent cells express a number of genes that are not typically expressed by differentiated cells. These are valuable tools to monitor whether the EC cytoplasm has reprogrammed the somatic nucleus. One such example is Oct4. In a preferred embodiment of the invention, the selected marker is Oct4 gene expression.

[0033] In still yet another aspect of the present invention, the selected marker is a cell surface marker. Preferably, the cell surface marker is SSEA-1 (-); SSEA-3
(+); SSEA-4 (+); TRA-1- 60 (+); TRA-1-81 (+); selected from the group comprising alkaline phosphatase (+). Alternatively or additionally, said pluripotent property comprises the presence of telomerase activity in said pluripotent cell. Ideally, telomerase activity correlates with telomere extension.

For clarity, telomerase enzymes add new repetitive DNA sequences to the ends of chromosomes. These ends are called telomeres. For example, telomeres on the human chromosome contain the sequence 5'TTAGGG3 ', which is repeated almost 1000 times at their termini. In young dividing cells, telomeres are relatively long. In old or non-dividing cells, telomeres become shorter and there is a strong correlation between telomere shortening and the ability to proliferate. Methods of increasing telomere length to increase proliferative capacity are known in the art and are described in WO 9513383. Alternatively or additionally, said pluripotent trait comprises the presence of a chromosomal methylation pattern characteristic of a pluripotent cell.

As is well known in the art, eukaryotic genomes are variably methylated by the addition of a methyl (—CH ₃ ) group attached to a cytosine residue in DNA, resulting in 5
Forms' methylcytosine (5'-mC). Methylation correlates with regulation of gene expression. Typically, genes that are hypomethylated tend to be highly expressed.
Hypermethylation correlates with reduced gene expression. As will be apparent to one of skill in the art, pluripotent cells will have a typical methylation pattern. This pattern can be analyzed at the genomic level or at the level of specific genes. Methods for analyzing the extent of methylation are well known in the art and include, but are not limited to: restriction digestion of DNA with a methylation sensitive restriction endonuclease followed by Southern blotting. Perform and probe with the appropriate gene probe (Umezawa et al., 1997).

Alternatively or additionally, said pluripotent property comprises the ability to induce a tumor when introduced into an experimental model of an animal, ideally a rodent. Even more ideally, the animal is immunosuppressed. According to a second aspect of the present invention there is provided a cell line comprising a cell according to the present invention. Ideally, the cell line is of human origin.

According to a third aspect of the present invention there is provided a method of producing a cytoplasm or portion thereof for use in producing a cell or cell line of the present invention, the method comprising the steps: i) at least one Providing two EC cells; ii) separating at least a portion of the cytoplasm from the nucleus of the EC cell; iii) isolating the cytoplasmic portion; and, optionally, iv) prior to using the isolated cytoplasmic portion. Storing. In a preferred method of the invention, said cytoplasmic part is a cytoplast.

As will be apparent to one of skill in the art, the cytoplast may be an aliquot isolated from at least one EC cell (eg, complete by micromanipulation techniques).
Aliquots extracted from EC cells) or, preferably, the cytoplasmic portion can be provided as an isolated cytoplast.

In a preferred method of the invention, said cytoplasmic portion is separated from said nucleus by exposure to a pharmacologically effective amount of at least one cytochalasin, ideally cytochalasin B. As is well known in the art, cytochalasin B is an example of a chemical that is effective in separating cell nuclei from the cytoplasm to form karyoplasm and cytoplast, respectively (Methods in Enzymology Vol. 151, pp. 221-237 19
87).

According to a fourth aspect of the present invention there is provided a method of producing a cell or cell line according to the present invention, comprising the steps: i) combining at least one EC cell with at least one somatic cell. Combining; ii) removing EC cell nuclei from the combined cells; iii) culturing the cells under conditions that promote proliferation and expansion of the cells; and, iv) adapting the cell culture as appropriate. Stored under suitable conditions.

As will be appreciated by those skilled in the art, there are micromanipulation methods that facilitate the removal of nuclei from selected cells. It is evident that this method of the invention is advantageously provided as follows: i) the continuous production of the factor produced by the EC cells, thereby requiring the reprogramming of the somatic cell nucleus. Maintain steady state levels of various factors; and ii) remove mitotic EC nuclei from the combined cells prior to mitosis so that oncogenes are not transferred. As will be apparent to one of skill in the art, the nature of the somatic cell chosen is not critical to the operation of the present invention, but should be optimized for successful production of the cell or cell line of the present invention,
Alternatively, select the cell type to maximize.

According to a fifth aspect of the present invention there is provided a method of combining at least a portion of the cytoplasm of an EC cell with a somatic cell, comprising the steps of: i) providing at least a portion of the cytoplasm of an EC cell. Ii) combining the cytoplasmic portion with at least one somatic cell; iii) growing the combined cells in culture; and, iv) storing the combined cells under appropriate storage conditions. Comprising;

In a preferred method of the present invention, said cytoplasmic portion is provided as a cytoplast. In yet another preferred method of the invention, the cytoplast is combined with the somatic cell via a cytoplast / somatic cell fusion. In the foregoing method, the EC cells and the somatic cells are ideally derived from a human. According to a sixth aspect of the present invention there is provided a cell culture comprising at least one cell according to the present invention.

