WO2006084229A2 - Use of nuclear material to therapeutically reprogram differentiated cells - Google Patents
Use of nuclear material to therapeutically reprogram differentiated cells Download PDFInfo
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- WO2006084229A2 WO2006084229A2 PCT/US2006/004077 US2006004077W WO2006084229A2 WO 2006084229 A2 WO2006084229 A2 WO 2006084229A2 US 2006004077 W US2006004077 W US 2006004077W WO 2006084229 A2 WO2006084229 A2 WO 2006084229A2
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- 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
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
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- C12N2501/235—Leukemia inhibitory factor [LIF]
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/04—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from germ cells
Definitions
- the present invention relates to the field of therapeutically reprogrammed cells.
- therapeutically reprogrammed cells are provided that are not compromised by the aging process, are immunocompatible and will function in the appropriate post-natal cellular environment to yield functional cells after transplantation.
- the present invention relates to therapeutic reprogramming cells with nuclear extracts that do not contain genetic material.
- Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to four divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus.
- cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number.
- An example of multipotent cells is hematopoietic cells - blood cells that can develop into several types of blood cells, but cannot develop into brain cells.
- At the end of the long chain of cell divisions that make up the embryo are "terminally differentiated" cells - cells that are considered to be permanently committed to a specific function. [0004]
- scientists had long held the opinion that differentiated cells cannot be altered or caused to behave in any way other than the way in which have had been naturally committed.
- scientists have been able to persuade blood stem cells to behave like neurons. Therefore research has also focused on ways to make multipotent cells into pluripotent types.
- Stem cells are a rare population of cells that can give rise to vast range of cells tissue types necessary for organ maintenance and function. These cells are defined as undifferentiated cells that have two fundamental characteristics; (i) they have the capacity of self-renewal, (ii) they also have the ability to differentiate into one or more specialized cell types with mature phenotypes.
- Each group of stem cells has their own advantages and disadvantages for cellular regeneration therapy, specifically in their differentiation potential and ability to engraft and function de novo in the appropriate or targeted cellular environment.
- stem cell biology Much of the understanding of stem cell biology has been derived from hematopoietic stem cells and their behavior after bone marrow transplantation.
- stem cells There are several types of adult stem cells within the bone marrow niche, each having unique properties and variable differentiation ability in relation to their cellular environment.
- mesenchymal stem cells which have a wide range of non-hematopoietic differentiation abilities, including bone, cartilage, adipose, tendon, lung, muscle, marrow stroma, and brain tissues.
- neural stem cells pancreatic, muscle, adipose, ovarian and spermatogonial stem cells have been found.
- the therapeutic utility of somatic or post-natal stem cells has been demonstrated and realized through the use of bone marrow transplants.
- adult somatic stem cells have genomes that have been altered by aging and cell division. Aging results in an accumulation of free radical insults, or oxidative damage, that can predispose the cell to forming neoplasms, reduce cell differentiation ability or induce apoptosis.
- Repeated cell division is directly related to telomere shortening which is the ultimate cellular clock that determines a cells functional life-span. Consequently, adult somatic stem cells have genomes that have sufficiently diverged from the physiological prime state found in embryonic and prenatal stem cells.
- stem cells must be induced to mature into the organ or cell type desired to be useful as therapeutics.
- the factors affecting stem cell maturation in vivo are poorly understood and even less well understood ex vivo.
- present maturation technology relies on serendipity and biological processes largely beyond the control of the administering scientist or recipient.
- embryonic stem cells As a source of totipotent or pluripotent immunologically privileged cells for use in cellular regenerative therapy.
- embryonic stem cells themselves may not be appropriate for direct transplant as they form teratomas after transplant, they are proposed as "universal donor" cells that can be differentiated into customized pluripotent, multipotent or committed cells that are appropriate for transplant.
- moral and ethical issues associated with the isolation of embryonic stem cells from human embryos.
- the present invention provides biologically useful pluripotent therapeutically reprogrammed cells having minimal oxidative damage and telomere lengths that compare favorably with the telomere lengths of undamaged, pre-natal or embryonic stem cells (that is, the therapeutically reprogrammed cells of the present invention possess near prime physiological state genomes). Moreover the therapeutically reprogrammed cells of the present invention are immunologically privileged and therefore suitable for therapeutic applications.
- a method for therapeutically reprogramming differentiated cells to yield pluripotent cells comprising isolating a differentiated cell, preparing a nuclear extract from a pluripotent stem cell; and incubating the differentiated cells with the nuclear extract to form reprogrammed pluripotent cells.
- the differentiated cell is any diploid (2N) cell derived from cells selected from the group consisting of a pre- embryonic, embryonic, fetal, and post-natal multi-cellular organisms or a primordial sex cell.
