Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
• • 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
• • 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 present invention combines the fields of cloning, developmental biology and tissue engineering to devise immune compatible tissues and cells for the purpose of transplantation.
• the invention discloses methods of generating therapeutic cells and tissues for transplantation using nuclear transfer techniques, and methods of verifying or evaluating the immune compatibility of such tissues.
• embryonic stem cells may be generated using the nucleus from an adult differentiated cell
• organ, cell and tissue transplantation There are currently thousands of patients waiting for a suitable organ donor, and face problems of both availability and incompatibility in their wait for a transplant If embryonic stem cells generated from the nucleus of a cell taken from a patient in need of a transplant could be made and induced to differentiate into the cell type required in the transplant, then the problem of transplantation rejection and the dangers of immunosuppressive drugs could be precluded
• Embryonic stem cells have been induced to develop into cells from the three different germ layers
• ICM inner cell masses
• embryonic discs from bovine and porcine blastocysts will develop into teratomas containing differentiated cell types from ectodermal, mesodermal and endodermal ongms when transplanted under the kidney capsule of athymic mice.
• Animal Repro Sci. 45- 231-240 (1996)
• the developmental signals that trigger cell differentiation are beginning to be deciphered.
• Gourdie et al. demonstrated the differentiation of embryonic myocytes into impulse- conducting Purkinje fiber cells. Proc Natl Acad. Sci. USA 95- 6815-6818 (June, 1998).
• tissues and organs could be designed from the differentiated cells, which could be used for transplantation.
• Shinoka et al. have designed viable pulmonary artery autografts by seeding cells in culture onto synthetic biodegradble
• the present invention addresses the uncertainties still to be overcome in the use of engineered cells and tissues for transplantation.
• the invention discloses methods of enginee ⁇ ng cloned, immune compatible, developmentally differentiated cells into tissues for transplantation, and methods of using such tissues to treat a patient in need of a transplant.
• tissue may be designed to express a therapeutic protein. Because the tissues and cells for transplantation are all generated from the same o ⁇ ginal donor cell through nuclear transfer, all the cells of the engineered tissue will express the heterologous gene of interest. The methods of the invention therefore additionally provide an invaluable alternative to tissue- targeted gene therapy.
• the present invention also provides methods for determining whether particular genetically engineered cells will provide immune compatible organs for transplantation.
• the present invention discloses methods of evaluating cloned cells for mitochond ⁇ al compatibility, and in particular, transgenic, developmentally differentiated cells, for immune compatibility in an animal model. Such evaluations will provide important information regarding the suitability of therapeutic tissues in transplantation, and will provide the foundation for controlling these parameters in order to provide immune compatible tissues Detailed Description
• the present invention is directed to methods of producing of immune compatible tissues using cloning technology
• the cells and engineered tissues produced by the disclosed methods are also encompassed in the present invention, as are the stable grafts produced by transplantation of the engineered tissues.
• a stable graft is defined as a graft that does not illicit an immune response or rejection when transplanted into a nuclear donor, or at least provides a substantial improvement in avoiding graft rejection over non-cloned control transplanted tissue.
• cloned cells generated by nuclear transfer are not completely identical with the donor cell or animal, e.g., they typically lack the mitochond ⁇ al DNA of the donor cell and gain the mitochondnal DNA of the recipient enucleated oocyte or other cell and typically are not produced in an in vivo environment that will perfectly mimic conditions present dunng embryogenesis, the question is raised as to whether such cells will be entirely immune-compatible when they are transplanted back into the donor animal.
• mice e.g., the ND1 peptide from the ammo terminus of NADH dehydrogenase and the MiHA peptide encoded by the amino terminus of the COI gene
• non-classical MHC class I molecules e.g., H-2M3a, m combination with beta-2-m ⁇ croglobuhn
• H-2M3a non-classical MHC class I molecules
• beta-2-m ⁇ croglobuhn Vyas et al., 1992, "Biochemical specificity of H-2M3a . . .,” J. Immunol.
• the present inventors have surpnsmgly found in performing the methods of the present invention that nuclear transfer generated cells having allogeneic mitochondria are not rejected when transplanted into the nuclear donor.
