WO2000049138A2 - Cellules 1 multipotentes - Google Patents

Cellules 1 multipotentes Download PDF

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Publication number
WO2000049138A2
WO2000049138A2 PCT/GB2000/000582 GB0000582W WO0049138A2 WO 2000049138 A2 WO2000049138 A2 WO 2000049138A2 GB 0000582 W GB0000582 W GB 0000582W WO 0049138 A2 WO0049138 A2 WO 0049138A2
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cell
cells
human
culture
pluripotential
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PCT/GB2000/000582
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WO2000049138A3 (fr
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Peter Andrews
Paul Kemp
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Intercytex Limited
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Priority to JP2000599864A priority Critical patent/JP2002536976A/ja
Priority to AU25641/00A priority patent/AU2564100A/en
Priority to CA002371895A priority patent/CA2371895A1/fr
Priority to EP00903892A priority patent/EP1155118A2/fr
Publication of WO2000049138A2 publication Critical patent/WO2000049138A2/fr
Publication of WO2000049138A3 publication Critical patent/WO2000049138A3/fr
Priority to HK02102271.4A priority patent/HK1040532A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/04—Cells produced using nuclear transfer

Definitions

  • the invention herein described relates to isolated pluripotential cells, comprising at least part of the cytoplasm from a teratocarcinoma cell and a nucleus of a somatic cell; methods to prepare such cells; therapeutic compositions of said cells; and uses thereof.
  • Animal embryonic development is a highly regulated development process that combines cell proliferation and cell/tissue differentiation to produce an intact organism.
  • the co-ordination of cell proliferation and differentiation is, and has been, the subject of intense research and the information derived from this has contributed to our understanding of cell function and disease. For example and not by way of limitation, regulation of gene expression, cell differentiation, oncology, teratology.
  • Mammalian embryonic development is remarkably conserved during the early stages. Post fertilisation the early embryo completes four rounds of cleavage to form a morula of 16 cells. These cells complete several more rounds of division and develope into a blastocyst in which the cells can be divided into two distinct regions; the inner cell mass, which will form the embryo, and the trophectoderm, which will form extra embryonic tissue, (eg placenta).
  • each cell has the developmental potential to form a complete embryo and all the cells required to support the growth and development of said embryo.
  • the cells that comprise the inner cell mass (ICM) are said to be pluripotential (ie each cell has the developmental potential to form a variety of tissues).
  • Embryonic stem cells may be principally derived from two embryonic sources. Pluripotential cells isolated from the inner cell mass are termed embryonic stem cells (ES cells). An alternate source of pluripotential cells is derived from primordial germ cells isolated from the mesenteries or genital ridges of days 8.5- 12.5 post coitum embryos which would ultimately differentiate into germ cells. These pluripotential cells are referred to as embryonic germ cells ( EG cells). Each of these types of pluripotential cell has the similar developmental potential with respect to differentiation into alternate cell types.
  • pluripotential cell eg either an ES or EG cell. Therefore a pluripotential cell has an increased commitment to terminal differentiation when compared to a totipotent cell.
  • pluripotential embryonic stem cells are those derived from primordial embryonal germ cells (EG cells) which are located in the mesenteries or genital ridges of embryos.
  • EG cells primordial embryonal germ cells
  • WO 98/43679 describes the isolation of EG cells from the gonadal or genital ridges of human embryos.
  • EG cells described in WO 98/43679 exhibit features in common with primate and human ES cells, (eg expression of cell surface markers, continuous proliferation in culture in an undifferentiated state, normal karyotype and the ability to differentiated into selected tissues under defined conditions).
  • pluripotential stem cells especially human cells
  • in vitro cultures of pluripotential stem cells has important ramifications for both basic research (eg as a model for studying gene expression and/or tissue differentiation) and in transplantation and/or replacement therapies for tissues which have been damaged either through injury or disease.
  • the establishment of in vitro cultures of human ES and EG cells is a major step toward realising the full potential of this technology; because of their pluripotent nature ES and EG cells may be capable of differentiating under controlled conditions into a variety of cell types and/or tissues and organs that could have a wide variety of applications.
  • replacement of damaged and/or diseased coronary and/or major arteries For example, and not by way of limitation, replacement of damaged and/or diseased coronary and/or major arteries; replacement of damaged and/or diseased organs (eg as a result of kidney disease, (eg cirrohosis), diabetes, various autoimmune diseases); replacement of damaged neurones ( eg Alzhiemers disease, Parkinsons disease, spinal injuries) or cancer.
  • diseases such as AIDS may benefit from from tissues derived from ES or EG cells. The depletion of T-cells through virus induced cell death is the major contributory factor to the immuno-compromised state of AIDS suffers.
  • amphibian somatic cell nuclei retain their ability to give rise to entire organisms when they are transplanted into egg cells which have had their nucleus removed or inactivated (Gurdon 1974). Thus determination of the pluripotent of these cells must be controlled by the egg cytoplasm which was able to in effect reprogramme the somatic cell nucleus into a totipotent state.
  • Mammalian somatic cell nuclei have also been shown to retain this placicity and can be reprogrammed when transferred to enucleated oocytes, (Campbell et al; Wakayama et al) Moreover nucleated mouse ES cells have been shown to be able to reprogramme somatic cell nuclei, although in this case, a heterokaryon was produced containing the cytoplasm and nuclei from both types of cells so it is difficult to determine the actual mechanism of action of the reprogramming state.
  • Embryonal carcinoma cells derived from teratomas are also able to reprogramme somatic cell nuclei in order to produce cells with pluroptential characteristics.