- the pluripotent stem cell is selected from the group consisting of pre-embryonic stem cells, embryonic stem cells, fetal stem cells and post-natal stem cells.
- the nuclear extract does not contain genetic material, does not contain chromosomes or chromatin or contain DNA.
- the step of preparing a nuclear extract from a pluripotent stem cell comprises obtaining pluripotent stem cells, isolating karyoplasts from the pluripotent stem cells, removing the genetic material from the karyoplasts, and preparing an extract from the genetic-material deficient karyoplasts.
- the method further comprises the step of cryopreserving the reprogrammed pluripotent cells.
- the method further comprises the step of transplanting the reprogrammed pluripotent cells into a patient in need thereof.
- the reprogrammed pluripotent cell is autologous with the patient.
- the differentiated cell is in G 0 .
- a pluripotent cell -useful for regenerative therapy in a patient in need thereof comprising a differentiated cell therapeutically reprogrammed by exposure to a nuclear extract.
- the nuclear extract does not contain genetic material, does not contain chromosomes or chromatin or contain DNA.
- the differentiated cell is any diploid (2N) cell derived from cells selected from the group consisting of a pre-embryonic, embryonic, fetal, and post-natal multi-cellular organisms or a primordial sex cell.
- the pluripotent stem cell is selected from the group consisting of pre-embryonic stem cells, embryonic stem cells, fetal stem cells and post-natal stem cells.
- the nuclear extract is prepared from a pluripotent stem cell.
- the pluripotent cell in another embodiment of the pluripotent cell of the present invention, is cryopreserved. In yet another embodiment, the reprogrammed pluripotent cell is autologous with the patient.
- chemical modification refers to the process wherein a chemical or biochemical is used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.
- Committed refers to cells which are considered to be permanently committed to a specific function. Committed cells are also referred to as “terminally differentiated cells.”
- Cytoplast Extract Modification refers to the process wherein a cellular extract consisting of the cytoplasmic contents of a cell are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.
- Dedifferentiation refers to loss of specialization in form or function. In cells, dedifferentiation leads to an a less committed cell.
- Differentiation refers to the adaptation of cells for a particular form or function. In cells, differentiation leads to a more committed cell.
- Donor Cell refers to any diploid (2N) cell derived from a pre-embryonic, embryonic, fetal, or post-natal multi-cellular organism or a primordial sex cell which contributes its nuclear genetic material to the hybrid stem cell.
- the donor cell is not limited to those cells that are terminally differentiated or cells in the process of differentiation.
- donor cell refers to both the entire cell or the nucleus alone.
- Embryo refers to an animal in the early stages of growth and differentiation that are characterized implantation and gastrulation, where the three germ layers are defined and established and by differentiation of the germs layers into the respective organs and organ systems.
- the three germ layers are the endoderm, ectoderm and mesoderm.
- Embryonic Stem Cell refers to any cell that is totipotent and derived from a developing embryo that has reached the developmental stage to have attached to the uterine wall. In this context embryonic stem cell and pre- embryonic stem cell are equivalent terms. Embryonic stem cell-like (ESC-like) cells are totipotent cells not directly isolated from an embryo. ESC-like cells can be derived from primordial sex cells that have been dedifferentiated in accordance with the teachings of the present invention.
- Fetal Stem Cell refers to a cell that is multipotent and derived from a developing multi-cellular fetus that is no longer in early or mid-stage organogenesis.
- Germ Cell refers to a reproductive cell such as a spermatocyte or an oocyte, or a cell that will develop into a reproductive cell.
- Host Cell refers to any multipotent stem cell derived from a pre-embryonic, embryonic, fetal, or post-natal multicellular organism that contributes the cytoplasm to a hybrid stem cell.
- Hybrid Stem Cell refers to any cell that is multipotent and is derived from an enucleated host cell and a donor cell, or nucleus thereof, of a multicellular organism. Hybrid stem cells are further disclosed in co-pending United States Patent Application No. 10/864,788.
- Karyoplast Extract modification refers to the process wherein a cellular extract consisting of the nuclear contents of a cell, lacking the DNA 1 are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation or receptive to the host cell cytoplasm.
- Maturation refers to a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation or de-differentiation. As used herein, maturation is synonymous with the terms develop or development when applied to the process described herein.
- Multipotent refers to cells that can give rise to several other cell types, but those cell types are limited in number.
- An example of a multipotent cells is hematopoietic cells - blood stem cells that can develop into several types of blood cells but cannot develop into brain cells.
- Multipotent adult progenitor cells refers to multipotent cells isolated from the bone marrow which have the potential to differentiate into mesenchymal, endothelial and endodermal lineage cells.