• the present invention provides methods and animal models for testing the immune compatibility of cloned cells or tissues in an animal model, and for enhancing the immune compatibility of such cells or tissues as needed.
• such methods compnse: a. obtaining a cell from a donor animal, b. transfernng the nucleus from said cell into a recipient oocyte or other suitable recipient cell to generate an embryo and optionally introducing a therapeutic heterologous DNA; c.
• a teratoma is defined as a group of differentiated cells containing de ⁇ vatives of mesoderm, endoderm, or ectoderm resulting from totipotent cells
• a control embryonic disc, inner cell mass, or stem cell is one which was not generated using a donor cell from the test animal (allogeneic or xenogeneic nuclear DNA), and therefore, the teratoma thus generated from such disc or cell is expected to be rejected in the donor animal, or alternatively, may never develop at all Teratomas generated using a nucleus from the donor animal (lsogenic) and an allogeneic recipient oocyte or other suitable recipient cell would also be expected to be rejected when used in transplantation due to presentation of mitochond ⁇ al alleles as histocompatibihty antigens
• the fact that such therapeutic tissues do not lead to transplant rejection is truly surp ⁇ sing indeed
• Donor and control embryonic discs, inner cell mass, and/or stem cells are generally injected intramuscularly, introduced under the renal capsule, subcutaneously or into the paralumbar fascia Where a teratoma is formed, it is removed and examined for the presence of germ layers, which may further be separated for the purpose of detecting or isolating specific cell types While teratoma formation may give an initial indication of immune compatibility, specific cell types may be generated and re-introduced into the donor animal to further test immune compatibility, particularly where the transfected heterologous gene is expressed from a cell-type specific promoter.
• this system is ideal for testing the affect of a transgene on tissue compatibility whereby the cell from said donor animal is transfected with a heterologous gene p ⁇ or to nuclear transfer.
• the methods of the invention may be performed using any cell from the donor animal.
• Suitable cells include by way of example immune cells such as B cells, T cells, dendritic cells, skin cells such as keratmocytes, epithelial cells, chondrocytes, cumulus cells, neural cells, cardiac cells, esophagial cells, primordial germ cells, cells of vanous organs including the liver, stomach, intestines, lung, kidneys, etc.
• immune cells such as B cells, T cells, dendritic cells, skin cells such as keratmocytes, epithelial cells, chondrocytes, cumulus cells, neural cells, cardiac cells, esophagial cells, primordial germ cells, cells of vanous organs including the liver, stomach, intestines, lung, kidneys, etc.
• the most appropriate cells are easily propagatable m tissue culture and can be easily transfected
• cell types for transfecting heterologous DNA and performing nuclear transfer are fibroblasts.
• the animal model may be any animal suitable for generating teratomas and studying immune compatibility.
• a preferred animal is an ungulate, and more preferred is a bovine.
• the animal may be a non-human primate, e.g , a baboon or cynomolgus monkey Large animals are preferred because they may give rise to larger teratomas, thereby providing more cells for immunological evaluation and for transplantation. Suitable animals include by way of example pigs, dogs, horses, buffalo and goats.
• Such cross-species models have particular relevance to the study of xenotransplantation, and would provide a convenient model for identifying the mitochondnal proteins that serve as histocompatibihty antigens. If the mitochondna of the recipient cell prove to be functionally, but not immunologically, compatible using the teratoma model, it will be possible to identify mitochondnal antigens and peptides which are displayed on the cell surface but may not exhibit allehc vanation within a single species Such a model will facilitate recombinant DNA methodology geared toward replacing the relevant mitochondnal antigens in the recipient cell with those from the nuclear transfer donor, in order to further enhance the immune compatibility of the cloned cells and tissues for transplantation therapy.
• steps may be taken in accordance with the invention to ensure that cloned cells and tissues are compatible with the nuclear donor, for instance, by selecting recipient cells which express compatible mitochondnal antigens, or by replacing the histocompatible mitochondnal epitopes.
• one group of researchers has reported complete replacement of endogenous mitochondnal DNA in one Drosophila species with the mitochondnal DNA of another (Niki et al. 1989. Complete replacement of mitochondnal DNA in Drosophila. Nature 341(6242): 551-2.
