WO2003062384A2 - Stem cell maturation for all tissue types - Google Patents
Stem cell maturation for all tissue types Download PDFInfo
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- WO2003062384A2 WO2003062384A2 PCT/US2003/001570 US0301570W WO03062384A2 WO 2003062384 A2 WO2003062384 A2 WO 2003062384A2 US 0301570 W US0301570 W US 0301570W WO 03062384 A2 WO03062384 A2 WO 03062384A2
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
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- 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
- C12N5/0608—Germ cells
- C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
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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
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
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- 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
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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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
- C12N2517/00—Cells related to new breeds of animals
- C12N2517/04—Cells produced using nuclear transfer
Definitions
- Stem cells are capable of long-term self renewal and can give rise to mature cell types with specific morphology and function.
- ES embryonic stem
- adult stem cells typically share at least two characteristics: i) they can make identical copies of themselves for long periods of time (long term self-renewal); and they can give rise to mature cell types that have characteristic morphologies and specialized functions.
- Stem Cells Scientific Progress and Future Research Directions, Dept. of Health and Human Services, Jun 2001; http://www.nih.gov/news/stemcell/scireport.htm.
- Adult stem cells are believed to be not as pluripotent as ES cells, however, at least one report has suggested that adult stem cells show more plasticity than previously conceived. Lagasse, E. et al. (2000), "Purified hematopoietic stem cells can differentiate into hepatocytes in vivo," Nat. Med. 6, 1229-34.
- an adult stem cell should give rise to fully differentiated cells that have mature phenotypes.
- the adult stem cells should also be fully integrated into their new tissue environment and be capable of specialized tissue functions, which are appropriate for that tissue.
- Stem Cells Scientific Progress and Future Research Directions, supra.
- the difficulty in studying adult stem cell plasticity is establishing that the adult stem cell arises out of one type of cell, or cell population.
- the best studied adult stem cells are based on bone marrow and brain cells.
- stem cells derived from the bone marrow i.e. hematopoietic stem cells (HSCs), stromal cells and/or endothelial cells
- the brain i.e.
- HSCs from the bone marrow are sorted using a cell sorter, which sorts the cells according to various cell surface markers. This methodology yields highly purified to partially purified cell types.
- purification of neuronal stem cells are difficult because these cells are located in different locations (i.e. olfactory bulb, hipppocampus and lateral ventricles of mice) and not in one convenient location or organ tissue.
- Altman, J. and Das, G.D. (1965) "Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats," J. Compl Neural., 124, 319-335; Altman, J. (1969), Autoradiographic and histological studies of postnatal neurogenesis.
- IV "Cell proliferation and migration into the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb," J. Compl Neural., 137, 433-457.
- Another type of adult stem cell is derived from germ cells, or primordial sex cells (PSC), residing in the lining of the seminiferous tubules of the testes and lining of the ovaries- the spermatogonia and oogonia, respectively.
- PSC primordial sex cells
- spermatogonia produce precursor cells that are involved in meiosis.
- type A spermatogonia are more spherical with a prominent nucleolus and uniformly scattered euchromatin.
- type B spermatogonia tend to be more irregular in shape and smaller with a lobed nucleus.
- Nicolas E, et al. 2001
- Proteome analysis of rat spermatogonia: reinvestigation of stathmin spatio-temporal expression within the testis Mol. Reprod. Dev., 60(4):439-45.
- Chiarini-Garcia, H. and Russell, L.D. (2002), "Characterization of mouse spermatogonia by transmission electron microscopy," Reproduction, 123(4): 567-77.
- adult germ cells are easy to locate and distinguished from other interstitial cells. [0009] Similar to other adult somatic stem cells, adult germ cells are diploidy (2n).
- germ cells spermatogonia and oogonia
- somatic cellular DNA is more damaged (i.e. free radicals) due to their age and low rate of replenishment.
- somatic stem cells finally succumb to the forces of differentiation that create the tissues of the body.
- methods comprising a stem cell consisting of undamaged DNA is preferred.
- a persistent problem with adult stem cell transplants in vivo is that of immune rejection.
- recipient's of stem cells are reliant on the right donors whose cells will not be rejected by the recipient's immune system.
- typically bone marrow transplants are allogeneic transplants (different host and donor) and in order for them to work, the recipient's immune system must accept rather than try to destroy the donated marrow. This is accomplished by making sure that the antigens on the donated marrow cells are identical, or very similar to, the antigens on the cells of the recipient.