- Pre-embryo refers to a fertilized egg in the early stage of development prior to cell division. During the pre-embryonic stage the initial stages of cleavage are occurring.
- Pre-embryonic Stem Cell See “Embryonic Stem Cell” above.
- Post-natal Stem Cell refers to any cell that is multipotent and derived from a multi-cellular organism after birth.
- Pluripotent refers to cells that can give rise to any cell type except the cells of the placenta or other supporting cells of the uterus.
- Primordial Sex Cell refers to any diploid cell that is derived from the male or female mature or developing gonad, is able to generate cells that propagate a species and contains a diploid genomic state. Primordial sex cells can be quiescent or actively dividing. These cells include male gonocytes, female gonocytes, spermatogonial stem cells, ovarian stem cells, oogonia, type-A spermatogonia, Type-B spermatogonia. Also known as germ-line stem cells.
- Primordial Germ Cell As used herein, “primordial germ cell” refers to cells present in early embryogenesis that are destined to become germ cells.
- Reprogamming refers to the resetting of the genetic program of a cell such that the cell exhibits pluripotency and has the potential to produce a fully developed organism.
- Responsive refers to the condition of a cell, or group of cells, wherein they are susceptible to and can function accordingly within a cellular environment. Responsive cells are capable of responding to and functioning in a particular cellular environment, tissue, organ and/or organ system.
- Somatic Cell As used herein, “somatic cell” refers to any cell in the body except gametes and their precursors.
- Somatic Stem Cells As used herein, “somatic stem cells” refers to diploid multipotent or pluripotent stem cells. Somatic stem cells are not totipotent stem cells.
- therapeutic Cloning refers to the cloning of cells using nuclear transfer methods including replacing the nucleus of an ovum with the nucleus of another cell and stem cells derived from the inner cell mass.
- Therapeutic Reprogramming refers to the process of maturation wherein a stem cell is exposed to stimulatory factors according to the teachings of the present invention to yield either pluripotent, multipotent or tissue-specific committed cells.
- Therapeutically reprogrammed cells are useful for implantation into a host to replace or repair diseased, damaged, defective or genetically impaired tissue.
- the therapeutically reprogrammed cells of the present invention do not possess non-human sialic acid residues.
- Totipotent refers to cells that contain all the genetic information needed to create all the cells of the body plus the placenta. Human cells have the capacity to be totipotent only during the first few divisions of a fertilized egg.
- whole cell extract modification refers to the process wherein a cellular extract consisting of the cytoplasmic and nuclear contents of a cell are used to induce genomic changes in the donor cell, or nucleus thereof, that allow the donor cell, or nucleus thereof, to be responsive during maturation and receptive to the host cell cytoplasm.
- the present invention provides biologically useful pluripotent therapeutically reprogrammed cells having minimal oxidative damage and telomere lengths that compare favorably with the telomere lengths of undamaged, pre-natal or embryonic stem cells (that is, the therapeutically reprogrammed cells of the present invention possess near prime physiological state genomes). Moreover the therapeutically reprogrammed cells of the present invention are immunologically privileged and therefore suitable for therapeutic applications.
- Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to four divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus.
- cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number.
- An example of multipotent cells is hematopoietic cells - blood cells that can develop into several types of blood cells, but cannot develop into brain cells.
- At the end of the long chain of cell divisions that make up the embryo are "terminally differentiated" cells - cells that are considered to be permanently committed to a specific function.
- primordial germ cells Upon migration and colonization of the genital ridge, the primordial germ cells undergo differentiation into male or female germ cell precursors (primordial sex cells).
- primordial sex cells For the purpose of this invention disclosure, only male primordial sex cells (PSC) will be discussed, but the qualities and properties of male and female primordial sex cells are equivalent and no limitations are implied.
- primordial stem cells become closely associated with precursor Sertoli cells leading to the beginning of the formation of the seminiferous cords.
- primordial germ cells When the primordial germ cells are enclosed in the seminiferous cords, they differentiate into gonocytes that are mitotically quiescent. These gonocytes divide for a few days followed by arrest at G 0 ZG 1 phase of the cell cycle. In mice and rats these gonocytes resume division within a few days after birth to generate spermatogonia! stem cells and eventually undergo differentiation and meiosis related to spermatogenesis.
- Primordial sex cells are directly responsible for generating the cells required for fertilization and eventually a new round of embryogenesis to create a new organism. Primordial sex cells are not programmed to die and are of a quality comparable to that of an embryonic state.