• mitochondnal phenotype for any particular nuclear transfer donor, or even a mixture of mitochondnal phenotypes, i.e., isogenic and allogeneic, or isogeneic and cross-species.
• Mitochondrial genes or DNA segments responsible for mitochondnal antigen histocompatibihty, particularly in cross-species models, may be readily identified using the methods of the present invention.
• isogenic nuclei from a designated mammalian nuclear donor can be transferred into different allogeneic mitochondrial backgrounds of a closely related species, and such cells may be used to immunize the nuclear transfer donor in order to isolate and identify antibodies and lymphocytes specific for mitochondrial epitopes.
• By comparing the specificities of the panels of antibodies and lymphocytes achieved by immunizing the nuclear donor it is possible to identify mitochondrial antigens and epitopes that result in immune recognition and possibly graft rejection in cross-species models.
• the present invention includes methods of identifying mitochondrial histocomptibility antigens using cross-species nuclear transfer, comprising: obtaining cells from a donor mammal; transferring nuclei from said donor mammal into at least two recipient oocytes or other suitable recipient cells of a mammalian species other than said nuclear donor to generate embryos, wherein said at least two recipient cells are allogeneic with regard to mitochondrial DNA; isolating embryonic discs and/or stem cells from said embryos; injecting said discs and/or stem cells separately back into said donor mammal as to generate a specific panel of antibodies and/or lymphocytes; and comparing panels of antibodies and/or lymphocytes generated in response to said allogeneic mitochondnal backgrounds in order to identify mitochondnal antigens and/or epitopes that are recognized
• the ability to re-clone cloned mammals and generate a line of cloned mammals that are isogenic for both nuclear and mitochond ⁇ al DNA allows for concurrent injection of the cross-species cloned cells containing allogeneic mitochondria into separate mammals, thereby facilitating the retrieval of panels of antibodies and lymphocytes specific for different mitochond ⁇ al backgrounds.
• Methods of recloning cloned mammals based on the observation that nuclear transfer can be used to rejuvenate senescent cells are disclosed in commonly assigned, copendmg Application Senal No. , filed concurrently herewith and incorporated by reference in its entirety.
• the methods of the present invention may also be performed wherein said discs and/or stem cells are injected into separate mammals which are isogenic to the nuclear donor with respect to both nuclear and mitochondnal DNA in order to isolate panels of antibodies and/or lymphocytes.
• the present invention also encompasses methods of generating therapeutic cloned tissue for transplant which express a heterologous protein.
• the heterologous DNAs to be used in the methods of the present invention may encode a therapeutic protein to be expressed in a transplant recipient, but may also be a reporter gene for the purpose of monitonng gene expression in the teratoma.
• the reporter gene may be any which is convenient for monitonng gene expression, but is preferably selected from the group consisting of green flourescent protein (GFP), beta-galactosidase, luciferase, vanants thereof, antibiotic resistance markers, or other markers
• GFP green flourescent protein
• beta-galactosidase beta-galactosidase
• luciferase vanants thereof
• antibiotic resistance markers or other markers
• tissue-specific promoters or tissue specific enhancers provides a means of selecting for expression of heterologous DNAs in desired tissue types.
• the cells may be selected based on the expression charactenstics of cell surface markers. For example, hematopoietic stem cells may be selected based on CD34 expression.
• the donor cell may also contain deletions and insertions into the genome that disrupt or modify the expression of native genes, preferably the donor cell is transfected with a heterologous gene that encodes a protein that is secreted and performs a therapeutic function in the intended transplant recipient, i.e., replaces a native gene which is mutated, or is not expressed.
• a heterologous gene that encodes a protein that is secreted and performs a therapeutic function in the intended transplant recipient, i.e., replaces a native gene which is mutated, or is not expressed.
• the animal used in the animal model to test immune compatibility may then be used for the evaluation of the immune response and the isolation of antibodies or cytotoxic T cell clones.