  • tumours that contain a wide range of more or less organised tissues have been known in humans for many hundreds of years. They typically occur as gonadal tumours of both men and women, but they also occur in other sites.
  • the gonadal forms of these tumours are generally believed to originate from germ cells, and the extra-gonadal forms, which typically have the same range of histology, are widely thought to arise from 'mis-placed' germ cells that have migrated incorrectly during embryogenesis.
  • a non-germ cell origin from persisting embryonic stem cells may be considered in some cases, especially for teratomas occurring in the new born.
  • teratomas are generally classed as germ cell tumours (GCT), which also manifest a range of other histological types, including seminoma (often called dysgerminoma in females), embryonal carcinoma (EC), yolk sac carcinoma and choriocarcinoma. GCT may contain any combination of these tissue types, with or without elements of teratoma. Combinations of teratoma and EC are frequently described as teratocarcinoma. Commonly, human GCT are divided into pure seminomas. in which none of the other histological types occur, and non-seminomatous GCT (NSGCT), which may contain any combination of histological types, with or without elements of teratoma. Confusingly,
  • NSGCT may also contain elements of seminoma.
  • Ovarian GCT are most commonly benign and contain only well differentiated somatic tissues that may include bone, muscle, nerve. Often well-organised tissues are found, including teeth and hair.
  • human testicular GCT are always malignant containing any combination of the tumour tissue types discussed above. Any teratoma elements present may be less well organised than in benign human ovarian teratomas, but somatic cell types such as nerve, muscle, bone and cartilage may be quite recognisable.
  • Testicular GCT are rare but have a peak incidence after puberty, being the most common form of cancer in young men between the ages of 20 - 35.
  • EC cells are pluripotent stem cells that are able to generate the whole range of differentiated cells found in teratocarcinomas. Subsequently it was found that similar teratocarcinomas and teratomas could be derived from embryos that have been transplanted to ectopic sites.
  • EC cell lines that can be maintained in vitro have been derived from several mouse teratocarcinomas. Some of these EC cell lines were found to retain a pluripotent phenotype and could be induced to differentiate into a range of cell types, either by transplantation back to a syngeneic mouse, in which case a teratocarcinoma would be formed, or by various manipulations in vitro. Some EC cell lines differentiated spontaneously when allowed to grow to confluence. Others required a feeder layer of irradiated or mitomycin-treated cells if they were to retain an EC phenotype, and they differentiated spontaneously if removed from the feeder cells.
  • EC cells have been shown to have many of the features which characterise ES and/or EG cells. EC cells are relatively easy to establish in culture, express cell surface markers associated with ES and/or EG cells, can be maintained in continuous culture in an undifferentiated state and have the potential to differentiate into selected tissues both in vivo and in vitro. However what is also evident is that EC cells contain a mutation, or mutations, in genes (oncogenes) which result in the additional undesirable feature that the cells retain the potential to form tumours.
  • karyoplasts and cytoplasts separate nuclear and cytoplasmic parts termed karyoplasts and cytoplasts, respectfully.
  • the separated parts of the cell may be reconstituted via cell fusion.
  • cytoplasts derived from EC cells fused the cytoplasts to form cybrids with selected somatic cells.
  • the aim of this approach is to reprogramme the differentiated somatic cell nucleus, through contact with factors located in the EC cytoplasm, so that, the cybrid de-differentiates and so takes on the characteristic features of a pluripotential cell.
  • This then provides the basis for the establishment of pluripotential cell lines which, upon exposure to various differentiation factors, can lead to the production of selected differentiated tissue for use in, amongst other things, transplantation therapy.
  • a cell comprising at least part of the cytoplasm derived from at least one embryonal carcinoma cell combined with at least the nucleus of at least one somatic cell.
  • said cell ideally a cybrid, is characterised by the possession of at least one pluripotential characteristic.
  • the cell of the invention may be derived, most preferably, by the creation of a cybrid; but an alternative option involves the fusion of a somatic cell with an EC cell. Clearly this latter option is not preferred because of the potentially oncogenic genome (gene) of the donating EC cell.
  • the further step of removing the EC nucleus is described hereinafter.
  • said pluripotential characteristic includes the ability to differentiate into at least one selected tissue type, preferably upon exposure to at least one differentiation factor.
  • said pluripotential characteristic includes the ability of said cell to proliferate in culture in an undifferentiated state.
  • said cell has the capacity to proliferate in continous culture in an undifferentiated state for at least 6 months and ideally 12 months.
  • said pluripotential characteristic includes the expression of at least one selected marker of pluripotential cells.
  • pluripotential cells express a number of genes not typically expressed by differentiated cells. These are valuable tools to monitor whether the EC cytoplasm has re-programmed a somatic cell nucleus.
  • One such example is Oct4.
  • said selected marker is expression of the Oct4 gene.
  • said selected marker is a cell surface marker.
  • said cell surface marker is selected from the group including SSEA-1 (-); SSEA-3 (+); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); alkaline phosphatase (+).
  • said pluripotential characteristic includes the presence of telomerase activity in said pluripotential cell.
  • said telomerase activity is correlated with extension of telomeres.
  • telomeres For the sake of clarity, telomerase enzymes add, de novo, repetitive DNA sequences to the ends of chromosomes. These ends are referred to as telomeres.
  • telomeres of human chromosomes contain the sequence '5 TTAGGG 3' repeated approximately 1000 times at their ends.
  • dividing cells the telomeres are relatively long.
  • aging, or non- dividing cells the telomeres become shortened and there is a strong correlation between telomere shortening and capacity to proliferate.
  • Methods to increase the length of telomeres to increase proliferative capacity are known in the art and are described in W09513383.