- Embryonic stem cells are cells derived from the inner cell mass of the pre- implantation blastocyst-stage embryo and have the greatest differentiation potential, being capable of giving rise to cells found in all three germ layers of the embryo proper. From a practical standpoint, embryonic stem cells are an artifact of cell culture since, in their natural epiblast environment, they only exist transiently during embryogenesis. Manipulation of embryonic stem cells in vitro has lead to the generation and differentiation of a wide range of cell types, including cardiomyocytes, hematopoietic cells, endothelial cells, nerves, skeletal muscle, chondrocytes, adipocytes, liver and pancreatic islets. Growing embryonic stem cells in co-culture with mature cells can influence and initiate the differentiation of the embryonic stem cells to a particular lineage.
- an embryo and a fetus are distinguished based on the developmental stage in relation to organogenesis.
- the pre-embryonic stage refers to a period in which the pre-embryo is undergoing the initial stages of cleavage.
- Early embryogenesis is marked by implantation and gastrulation, wherein the three germ layers are defined and established.
- Late embryogenesis is defined by the differentiation of the germ layer derivatives into formation of respective organs and organ systems.
- the transition of embryo to fetus is defined by the development of most major organs and organ systems, followed by rapid fetal growth.
- Embryogenesis is the developmental process wherein an oocyte fertilized by a sperm begins to divide and undergoes the first round of embryogenesis where cleavage and blastulation occur. During the second round, implantation, gastrulation and early organogenesis takes place. The third round is characterized by organogenesis and the last round of embryogenesis, wherein the embryo is no longer termed an embryo, but a fetus, is when fetal growth and development occurs. [0062] During embryogenesis the first two tissue lineages arising from the morulae post-cleavage and compaction are the trophectoderm and the primitive endoderm, which make major contributions to the placenta and the extraembryonic yolk sac. Shortly after compaction and prior to implanting the epiblast or primitive ectoderm begins to develop.
- the epiblast provides the cells that give rise to the embryo proper. Blastulation is complete upon the development of the epiblast stem cell niche wherein pluripotent cells are housed and directed to perform various developmental tasks during development, at which time the embryo emerges from the zona pellucida and implants to the uterine wall.
- Implantation is followed by gastrulation and early organogenesis.
- organogenesis By the end of the first round of organogenesis, all three germ layers will have been formed; ectoderm, mesoderm and definitive endoderm and basic body plan and organ primordia are established.
- embryogenesis is marked by extensive organ development at which time completion marks the transformation of the developing embryo into a developing fetus which is characterized by fetal growth and a final round of organ development.
- the gestation period is ended by birth, at which time the organism has all the required organs, tissues and cellular niches to function normally and survive post-natally.
- the process of embryogenesis is used to describe the global process of embryo development as it occurs, but on a cellular level embryogenesis can be described and/or demonstrated by cell maturation.
- Fetal stem cells have been isolated from the fetal bone marrow (hematopoietic stem cells), fetal brain (neural stem cells) and amniotic fluid (pluripotent amniotic stem cells). In addition, stem cells have been described in both adult male and fetal tissues. Fetal stem cells serve multiple roles during the process of organogenesis and fetal development, and ultimately become part of the somatic stem cell reserve.
- Maturation is a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation and/or dedifferentiation.
- a cell, or group of cells interacts with its cellular environment during embryogenesis and organogenesis. As maturation progresses, cells begin to form niches and these niches, or microenvironments, house stem cells that direct and regulate organogenesis. At the time of birth, maturation has progressed such that cells and appropriate cellular niches are present for the organism to function and survive post- natally. Developmental processes are highly conserved amongst the different species allowing maturation or differentiation systems from one mammalian species to be extended to other mammalian species in the laboratory.
- a single stem cell clone can contribute to generations of lineages such as lymphoid and myeloid cells for more than a year and therefore have the potential to spread mutations if the stem cell is damaged.
- the body responds to a compromised stem cell by inducing apoptosis thereby removing it from the pool and preventing potentially dysfunctional or tumorigenic properties.
- Apoptosis removes compromised cells from the population, but it also decreases the number of stem cells that are available for the future. Therefore, as an organism ages, the number of stem cells decrease. In addition to the loss of the stem cell pool, there is evidence that aging decreases the efficiency of the homing mechanism of stem cells.
- Telomeres are the physical ends of chromosomes that contain highly conserved, tandemly repeated DNA sequences.
- Telomeres are involved in the replication and stability of linear DNA molecules and serve as counting mechanism in cells; with each round of cell division the length of the telomeres shortens and at a pre-determined threshold, a signal is activated to initiate cellular senescence.
- Stem cells and somatic cells produce telomerase, which inhibits shortening of telomeres, but their telomeres still progressively shorten during aging and cellular stress.
- stem cells can be differentiated into particular cell types in vitro and shown to have the potential to be multipotent by engrafting into various tissues and transit across germ layers and as such have been the subject of much research for cellular therapy.