- an improved stem cell transplant method which eliminates concerns regarding immune rejection is highly advantageous.
- a general object of the present invention is to provide a modified germ cell comprising a primordial sex cell (PSC), or nucleus thereof, translocated into an enucleated ovum, wherein the PSC and the ovum are derived from the same species of animal.
- PSC primordial sex cell
- Another general object of the present invention is to provide a method for preparing a modified germ cell comprising: (a) obtaining a PSC from a first donor animal; (b) obtaining an ovum from a second donor animal of the same species as the first donor animal; (c) enucleating the ovum; and (d) translocating the PSC, or nucleus thereof, into the enucleated ovum.
- Another general object of the present invention is to provide for maturing a modified germ cell comprising: (a) expanding the modified germ cell in a first medium; (b) placing the modified germ cell into a second medium; (c) screening for membrane receptors on the modified germ cell; (d) repeating steps b and c until the number of receptors on the modified germ cell are sufficient to be a primed modified germ cell; and (f) translocating the primed modified germ cell into the tissue of the animal
- Another general object of the present invention is to provide a method for preparing a mammalian modified germ cell comprising: (a) obtaining a PSC from a first donor mammal; (b) obtaining an ovum from a second donor animal of the same species as the first donor animal; (c) enucleating the ovum; (d) translocating the
- PSC PSC, or nucleus thereof, into the enucleated ovum; (e) expanding the modified germ cell in a first medium; (f) placing the modified germ cell into a second medium; (g) screening for membrane receptors on the modified germ cells; (h) repeating steps b and c until the number of receptors on the modified germ cell are sufficient to be a primed modified germ cell; and (i) translocating the primed modified germ cell into the tissue of the animal.
- Another general object of the present invention is to provide a method for preparing a modified germ cell comprising: (a) obtaining a PSC from a first donor animal; (b) obtaining an ovum from a second donor animal of the same species of the first donor animal; (c) enucleating the ovum; (d) fusing the PSC and the enucleated ovum to form a fused cell; and (e) activating the fused cell.
- Another general object of the present invention is to provide a method for inducing the modified germ cell to produce the precursor cell that functions in the adult tissue environment, comprising: (a) obtaining at least one modified germ cell; and (b) culturing the modified germ cells in the presence of maturing factors under suitable conditions, and for a time sufficient, to induce the modified germ cells to produce receptor sites.
- Another general object of the present invention is to provide a cell culture chamber comprising: (a) at least one isolation chamber; (b) tubing connecting the chamber; and (c) at least one pump.
- Still another general object of the present invention is to provide a cell culture chamber comprising: (a) at least one isolation chamber; (b) tubing connecting the chamber; (c) a carbon dioxide source; (d) an oxygen source; (e) a molecular filter; and (f) at least one pump.
- a cell culture chamber comprising: (a) at least one isolation chamber; (b) tubing connecting the chamber; (c) a carbon dioxide source; (d) an oxygen source; (e) a molecular filter; and (f) at least one pump.
- spermatogonia used herein means a primordial male sex cells that give rise to progenitors of primary spermatocytes.
- oogonia used herein means a primordial female sex cells that serves as a source of ova.
- ovum used herein means the female gamete, a haploid unfertilized egg, which is capable of developing into a new animal when fertilized by a spermatozoon.
- oocyte used herein means a developing egg cell in oogenesis and upon undergoing meiosis forms the ovum.
- biomass used herein means a specialized chamber to grow, expand, maintain, sustain and mature cells in vitro.
- modified germ cell means a cell comprised of an ovum cytosol from one animal and a nucleus from either an oogonium or a spermatogonium of the same species animal as that of the ovum, or a different animal.
- the term "primed MGC” used herein means modified germ cell(s) that ready for translocation into the host animal in vivo or host tissue in vitro.
- the invention provides a totiipotential modified germ cell derived from an animal or mammal donor, which can be expanded, grown, maintained, sustained and matured in a specialized bioreactor chamber. Only those modified germ cells which have the sufficient number of membrane receptors described by the methods herein are within the scope of the invention. Given the methods described herein, the modified germ cells can be made from any animal and translocated into any animal. However, mammals are preferred because there are many therapeutics and beneficial effects of stem cells for mammals.