• reporter gene constructs designed with putative developmental promoters, enhancers, repressors or other gene control sequences may be inserted into the donor nucleus p ⁇ or to nuclear transfer, and the teratomas may then be monitored visually or by other means to see at which stage reporter gene expression is turned on.
• the differentiated teratoma cells may be separated and used to individually test the immune compatibility of a particular cell or tissue. Once a particular cell type of interest is identified, it may be used to engineer a tissue using the methods descnbed herein and known in the art.
• the animal models disclosed find particular use in testing new matrix materials in tissue engineering for immune compatibility.
• Preferred engineered tissues of the present invention are selected from the group consisting of smooth muscle, skeletal muscle, cardiac muscle, skin, kidney and nervous tissue.
• the present invention also concerns methods of generating immune compatible tissues for transplantation, compnsing a. obtaining a donor cell from an intended transplant recipient; b. transfernng the nucleus from said cell into a recipient oocyte or other suitable recipient cell to generate an embryo; c. isolating an embryonic disc, inner cell mass, and/or stem cell from said embryo; d injecting said disc, inner cell mass, and/or stem cell into an immune compromised animal; e. isolating the resulting teratoma; f. isolating from the teratoma a cell of the type required for transplantation, and optionally expanding said cells in vitro using a growth factor; and g. enginee ⁇ ng a tissue from said cells or combinations of cells.
• Tissue enginee ⁇ ng may be effected, e.g., using three-dimensional scaffolds or biodegradable polymers such as are used in the construction of dissolvable sutures. Such methods have been well reported in the patent and non-patent literature by companies such as Tissue Engineering, Inc. and Organogenesis.
• the immune-compatible tissues and cells generated are useful in methods of providing a patient in need of a transplant with an immune-compatible transplant.
• Such a method further comprises, in addition to the above steps, transplanting said engineered tissue into a patient.
• transplanting said engineered tissue into a patient has particular relevance for transplants which replace native cells suffering from mitochondrial damage, for instance as in amythrophic lateral sclerosis (ALS), or Leber's hereditary optic neuropathy (LHON).
• ALS amythrophic lateral sclerosis
• LHON Leber's hereditary optic neuropathy
• tissue having isogenic nuclear DNA and allogeneic mitochondrial DNA that does not induce an immune reaction is the most ideal tissue for transplantation in that such tissue not only provides the closest histocompatibility match, but it also effectuates mitochondrial gene therapy in that tissue containing damaged mitochondria is replaced.
• cloned tissues having allogeneic "young" mitochondrial DNA might provide an advantage over the patient's own cells by virtue of the absence of age-related mitochondrial mutations.
• Mitochondrial DNA is believed to be more susceptible to age-related mutations than is genomic DNA because of the relative lack of DNA repair mechanisms and histones (Dhaliwal et al. 2000).
• hereditary mitochondrial mutations transmitted maternally that manifest themselves in particular tissues, that would benefit by the cloning and tissue engineering techniques in the present application.
• LHON Leber's hereditary optic neuropathy
• the cloning, tissue engineenng, and transplantation techniques of the present invention will be especially valuable for replacing diseased tissue linked to mitochondnal mutations in that the cloned tissues will typically possess isogenic nuclear DNA, but allogeneic mitochondnal DNA. Therefore, for instance, engineered nervous tissue for transplantation into LHON patients will effectuate gene therapy of the mitochondnal DNA while at the same time, replace the diseased optic nerve tissue.
• said donor cell may be genetically altered pnor to nuclear transfer by the transfection of at least one heterologous gene, or the disruption or replacement of at least one native gene.
• a genomic modification is particularly useful where the transplant recipient's own genome fails to express a required protein, or expresses a mutated protein such that the onginal tissue or organ failed to function properly Alternatively or additionally, if p ⁇ or tests of immune compatibility suggest some rejection is anticipated, e g , due to allogeneic or xenogeneic differences in mitochondnal DNA, the donor cell may be transfected with genes expressing proteins that deter or decrease immune rejection p ⁇ or to nuclear transfer.