- immune rejection is the limiting factor for cellular therapy.
- the recipient individual's phenotype and the phenotype of the donor will determine if a cell or organ transplant will be tolerated or rejected by the immune system.
- the present invention provides methods and compositions for providing functional immunocompatible stem cells for cellular regenerative/reparative therapy.
- therapeutically reprogrammed cells are provided.
- Therapeutic reprogramming refers to a maturation process wherein a stem cell is exposed to stimulatory factors according the teachings of the present invention to yield pluripotent, multipotent or tissue-specific committed cells.
- the process of therapeutic reprogramming can be performed with a variety of stem cells including, but not limited to, therapeutically cloned cells, hybrid stem cells, embryonic stem cells, fetal stem cells, multipotent adult progenitor cells, adipose-derived stem cells (ADSC) and primordial sex cells.
- ADSC adipose-derived stem cells
- Therapeutic reprogramming takes advantage of the fact that certain stem cells are relatively easily to obtain, such as spermatogonia! stem cells and adipose-derived stem cells, and epigenetically reprograms these cells by exposure to stimulatory factors. These therapeutically reprogrammed cells have changed their maturation state to either a more committed cell lineage or a less committed cell lineage. Therapeutically reprogrammed cells are therefore capable of repairing or regenerating disease, damaged, defective or genetically impaired tissues.
- Therapeutic reprogramming uses stimulatory factors including, without limitation, chemicals, biochemicals and cellular extracts to change the epigenetic programming of cells. These stimulatory factors induce, among other results, genomic methylation changes in the donor DNA.
- stimulatory factors including, without limitation, chemicals, biochemicals and cellular extracts to change the epigenetic programming of cells.
- These stimulatory factors induce, among other results, genomic methylation changes in the donor DNA.
- Quiescent spermatogonia! stem cells (SSC) are particularly suitable for therapeutic reprogramming with the nuclear factors of the present invention.
- the quiescent SSCs are highly demethylated and therefore they are available for programming (patterning) or differentiation into any cell type.
- Embodiments of the present invention include methods for preparing cellular extracts from whole cells, cytoplasts, nuclei and karyoplasts, although other types of cellular extracts are contemplated as being within the scope of the present invention.
- the cellular extracts of the present invention are prepared from stem cells, specifically embryonic stem cells. Donor cells are incubated with the chemicals, biochemicals or cellular extracts for defined periods of time, in a non-limiting example for approximately one hour to approximately two hours, and those reprogrammed cells that express embryonic stem cell markers, such as OctA, after a culture period are then ready for transplantation, cryopreservation or further maturation.
- primordial sex cells are therapeutically reprogrammed.
- Primordial sex cells residing in the lining of the seminiferous tubules of the testes and the lining of the ovaries (the spermatogonia and oogonia, respectively) have been determined to possess diploid (2N) genomes remarkably undamaged by to the effects of aging and cell division.
- 2N diploid
- PSCs possess genomes in a nearly physiologically prime state.
- a non-limiting example of a PSC particularly useful in an embodiment of the present invention is a spermatogonia! stem cell.
- therapeutically reprogrammed PSC cells are prepared for the maturation process using means similar to that experienced by stem cells present in the developing embryo and fetus during embryogenesis and organogenesis.
- Therapeutically reprogrammed cells made in accordance with the teachings of the present invention can be used for therapeutic purposes as is, they can be cryopreserved for future use or they can be further matured into a more committed cell lineage.
- Embodiments of the present invention provide methods for further maturing or differentiating therapeutically reprogrammed cells, stem cells and primordial sex cells into more committed cell lineages in a post-natal environment to provide more committed cells for use in cellular regenerative/reparative therapy.
- the maturation and differentiation process provides therapeutic cells that can be used to treat or replace damaged cells in pre- and post-natal organs.
- the therapeutically reprogrammed cells made in accordance with the teachings of the present invention are useful in a wide range of therapeutic applications for cellular regenerative/reparative therapy.
- the therapeutically reprogrammed cells of the present invention can be used to replenish stem cells in animals whose natural stem cells have been depleted due to age or ablation therapy such as cancer radiotherapy and chemotherapy.
- the therapeutically reprogrammed cells of the present invention are useful in organ regeneration and tissue repair.
- therapeutically reprogrammed cells can be used to reinvigorate damaged muscle tissue including dystrophic muscles and muscles damaged by ischemic events such as myocardial infarcts.
- the therapeutically reprogrammed cells disclosed herein can be used to ameliorate scarring in animals, including humans, following a traumatic injury or surgery.