  • said pluripotential characteristic includes the presence of a chromosomal methylation pattern characteristic of pluripotential cells.
  • Methods to analyse the extent of methylation include, by example and not by way of limitation, restriction enzyme digestion of DNA with methylation sensitive restriction endonucleases followed by Southern blotting and probing with suitable gene probes ( Umezawa et al 1997).
  • said pluripotential characteristic includes the ability to induce tumours when introduced into an animal, ideally a rodent experimental model. More ideally still said animal is immunosupressed.
  • a cell-line comprising cells according to the invention. Ideally, said cell-line are of human origin.
  • a method for preparing a cytoplast, or part thereof, for use in the production of the cell or cell line of invention comprising;
  • said cytoplasmic part is a cytoplast.
  • said cytoplast may be provided either as an aliquot isolated from at least one EC cell (eg an aliquot extracted from an intact EC cell via micromanipluation techniques) or alternatively, and preferably, said cytoplasmic part may be provided as an isolated cytoplast.
  • said cytoplast part is separated from said nucleus by exposure to a pharmacologically effective amount of at least one cytochalasin.
  • cytochalasin B a pharmacologically effective amount of at least one cytochalasin.
  • cytochalasin B is an example of a chemical effective at separating the nucleus of a cell from the cytoplasm to form a karyoplast and cytoplast respectively, (Methods in Enzymology Vol 151 , p221-237 1987).
  • a method for preparing a cell or cell-line in accordance with the invention comprising; i) combining at least one EC cell with at least one somatic cell; ii) removing from said combined cell, the EC cell nucleus; iii) culturing said cell under conditions conducive to proliferation and expansion of said cell; and. optionally iv) storing said cell culture under suitable conditions.
  • the factors produced by the EC cell are continually produced thereby maintaining a steady-state level of factors necessary to reprogramme the somatic cell nucleus; and ii) the EC cell nucleus is removed from the combined cell prior to mitosis ensuring oncogenic genes are not transferred.
  • somatic cell selected is not critical to the operation of the invention although the cell-type will be selected so as to optimise or maximise success in terms of prodcution of a cell or cell-line of the invention.
  • a method of combining at least part of the cytoplasm of an EC cell with a somatic cell comprising; i) providing at least part of the cytoplasm of an EC cell; ii) combining said cytoplasmic part with at least one somatic cell; iii) growing said combined cell in culture; and, optionally iv) storing said combined cell under suitable storage conditions.
  • said cytoplasmic part is provided as a cytoplast.
  • said cytoplast is combined with said somatic cell via cytoplast/somatic cell fusion.
  • the EC cell and somatic cell are, ideally of human origin.
  • a cell culture comprising at least one cell according to the invention.
  • a method for inducing differentiation of at least one cell of the invention comprising:
  • said culture conditions are selected so as to provide a tissue type, by example and not by way of limitation, that is, neuronal, muscle (eg smooth, striated, cardiac), bone, cartilage, liver, kidney, respiratory epithelium, haematopoietic cells, spleen, skin, stomach, intestine.
  • a tissue type by example and not by way of limitation, that is, neuronal, muscle (eg smooth, striated, cardiac), bone, cartilage, liver, kidney, respiratory epithelium, haematopoietic cells, spleen, skin, stomach, intestine.
  • tissue type or organ comprising at least one cell according to the invention.
  • differentiated tissue according to the invention may have extensive application with respect to transplantation therapy. For example, and not by way of limitation, replacement of damaged and/or diseased coronary and/or major arteries; replacement of damaged and/or diseased organs (eg as a result of kidney disease, (eg cirrohosis), diabetes, various autoimmune diseases); replacement of damaged neurones (eg Alzhiemers disease, Parkinsons disease, spinal injuries) or cancer.
  • diseases such as AIDS may benefit from from tissues derived from the cells of the invention.
  • T-cells through virus induced cell death is the major contributory factor to the immuno-compromised state of AIDS suffers.
  • the provision of a non-exhaustive supply of T-cells derived from a non-infected somatic cell from the patient has obvious benefits.
  • tissue rejection due to host cell immune responses are likely to be negligible since the somatic nucleus used in the cybrid would ideally be derived from the patient requiring the replacement tissue or organ.
  • a therapeutic composition comprising at least one cell of the invention including a suitable excipient, diluant or carrier.
  • said therapeutic composition is provided for use in tissue transplantation.
  • a method to treat conditions or diseases requiring transplantation of tissue comprising;
  • tissue type or organ i) providing at least one tissue type or organ according to the invention; ii) surgically introducing said tissue type or organ to a patient to be treated; and iii) treating said patient under conditions which are conducive to the acceptance of said transplanted tissue by said patient.
  • kits comprising; at least one cell according to the invention; instructions with respect to the maintenance of said cell in culture; and. optionally, factors required to induce differentiation of said cell to at least one desired tissue type or organ.
  • Table 1 represents a summary of the human EC cell lines and some of the characteristic features of said human EC cell-lines ;
  • Table 2 represents a summary of the murine EC cell lines used
  • Figure 1 shows various human teratocarcinoma-derived cell lines.
  • the characteristic embryonal carcinoma (EC) morphology of several human EC cell lines derived from testicular (a-e) and extragonadal (f-g) teratocarcinomas: (a) 1156QE, (b) TERA-1, (c) SuSa, (d) 833KE, (e) 1777NRpmet, (f) 1618K, (g) NCCIT. Bar 50 ⁇ m;
  • Figure 2 shows a flow cytofluorimetric analyses of surface antigen expression by the human EC cell line 2102Ep;
  • Figure 3 shows differentiation of several human EC cell lines caused by alteration in growth conditions: (a) 2102Ep cells placed at 10 5 per 75 cm 2 flask
  • Figure 6 shows cytoplasts derived from NTERA-2 cl Dl human EC cells following enucleation with cytochalasin B. A remaining nucleated cell (top right) has been included in the field for comparison;
  • Figure 7 shows PCR amplification of Oct4 mRNA from a human EC x somatic cell ( thymocyte) heterokaryon.