• PBC primary biliary cirrhosis
• PDC pyruvate dehydrogenase complex
• one theory as to how PBC is initiated is that a nuclear genetic alteration affects the transport of PDC-E2 to the mitochondria, i.e , such as mutations in the leader sequence that direct E2 to the outer membrane (Bj ⁇ rkland and T ⁇ tterman, 1994, "Is pnmary biliary cirrhosis an autoimmune disease?" Scand. J. Gastroenterol. 29 Suppl. 204. 32-9).
• the nuclear transfer-generated cells can be corrected for the nuclear defects that lead to the autoimmune disease pnor to generation of the liver cells and tissues for transplantation, i.e., by replacing the mutated leader sequence.
• the cloned cells and tissues used for transplantation into a PBC patient would not only provide the closest immune compatible tissue to avoid rejection, but also effectuate gene therapy which repairs a nuclear gene linked to the autoimmune disease itself.
• the methods of the present invention are equally as valuable for the transplantation and gene therapy of any diseased tissue where the nuclear mutations associated with the disease process have been identified, e.g., for the treatment of burns, blood disorders, cancer, chronic pain, diabetes, dwarfism, epilepsy, heart disease such as myocardial infarction, hemophihc, infertility, kidney disease, liver disease, osteoarth ⁇ tis, osteoporosis, stroke, affective disorders, Alzheimer's disease, enzymatic defects, Huntington's disease, hypocholesterolemine, hypoparathyroidase, immunodeficiencies, Lou Geh ⁇ g s disease, macular degeneration, multiple sclerosis, muscular dystrophy, Parkinson's disease, rheumatoid arthritis, and spinal cord inju ⁇ es.
• heart disease such as myocardial infarction, hemophihc, infertility, kidney disease, liver disease, osteoarth ⁇ tis, osteoporosis, stroke, affective disorders, Alzheimer's disease, enzy
• a preferred transplant recipient is a human
• teratomas may be formed following nuclear transfer, i.e., of a fibroblast nucleus, from said human into any human recipient oocyte, because it is the genome of the donor (intended transplant recipient) that reprograms the cell for development.
• Teratomas generated from human nuclear donors and recipients may be formed in and isolated from an immune compromised animal, such as a skid or nude mouse.
• the teratomas generated may be removed and examined for the formation of germ layers, and such germ layers may be further separated or differentiated into distinct cell types. Distinct cell types may then be used to engineer tissues for transplantation.
• said tissues are selected from the group consisting of smooth muscle, skeletal muscle, cardiac muscle, skin, kidney and nervous tissue.
• the tissues and cells generated by the disclosed methods are also encompassed.
• the concept of human "therapeutic cloning" is to transfer the nucleus from one of the patient's cells, i.e., a fibroblast cell, into an enucleated recipient oocyte or other suitable recipient cell. After reprogramming, the donated somatic nucleus regains its totipotency and is able to initiate a round of embryonic development.
• Plunpotent stem cells derived from the resulting embryo carry the nuclear genome of the patient, and are then induced to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue, insulin-producing n-cells for patients with diabetes, chrondrocytes for osteoarthntis, or dopaminergic neurons to treat Parkinson's disease.
• replacement cells such as cardiomyocytes to replace damaged heart tissue, insulin-producing n-cells for patients with diabetes, chrondrocytes for osteoarthntis, or dopaminergic neurons to treat Parkinson's disease.
• the methods of the invention should eliminate or at least substantially alleviate the immune responses associated with transplantation of these vanous tissues, and therefore abrogate the requirement for immunosuppressive drugs, such as cyclospo ⁇ ne, imoran, FK-506, glucocorticoids, and vanants thereof, which carry the nsk of a wide variety of senous complications, including cancer, infection, renal failure and osteoporosis
• immunosuppressive drugs such as cyclospo ⁇ ne, imoran, FK-506, glucocorticoids, and vanants thereof, which carry the nsk of a wide variety of senous complications, including cancer, infection, renal failure and osteoporosis
• anti-rejection agents at least initially.
• the transplanted cells may not be immunologically identical to the transplant recipient's cells, even though the nucleus of one of the recipient's cells served as the donor This could be caused by mitochondnal DNA differences particularly in the case of xenogeneic mitochondna, or antigenic differences that may result from transfected heterologous DNA or because of the artificial environment used to affect nuclear transfer In particular, such an environment does not mimic identically the cellular environment that exits dunng embryonic development For example, it is known that cells cultured for prolonged periods may be antigenically different as a result of cultunng (a phenomenon known as "antigenic d ⁇ ft").