According to a seventh aspect of the present invention there is provided a method of inducing differentiation of at least one cell of the present invention, the method comprising the steps of: i) providing a cell according to the present invention; Culturing the cells under conditions that promote differentiation of the cells into at least one tissue; and, optionally, iii) storing the differentiated tissues under suitable storage conditions prior to use. . Ideally, the culture conditions are selected to provide a tissue type, where tissue types include, but are not limited to: nerves, muscles (eg, smooth muscle, striated muscle, Myocardium), bone, cartilage, liver, kidney, airway epithelium, hematopoietic cells, spleen, skin, stomach, intestine.

According to an eighth aspect of the present invention there is provided at least one tissue type or organ comprising at least one cell according to the present invention. As will be apparent to those skilled in the art, differentiated tissue according to the present invention may have a wide range of uses for transplantation therapy. Examples include, but are not limited to: replacement of coronary and / or major arteries with injury and / or disease; organs with injury and / or disease (eg, kidney disease (eg, cirrhosis)
, Diabetes, the result of various autoimmune diseases); damaged neurons (eg,
Alzheimer's disease, Parkinson's disease, spinal cord injury) or cancer replacement.

Also, as will be apparent to those skilled in the art, diseases such as AIDS can benefit from tissues derived from the cells of the present invention. T cell depletion through virus-induced cell death is a major contributor to the immune-compromised state of AIDS patients. Obtaining an inexhaustible supply of T cells from non-infected somatic cells from the patient has clear advantages. Moreover, tissue rejection for the host cell's immune response appears negligible. This is because the somatic nuclei used in the hybrids are ideally derived from patients in need of tissue or organ replacement.

According to a ninth aspect of the present invention, at least one cell of the present invention and a suitable excipient,
A therapeutic composition comprising a diluent or carrier is provided. In a preferred embodiment of the invention, said therapeutic composition is provided for use in tissue transplantation.

According to a tenth aspect of the present invention there is provided a method of treating a condition or disease requiring tissue transplantation, comprising the steps: i) at least one tissue type or organ according to the present invention Ii) surgically introducing the tissue type or organ into the patient to be treated; and iii) treating the patient under conditions that facilitate acceptance of the transplanted tissue by the patient. It becomes.

According to an eleventh aspect of the invention, at least one cell according to the invention; instructions for the maintenance of said cell in culture; and, optionally, at least one desired tissue type or organ. A factor necessary for inducing the differentiation of the cell. Embodiments of the present invention will now be described, by way of example only, with reference to the following tables and drawings. Table 1 represents a summary of some of the human EC cell lines and the characteristic features of said human EC cell lines. Table 2 represents a summary of the murine EC cell lines used.

Materials and Methods Preparation of Mouse Thymocytes Thymus removed from 4-6 week old male mice (Swiss strain) was minced and 10 ml of 10
Thymocytes were obtained by suspending the released cells in medium (DMEM) containing 5% fetal calf serum (FCS). After settling large fragments of thymus for 2-3 minutes, the supernatant was removed and centrifuged at 1500 rpm for 5 minutes to pellet suspended thymocytes.

The thymocytes were resuspended in fresh medium without FCS and pelleted again by centrifugation; after this was repeated once more, the cells were resuspended in fresh serum-free medium, Counted. Human EC cells were obtained by trypsinizing confluent cultures as described previously (Andrews et al., 1980; 1982). 2 in serum-free DMEM
After washing and counting, human EC cells were mixed with mouse thymocytes at a 1:10 EC cell / thymocyte ratio. The mixed cells were pelleted by centrifugation at 1500 rpm for 5 minutes.

Heterokaryon fusion of human EC cells and mouse thymocytes and extraction of RNA Cells were fused using polyethylene glycol (PEG) (Kennett, 1979).
). Pelletization (2 × 10 ⁶ EC cells and 2 × 10 ⁷ thymocytes in experiment 1; 3 × 10 ⁶ EC cells and 3 × 10 ⁷ thymocytes in experiment 2) was performed with 200 μl of 75 mM HEPES, pH 8.0 (
Resuspended in 50% (w / v) PEG 1500 in Boehringer Mannheim, 37 ° C
For 15 minutes. Then, a serum-free medium pre-warmed to 37 ° C. was gradually added over 5 minutes. The cells were then pelleted by centrifugation at 1500 rpm for 5 minutes and resuspended in 5 ml DMEM containing 20% fetal calf serum.
The cells were then placed in T25 flasks and placed in a humidified incubator (10% CO _{2 in} air) at 37 ° C. for 2 days.

After 2 days, unbound cells were aspirated. The remaining bound cells were collected by trypsinization and washed twice in DEPC-treated PBS to remove serum. The pellet was then resuspended in Tri reagent (1 ml) to isolate RNA (Sigma-Aldrich Chemical
Co., Sigma Technical Bulletin MB-205). The isolated RNA was quantified by measuring optical density, and the absence of contaminating DNA was determined by PCR using β-actin and HPRT primers in another sample (Wa
keman et al., 1998). If no DNA was present, the RNA was then used for RT PCR analysis of Oct4 expression.

PCR amplification of Oct4 from human EC × mouse thymocyte heterokaryons In one experiment (2102Ep and thymocytes), a control was prepared consisting of cells treated similarly for fusion except that incubation with PEG was omitted— Thus, it was predicted that 2102Ep × thymocyte heterokaryon was not formed. In other experiments, RNA was isolated from thymocytes alone and from a mouse EC line (PCC4azal, clone 3) to further prepare negative and positive controls for mouse Oct4 expression.