  • the thymocytes were obtained by mincing a thymus removed from a 4-6 week old male mouse (Swiss strain) and suspending the released cells in 10 ml medium (DMEM) with 10% foetal calf serum (FCS). After standing for 2-3 minutes to allow large fragments of thymus to settle, the supernatant was removed and centrifuged at 1500 rpm for 5 min to pellet the suspended thymocytes. The thymocytes were resuspended in fresh medium without FCS, and pelletted again by centrifugation; this was repeated a second time after which the cells were resuspended in fresh serum free medium and counted.
  • DMEM 10 ml medium
  • FCS foetal calf serum
  • Human EC cells were obtained by trypsinisation of confluent cultures as previously described (Andrews et al , 1980; 1982). After washing two times in serum free DMEM, and counting, the human EC cells were mixed with the mouse thymocytes in a ratio of 1 EC cell to 10 thymocytes. The mixed cells were pelletted by centrifugation at 1500 rpm for 5 min.
  • the cells were fused using polyethylene glycol (PEG) (Kennett, 1979).
  • the pellet in Experiment 1, 2 x 10 EC cells and 2 x 10 thymocytes; in Experiment 2, 3 x 10 EC cells and 3 x 10 thymocytes
  • PEG polyethylene glycol
  • the pellet was resuspended in 200 ⁇ l 50% (w/v) PEG 1500 in 75 mM HEPES, pH8.0 (Boehringer Mannheim) and incubated at 37° C for 1.5 min. Serum free medium, pre- warmed to 37° C, was then added gradually over 5 min.
  • the cells were then pelletted by centrifugation at 1500 rpm for 5 min. and resuspended in 5 ml DMEM with 20% foetal calf serum. These cell were then plated into a T25 flask and placed in a humidified incubator (10% C0 2 in air) at 37°C for 2 days.
  • RNA was quantified by optical density measurements and the absence of contaminating DNA was determined by PCR using ⁇ -actin and HPRT primers in separate samples (Wakeman et al.,
  • RNA was then used for RT.PCR analysis of Oct4 expression.
  • RT reverse transcriptase
  • PCR was then performed using oligonucleotide primers specific for human and mouse Oct 4, a marker of pluripotent cells under the standard PCR conditions described in Wakeman et al. (1998) with an annealing temperature of 61°C. These products were then subjected to electrophoresis and separated DNA fragments detected by ethidium bromide staining ( Figure 7). Molecular size of the amplified fragments was determined by using a lkb DNA step ladder.
  • RPES cells Re- Programmed Embryonic Stem Cells
  • cytochalasin B to generate enucleated EC cells (EC cytoplasts) as the cytoplasm donor
  • 'karyoplasts' also called "mini-cells'
  • Cytochalasin B is well-known to induce cells to extrude their nuclei (Carter, 1967) and has been employed by numerous authors to induce enucleation of a wide range of cells of a variety of 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). Such enucleation results in a cell lacking a nucleus, but is otherwise intact and viable for a number of days (Goldman et al 1973); these enucleated cells have been called anucleate cells (Poste 1972) or cytoplasts (Veomett et al 1974).
  • 'karyoplasts' Veomett et al 1974
  • 'mini- cells' Ege and Ringertz 1975.
  • Enucleation of cells to yield both cytoplasts and karyoplasts may be achieved by well-established techniques in which cells growing attached to a plastic disc are inverted over a solution of cytochalasin B in a centrifuge tube and centrifuged; the cytoplasts remain attached to the plastic disc, while the karyoplasts are pelleted at the bottom of the centrifuge tube (Prescott et al 1972).
  • cells in suspension may be centrifuged through a density gradient, typically composed of Ficoll, containing cytochalasin B (Wigler and Weinstein 1975).
  • cytoplasts and karyoplasts are formed and may be recovered from different parts of the gradient after centrifugation.
  • mixtures of cells that it is desired to fuse are incubated with a fusogenic agent, such as Sendai virus or PEG, often with centrifugation or agitation to encourage clumping and close apposition of the cell membranes; variables such as time, temperature, cell concentration and fusogenic agent concentration are optimised for each cell combination.
  • a fusogenic agent such as Sendai virus or PEG
  • variables such as time, temperature, cell concentration and fusogenic agent concentration are optimised for each cell combination.
  • An example of cell fusion to produce heterokaryons is presented in Figure 6.
  • Cell fusion was carried out using Polyethylene Glycol 1000, as described by Kennett 1979. Following fusion, the cells were seeded into tissue culture dishes, and incubated in fresh medium overnight. They were then fixed with methanol and stained with haematoxylin and cosin.
  • the production technique may, in some cases, be optimised by pre-treatment of the differentiated cells, or contemporaneous treatment of the differentiated cell/ EC cell fused products, with various agents such as, but not limited to, inhibitors of DNA methylation, to enhance the ability of the differentiated cell nucleus to be re-programmed.
  • agents such as, but not limited to, inhibitors of DNA methylation, to enhance the ability of the differentiated cell nucleus to be re-programmed.
  • additional methods are required to propagate the cells, to characterise their properties and to induce them to differentiate into required somatic cell types.