- the present invention also provides for a composition
- a composition comprising: a mammalian primordial sex cell (PSC), or nucleus thereof, translocated into an enucleated ovum, wherein the PSC and the ovum are derived from the same species of animal or mammal or different animal or mammal.
- PSC primordial sex cell
- Another term for this composition is a modified germ cell or MGC.
- the PSC is a mammalian spermatogonium, or nucleus thereof. In another aspect, the PSC is a mammalian oogonium, or nucleus thereof.
- Alternative methods of enucleation and nucleation are contemplated within the scope of the present invention including mechanical methods as well as methods utilizing electrical stimuli.
- the nucleus from any precursor cell from the spermatogonia or oogonia prior to the DNA being divided into the haploid state can be used.
- the MGC of the present invention is totipotent, pluripotent, multipotent or bipotent. That is the MGC is capable of forming at least one type of tissue, more particularly, the MGC is capable of forming at least more than one type of tissue.
- the MGC Once the MGC is established, it can be manipulated by various methods described herein to produce desired characteristics. For example, the MGC can be expanded and maintained in a particular medium. For another example, the MGC can be matured in a step-wise manner to particular stages of development typical of a mature stem cell.
- the MGC is first expanded to about a 6-cell stage in one chamber of the multi-chamber bioreactor.
- the MGC can be expanded to more than a 6-cell stage, however, beyond the 10-cell stage, germ cells make progenitor or precursor cells.
- the 6-cell stage MGC is then matured in a step-wise fashion using cells isolated from different gestation to post-natal stages, which are being maintained in a nearby chamber. At least one group of cells from a gestational to post-natal donor is used to facilitate maturing of the MGC. However, more than one group of cell(s) from a gestational to post-natal stage may be used to mature the MGC.
- the mature MGC is now termed a primed MGC.
- the primed MGC has sufficient receptors that upon translocation into a host animal or tissue, in vivo or in vitro, the primed MGC behaves similar to that of a mature stem cell.
- a method for screening MGCs which have acquired certain receptors is described herein using a resonance energy transfer method, in particular, Fluorescence Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET; Packard BioScience, BioSignal Packard Inc., Meriden.CT). This method helps to determine which MGCs are ready to be translocated into the host animal.
- FRET Fluorescence Resonance Energy Transfer
- BRET Bioluminescence Resonance Energy Transfer
- Other methods to screen for the number of receptors are possible and although not described herein, are within the scope of this invention in that it is used to determine whether the MGC is primed.
- a specialized apparatus for expanding, growing, maintaining, sustaining and maturing cells.
- the specialized apparatus is termed a bioreactor chamber, containing at least one chamber, in particular, at least two chambers.
- the chambers of the bioreactor are connected by tubing that allows bidirectional flow of fluid between the chambers.
- the pressure driving the bidirectional flow of fluid is provided by at least by one peristaltic pump with a multiple head, or two or more stand-alone pumps.
- Other ancillary systems not described herein in FIG. 1 , are also used including a micro-oxygenator and pump, a C0 2 reserve and some type of molecular sieve filtration system.
- pumps are associated with each chamber for even flow distribution.
- the invention provides MGCs generated by any animal, the invention provides methods of using the MGCs to contribute to therapeutics in vivo and in vitro comprising, injecting the primed MGCs into the host animal or mammal.
- Preparations of the MGCs can be derived from the same species or they can be derived from different species. Translocation of the primed MGCs can be into the same species host or a different species host.
- the primed MGCs can be used to derive cells for therapeutics to treat abnormal conditions and tissue repair.
- PSCs Primordial Sex Cells
- the mammal or animal is anesthetized and the gonads are removed and transected.
- the primary sex cells (PSCs) are isolated with the aid of a microscope.
- a biopsy punch of the gonads can also be used and the PSCs isolated with the aid of a microscope.
- the PSCs have stem cell morphology (i.e. large, round and smooth) and are mechanically retrieved from the gonads.
- the spermatogonia and oogonia are retrieved from the gonads.
- type A and type B spermatogonia are retrieved.
- an ova/ovum the animal is superovulated, and at least one ovum is retrieved and placed in nutritive media to keep it viable.
- the ova is held in place using a micropipette and with another micropipette (i.e. patchman) enter the ova until the tip is adjacent to the ova nucleus.