• tolenze the cells or tissues pnor to transplantation e.g by treatment with soluble CD40, CD40-hgand antagonists, low temperature culture, use of antibodies that mask donor antigens, or by expression of UV light (e.g., islets).
• Isolated embryos having at least one cell, or embryonic discs/ inner cell mass or stem cells generated from bovine blastocysts/ stem cells are then injected into the paralumbar fascia of the donor steers (two sites with experimental (same animal) stem cells, two sites with expenmental (same animal) embryonic discs, two sites with inner cell mass, and four sites with control (different animal) stem cells, per animal). After two months, the muscle is examined for teratoma formation. Any tumors identified are removed for histological analysis.
• the procedure is performed on the standing animal using 20 mg Xylazine/ 8 mg Butorphanol Tatrate administered IV in the tail vein.
• the paralumbar fascia area is clipped and surgically prepared, using 100 ml of 2% Lidocaine as a local anesthetic administered as a paralumbar block.
• the animals should be given antibiotics for three days post-surgically as a precautionary measure (Cefilofur Hcl 50 mg/cc @ lcc/100 pounds).
• a single injection intramuscularly or under the kidney capsule of Flunixin Meglumine @ lcc/100 pounds may be given to control pain and swelling at the surgical site. If teratoma formation does not occur at the paralumbar fascia, other sites may be analyzed, i.e,. subcutaneously.
• stem cells will survive in the recipient (donor of nucleus) animal in contrast to “different animal” stem cells, or survive at least better or longer depending on the cytotoxic T cell response or other immune reaction to foreign mitochondnal peptides. Furthermore, it is expected that cells from all three germ layers, i.e., ectoderm, mesoderm, and endoderm, will be observed in "same animal” teratomas.
• This example was designed to test teratoma formation in an immune- compromised animal model. This example is relevant to the methods whereby cloned, nuclear transfer-generated cells from a patient in need of a transplant may be grown in a SCID mouse or other immune-compromised animal in order to generate differentiated cells for isolation and design of engineered tissues for transplant.
• ES cells transfected with GFP were de ⁇ ved from two adult Holstein steers (two different ES cell lines were denved from each animal). ICMs were derived from 12-day-old blastocysts.
• Cells were cut into pieces (sections of no more than about 100 cells each) and loaded into a 1 ml syringe, no more than 200 microhters each, and preferably 100 microhters.
• ICMS were mechanically isolated and loaded into a 1-ml synnge 100 to 150 microhters.
• Cells were kept at room temperature in HECM-Hepes. Twenty-two-gauge needles were used for injection procedures Cells were injected into the skeletal muscle of the hind leg of SCID mice.
• Bovme stem cells and ICMs that were injected into the skeletal muscle of the SCID mice were ret ⁇ eved after 7-8 weeks (although it is possible to let the cells go longer, or remove them sooner).
• a small nodular lesion was identified in two of the mice which received ES cell injections (mice #s 7 and 9)
• a 2X2 mm sized milky white nodule was retneved from the nght hind leg near the sciatic nerve of mouse #7 This corresponds with the injection of three plates of ES 22.
• C. A IX 1 mm sized milky white nodule was identified within the muscle tissue of mouse # 9 which corresponds with the injection of the three plates of ES 25.F.
• Histologic Analysis Mouse #7 Histologic sections of the teratoma were analyzed with hematoxyhn and eosin (H&E), safran ⁇ n-0 and lmmunocytochemistry using cytokeratin (AE1/AE3) and alpha smooth muscle actin antibodies
• the injected cells formed a round tissue mass within the skeletal muscle tissue
• the teratoma consisted of four different sized compartments with cellular debns in the center Tissue formation was noted on the wall of each compartment (data not shown)
• Epithelial (round nuclei) and stromal cells (spindle- shaped nuclei) were observed in the teratoma tissue (data not shown)
• There was no evidence of cartilage, bone or adipose tissue Safranin-0 Negative staining was obtained, which indicates the absence of cartilage tissue formation.