  • somatic cells derived from any tissue or organ of an adult mammal or human, or from embryos or foetuses, or from extra-embryonic tissues such as the trophoblast or yolk sac may be used as a source of nuclei for reprogramming.
  • somatic cell types include but are not limited to thymocytes, peripheral blood lymphocytes, epidermal cells such as from the bucal cavity, cumulus cells, or other stem cells isolated from biopsies of various tissues, such as the bone marrow, the nervous system and the gut.
  • the technique may also be applied to various established cell lines, such as those derived from various tumours including, for example, but not limited to lymphoblastoid cell lines.
  • the selected somatic cells used for the re- programming procedure may be used directly upon isolation or they may be cultured for a short time before further manipulation.
  • somatic cells may be combined entirely with EC cells as described below, or nuclei or karyoplasts may first be isolated from them, for example using agents such as cytochalasin B, as discussed above, or by other methods.
  • nuclei may also be isolated using established micromanipulation procedures, or other established cell fractionation procedures.
  • EC cell lines to be used as Cytoplasm Donors A large number of EC cell lines have been isolated from human teratocarcinomas, which occur predominantly, but not exclusively, as testicular germ cell tumours. Examples of available human EC cell lines are shown in Table 1. Similarly, EC cell lines derived from teratocarcinomas of the laboratory mouse 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 any other species, but if they were derived in the future we would anticipate that they should behave in a manner similar to the existing human and mouse EC cells which resemble one another. Thus, newly isolated EC cells of other species, or from human or mouse sources, should also be able to re-program differentiated cells to RPES cells as described in its proposal for human and mouse EC cells.
  • Human EC cells can be readily recognised by a combination of features that include their morphology (see Figure 1), their expression of the cell surface antigens SSEA3 (Shevinsky et al 1982, Andrews et al 1982, 1984b, 1996), SSEA4 (Kannagi et al 1983, Andrews et al 1984b, 1996), and TRA-1-60 and TRA-1-81 (Andrews et al 1984a, 1984b, 1996), and typically by low expression or absence of SSEA1 (Andrews et al 1980, 1982, 1984b, 1996) (see Figure 2).
  • SSEA3 Shevinsky et al 1982, Andrews et al 1982, 1984b, 1996)
  • SSEA4 Kernagi et al 1983, Andrews et al 1984b, 1996)
  • TRA-1-60 and TRA-1-81 and typically by low expression or absence of SSEA1 (Andrews et al 1980, 1982, 1984b, 1996)
  • mouse EC cells typically express SSEA1 (Solter and Knowles, 1978) but not 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 like mouse EC cells express high levels of alkaline phosphatase (ALP) (Bernstine et al 1973, Benham et al 1981); in the case of human EC cells most ALP activity is due to expression of the liver/bone/kidney isoform which can be detected as a cell surface antigen by monoclonal antibodies TRA-2-59 and TRA-2-54 (Andrews et al 1984c).
  • ALP alkaline phosphatase
  • TRA-2-59 and TRA-2-54 In common with ES cells and primordial germ cells, EC cells also typically express the transcription factor Oct3/4 (Rosfjord and Rizzino 1994; Brehm et
  • Several methods may be used to combine the cytoplasm of an EC cell and the nucleus of a differentiated cell to yield an RPES containing the nuclear genome of the differentiated cell but not the EC cell.
  • Cells may be fused by use of chemical agents such as polyethylene glycol (PEG) or viruses such as Sendai virus, or by passing an electric current through a mixture of cells. As discussed above, these methods are well known and may be readily applied. These methods may be used to fuse:
  • chemical agents such as polyethylene glycol (PEG) or viruses such as Sendai virus
  • a differentiated cell with an EC cell or 3. a differentiated cell with one or more cytoplasts isolated from
  • the result will initially be a heterokaryon containing two nuclei, one from each parental cell. If this heterokaryon were allowed to divide the result would be a hybrid cell containing a single nucleus with a complete or partial genome from each parental cell. However, in our method of producing RPES cells, the EC nucleus is removed prior to cell division of the hybrid cell, so that the derivative dividing cell population retains only the genome of the parental differentiated cell.
  • the EC nucleus is removed from the EC cell before fusion, for example by enucleation with cytochalasin B as discussed above, so that the resulting product contains only the differentiated cell nucleus and cytoplasm from the EC cell parent.
  • the resulting RPES cells that continue to proliferate retain only the nuclear genome of the differentiated parental cell, which is now reprogrammed to express a new pattern of gene activity.
  • the EC cell nucleus is removed from the heterokaryon in one of several ways that include, but are not limited to, partial enucleation using drugs such as cytochalasin B, applied in the same manner as described above for enucleating EC cells and generating cytoplasts for fusion.
  • drugs such as cytochalasin B
  • some heterokaryons lose both nuclei, in which case they do not proliferate, some heterokaryons lose the differentiated cell nucleus, in which case they retain the parental EC nucleus and continue proliferating, some heterokaryons lose the EC cell nucleus, in which case they continue proliferating as RPES cells, and some heterokaryons retain both nuclei and eventually continue proliferating as hybrid cells.
  • the proliferating cells are cloned by established techniques (e.g. by picking single cells with a micropipette - see Andrews et al 1982, 1984b), and individual clones are screened using genetic markers for those that retain an EC genome. The latter cells are discarded, whereas those that retain only a differentiated cell genome but not an EC cell derived genome, and express an RPES phenotype, are retained.
  • Standard DNA genotyping techniques using well established DNA fingerprinting technology may be used to identify whether the nuclear genome of any proliferating cells is derived from either the EC cell or differentiated cell parent, or both.