[0055] cDNA was then produced from the sample using reverse transcription (RT) (Wakeman et al., 1998).
. Then, using an oligonucleotide primer specific for human and mouse Oct4, a marker of pluripotent cells, under standard PCR conditions described in Wakeman et al. (1998) at an annealing temperature of 61 ° C. Was carried out. Next, these products were subjected to electrophoresis to separate DNA fragments detected by ethidium bromide staining (FIG. 7). The molecular size of the amplified fragment was determined using a 1 kb DNA step ladder.

[0056]

[Table 1]

These primers were designed using the PrimerSelect module of the program Lasergene suite (DNAStar Inc., USA). Mouse primers would not be expected to amplify human Oct4.

One of the techniques used in our method of producing enucleated reprogrammed embryonic stem cells (RPES cells) of cells that give rise to “cytoplasts” and “karyopses” or “ minicells” is a cytoplasmic donor. Uses cytochalasin B to generate "nuclear bodies" (also called "minicells") from enucleated EC cells (EC cytoplasts) and differentiated or committed cells as nuclear donors That is. It is well known that cytochalasin B induces cells to extrude nuclei (Carter, 1967), and induces enucleation in a wide range of cells of various species, including both mouse and human cells (Poste, 1972; Prescott et al., 1972; Goldman et al., 1973; Wright and
Hayflick, 1973; Ege and Ringertz, 1974a; Wigler and Weinstein, 1975)
.

[0059] Such enucleation results in cells that lack nuclei but are otherwise complete and viable for many days (Goldman et al., 1973); these enucleated cells are enucleated cells (Pos
te, 1972) or the cytoplast (Veomett et al., 1974). The nucleus extruded from the cells retains the thin rim of the cytoplasm and is surrounded by the plasma membrane; these structures may be "nuclear" (Veomett et al., 1974) or "minicells" (Ege and Ringertz,
1975).

[0060] Enucleation of cells that give rise to both cytoplasts and karyotypes can be achieved by well-established techniques, wherein cells growing in combination with plastic discs are treated with a solution of cytochalasin B in a centrifuge tube. The cytoplast remains attached to the plastic disc, but the nucleolus is pelleted at the bottom of the centrifuge tube (Prescott et al., 1972). Alternatively, the suspended cells can be centrifuged through a density gradient containing cytochalasin B, typically composed of Ficoll (Wigler and Weinstein, 1975). In this case, cytoplasts and nuclei form, which can be recovered after centrifugation from different parts of the gradient.

Using the method described by Prescott et al., 1972, in a 50 ml centrifuge tube, a solution of 7.5 μg / ml cytochalasin B in phosphate buffer containing 10% fetal calf serum. Next, NTERA-2 cl D1 human EC cells growing on plastic disks were inverted and centrifuged for 30 minutes at 12,000 rpm and 37 ° C. in a J2 Bectman centrifuge using a JA20 rotor. Special cells that escaped enucleation also remain (
See Fig. 6). After overnight incubation in medium without cytochalasin, the disks were fixed in methanol and stained with hematoxylin and cosin (
Bar = 50 μm).

Methods for Combining (Fusing) the Cytoplasm of One Cell with the Nucleus of Another Cell Methods for making hybrid cells by fusing two or more cells of different origins are very well established and widely used. Are known. For an overview of commonly used methods based on Sendai virus-induced cell fusion or cell fusion induced by polyethylene glycol (PEG), see Kennett (1979).
reference.

Briefly, a mixture of cells to be fused is incubated with a fusogenic agent, such as Sendai virus or PEG, often with centrifugation or agitation to aggregate and tightly attach cell membranes; For example, time, temperature, cell concentration and fusogen concentration are optimized for each cell combination. One example of cell fusion producing heterokaryons is shown in FIG. Cell fusion, Ke
This was performed using polyethylene glycol 1000 as described by nnett (1979). After fusion, cells were seeded in tissue culture dishes and incubated overnight in fresh medium. They were then fixed with methanol and stained with hematoxylin and cosin. Several heterokaryons containing two and three nuclei can be seen in this field. (Bar = 50 μm).

These techniques also enable fusion of cytoplasts prepared by cytochalasin B-induced enucleation with whole cells or karyotypes induced by cytochalasin B-induced enucleation. (Poste and Reeve, 1971; Wright and Hayf
lick, 1975; Shay, 1977). Another technique that is now well established and widely used to induce cell fusion, "electrofusion", involves passing a short electrical pulse through a mixture of cells (Neil and Zimmermann, 1993).

Production of RPES cells Production of RPES cells requires several steps: 1. Selection of suitable differentiated cells (nuclear donors) and, if necessary, isolation of their nuclei. Selection of EC cells (cytoplasmic donor), 3. Fusion of differentiated cell nuclei with EC cells, and 4. Removal of EC nuclei before or after fusion.

In some cases, pre-treating differentiated cells or simultaneously treating differentiated cell / EC cell fusion products using various factors to enhance the possibility of reprogramming the differentiated cell nuclei Thereby, production techniques can be optimized, such factors including but not limited to inhibitors of DNA methylation. After production of RPES cells, additional methods are required to grow the cells, characterize the cells, and induce the cells to differentiate into the required somatic cell type.

Differentiated cells to be used as nuclear donors Any adult mammal or human tissue or organ, or embryo or fetus, or extraembryonic tissue, such as trophoblasts or a large range of bodies from the yolk sac Cells can be used as a nuclear source for reprogramming. Particular somatic cell types include, but are not limited to: thymocytes, peripheral blood lymphocytes, epidermal cells, eg, epidermal cells from the oral cavity, cumulus cells, or various tissues, eg, bone marrow , Other stem cells isolated from the nervous system and intestinal biopsies. Also,
This technique can be applied to various tumors, including, but not limited to, various established cell lines, eg, lymphoblast cell lines.