- Enucleating the ova is possible by applying a small vacuum to the micropipette.
- Enucleation methods (above) are repeated with the PSCs (i.e spermatogonia and/or oogonia), except this time the nucleus is retained and the cytosol is discarded.
- Other methods of enucleation and nucleation are contemplated within the scope of the present invention including other mechanical methods as well as methods utilizing electrical stimuli.
- MGC Modified Germ Cell
- EXAMPLE 2 Expanding the MGC. Take the MGC and place in a nutritive media comprising at least M15:high glucose DMEM (minus pyruvate and minus glutamine), about 15-20% fetal bovine serum (FBS), 1X 1-glutamine, 1X penicillin/streptomycin, 1X non-essential amino acids, 1X ribonucleosides, 1X b-mercaptoethanol (bME) and 1:1000 leukemia inhibitory factor (LIF).
- the MGCs are prohibited from aggregating past a certain cell size, for example, the 6-cell stage. Mitotic divisions of the MGC can be observed using a dissecting scope.
- the MGCs can be expanded on a layer of feeder cells. However, typically the cells are kept in suspension when maintained in the bioreactor chamber. At the 6-cell stage, the MGCs are mechanically separated. Alternatively, a sugar residue that binds to the surface molecules on the membrane of the MGCs will prevent aggregation of cells. Other methods of separating the MGCs are also within the scope of the present invention.
- the MGCs at this 6-cell stage are now grown or expanded to lesser to equal 10 3 to greater and equal to 10 8 cells, preferably 10 5 cells.
- the MGCs should be suspended in a multicellular or unicellular state in the bioreactor chamber(s). Upon expanding to certain number, the MGCs are then placed in a different bioreactor chamber containing a different medium to start the maturation process.
- FIG. 1 describes an example of one modification of the bioreactor chamber.
- the bioreactor is comprised of at least one chamber, preferably at least two chambers.
- the chamber is used to grow, expand, maintain, sustain and mature cells, generally, or in the case of the present invention, MGCs.
- Figure 1 is an example of a multi-chambered bioreactor 1.
- the chambers 5 can be limited to one, but preferably there are at least two chambers 5.
- the chambers 5 are comprised of silicon oxide or glass. However, other materials used to construct similar biological chambers can be used.
- the chambers 5 are connected by tubing 10 to each other, and further connected by tubing 10 to various ancillary systems including peristaltic pumps 15, micro-oxygenators, C0 2 reserves and molecular sieve filters (not shown in FIG. 1).
- the tubing 10 is comprised of neoprene or other similar made materials for use in biological systems.
- the tubing 10 can have various diameters from 1/8 of an inch to 1/3 of an inch. However, smaller or greater diameter tubing for similar uses is possible.
- the different size tubings 10 are accommodated by different size fittings 20 of the chamber(s) 5.
- the tubing 10 allows flow of fluid media in the chambers comprising of nutrients, further comprising of macro and micromolecules, between the chambers 10.
- the flow of the nutrients is driven by two peristaltic pumps 15; or alternatively by at least one pump with multiple heads (not shown).
- Each peristaltic pump 15 or each head of a multi-head peristaltic pump drives fluid flow in one direction. However, using at least two pumps 15 allows for bi-directional fluid flow into and out of the chambers 5.
- a pH sensor 25 and pH meter 30 Also shown in FIG. 1 is a pH sensor 25 and pH meter 30.
- the pH sensor 25 is first connected to a pH meter 30 is secondly connected or immersed below the surface of the media in the chamber 5.
- a pH sensor 25 is optionally immersed below the surface of the media in all chambers 5 of the bioreactor 1 and further connected to a pH meter 30.
- the pH sensor 25 detects drops and rises in pH in the media in the chamber 5, and will send a stimulus to the pH meter 30.
- the pH meter 30 in turn contains wires connected to C0 2 valves 35 further connected by fittings 20 on the chambers 5. For example, when the pH of the media in the chambers 5 is low, a stimulus back to the pH meter 35 to open the C0 valve(s) 35, thereby allowing C0 2 from the C0 2 reserve to flow into the chamber 5.