  • the EC cell parent is genetically marked by insertion of a gene that will allow selection against any cell carrying that gene; for example, the EC cell can be stably transfected with a vector encoding the Herpes Simplex Virus- 1
  • Tk gene HSVl-Tk
  • acyclovir 9- [(2-hydroxyethoxy)methyl]guanine
  • FIAU l-(2-deoxy-2-fluoro- ⁇ - D-arabinofuranosyl)-5-iodouracil
  • gancyclovir Rubinstein et al 1993; McCarrick and Andrews
  • FACS fluorescence activated cell sorting
  • the EC cell parent is incubated prior to fusion, with a drug that irreversibly inactivates its nucleus and prevents its replication, for example, topoisomerase inhibitors such as etoposide (Downes et al 1991 ; Fulka and Moor 1993).
  • a drug that irreversibly inactivates its nucleus and prevents its replication for example, topoisomerase inhibitors such as etoposide (Downes et al 1991 ; Fulka and Moor 1993).
  • etoposide etoposide
  • This approach may also be combined with the preceding 'partial enucleation of heterokaryons' approach to ensure complete loss of the EC genome.
  • the EC cell nucleus is removed by micro-manipulation.
  • the nucleus of the differentiated cell is combined with an EC cell parent by micro-manipulation.
  • the nucleus of the differentiated cell is withdrawn using a micropipette inserted through the cell membrane. It is then injected either into an inoculated EC cell, or into an intact EC.
  • the EC cell nucleus is then removed by a similar technique, or by one of the techniques described above, before nuclear fusion and cell division occurs.
  • the differentiated cell and EC cell are synchronised with respect to position in the cell cycle, by use of reversible inhibitors that arrest the cell cycle at specific stages (e.g. nocodazole), or by the use of conditions such as low serum to arrest cells in Gl, or by selection of cells at specific stages of the cell cycle by using vital DNA stains and flow microfluorimetry (Fluorescence Activated Cell Sorting) (Ashihara and Baserga 1979; Andrews et al 1987; Crissman 1995; Stein et al 1995).
  • the differentiated cell or the immediate fusion product is cultured in the presence of drugs that inhibit methylation or promote demethylation (e.g. 5-azacytidine) (e.g. Taylor and Jones 1979; Jones 1985; Keshet et al 1986), or alter the structure of chromatin, for example butyrate, spermine, trichostatin A or trapoxin which inhibit deacetylation and promote acetylation of histones, which plays a role in X chromosome inactivation, gene imprinting and regulation of gene expression (Caldarera et al 1975; McKnight et al
  • the period of time between production of heterokaryons and the removal of the EC cell nucleus is made as long as possible without permitting nuclear fusion. This period can be elongated by culturing the heterokaryons under conditions that reversibly inhibit progress through the cell cycle (e.g. thyrnidine block - Stein et al 1995), or by altering growth conditions, such as serum starvation or lowered temperature, that retard cell division but permit reprogramming to proceed.
  • the cells are cultured in standard cell culture media that include but are not restricted to Dulbecco's modified Eagle's Medium (DME, high glucose formulation) or Ham's F12, supplemented in some cases with foetal bovine serum or with other additives (e.g. see Andrews et al 1980, 1982, 1984, 1994).
  • DME Dulbecco's modified Eagle's Medium
  • Ham's F12 supplemented in some cases with foetal bovine serum or with other additives (e.g. see Andrews et al 1980, 1982, 1984, 1994).
  • the growth of the resulting cells may be optimised culture on feeder layers of cells that include, but are not restricted to, irradiated or mitomycin C treated STO cells, or embryonic fibroblasts of various species, including humans (see Robertson 1987a; Thomson et al 1998).
  • the cells may be cultured in the presence of various growth factors or other tissue culture additives, that include but are not restricted to LIF, FGF, SCF.
  • the RPES cells acquire pluripotent properties that closely resemble those of embryonic stem cells, so that the RPES cells are able to differentiate and to initiate differentiation pathways that result in the formation of any cell type that may be found in the adult, embryo or in extra- embryonic tissues, given appropriate conditions.
  • the maintenance of an EC cell state can be monitored by assay of various markers that include the cell surface antigens
  • SSEA3, SSEA4, TRA- 1-60, TRA-1-81 by their expression of alkaline phosphatase and by expression of Oct3/4, as discussed above.
  • the RPES cells typically retain their stem cell phenotype when cultured on appropriate feeder cells. However, they can initiate differentiation under a variety of circumstances.
  • pluripotent stem cells may also be initiated by altered conditions affecting cell density and aggregation (e.g. seeding at low cell densities or trypsinisation) or exposure to various agents that include but are not restricted to retinoic acid, and other retinoids, hexamethylene bisacetamide, and the bone morphogenetic proteins
  • pluripotent stem cell lines have been derived from early embryos (Robertson, 1987b; Thomson et al 1995, 1998), primordial germ cells (Matsui et al 1992; Shamblott et al 1998) and from germ cell tumours (reviewed, Andrews, 1998) of various species, including the laboratory mouse, rhesus monkeys and humans, and nuclei from differentiated somatic adult cells have been re-programmed to yield embryonic stem cells by transplantation to enucleated oocytes (Campbell et al 1996; Wakayama et al 1998), there are no reports that pluripotent stem cells, resembling embryonic stem cells with the capacity to differentiate into a variety of functional somatic cell types, can be produced by the re-programming of differentiated or committed embryonic or adult somatic cells, or extra-embryonic cells, without the use of oocytes.