The somatic cells selected for use in reprogramming can be used directly at the time of isolation or can be cultured for a short time before further manipulation. In some cases, such somatic cells are completely combined with EC cells, as described below, or the nucleus or karyotype is used, for example, using factors such as cytochalasin B, as described above, or By other methods, they can first be isolated from them. For example, nuclei can also be isolated by established micromanipulation techniques, or other established cell fractionation techniques.

Parental EC Cells to be Used as Cytoplasmic Donors A large number, but not exclusively, of EC cell lines have been isolated from human teratocarcinomas that arise as testicular germ cell tumors. Examples of available human EC cell lines are shown in Table 1. Similarly, EC cell lines derived from laboratory mouse teratocarcinomas have also been derived and are readily available. Examples of available mouse EC cell lines are shown in Table 2. To date, EC cell lines have not been described from other species, but we predict that if they are derived in the future, they will behave in a manner similar to existing human and mouse EC cells that are similar to each other. I do. Thus, freshly isolated EC cells from other species or from human or mouse sources can also reprogram differentiated cells into RPES cells, as described in that proposal for human and mouse EC cells. can do.

Human EC cells can be expressed in their morphology (see FIG. 1), cell surface antigen SSEA3 (Shevinsky et al., 1982, Andrews et al., 1982, 1984b, 1996), SSEA4 (Kannagi et al., 1983, Andrews).
TRA-1-60 and TRA-1-81 (Andrews et al., 1984a, 1).
984b, 1996), the combination of features encompassing their expression typically results in SSEA
One can be easily recognized by the low expression or absence of 1 (Andrews et al., 1980, 1982, 1984b, 1996) (see FIG. 2). In contrast, mouse EC cells are typically
It expresses SSEA1 (Soltr and Knowles, 1978) while SSEA3 (Shevinsky et al., 1982)
), SSEA4 (Kannagi et al., 1983) or TRA-1-60 or TRA-1-81 (Andrews et al., 1984a).

Human EC cells express high levels of alkaline phosphatase (ALP) in mouse EC cells (Bernstine et al., 1973, Benham et al., 1981); in the case of human EC cells, the majority of ALP activity is liver / bone / Due to expression of the kidney isoform. Liver / bone /
Kidney isoforms can be detected as cell surface antigens by monoclonal antibodies TERA-2-59 and TERA-2-54 (Andrews et al., 1984c). In common with ES cells and primordial germ cells, EC cells also typically express the transcription factor Oct3 / 4 (Rosfjor
d and Rizzino, 1994; Brehm et al., 1998).

Fusion of Parental Differentiated and Parental EC Cells Producing RPES Cells Combining the cytoplasm of EC cells and the differentiated cells using several methods,
RPES containing the nuclear genome of differentiated cells, but not EC cells, can be produced. A. Chemical agents such as polyethylene glycol (PEG) or virus,
For example, cells can be fused using Sendai virus or by passing an electric current through a mixture of cells. As mentioned above, these methods are well known and can be easily applied. Using these methods,

1. fusing the differentiated cells with EC cells, or 2. fusing a karyotype from the differentiated cells with EC cells, or 3. one or more of the differentiated cells isolated from EC cells. The fusion can be with the above cytoplasts, or 4. The karyotype from the differentiated cells can be fused with one or more cytoplasts isolated from EC cells.

In cases (1) and (2), a heterokaryon containing two nuclei, one from each parent cell, will first occur. When dividing this heterokaryon, a hybrid cell containing a single nucleus with a complete or partial genome from each parent cell will result. However, in our method of producing RPES cells, the EC nucleus is removed prior to cell division of the hybrid cells, so that the derivative dividing cell population retains only the genome of the parent differentiated cells.

In cases (3) and (4), the EC nuclei are removed from the EC cells prior to fusion, for example by enucleation using cytochalasin B as described above, so that the resulting product is a differentiated cell Contains only the nucleus and cytoplasm from the EC cell parent. In each of these cases, the resulting RPES cells continue to proliferate and contain only the nuclear genome of the differentiated parental cell, which is now reprogrammed to express a new pattern of gene activity.

In cases (1) and (2), using an agent such as cytochalasin B, applied in the same manner as described above for the generation of cytoplasts for enucleation and fusion of EC cells In one of several methods, including but not limited to partial enucleation, EC cell nuclei are removed from heterokaryons. In the case of the present invention in which enucleation is performed after fusion, some heterokaryons lose both nuclei, in which case heterokaryons do not grow and some heterokaryons lose nuclei in differentiated cells. However, in this case, the heterokaryon retains the parent EC nucleus and continues to proliferate, some heterokaryons lose the EC cell nucleus, in which case the heterokaryon continues to proliferate as RPES cells, and some Heterokaryons retain both nuclei and ultimately continue to grow as hybrid cells.

Several methods are used to select RPES cells to eliminate any of the cells that carry the EC cell genome, or to eliminate cells that carry a somatic nucleus that could not be reprogrammed. I do. In one method, proliferating cells are established using established techniques (eg, picking up single cells with a micropipette-Andrews et al., 1982).
, 1984b) and individual clones are screened by genetic markers for clones carrying the EC genome. The latter cells are discarded, but do not retain the EC cell-derived genome, but retain only the differentiated cell genome and those that express the RPES phenotype. Using standard DNA genotyping (Jeffreys et al., 1985; Yen et al., 1996) using well-established DNA fingerprinting techniques, the nuclear genome of proliferating cells is the parent of EC cells or differentiated cells, or Whether it is derived from both can be identified.