- the therapeutically reprogrammed cells of the present invention are administered systemically, such as intravenously, and migrate to the site of the freshly traumatized tissue recruited by circulating cytokines secreted by the damaged cells.
- the therapeutically reprogrammed cells can be administered locally to a treatment site in need or repair or regeneration.
- Stem cells are not universally susceptible to the maturation process of the present invention. Therefore the present inventors have developed a therapeutic reprogramming process whereby stem cells are induced into a state whereby they are susceptible to maturation factors. This therapeutic reprogramming process can be accomplished by incubation with stimulatory factors under suitable conditions and for a time sufficient to render the donor cell susceptible for maturation.
- differentiated cells are reprogrammed by incubation with nuclear factors from pluripotent cells.
- the source of these nuclear factors can be any multipotent stem cell including, but is not limited to, multipotent stem cells isolated from pre-embryonic, embryonic, fetal or post-natal multicellular organisms.
- Nuclear factors for reprogramming are isolated from multipotent stem cell karyoplasts.
- the nuclear extracts contain the nuclear contents with genetic material removed.
- the nuclear extracts lack intact or functional chromosomes.
- the scope of the present invention includes multipotent stem cell nuclear extracts having DNA and/or chromatin removed.
- Methods for preparing the nuclear extracts of the present invention are disclosed in Examples 3 and 4. Additional methods for removing genetic material from cells or karyoplasts include, but are not limited to, centrifugation, enzymatic treatment, precipitation, chromatography and other methods that are known to persons skilled in the art.
- the nuclear extracts made according to the teachings of the present invention can be cryopreserved after preparation.
- Differentiated cells are treated with the nuclear extracts of the present invention by methods known to persons skilled in the art.
- methods to treat differentiated cells with nuclear extracts include co-culture of differentiated cells with nuclear extracts and micro-injection of nuclear extracts into the nucleus of differentiated cells.
- Pluripotentiality of extract-treated cells is determined by measuring the expression of the stem cell marker gene Oct4.
- OcU has an essential role in the control of developmental pluripotency and can activate or repress the expression of various genes, it is not known how Oct4 controls the pluripotent epigenotype but cells which have lost Oct4 expression are not plutipotent.
- Nuclear extract-treated cells which express Oct4 are considered reprogrammed with a pluripotent epigenotype and are capable of differentiating into many cell types upon presentation with the appropriate differentiation factors in vitro or in vivo.
- testes were excised and decapsulated.
- Testicular tissue was minced using fine scissors and transferred into culture medium (DMEM/F12) containing 1 mg/mL collagenase type I (Sigma) and 0.5 mg/mL DNase (Sigma). Digestion was performed at 37°C for 10 min in a shaking water bath operated at 110 cycles/min. Interstitial cells are separated by sedimentation at unit gravity for 10 min and washed in DMEM/F12.
- culture medium DMEM/F12
- a final digestion of the basal lamina components of the testicular tissue was carried out in a mixture of collagenase type I (1 mg/mL), DNase (0.5 mg/mL), and hyaluronidase (Sigma; 0.5 mg/mL) under the same conditions as for the first digestion step.
- the single-cell suspension obtained was washed successively with medium and PBS containing 1 mM EDTA (Sigma) and 0.5% fetal calf serum.
- the undigested remains of the tunica albuginea were eliminated by filtering the cell suspension through a 50 ⁇ m nylon mesh. All cells were kept at 5°C throughout the procedure.
- the dissociated testicular cells were suspended (5x10 6 cells/mL) in PBS containing 0.5% FBS (PBS/FBS). The cells were then incubated with primary antibodies for 20 min on ice, washed twice with excess PBS/FBS, and used for FACS analysis.
- Primary antibodies include R-phycoerythrin (PE)- conjugated anti- ⁇ 6-integrin, allophycocyanin (APC)-conjugated anti-c-kit, and biotinylated anti- ⁇ v-integrin.
- PE R-phycoerythrin
- APC allophycocyanin
- biotinylated anti- ⁇ v-integrin biotinylated anti- ⁇ v-integrin.
- secondary reagents cells were further incubated for 20 min with APC-conjugated streptavidin to detect biotinylated antibody. All antibodies or secondary reagents were used at 5 ⁇ g/mL.
- Control cells were not treated with antibodies. After the final wash, the cells were resuspended (10 7 cells/mL) in 2 mL PBS/FBS containing 1 ⁇ g/mL propidium iodide (Sigma), filtered into a tube through a 35 ⁇ m pore-size nylon screen, and kept in the dark on ice until analysis. The cells were sorted based on antibody staining and their relative granularity or internal complexity (side scatter, SSC). Cell sorting was performed by a dual-laser FACStar Plus (Becton Dickinson) equipped with 488-nm argon (200 mW) and 633-nm helium neon (35 mW) laser.