  • embryonal carcinoma (EC) cells derived from teratocarcinomas
  • EC embryonal carcinoma
  • RPES cells 'Re-programmed Embryonic Stem Cells'
  • RPES cells 'Re-programmed Embryonic Stem Cells'
  • RPES cells resemble embryonic stem cells derived directly from early embryos, and can be induced to differentiate into a broad range of functional, differentiated cell types that include, but are not limited to, neurons, muscle (including skeletal and cardiac muscle) and haematopoietic cells.
  • RPES cells are diploid with a normal karyotype, and isogenic with the differentiated parental cells from which they are derived. They may be used to generate differentiated cells for transplantation and use in cell and tissue replacement therapies.
  • only partial reprogramming occurs with, for example, the activation of several genes that are not active in the parental differentiated nuclear donor cell.
  • Such cells are also of use in a variety of these same circumstances.
  • Oct4 has previously been reported to be characteristically expressed by undifferentiated EC and ES cells (Brehm et al, 1998). Therefore, to test the ability of human EC cell cytoplasm to reprogra somatic cells, isolated mouse thymocytes were fused with human EC cells, (2102Ep, clone 4D3 (Andrews et al . 1982) or TERA1 (Fogh and Trempe, 1975; Andrews et al, 1980)). to produce heterokaryons which were tested after 2 days for activation of Oct4 expression from the thymocyte genome.
  • an amplified band (573 bp), corresponding to human Oct4 expression was detected similarly in RNA preparations from the 2102Ep x thymocyte fusion in the presence of PEG, and in the mock fusion in the absence of PEG, consistent with its expression by 2102Ep human EC cells.
  • a band corresponding to mouse Oct4 (415 bp) was only detected in the RNA preparation from the 2102Ep x thymocyte fusion in the presence of PEG, when heterokaryons were expected to be present.
  • RPES cells are created by combining the nucleus from a differentiated or committed cell (the Nuclear donor), whether from adults or from embryos, with the cytoplasm from an EC cell (the Cytoplasm donor), from which the nucleus is removed.
  • the Nuclear donor the nuclear donor
  • the Cytoplasm donor the cytoplasm from an EC cell
  • Several methods can be used to combine the nucleus from the differentiated cell and the cytoplasm from the EC cell; in some methods the EC cell nucleus is removed prior to combination of the cytoplasm with the donated nucleus, and in other methods the EC cell nucleus is removed after combination. If EC cells and differentiated cells from the same species are used, then the resulting RPES cells retain cytoplasmic genetic determinants (e.g. the mitochondrial genome) and a nuclear genome from the same species.
  • cytoplasmic genetic determinants e.g. the mitochondrial genome
  • embryonic stem-like cells produced by transplantation of somatic cells into enucleated oocytes of other species will continue to harbour mitochondria of that other species.
  • RPES cells and their differentiated derivatives for transplantation into a human host the maintenance of a human nuclear and human cytoplasmic genome could be a distinct advantage.
  • RPES cells that are isogenic with the anticipated human host can be produced by this technique without resort to any embryo, so avoiding practical difficulties that may be associated, for example, with immune rejection upon transplantation to the human host, and also obviating ethical difficulties inherent in the use of human embryos.
  • the method that we describe incorporates the techniques for maintaining and propagating the RPES cells produced, and the techniques for inducing them to differentiate into a range of differentiated, functional cell types.
  • Andrews P.W. Bronson D.L., Benham F., Strickland S. and Knowles B.B. (1980) A comparative study of eight cell lines derived from human testicular teratocarcinoma. Int. J. Cancer 26: 269-280. Andrews P.W., Goodfellow P.N., Shevinsky L., Bronson D. L. and Knowles B.B. (1982) Cell surface antigens of a clonal human embryonal carcinoma cell line: Morphological and antigenic differentiation in culture. Int. J. Cancer 29: 523-531.
  • Cytomegalovirus replicates in differentiated but not undifferentiated human embryonal carcinoma cells. Science 224, 159-161.
  • Monclonal antibody to murine embryo defines a stage specific embryonic antigen expression on mouse embryos and human teratocarcinoma cells. Cell 30, 697- 705.
  • Veomett G., Prescott, D.M., Shay, J., Porter, K.R. (1974). Reconstruction of mammalian cells from nuclear and cytoplasmic components separated by treatment with cytocholasin B. Proc Nat Acad Sci, 71, 1999-2002.
  • Glycolipids of germ cell tumours extended globo-series glycolipids are a hallmark of human embryonal carcinoma cells. Int. J. Cancer., 58, 108-1 15.
  • the phenotype of cells in culture is based upon observations of morphology, growth patterns and antigen expression (see, Andrews et al 1996).
  • Human EC cells can generally be maintained in Dulbecco's Modified Eagles medium (DMEM), high glucose formulatio supplemented with glutamine and 10% foetal bovine serum, but other media have also been used (e.g. RPMI for NCCIT) (see Andrews and Damjan 1994). High cell densities (75 x 10 6 per 75 cm 2 tissue culture flask) are optimal for maintaining an EC phenotype (see Andrews et al 1982, 198 Andrews and Damjanov 1994).
  • DMEM Dulbecco's Modified Eagles medium
  • RPMI for NCCIT
  • High cell densities 75 x 10 6 per 75 cm 2 tissue culture flask
  • are optimal for maintaining an EC phenotype see Andrews et al 1982, 198 Andrews and Damjanov 1994).