In another method, the parent of an EC cell is genetically marked by insertion of a gene that allows selection for cells carrying that gene before use as a fusion partner; eg, herpes simplex virus EC cells are stably transfected with a vector encoding the -1 Tk gene (HSV1-Tk), and thus acyclovir (9- [
(2-Hydroxyethoxy) methyl] guanine) or FIAU (1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-iodouracil) (Borrell
i et al., 1988; Hasty et al., 1991) or Ganciclovia (Rubinstein et al., 1993; Mc)
By culturing in the presence of a number of agents, including Carrick and Andrews, 1992), cells carrying the gene can be killed.

In this method, after partial enucleation, the remaining heterokaryon is cultured in a medium containing the drug, and the heterokaryon that has lost the EC nucleus survives. Other selectable genetic systems can also be used. By cloning surviving cells or selecting RPES cells by markers for specific surface antigens, persistent parental differentiated cells that have not been reprogrammed are removed.

Such markers include SSEA3, S described above as characteristic markers of EC cells.
Including but not limited to SEA4, TRA-1-60 or TRA-1-81. These same markers have been shown to be expressed by ES cells directly derived from human embryos (Thomson et al., 1998; Shamblott et al., 1998). Regarding the latter approach,
Fluorescence activated cell sorting (FACS), a widely used method for separating a subset of cells, can be used (eg, Andrews et al., 1982, 1987; Acker
man et al., 1994; Williams et al., 1988).

In another method, agents that irreversibly inactivate the parent nucleus of EC cells and prevent their replication, such as topoisomerase inhibitors, such as etoposide, and EC
Incubate the cell parent prior to fusion (Downes et al., 1991; Fulka and Moor, 1
993). The resulting heterokaryon naturally eliminates this treated nucleus before cell division, so that the resulting dividing cell population only contains the genome from the parent differentiated cells. This approach can also be combined with the preceding "partial enucleation of heterokaryons" to ensure complete loss of the EC genome. In another method, after cell fusion producing heterokaryons, EC cell nuclei are removed by micromanipulation.

B. Rather than chemically, virally or electronically induced fusion, the nuclei of the differentiated cells are combined with EC cells by micromanipulation. In this method, the nuclei of the differentiated cells are extracted using a micropipette inserted through the cell membrane. It is then injected into the inoculated EC cells or into intact EC. In the latter case, the EC cell nucleus is then removed by a similar technique or by one of the techniques described above before fusion and cell division occur.

After fusion of combining RPES cell growth and selectively differentiated cells and EC cells, prior to or following removal of EC cell nuclei, the nuclei of the differentiated cells are reprogrammed and the resulting RPES cells are subsequently expanded Therefore, it is necessary to prepare appropriate conditions.

Several methods are used to enhance the efficiency of reprogramming: 1. Prior to fusion, a reversible inhibitor (eg, nocodazole) or a cell at G1 that arrests the cell cycle at a particular stage. Use conditions such as low serum, or use biological DNA lines (microfluorometry) (fluorescence-activated cell sorting) (Ashihara and Baserga, 1979; Andrews et al., 1987; Cr
Synchronizing differentiated cells and EC cells with respect to their location in the cell cycle by selecting cells at specific stages of the cell cycle using issman, 1995; Stein et al., 1995).

2. Agents that inhibit methylation or promote demethylation (eg, 5-azacytidine) (eg, Taylor and Jones, 1979; Jones, 1985; Keshet et al., 1986)
Or agents that alter the structure of chromatin, such as butyrate, spermine,
In the presence of trichostatin A or trapoxin (a drug that inhibits histone deacetylation and promotes acetylation and plays a role in X chromosome inactivation, gene impinging and regulation of gene expression) Culture differentiated cells or immediate fusion proteins (Caldarera et al., 1975; McKnight et al., 1980; S
tein et al., 1997; Hu et al., 1998; Volffe and Pruss, 1996).

3. The time between heterokaryon production and removal of EC cell nuclei is maximized without fusion of the nuclei. Heterokaryons are cultured under conditions that reversibly inhibit progress through the cell cycle (eg, thymidine block-Stein et al., 1995) or growth conditions that delay cell division but allow reprogramming to proceed, eg, This period can be extended by altering serum starvation or lowering of temperature. 4. Any or all of these combinations.

In all of these experiments, cells were cultured in standard cell media and such media include, but are not limited to: Dulbecco's modified Eagle's medium (DME, high glucose formulation) or Ham's F12, in some cases supplemented with fetal calf serum or other additives (eg, Andrews et al., 1980, 1
982, 1984, 1994). Subsequent to fusion and reprogramming, the resulting cell growth can be an optimized medium on the cell's feeder layer, such feeder layers including but not limited to: irradiation STO cells isolated or treated with mitomycin C, or embryonic fibroblasts of various species, including humans (Robertson, 1987a; Thomson et al., 1998). Including LIF, FGF, SCF,
Cells can be cultured in the presence of various growth or other tissue culture additives, including, but not limited to.

Differentiation of RPES cells In the best case, since RPES cells acquire pluripotent properties similar to those of embryonic stem cells, RPES cells differentiate and, given the appropriate conditions, initiate the differentiation pathway. Any cell type that can be found in an adult, embryo or extraembryonic tissue can be formed. As described above, the cell surface antigens SSEA3, SSEA4, TRA-1-60, TRA
The maintenance of EC cell status can be monitored by assays for various markers, including -81, expression of alkaline phosphatase and Oct3 / 4. When cultured on suitable feeder cells, RPES cells typically retain their stem cell phenotype. However, they can initiate differentiation under various circumstances.