- FACStar Plus Becton Dickinson
- An argon laser was used to excite PE and propidium iodide, and emissions were collected with a 575 DF 26 filter for PE and a 610 DF 20 filter for propidium iodide.
- a neon laser was used to excite APC, and emission was detected with a 675 DF 20 filter.
- Dead cells were excluded by eliminating propidium iodide-positive events at the time of data collection.
- Cells were sorted into 5 mL polystyrene tubes containing 2 mL of ice-cold DMEM supplemented with 10% FBS (DMEM/FBS). The ⁇ 6-integrin hi /SSC l0 /c-kit(-) population was used as the donor cell.
- primordial sex cells can be isolated from a punch biopsy of the ovaries. The PSCs are then isolated with the assistance of a microscope. Primordial sex cells have stem cell morphology (i.e. large, round and smooth) and are mechanically retrieved from the ovaries.
- This example describes the therapeutic reprogramming of a PSC so that it is functional and responds appropriately during maturation by inducing genomic modifications using nuclear (karyoplast) extracts from embryonic stem cells.
- ESCs embryonic stem cell nuclear
- the ESC karyoplasts are prepared using a discontinuous density gradient of Ficoll-400 (30%, 25%, 22%, 18% and 15%) containing 10 //g/mL cytochalasin B.
- Ten million ESCs in 12.5% Ficoll-400 are carefully layered on top of the gradient and centrifuged at 40,000 rpm at 36 0 C for 30 min.
- the karyoplasts are collected from the 30% level.
- the karyoplasts are then washed three times with ice-cold PBS followed by a wash in cell lysis buffer.
- the karyoplasts are then centrifuged at 350xg and resuspended in 1.5 volumes of cell lysis buffer containing protease inhibitors and incubated on ice for 45 min.
- the karyoplasts are then homogenized by pulse sonication and then the karyoplasts are centrifuged at 16,000xg for 20 min at 4°C. The supernatant is then collected and protein concentration determined to be approximately 6 mg/mL.
- the previously isolated PSCs are washed three times with ice-cold PBS, followed by a two washes in HBSS.
- the cells are then centrifuged at 350xg for 5 min at 4 0 C and resuspended at 10,000 cells per 14 ⁇ l_ of ice-cold HBSS.
- the cells are then incubated at 37°C for 2 min followed by the addition of streptolysin O (SLO; Sigma) at a final concentration of 115 ng/mL to 230 ng/mL depending on cell number and incubated for 50 min at 37°C with constant shaking to keep the cells from sedimenting.
- the cells are then centrifuged at 500xg for 5 min at 4 0 C and the supernatant removed.
- the PSCs are then incubated with 50 ⁇ L of previously prepared embryonic stem cell extract containing an ATP- regenerating system and 1mM of each of the four nucleoside triphosphates (NTP) at 37 0 C for 1-2 hours.
- the cells are then resuspended in solution of 2 mM CaCI2 in preparation media (1% nonessential amino acids, 1% L-glutamine, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.1 mM ⁇ -mercaptoethanol, 3,000 units/mL of leukemia inhibitory factor (LIF) in DMEM/20% FBS) and placed into one well of a 48-well dish pre-treated with 0.1% gelatin containing a mitomycin C-inactivated primary embryonic fibroblast (PEF) layer.
- LIF leukemia inhibitory factor
- the extract-treated PSCs in a 48-well dish pre-treated with 0.1% gelatin containing a mitomycin C-inactivated PEF layer and 50% confluent ESCs. After 24 hours, cells that were not attached to the feeder layer were removed and the extract exposure procedure was repeated a second time with the unattached cells.
- the reprogrammed cells (attached cells) are cultured and assayed for embryonic stem cell specific markers (i.e. REX1 , OCT4). Additionally the extract-treated (reprogrammed) cells can be tested for in vitro differentiation potential prior to being exposed to a maturation process.
- the stem cells are plated two days prior to extract preparation and allowed to reach maximum monolayer growth in preparation media on 25x75 mm tissue culture slides pre-treated with 10 ng/mL fibronectin at 37 0 C for 1 hour.
- 2 ⁇ g/mL of cytochalasin D (final concentration) is added to the media and the slides incubated for 120 min at 37°C.
- the slides are centrifuged in a swinging bucket centrifuge at 10,000xg for 1 hour in preparation media containing 2 ⁇ g/mL of cytochalasin D. Prior to centrifugation, the rotor and centrifuge are pre-warmed to 37°C.
- the pellet containing the nuclei is washed three times with ice-cold PBS, followed by one wash in cell lysis buffer.