• lmmunocytochemistry with AE1/AE3 antibodies The teratoma section showed positively stained epithelial cells (data not shown) lmmunocytochemistry with alpha smooth actm antibodies Small islands of positively stained muscle tissue was observed within the teratoma (data not shown).
• the retneved tissue demonstrated epithelial, smooth muscle and stromal tissue components. Cartilage, bone and adipose tissues were not identified in the teratoma.
• myocardial infarction is one of the most common diagnoses occurring in hospitalized patients in western countries. While injection of individual or small groups of cardiomyocytes could aid in the treatment of some localized infarcts, this approach is unlikely to be of value in patients with more extended lschemic injury, where the risk of scar formation, cardiac rupture and other complications is much greater Tissue engineering offers the possibility of organizing the cells into three-dimensional myocardial "patches" which could be used to repair the damaged portions of the heart. For myocardium and other relatively simple tissues, such as skin and blood vessel substitutes, this may involve seeding cells onto masses or sheets of polymeric scaffold. Creating more complex, vital organs, such as the kidney, liver, or even an entire heart will require assembling different cell types and materials in greater combinatorial complexity.
• bovine inner cell mass/embryonic discs/stem cells may be generated as described above, and injected into the rear leg muscles of nude or SCID mice. Seven to eight weeks after injection, the resulting teratomas are removed and various cell types are isolated and grown in culture.
• a number of tissues may be generated from the cloned cells, including smooth and/or skeletal muscle, sheets or "patches" of cardiomyocytes, elastic cartilage, skin, (including the placement of hair follicles), and kidney, including miniature kidneys that excrete urine. These tissues/ organ constructs are then transplanted back into the original adult animal from which the donor cell biopsy was obtained.
• Kidney Cells from bovine kidney, heart, skeletal muscle, cartilage and skin were harvested from cloned and allogenic (control) 40 day old fetuses, and expanded separately in vitro. Kidney:
• the kidney tissue was cut into small pieces (1 mm " ) using sha ⁇ tenotomy scissors.
• the kidney tissue fragments were digested using collagenase dispase (1 mg/ml) at 37° C for 30 minutes.
• the recovered cells were washed with phosphate buffered saline and plated in culture dishes.
• the cells were grown in medium consisting of DMEM, HEPES 3 1 g/1, Pen/Strep (5 ml/500 ml), L-glutamine 146 mg/L and FBS 10% (Sigma, St. Louis, MO).
• Muscle- Cardiac and skeletal muscle cells were processed by the tissue explant technique using Dulbecco's Modified Eagle's Medium (DMEM; HyClone
• Unwoven sheets of polyglycohc acid polymers (1 x 2 cm) were used as cell delivery vehicles.
• the polymer meshes were composed of fibers of 15 ⁇ m in diameter and an interfiber distance between 0 - 200 um with 95% porosity.
• the scaffold was designed to degrade via hydrolysis in 8-12 weeks.
• the polymers were ste ⁇ zed in ethylene oxide and placed under stenle conditions until cell delivery.
• hematoxyhn and eosin were performed using specific antibodies in order to identify the cell type of the retrieved tissues Histochemical analyses using aldehyde fuschin-alcian blue, and immunocytochemical studies using monoclonal anti-collagen II (Chemicon, St. Louis, MO) were used to identify the engineered cartilage structures. Monoclonal sarcomenc tropomyosm (Sigma, St Louis, MO) and tropomn I (Chemicon, Temecula, CA) antibodies were used to detect skeletal and cardiac muscle fibers, respectively. Immunolabehng was performed using he avidin-biotm detection system. Sections were counterstained with methyl green. Implantation in the Steer Immunocytochemical and histological analyses
• hematoxyhn and eosm Five micron sections of formalin fixed paraffin embedded tissue were cut and stained with hematoxyhn and eosm (H&E). Immunocytochemical analyses were performed using specific antibodies in order to identify the cell type of the retneved tissues. Histochemical analysis using Penodic Acid Schiff (Sigma, St. Louis, MO), and immunocytochemical studies using polyclonal anti-alkaline phosphatase and anti-osteopontin (Chemicon, Temecula, CA) were used to identify renal cells. Monoclonal sarcomenc tropomyosm (Sigma, St.