Thus, removal from feeder cells, or suspension culture, and subsequent replating in the absence of feeder cells in a suitable tissue culture flask, can result in a variety of neurons, various types of muscle and hematopoietic cells, (See Robertson, 1987a). Alteration of conditions affecting cell density and aggregation (eg, seeding at low cell densities or trypsinization) or exposure to various factors may also lead to differentiation of pluripotent stem cells (eg, FIGS. 3 and 4). (See Figure), where such factors include, but are not limited to: retinoic acid, and other retinoids, hexamethylenebisacetamide, and bone morphogenic proteins (see below). See Robertson, 1987a; Andrews, 1984; Andrews et al., 1982, 1990, 1994,
1996; Thomson et al., 1998). The type of cells that develop will depend on the nature of the inducer and the culture conditions, including the presence or absence of the particular factor or other molecule.

DISCUSSION Pluripotent stem cell lines include early embryos (Robertson, 1987b; Thomson et al., 1995, 1998),
Germ cell tumors of various species derived from primordial germ cells (Matsui et al., 1992; Shamblott et al., 1998) and including laboratory mice, rhesus monkeys and humans (Overview, Andr
ews, 1998) and nuclei from differentiated adult somatic cells have been reprogrammed to give rise to embryonic stem cells by transplantation into enucleated oocytes (Campbell et al., 19).
96; Wakayama et al., 1998) differentiated or committed embryonic or adult somatic cells, or embryos without using oocytes, similar to embryonic stem cells having the ability to differentiate into various functional somatic cell types There are no reports that pluripotent stem cells can be produced by reprogramming extracorporeal cells.

[0091] Embryonic carcinoma (EC) cells derived from teratocarcinomas are used to reprogram various somatic cells, differentiated cells, or other embryonic or extraembryonic cell types, and then convert them to parent cells. We now describe methods that can be brought into a state that can be induced to differentiate into one or more functionally differentiated cell types that are distinguished from.

In the best case, but not necessarily in all cases, the reprogrammed cells, “reprogrammed embryonic stem cells” (RPES cells) produced by this technique are directly derived from the early embryo Similar to embryonic stem cells, neurons,
It can be induced to differentiate into a wide range of functional, differentiated cell types, including but not limited to muscle (including skeletal and cardiac muscle) and hematopoietic cells. These RPES cells are diploid with a normal karyotype and are isogenic to the differentiated parent cell from which they were derived. They are for transplantation,
And can be used to generate differentiated cells for use in cell and tissue replacement therapy.

In some cases, only partial reprogramming occurs, for example, with the activation of some genes that are inactive in parental differentiated nuclear donor cells.
Such cells are also used in a variety of these same environments.

One example of such a gene is Oct4. Oct4 was previously reported to be characteristically expressed by undifferentiated EC cells and embryonic stem cells (Brehm et al., 1998).
Therefore, to test the cytoplasmic ability of human EC cells to reprogram somatic cells, isolated mouse thymocytes were transformed into human EC cells (2102Ep, clone 4D3 (And
rews et al., 1982) or fused with TERA1 (Fogh and Trempe, 1975 Andrews et al., 1980)) to produce heterokaryons, which were tested two days later for activation of Oct4 expression from the thymocyte genome. .

Evidence of such activation is not only due to the ability of human EC cells to reprogram somatic nuclei to an ES / EC cell-like state, but also among mammalian species in which the regulators involved are different. Will show that you can work. Thus, human
If EC cells can reprogram mouse somatic cells, human EC cells can not only reprogram human somatic cells, but also
We predict that EC cells will be able to reprogram human somatic cells as well. Similarly, given the similarity of EC cells and embryonic stem cells, it would be expected that embryonic stem cells would be able to reprogram somatic cells in the same manner as EC cells.

In Experiment 1, as expected, the amplified band (573 bp), corresponding to human Oct4 expression, was expressed in RNA preparations from 2102 Ep × thymocyte fusions in the presence of PEG and non-PEG Similarly detected in mock fusions in the presence of 2102Ep human EC
Consistent with its expression by cells. However, the band corresponding to mouse Oct4 (
415 bp) was only detected in RNA preparations from a 2102Ep × thymocyte fusion in the presence of PEG when the presence of heterokaryons was expected. Mouse O from mock fusion
The corresponding absence of ct4 indicates the absence of Oct4 expression from mouse thymocytes in this experiment, and indicates that heterokaryon formation is required for its activation from the thymocyte genome by 2102Ep cytoplasm . No product was found in the "water" control, indicating the absence of contaminants.

In a second experiment, 2102Ep and TERA1 human EC cells were fused with mouse thymocytes in the presence of PEG, mouse Oct4 was only detected in the 2102Ep fusion, and again activation of Oct4 expression resulted in mouse thymocytes Although the ability to reprogram was confirmed, this experiment suggested that the TERA1 cytoplasm did not activate reprogramming.
In both cases, human Oct4 was detected as expected and 2102Ep or TERA
One was consistent with its expression by human EC cells. In a further control, mouse Oct4 in RNA prepared from isolated mouse thymocytes not used for fusion
Was not detected. However, a PCR band of a size similar to that detected in the 2102Ep × thymocyte fusion sample, corresponding to mouse Oct4, was detected in mouse PCC4 EC cells, as expected.