- the cells are then centrifuged at 350xg and resuspended in 1.5 volumes of cell lysis buffer containing protease inhibitors and the nuclei are incubated on ice for 15-45 min.
- the nuclei are homogenized by pulse sonication.
- the lysates are then centrifuged at 16,000xg for 20 min at 4°C. The supernatant is then collected and protein concentration determined.
- the previously isolated stem or somatic cells are washed three times with ice- cold PBS, followed by a two washes in HBSS.
- the cells are then centrifuged at 350xg for 5 min at 4°C and resuspended at 10,000 cells per 14 ⁇ L of ice-cold HBSS.
- the cells are then incubated at 37 0 C for 2 min followed by the addition of streptolysin O at a final concentration of 115 ng/mL to 230 ng/mL depending on cell number and incubated for 50 min at 37°C with constant shaking to keep the cells from sedimenting.
- the cells are then centrifuged at 500xg for 5 min at 4°C and the supernatant removed.
- the stem or somatic cells are then incubated with 50 ⁇ L of previously prepared stem cell nuclear extracts containing an ATP- regenerating system and 1mM of each of the four NTPs at 37 0 C for 1-2 hours.
- the cells are then resuspended in solution of 2 mM CaCI 2 in preparation media (1% nonessential amino acids, 1% L-glutamine, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.1 mM ⁇ - mercaptoethanol, 3,000 units/mL of LIF in DMEM/20% FBS) and placed into one well of a 48-well dish pre-treated with 0.1% gelatin containing a mitomycin C-inactivated primary embryonic fibroblast layer.
- the reprogrammed cells are cultured are then available for transplant directly into a recipient, cryopreserved for future use, subjected to a maturation process or fused with appropriate host cells to generate hybrid stem cells.
Abstract
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AU2006210463A AU2006210463A1 (en) | 2005-02-02 | 2006-02-02 | Use of nuclear material to therapeutically reprogram differentiated cells |
EP06734404A EP1844137A2 (en) | 2005-02-02 | 2006-02-02 | Use of nuclear material to therapeutically reprogram differentiated cells |
CA002595750A CA2595750A1 (en) | 2005-02-02 | 2006-02-02 | Use of nuclear material to therapeutically reprogram differentiated cells |
JP2007554290A JP2008528059A (en) | 2005-02-02 | 2006-02-02 | Use of nuclear material to reprogram differentiated cells for therapy |
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US11/060,131 US20050170506A1 (en) | 2002-01-16 | 2005-02-16 | Therapeutic reprogramming, hybrid stem cells and maturation |
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Cited By (6)
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US8048999B2 (en) | 2005-12-13 | 2011-11-01 | Kyoto University | Nuclear reprogramming factor |
US8058065B2 (en) | 2005-12-13 | 2011-11-15 | Kyoto University | Oct3/4, Klf4, c-Myc and Sox2 produce induced pluripotent stem cells |
US8129187B2 (en) | 2005-12-13 | 2012-03-06 | Kyoto University | Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2 |
US8211697B2 (en) | 2007-06-15 | 2012-07-03 | Kyoto University | Induced pluripotent stem cells produced using reprogramming factors and a rho kinase inhibitor or a histone deacetylase inhibitor |
US9213999B2 (en) | 2007-06-15 | 2015-12-15 | Kyoto University | Providing iPSCs to a customer |
US9499797B2 (en) | 2008-05-02 | 2016-11-22 | Kyoto University | Method of making induced pluripotent stem cells |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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US8048999B2 (en) | 2005-12-13 | 2011-11-01 | Kyoto University | Nuclear reprogramming factor |
US8058065B2 (en) | 2005-12-13 | 2011-11-15 | Kyoto University | Oct3/4, Klf4, c-Myc and Sox2 produce induced pluripotent stem cells |
US8129187B2 (en) | 2005-12-13 | 2012-03-06 | Kyoto University | Somatic cell reprogramming by retroviral vectors encoding Oct3/4. Klf4, c-Myc and Sox2 |
US8211697B2 (en) | 2007-06-15 | 2012-07-03 | Kyoto University | Induced pluripotent stem cells produced using reprogramming factors and a rho kinase inhibitor or a histone deacetylase inhibitor |
US8257941B2 (en) | 2007-06-15 | 2012-09-04 | Kyoto University | Methods and platforms for drug discovery using induced pluripotent stem cells |
US9213999B2 (en) | 2007-06-15 | 2015-12-15 | Kyoto University | Providing iPSCs to a customer |
US9714433B2 (en) | 2007-06-15 | 2017-07-25 | Kyoto University | Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue |
US9499797B2 (en) | 2008-05-02 | 2016-11-22 | Kyoto University | Method of making induced pluripotent stem cells |
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