• troponm I antibodies were used to detect skeletal and cardiac muscle fibers, respectively Aldehyde fuschin-alcian blue and monoclonal anti-collagen II (Chemicon, St. Louis, MO) were used to stain cartilage tissue implants.
• Anti-cytokeratms 5/6, AE1/AE3 were employed in order to identify keratmocytes.
• Bronchial ciliary antibodies were used in order to detect respiratory epithelium.
• Ant ⁇ -CD6 antibodies were used in order to identify immune T and B cells Immunolabehng was performed using the avidin-biotm detection system Sections were counterstained with methyl green RESULTS
• Implants retrieved from the steer were implanted in the animals with the polymer scaffolds, and retrieved without complications. At retrieval, the implants maintained their initial size without any evidence of fibrosis. Implants retrieved from the steer:
• the mtDNA of the cloned tissues was from the recipient oocyte, the mtDNA of the nuclear donor and that of the cloned embryo were sequenced. Sequence data confirmed that the mtDNAs were indeed different, particularly in the d-loop region where there were four different corresponding nucleotides in the cloned tissues in comparison with the nuclear donor.
• EXAMPLE 4 The above results suggest that it is possible to generate cloned tissues for transplantation by nuclear transfer into an allogeneic background, and that differentiated cells and tissues isolated or constructed from cloned teratomas or cultures of embryonic cells can be transplanted back into the donor animal without significant signs of rejection. To further confirm that nuclear transfer technology has the potential to eliminate the immune responses associated with the transplantation of cells and organs despite mitochond ⁇ al mismatch, the inventors will next perform transplants between full grown clones having different mitochondnal backgrounds.
• reciprocal skin grafts Two groups of animals were assembled to test reciprocal skin grafts: (1) four cloned cows (animals CL53-8, CL53-9, CL53-10, and CL53-11) at Trans Ova and (2) five cloned goats at LSU
• reciprocal skin grafts (approximately 2-3 cm diameter) are exchanged between the two groups of animals. Self-grafts will serve as positive controls, whereas grafts from genetically unrelated animals will serve as negative controls. The grafts are monitored for signs of immune rejection, and will be removed if and when they become necrotic and the sites patched. If rejection is observed, second-set grafts would then be transplanted in order to confirm the results, which should be rejected in an accelerated fashion.
• in vitro assays will be performed to identify target peptides, and the associated mtDNAs which encode the peptides will be isolated.
• the experiments aimed at determining mitochondnal DNA polymo ⁇ hisms will also reveal information about chime ⁇ sm levels in mtDNA in general For instance, once the sequences of the mtDNAs are known, a region of maximal polymo ⁇ hism will be selected, most likely the D-loop, and this segment will be amplified and cloned. A range of clones may then be sequenced to the determine extent of vanation in this region.
• the present invention demonstrates that it is possible to obtain cloned differentiated cells and tissues for the pu ⁇ ose of tissue enginee ⁇ ng and transplantation
• the present invention also demonstrates that stable grafts can be achieved with nuclear transfer-generated cloned cells having allogeneic mitochondria, despite the fact that transplantation rejection would be expected due to foreign mitochondnal peptides.
• H-2M3a the mouse class I molecule that presents the Mtf and MiHA peptides
• M3 gene is encoded by the M3 gene at the telome ⁇ c end of the H-2 complex on mouse chromosome 17 (Fischer Lindahl et al., "Maternally transmitted antigen of mice: a model transplantation antigen,” Annu. Rev. Immunol 1991;9.351-72).
• the present invention confirms the therapeutic utility of nuclear transfer-generated cloned tissues in the context of transplantation. Further, by providing a model for testing the immune compatibility of allogeneic and xenogeneic mitochondrial proteins in an isogenic nuclear background, the present invention paves the way for deciphering the immune regulatory systems that exist in and between mammals, which contribute to mitochondrial stability and the separate evolution of species.

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