  • the cell lines may be harvested for passage using 0.25% trypsin and 2mM EDTA, in Ca 2+ /Mg z+ - free Dulbecc phosphate buffered saline, but in some cases (TERA-2 and derivatives, and SuSa) clumping of cells is preferable for best maintenance of an phenotype. In the latter cases, cells are harvested for passage by scraping, for example with glass beads, rather than by use of trypsin (see Andrews et 1984b, Andrews and Damjanov 1994). ⁇
  • SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 expression is characteristic of human EC cell lines but the level of expression ⁇ tthheessee aannttiiggeennss iiss vvaarriiaabbllee aanndd aappppeeaarrss t too rreeflect their state of differentiation (e.g. see Andrews et al 1996, Andrews et al 1982, Kannagi et al 19
  • W TRA-2-54 are also characteristic of human EC cells (Benham et al 1981, Andrews et al 1984c, Andrews et al 1996). m
  • C changes appear to represent a limited capacity for differentiation.
  • Other lines differentiate extensively if exposed to agents such as retinoic acid (m NTERA-2, Andrews 1984; NCCIT, Teshima et al 1988), hexamethylene bisacetamide (e.g. NTERA-2, Andrews et al 1986, 1990), and the b
  • N> morphogenetic proteins e.g. NTERA-2, Andrews et al 1994. Differentiation in the latter cases is marked by loss of the characteristic EC mar antigens, appearance of new antigens (e.g. see, Fenderson et al 1987), activation of new genes (e.g. ox genes, Mavilo et al 1988; Mai, Wakeman e

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Abstract

La présente invention concerne des cellules multipotentes isolées qui comprennent au moins une partie du cytoplasme d'une cellule de tératocarcinome et un noyau de cellule somatique ; ainsi que des procédés de préparation de ces cellules.
PCT/GB2000/000582 1999-02-20 2000-02-18 Cellules 1 multipotentes WO2000049138A2 (fr)

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JP2000599864A JP2002536976A (ja) 1999-02-20 2000-02-18 多能性細胞−１
AU25641/00A AU2564100A (en) 1999-02-20 2000-02-18 Pluripotential cells-1
CA002371895A CA2371895A1 (fr) 1999-02-20 2000-02-18 Cellules 1 multipotentes
EP00903892A EP1155118A2 (fr) 1999-02-20 2000-02-18 Cellules 1 multipotentes
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014337A2 (fr) * 2001-08-03 2003-02-20 Intercytex Limited Fusion de cellules
EP1465992A2 (fr) * 2001-12-18 2004-10-13 Acceptys, Inc. Cellules souches multipotentes derivees embryons ni tissu foetal
WO2007026255A2 (fr) * 2005-06-22 2007-03-08 Universitetet I Oslo Cellules dedifferenciees et procedes permettant de realiser et d'utiliser des cellules dedifferenciees
US7785876B2 (en) 2002-11-14 2010-08-31 Aderans Research Institute, Inc. Cultivation of hair inductive cells
US7922688B2 (en) 2007-01-08 2011-04-12 Restoration Robotics, Inc. Automated delivery of a therapeutic or cosmetic substance to cutaneous, subcutaneous and intramuscular tissue regions

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FULKA J ET AL: "CLONING BY SOMATIC CELL NUCLEAR TRANSFER" MICROBIOLOGICAL REVIEWS,US,AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC, vol. 20, 1998, pages 847-851, XP002900701 ISSN: 0146-0749 *
J A THOMSON ET AL: "EMBRYONIC STEM CELL LINES DERIVED FROM HUMAN BLASTOCYSTS" SCIENCE,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,,US, vol. 282, 6 November 1998 (1998-11-06), pages 1145-1147, XP002121340 ISSN: 0036-8075 *
TOSU M ET AL: "CLONAL ISOLATION AND CHARACTERIZATION OF MYOBLAST-LIKE RECONSTITUTED CELLS FORMED BY FUSION OF KARYOPLASTS FROM MOUSE TERATOCARCINOMA CELLS WITH RAT MYOBLAST CYTOPLASTS" CELL STRUCTURE AND FUNCTION, vol. 13, no. 3, 1988, pages 249-266, XP000939352 ISSN: 0386-7196 *
WANGH LAWRENCE J ET AL: "Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts." JOURNAL OF CELL SCIENCE, vol. 108, no. 6, 1995, pages 2187-2196, XP000939319 ISSN: 0021-9533 *
YEOM ET AL: "Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells" DEVELOPMENT,GB,COLCHESTER, ESSEX, vol. 122, 1996, pages 881-894, XP002094592 ISSN: 0950-1991 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014337A2 (fr) * 2001-08-03 2003-02-20 Intercytex Limited Fusion de cellules
WO2003014337A3 (fr) * 2001-08-03 2003-05-30 Intercytex Ltd Fusion de cellules
EP1465992A2 (fr) * 2001-12-18 2004-10-13 Acceptys, Inc. Cellules souches multipotentes derivees embryons ni tissu foetal
EP1465992A4 (fr) * 2001-12-18 2005-06-01 Acceptys Inc Cellules souches multipotentes derivees embryons ni tissu foetal
US7785876B2 (en) 2002-11-14 2010-08-31 Aderans Research Institute, Inc. Cultivation of hair inductive cells
WO2007026255A2 (fr) * 2005-06-22 2007-03-08 Universitetet I Oslo Cellules dedifferenciees et procedes permettant de realiser et d'utiliser des cellules dedifferenciees
WO2007026255A3 (fr) * 2005-06-22 2008-01-03 Uni I Oslo Cellules dedifferenciees et procedes permettant de realiser et d'utiliser des cellules dedifferenciees
US7922688B2 (en) 2007-01-08 2011-04-12 Restoration Robotics, Inc. Automated delivery of a therapeutic or cosmetic substance to cutaneous, subcutaneous and intramuscular tissue regions

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