- Ancillary systems not shown in FIG.1 include a C0 2 reserve which supplies C0 2 via the C0 2 valve 35. Also not shown in FIG. 1 is a micro-oxygenator (Aqua Pro) and pump. The micro-oxygenator is connected similar to the C0 2 reserve via a valve and tubing 10. Fluid from the tubing 10 flows through the micro-oxygenator and is oxygenated by side ports or inlets which inject oxygen into the space; thereby aerating the fluid for improved viability of the cells.
- Aqua Pro micro-oxygenator
- a molecular dialysis filter Similar to the micro- oxygenator and attachment, fluid flows through the filter and particular sized molecules are restricted, for example, molecules at least about 60 KDa are restricted from the fluid.
- the dialysis filter works on counter-current system and uni-directional current system .
- highly purified water i.e., ionized, UV treated and microfiltered
- the highly purified water can be added to the media in the chambers 5 by any sterile means available.
- the media used in the chambers 5 is artificial blood comprised of a perfluorocarbon, which affords increased oxygen to the cells.
- Fetal blood from different stages of development can be used as media and/or supplements to the media.
- fetal blood is used to bathe the stem cells in vivo by placing MGCs in an anchored semi- permeable membrane.
- the proteins and macro- and micro-molecules can be extracted using electrophoresis and filtration methods standard in the art. Additionally, the proteins and macro- and micro-molecules can also added to the fluid media in the chambers for the purposes of maturing in an acellular environment.
- blastula cells from the blastula are removed from the donor mammal or animal, and placed in a chamber 5 containing ES cell media. These blastula cells continue to divide and differentiate. During this developmental process, these blastula cells are secreting various macro and micromolecules (i.e. cytokines, GFs, different proteins) which then diffuse to the nearby chamber 5 wherein the MGCs are being sustained. This free flow of messengers facilitates the maturation of the MGCs, such that over a period of time, the MGCs develop various receptors or other surface markers to respond to the messengers secreted by the fetal cells in the nearby chamber 5. [0064] This procedure can be repeated several times with different fetal cells from different stages.
- macro and micromolecules i.e. cytokines, GFs, different proteins
- the chambers 5 When replacing the fetal cells with different fetal cell types, the chambers 5 are closed off by means of a valve on the fittings 20.
- the ancillary systems including the C0 2 reserve, the micro-oxygenator and the molecular dialysis filter are shut off.
- the cells and media are removed from all chambers 5 in use, and new media and new fetal cells are replaced.
- the maturing MGCs from the previous process with the first fetal cell stage are placed back in a nearby chamber 5.
- the valves are re-opened and the ancillary systems and pumps turned back on. Fluid again fluid flows freely between the chambers 5 and driven by the peristaltic pumps 15.
- EXAMPLE 4 Screening the MGCs. To screen for receptors on the MGCs, samples are taken from the chambers 5 containing the MGCs. Samples can be taken at different intervals for the purpose of determining the level of maturation MGCs. The level of maturation of the MGCs is determined by the number of receptor sites on the membrane of the MGCs and location of the receptor sites.
- FRET Fluorescence Resonance Energy Transfer
- BRET Bioluminescence Resonance Energy Transfer
- the typical effective distance between the donor and acceptor is 10 to 100 angstroms and this range correlates with most biological interactions.
- BRET Packard BioScience, BioSignal Packard Inc., Meriden, CT.
- BRET and FRET technologies screen for MGCs with certain numbers of receptors and their location on the cell. Visual identification of receptors using BRET and FRET can be viewed on a larger screen or monitor. These projection systems are standard in the art.
- other methods to screen for receptors sites in contemplated within the present invention, although not described herein.
- the MGCs have developed all, or nearly all, or mostly all the receptor sites as that observed on a mature stem cell. Often the maturing and screening procedures are repeated many times depending on the number of types of fetal cells utilized and the length of growth or development in any one media. That is the number of steps is on a case by case basis.
- EXAMPLE 5 Translocation into the Host.
- the MGCs Once the MGCs have acquired the critical mass of receptors to develop into a mature stem cell (primed MGCs), they are subsequently translocated into the host tissue, in vivo or in vitro.
- the primed MGCs are injected systemically into the circulatory system of the host in vivo. In the circulatory system, the primed MGCs typically migrate to the bone marrow where other adult stem cells reside. However, similar to other adult stem cells in the bone marrow, the primed MGCs travel systemically and will relocate where they are needed.
- the mature MGCs are injected subcranially into the CNS.