In our method, nuclei from differentiated or committed cells (nuclear donors), whether from adults or embryos, are combined with cytoplasms from denucleated EC cells (cytoplasmic donors). Create RPES cells by combining them. Several methods can be used to combine nuclei from differentiated cells and cytoplasm from EC cells; in some methods, remove EC nuclei before combining cytoplasm with donated nuclei; In another method, the EC nuclei are removed after the combination. If EC cells and differentiated cells from the same species are used, the resulting RPES cells will carry cytoplasmic genetic determinants (eg, mitochondrial genome) and nuclear genome from the same species.

In contrast, embryonic stem-like cells produced by transplanting somatic cells into enucleated oocytes of other species will continue to harbor mitochondria of other species. For transplantation into a human host, particularly for producing human RPES cells and their differentiated derivatives, maintenance of the human nuclear and cytoplasmic genomes can have distinct advantages. In addition, RPES cells that are isogenic to the predicted human host can be produced by this technique without relying on any embryos, such as those associated with immune rejection upon transplantation into a human host. Avoid practical difficulties and eliminate the inherent ethical difficulties in using human embryos.

The methods we describe incorporate techniques for maintaining and expanding RPES cells produced, and for inducing RPES cells to differentiate into a range of differentiated, functional cell types.

[0101]

[Table 2]

[0102]

[Table 3]

Note 1 The phenotype of the cells in culture is based on observations of morphology, growth patterns and antigen expression (see Andrews et al., 1996). 2 Culture conditions: Human EC cells can generally be maintained in Dulbecco's Modified Eagle's Medium (DMEM), a high glucose formulation supplemented with glutamine and 10% fetal calf serum, but other media were also used (eg, , RPMI about NCCIT) (And
rews and Damjanov, 1994). High cell density (75 × 10 ⁶ / 75cm ² tissue culture flasks) is optimal maintenance of the EC phenotype (see Andrews other, 1982,1984b, Andrews and Damjanov, 1994). All cell lines can be harvested for subculture using 0.25% trypsin and 2 mM EDTA in Ca ²⁺ / Mg ²⁺ -free Dulbeccholine buffer, but in some cases (TERA-2 and derivatives, and S
uSa) Clamping of cells is preferred for best maintenance of the EC phenotype. In the latter case, cells are harvested for subculture, for example by scraping with glass beads, rather than using trypsin (Andrews et al., 1984b, Andrews
And Damjanov, 1994).

3 Surface antigen expression: The expression of SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 is characteristic of a human EC cell line, but the expression levels of these antigens are variable. And appears to reflect these states of differentiation (see, eg, Andrews et al., 1996, Andrews et al., 1982, Kannagi et al., 1983, Andrews et al., 1984a.
, Shevinsky et al., 1982). 4 High levels of liver / bone / kidney isoforms of alkaline phosphatase (L-ALP) detected as cell surface antigens by monoclonal antibodies TERA-2-49 and TERA-2-54 are also characteristic of human EC cells. (Benham et al., 1981, Andrews et al., 1984c, Andrews et al., 1996).

5 Decreased differentiation and EC phenotype: When cultured at low cell density and well dispersed,
Many human EC cells undergo morphological changes and alter surface antigens, notably SSEA
Induces -1 and down regulates SSEA-3 (Andrews et al., 1982, 1984b).
These changes appear to represent a limited capacity for differentiation. Other strains include factors such as retinoic acid (eg, NTERA-2, Andrews, 1984; NCCIT, Te
shima et al., 1988), hexamethylene bisacetamide (eg, NTERA-2, Andre
ws, 1986, 1990), and bone morphogenetic proteins (eg, NTERA-2, Andrews
, 1984) differentiate extensively. Differentiation in the latter case is characterized by a reduction in characteristic EC marker antigens, the appearance of new antigens (see, eg, Fenderson et al., 1987), activation of new genes (eg, Hox gene, Mavilo et al., 1988; Mal, Wakema
n et al., 1977; Wnt-13, Wakeman et al., 1998).

[0106]

[Table 4]

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[Brief description of the drawings]

FIG. 1 shows various human teratocarcinoma-derived cell lines. Characteristic embryonic carcinoma (EC) morphology of several human EC cell lines derived from testicular (ae) and extragonadal (fg) teratocarcinoma: (a) 1156QE, (b) TERA -1, (c) SuSa, (d) 833
KE, (e) 1777NRpmet, (f) 1618K, (g) NCCIT. Bar = 50 μm.

FIG. 2 shows a flow cytometric analysis of the expression of surface antigens by the human EC cell line 2102Ep.

[Figure 3] Figure 3 is caused by changing the growth conditions, some showing the differentiation of human EC cell ^{lines: (a) 10 5 / 75cm} 2 flask 2102Ep cells arranged in (low density);
(B) spontaneous differentiation of a few cells in culture of 1156QE; (c) culture of SuSa cells subcultured by trypsinization; (d) 1777NRp by low density repetitive subculture.
Culture of 1777NRp differentiated cells derived from met EC cells. Bar = 50 μm.

FIG. 4 shows the differentiation of TERA-2-derived human EC cells: (a) NTERA-2 cl D1 human EC cells; (b) parental TERA-2 cultures, EC cells and non-EC cells. Mixed patch; (c)
Neurons and other differentiated cells developed in culture of NTERA-2 cl D1 cells after induction with retinoic acid (Andrews 1984); (d) Non-neural differentiated NTERA-2 induced by exposure to hexamethylene bisacetamide cl D1 cells. (Bar = 50 μm).

FIG. 5 shows the 2102Ep cl 4D3 human EC cell and human T cell leukemia cell line, MOL
2 shows heterokaryons obtained by fusion of T4. Several heterokaryons containing two or three nuclei can be seen in this field. (Bar =
50 μm).