- the primed MGCs are translocated in the host of the same species as the donor which contributed the PSCs to create the MGC. This approach is preferred since there is no immune rejection from that of the recipient host against the donor primed MGCs.
- EXAMPLE 6 Retrieving the primed MGCs.
- the MGCs are first placed in a semi- permeable membrane compartment. Affixed to the membrane compartment is a thin tagged thread.
- the semi-permeable bag with the thread affixed is translocated into an animal in vivo or a tissue in vitro.
- the thread is tagged by fluorescence, so when exposed to UV light, the thread fluoresces and is visualized.
- the compartment is retrieved by locating the tagged thread.
- Subsequent screening of the primed MGCs is optionally performed to determine their level of development.
- the membrane compartment or the thread can be comprised of a hollow fiber.
- Another bioassay to screen for the number of receptors on MGCs or primed MGCs is accomplished by placing the MGCs in post-natal tissue (i.e. skin) and determining the linearity of the precursor cells that have developed from the MGCs in the tissue type.
- post-natal tissue i.e. skin
- the invention is not limited to the precise embodiments described in detail hereinabove.
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EP03731989A EP1468088A4 (en) | 2002-01-16 | 2003-01-16 | Stem cell maturation for all tissue types |
CA002474766A CA2474766A1 (en) | 2002-01-16 | 2003-01-16 | Stem cell maturation for all tissue types |
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US34852402P | 2002-01-16 | 2002-01-16 | |
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US36716102P | 2002-03-26 | 2002-03-26 | |
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Cited By (4)
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WO2005013679A1 (en) * | 2003-08-07 | 2005-02-17 | Ivan De Weber | Reproductive and non-reproductive cloning method |
WO2005123901A2 (en) * | 2004-06-08 | 2005-12-29 | Primegen Biotech Llc | Stem cell maturation for all tissue lines |
WO2005123123A2 (en) * | 2004-06-08 | 2005-12-29 | Primegen Biotech Llc | Therapeutic reprogramming, hybrid stem cells and maturation |
CN110106257A (en) * | 2019-05-08 | 2019-08-09 | 绵阳师范学院 | The construction method of kamao phylogenetic tree |
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2003
- 2003-01-16 CA CA002474766A patent/CA2474766A1/en not_active Abandoned
- 2003-01-16 EP EP03731989A patent/EP1468088A4/en not_active Withdrawn
- 2003-01-16 WO PCT/US2003/001570 patent/WO2003062384A2/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5888807A (en) * | 1989-06-15 | 1999-03-30 | The Regents Of The University Of Michigan | Devices for maintaining and growing human stem and/or hematopoietics cells |
US5882918A (en) * | 1995-08-08 | 1999-03-16 | Genespan Corporation | Cell culture incubator |
US6194635B1 (en) * | 1996-01-09 | 2001-02-27 | The Regents Of The University Of California | Embryonic germ cells, method for making same, and using the cells to produce a chimeric porcine |
WO1999035906A1 (en) * | 1998-01-16 | 1999-07-22 | Agrobiogen Gmbh | Efficient nuclear transfer using primordial germ cells |
Non-Patent Citations (1)
Title |
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See also references of EP1468088A2 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005013679A1 (en) * | 2003-08-07 | 2005-02-17 | Ivan De Weber | Reproductive and non-reproductive cloning method |
WO2005123901A2 (en) * | 2004-06-08 | 2005-12-29 | Primegen Biotech Llc | Stem cell maturation for all tissue lines |
WO2005123123A2 (en) * | 2004-06-08 | 2005-12-29 | Primegen Biotech Llc | Therapeutic reprogramming, hybrid stem cells and maturation |
WO2005123123A3 (en) * | 2004-06-08 | 2006-03-02 | Primegen Biotech Llc | Therapeutic reprogramming, hybrid stem cells and maturation |
WO2005123901A3 (en) * | 2004-06-08 | 2006-03-09 | Primegen Biotech Llc | Stem cell maturation for all tissue lines |
CN110106257A (en) * | 2019-05-08 | 2019-08-09 | 绵阳师范学院 | The construction method of kamao phylogenetic tree |
Also Published As
Publication number | Publication date |
---|---|
EP1468088A2 (en) | 2004-10-20 |
EP1468088A4 (en) | 2006-02-15 |
CA2474766A1 (en) | 2003-07-31 |
WO2003062384A3 (en) | 2004-02-05 |
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