FIG. 6 shows a cytoplast derived from NTERA-2 cl D1 human EC cells after enucleation with cytochalasin B. The remaining nucleated cells (top right) were included in this field for comparison.

FIG. 7 shows PCR of Oct4 mRNA from human EC × somatic (thymocyte) heterokaryon.
Shows amplification.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 ) Inventor Kemp, Paul UK, Romilet Sk. 6 3 J. Wai, Chad Kirk Road 16 F-term (reference) 4B024 AA11 AA20 DA03 GA11 HA17 4B065 AA93X AA93Y AB01 AC20 BA01 4C081 AB02 AB18 AB19 BA12 CD34 4C087 AA03 ZA02A ZA51 ZA59 ZA66 ZA75 ZA81 ZA89 ZA94 ZA96

Claims (29)

[Claims] 1. A cell comprising at least a portion of the cytoplasm derived from an embryonic teratocarcinoma cell in combination with a somatic cell nucleus. 2. The cell of claim 1, wherein said cell is a cybrid characterized by having at least one pluripotent property. 3. The cell of claim 2, wherein said pluripotent property is the ability to differentiate into at least one selected tissue type. 4. The cell of claim 2, wherein said pluripotent property comprises the ability of said cell to grow in culture in an undifferentiated state. 5. The cell of claim 4, wherein said cell has the ability to proliferate when continuously cultured in a non-differentiated state for at least 6 months, ideally 12 months.
The cell according to 1. 6. The cell according to claim 2, wherein said pluripotent property comprises the expression of at least one selected marker. 7. The method of claim 6, wherein the pluripotent property is Oct4 expression. 8. The cell according to claim 6, wherein the selected marker is a cell surface marker. 9. The cell surface marker may be SSEA-1 (-); and / or SSEA-3 (+); and / or SSEA-4 (+); and / or TRA-1-60 (
+); And / or TRA-1-81 (+); and / or alkaline phosphatase (+). 10. The cell according to any one of claims 2 to 9, wherein said pluripotent property comprises the presence of telomerase activity. 11. The cell of claim 2, wherein said pluripotent property comprises the presence of a chromosomal methylation pattern characteristic of a pluripotent cell. 12. The cell according to claim 2, wherein said pluripotent property comprises the ability to induce a tumor when introduced into an animal. 13. A cell line comprising the cell according to any one of claims 1 to 12. 14. The cell line of claim 12, wherein said cell line is of human origin. 15. A method comprising: (i) providing at least one embryonic teratocarcinoma cell; (ii) isolating at least a portion of a cytoplasm from a nucleus of the cell; (iii) isolating the cytoplasmic portion; 13. The cell of any one of claims 1 to 12, or the cell line of claim 13 or 14, comprising, optionally, (iv) storing the isolated cytoplasmic portion prior to use. A method for producing a cytoplasmic portion for use in the production of 16. The method according to claim 15, wherein said cytoplasmic part is a cytoplast. 17. A method comprising: (i) combining at least one embryonic teratocarcinoma cell with at least one somatic cell; (ii) removing the nucleus of the embryonic teratocarcinoma from the combined cells; (iii) Culturing the cells under conditions that promote the growth and expansion of the cells;
And (iv) storing the cell culture under appropriate conditions, if necessary. The cell according to any one of claims 1 to 12, or the cell according to claim 13 or 14, A method for producing a cell line. 18. A method comprising: (i) providing at least a portion of the cytoplasm of an embryonic teratocarcinoma cell; (ii) combining the cytoplasmic portion with at least one somatic cell; (iii) culturing the combined cell And optionally, (iv) storing the combined cells under suitable storage conditions prior to use, wherein at least a portion of the cytoplasm of the embryonic teratocarcinoma cells comprises a somatic cell. How to combine. 19. The method according to claim 18, wherein the cytoplasmic portion is provided as a cytoplast. 20. The method according to claim 18 or 19, wherein said cytoplast is combined with said somatic cell via a cytoplast / somatic cell fusion. 21. The method according to claim 18, wherein the embryonic carcinoma cells and the somatic cells are derived from a human. 22. A cell culture comprising at least one cell according to the invention. 23. Step: (i) providing a cell according to any one of claims 1 to 12; (ii) culturing the cell under conditions that promote differentiation of the cell into at least one tissue. Culturing; and, if necessary, (iii) storing the differentiated tissue under appropriate storage conditions. 13. The differentiation of at least one cell according to any one of claims 1 to 12, comprising: How to induce. 24. Provide a tissue type selected from at least one of nerve, smooth muscle, striated muscle, myocardium, bone, cartilage, liver, kidney, airway epithelium, hematopoietic cells, spleen, skin, stomach, and intestine. The method according to claim 23, wherein the culture conditions are selected as described above. 25. At least one tissue type or organ comprising at least one cell according to any one of claims 1 to 12. 26. A therapeutic composition comprising at least one cell according to any one of claims 1 to 12 and a suitable excipient, diluent or carrier. 27. The therapeutic composition according to claim 26 for use in tissue transplantation. 28. Steps: (i) providing at least one tissue type or organ according to the present invention; (ii) surgically introducing said tissue type or organ into a patient to be treated; and (iii) said implanting Treating said patient under conditions that promote the acceptance of said tissue by said patient, comprising the steps of: 29. at least one cell according to the invention; instructions for the maintenance of said cell in culture; and, optionally, for inducing differentiation of said cell into at least one desired tissue type or organ. A kit comprising: