WO2002073188A9 - Method for generating replacement cells and/or tissues - Google Patents
Method for generating replacement cells and/or tissuesInfo
- Publication number
- WO2002073188A9 WO2002073188A9 PCT/US2002/007394 US0207394W WO02073188A9 WO 2002073188 A9 WO2002073188 A9 WO 2002073188A9 US 0207394 W US0207394 W US 0207394W WO 02073188 A9 WO02073188 A9 WO 02073188A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- tissues
- cell
- replacement
- selectable marker
- Prior art date
Links
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Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C12N2517/02—Cells from transgenic animals
-
- 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 is concerned with engineering replacement cells and tissues that may be used for transplantation into patients in need of such tissues.
- the present invention provides a means for producing isogenic replacement tissues using nuclear transfer, without requiring in vitro isolation and differentiation of embryonic stem cells.
- Embryonic stem (ES) cell technology is one of the few known means for generating large numbers of replacement cell types from pre-implantation stage embryos.
- Nuclear transfer technology in particular enables the isolation of ES and inner cell mass (ICM) cells from a differentiated somatic cell nuclear donor.
- ICM inner cell mass
- BMSC human or mouse bone marrow stromal cells
- Klug et al have described an approach for isolating relatively pure cardiomyocytes from differentiating murine embryonic stem (ES) cells whereby ES cells are stably transfected with a fusion gene consisting of a selectable marker, aminoglycoside phosphotransferase, linked to the alpha-cardiac myosin heavy chain promoter.
- the present invention solves the problems of the prior art by providing a method whereby differentiated cells and adult stem cells may be generated in vivo, by exposing nuclear transfer-generated ICM cells and other pluripotent embryonic cells to the appropriate cellular or tissue-type environment to encourage development of said pluripotent cells along a desired path.
- ICM cells can be injected into adult animals (i.e., SCID or nude mice), or more preferably into animal-embryos or fetuses at various stages of development. These cells could also be implanted into different sites to encourage differentiation into certain cell lineages. Injecting/implanting pluripotent stem cells into fetal environments may foster and encourage the cells to differentiate into cell types, such as pancreatic beta cells, that may not occur efficiently or completely in adult animals or in embryoid bodies in vitro.
- the methods of the invention also encompass a multi-level targeted differentiation approach, whereby cells may first be encouraged to develop along a particular cell lineage path, such as an endodermal, mesodermal or ectodermal cell lineage, by controlled expression of markers specific for a particular lineage path.
- specific cell types and tissues may be isolated from lineage specific partially differentiated cells.
- cells can be directed down an endodermal path, from which islets may be then be isolated.
- Such a multi-level approach has the advantage of focusing a majority of the implanted or injected cells in the desired direction, and facilitates purification of cells following differentiation into the desired cell type.
- the present invention also is directed to the ex vivo or in vitro production of replacement cells and/or tissues, preferably mammalian cells or tissues, by inducing the differentiation of primordial stem cells or other embryonic pluripotent cell or cells, e.g., an inner cell mass, morula, ES cells, or blastocyst, wherein said cells contain at least one selectable marker operatively linked to a cell or tissue specific promoter, and wherein induction of differentiation is achieved by contacting the primordial stem cells or embryonic pluripotent cells to factors that induce differentiation.
- factors include by way of example hormones, growth factors, salts, cells that promote differentiation, membranes cytoplasm, or other soluble and insoluble substituents isolated from said cells, and nucleic acid sequences that promote differentiation.
- this may be accomplished in vitro, by expressing such cells to appropriate factors in tissue culture. Alternatively, it may be accomplished in an apparatus that mimics in vivo conditions that promote tissue and organ development.
- the invention further embraces the isolation of differentiated cells, tissues and organs produced by such procedures and the use thereof in transplantation therapies.
- the pluripotent cells used in the invention will be produced by nuclear transfer, i.e., by transfer of a suitable donor cell, nucleus or chromosomes, into a suitable recipient cell, e.g., a blastomere, ES cell, or oocyte that is enucleated prior, concurrent or after transfer.
- the donor cell can be of any cell cycle, i.e., GO, G1 , G2, S and M, including quiescent cells produced by serum starvation and other methods.
- the donor is a vertebrate cell, nucleus or chromosomes therefrom, e.g., mammalian, amphibian, reptilian, or avian but preferably mammalian.
- the donor cell may be any somatic or germ cell, but preferably will be a somatic cell, preferably human or non-human promote.
- pluripotent cells may be produced from parthenogentically desired embryos, e.g., produced by activation of unfertilized germ cells, i.e., oocytes.
- FIG. 1 Illustration of renal unit. Renal cells were expanded in vitro, and then seeded onto collagen-coated cylindrical polycarbonate membranes. Devices with collecting systems were constructed by connecting the ends of three membranes with Silastic catheters that terminated in a reservoir made from polyethylene.
- FIG. 1 Tissue-engineered renal units retrieved 3 months after implantation.
- A Unseeded control.
- B Seeded with allogeneic control cells.
- C & D Seeded with cloned cells, showing the accumulation of urinelike fluid.
- Figure 3 Characterization of tissue retrieved 12 weeks after implantation.
- A Cloned cells stained positively with synaptopodin antibody (A) and AQP1 antibody (B). The allogeneic controls displayed a foreign body reaction with necrosis (C).
- Cloned explant shows organized glomeruli (D) and tubule (E)-like structures. H&E, reduced from 400x. Immunohistochemical analysis using factor VIII antibodies identifies vascular structure within D (F). Reduced from x400.
- FIG. 4 Figure 4 RT-PCR analyses (upper) confirming the transcription of AQP1 , AQP2, Tamm-Horsfall protein and synaptopodin genes exclusively in the cloned group (Cls).
- Western blot analysis (lower) confirms high protein levels of AQP1 and AQP2 in the cloned group, whereas expression intensities of CD4 and CD8 were significantly higher in the control allogeneic group (Co1&2).
- FIG. 5 Retrieved muscle tissues: A. Cloned cardiac muscle tissue retrieved shows a well-organized cellular orientation 6 weeks after implantation. H & E, reduced from 200x. B. Immunocytochemical analysis using troponin I antibodies identifies cardiac muscle fibers within the implanted constructs 6 weeks after implantation. Reduced from 200x. C. Cardiac muscle cell implant in control group shows fibrosis and necrotic debris in 6 weeks. H & E, reduced from 100x. D. Cloned skeletal muscle cell implants shows well-organized bundle formation. H & E, reduced from 40x. E. Retrieved skeletal muscle cell implant with polymer fibers. H & E, reduced from 200x. F.
- Immunohistochemical analysis using sarcomeric tropomyosin antibodies identifies skeletal muscle fibers within the implanted second-set constructs 12 weeks after implantation. Reduced from 40x.
- G. Retrieved cloned skeletal muscle cell implants show spatially oriented muscle fiber 12 weeks after implantation.
- H & E reduced from 100x.
- H. Retrieved control skeletal muscle cell implant shows fibrosis with increased inflammatory reaction in 12 weeks.
- H & E reduced from 40x.
- I. Skeletal muscle cell implant in control group shows an increased number of inflammatory cells, fibrosis, and necrotic debris in 12 weeks. H & E, reduced from 100x. J.
- FIG. 7 Western blot analysis of the implants confirmed the expression of specific proteins in the skeletal and cardiac muscle tissues.
- A. Skeletal muscle tissues. Mean expression intensities of desmin were calculated with NIH image software (CO 6; 30.58, CL 6; 85.14, CO 12; 52.61 , CL 12; 79.48).
- Figure 8 shows the formation of differentiated cells (myocardial cells) produced by co-culture of rabbit ICM (parthenogenic) on an endothelial cell monolayer.
- Figure 9 depicts a bioreactor co-culture system used to produce differentiated cells (e.g. myocardial cells) by co-culture of undifferentiated cells (e.g., ICM or ES cells) and helper cells (endothelial cells) according to the invention.
- differentiated cells e.g. myocardial cells
- undifferentiated cells e.g., ICM or ES cells
- helper cells endothelial cells
- Figure 10 depicts another bioreactor co-culture system used to produce differentiated cells (e.g., myocardial cells) by co-culture of undifferentiated cells (e.g., ES or ICM cells) and helper cells or other differentiation inducers (e.g., endothelial and stromal ceil inducers) according to the invention.
- differentiated cells e.g., myocardial cells
- undifferentiated cells e.g., ES or ICM cells
- helper cells or other differentiation inducers e.g., endothelial and stromal ceil inducers
- the present invention provides methods for encouraging the development of pluripotent cells along a particular path of differentiation and development by exposing such cells to environmental cues.
- Preferred pluripotent cells of the present invention are inner cell mass (ICM), intact blastocyst, morula and cells isolated therefrom wherein such cells include cells derived or isolated from an ICM or morula that have partially differentiated although they are still pluripotent.
- ES cells and other pluripotent cells may also be used.
- the invention includes a method of producing replacement cells and/or tissues for a mammal in need of such replacement cells and/or tissues, comprising:
- the pluripotent cells employed in the present invention are human pluripotent cells.
- Such cells may be isolated using nuclear transfer of a human somatic cell nucleus into a mammalian enucleated oocyte or other suitable recipient cell using methods known in the art.
- cross- species nuclear transfer is disclosed in PCT US/97/12919 and PCT US/99/04608 which are both herein incorporated by reference in its entirety.
- cross-species nuclear transfer will be used to provide human pluripotent cells by transplantation of a human cell, nucleus or chromosomes into an oocyte of another species, e.g., a bovine, non-human primate, or rabbit oocyte.
- Such methods as applied to human embryonic pluripotent cells are useful for generating replacement cells and tissues to be used for transplantation and to treat various diseases, i.e., heart disease, cancer and diabetes to name a few.
- Nuclear transfer methods are disclosed in U.S.
- Replacement cells and/or tissues are any desired cell type, but are preferably selected from the group consisting of pancreatic beta cells, brain cells, neurons, cardiomyocytes, fibroblasts, skin cells, liver cells, kidney cells and islets.
- pluripotent cells can be induced to differentiate along certain development pathways, i.e., endodermal lines, mesodermal lines and ectodermal lines.
- Specific cell types and adult stem cells could then be isolated from a particular line of partially differentiated cells. For instance, islets could be isolated by encouraging further differentiation of an endodermal line of partially differentiated cells.
- nerve stem cells or hematopoietic stem cells or other adult stem cells could be obtained which have the potential to differentiate into a variety of cell types.
- the pluripotent cells of the invention may be placed into a developing mammal at any appropriate age to facilitate directed differentiation and development.
- the cells are generally placed in the vicinity of the cell or tissue type desired.
- the cells may be placed into the heart muscle wall of the developing fetus.
- Cells may be implanted or injected as a mixture of individual cells, or could be arranged onto a synthetic scaffold or other extracellular matrix material using tissue engineering techniques.
- cells to be induced to develop into cardiomyocytes can be arranged on a scaffold and patched onto the heart muscle. Similar patches can be constructed for cells implanted into other organs, i.e., the brain or liver.
- the cells and formed tissues are readily retrievable following differentiation.
- Alternative development may be performed in vitro in the presence of cells isolated from a mammal.
- cells may be mixed with a matrigel substance, or other suitable extracellular matrix material which causes cells to aggregate, in order to encourage tissue formation, either for in vivo implantation or in vitro development in the presence of mixed cell types.
- the cells are inserted into a developing mammalian fetus that has not yet developed self recognition immune function.
- a developing fetal sheep does not begin to develop self recognition until the age of 60 days (continuing to about 85 days), so it is possible to introduce human cells before about day 55 to 60 and have the animal be tolerized to the implanted human cells.
- pluripotent cells according to the invention which comprise a selectable marker operably linked to a tissue specific promoter may be induced to differentiate in tissue culture.
- tissue cultures comprise suspension and non-suspension cultures.
- the culture sequences may or may not comprise a feeder layer.
- Tissue development may be engineered vitro as described above.
- the pluripotent cells may be genetically modified such that they are incapable of giving rise to some cell lineage.
- pluripotent cells e.g., an inner cell mass or morula may be cultured in vitro in the presence of factors that "turn on" the particular tissue or lineage specific promoter.
- Cells which differentiate into the appropriate lineage can be isolated based on their expression of the selectable marker. This may be achieved by exposing the cells to growth factors, hormones, salts, and other substituents that allow for the production of the desired differentiated cells.
- the pluripotent or lineage-deficient "pluripotent" cells may be placed in a tissue culture apparatus that mimics the in vivo conditions necessary for production of the desired differentiated cells, tissues or organs.
- tissue culture apparatus mimics the in vivo conditions necessary for production of the desired differentiated cells, tissues or organs.
- tissue culture apparatus mimics the in vivo conditions necessary for production of the desired differentiated cells, tissues or organs.
- such cells may be produced by culturing such cells in an apparatus containing a polymer or membrane useful for tissue engineering, e.g., one completed of poly(glycolic acid) that acts as a biodegradable scaffold for the resultant differentiated cells.
- the pluripotent cells will be placed on the polymer or membrane in a 3-dimensional bioreactor, and the cells will be contacted with factors that produce for differentiation into the desired cell, tissue or organ types.
- bioreactor will be infused with factors that promote development such as hormones, growth factors, salts, etc., that allow for tissue development and growth and activation of the particular tissue or lineage specific promoter.
- factors that promote development such as hormones, growth factors, salts, etc., that allow for tissue development and growth and activation of the particular tissue or lineage specific promoter.
- desired tissues or artificial organs may be produced in the cell culture apparatus, and recovered for use in cell and tissue transplantation therapies.
- the bioreactor may contain a of different pluripotent cells comprising different lineage specific promoter operably linked to different selectable marker, allowing for the production of different cell lineages, tissues or organs in the bioreactor.
- the invention also includes variations of the method discussed above as would be envisioned by the skilled artisan.
- antigens specific to the donor cells could be injected prior to the time period during which self recognition develops in the host fetus in order to tolerize the fetus to the foreign antigens.
- cells to be encouraged along particular developmental pathways can be injected either during or after the development of self recognition without adverse immune response.
- This variation is particularly useful for encouraging differentiation into cell types found in organs that do not fully develop until after the period in which self recognition develops, i.e., thymus cells.
- the embryonic pluripotent cells may be transfected with a selectable marker either prior to implantation, or prior to nuclear transfer, in order to help select cells which have differentiated along the proper pathway.
- the selectable marker may be any marker that may be employed in mammalian cells.
- the selectable marker may be selected from the group consisting of aminoglycoside phosphotransferase, puromycin, zeomycin, hygromycin, GLUT-2 and non-antibiotic resistance selectable marker systems.
- U.S. Patent No. 6,162, 433 discloses non- antibiotic selectable markers suitable for mammalian use, and is herein incorporated by reference in its entirety.
- a preferred selectable marker is aminoglycoside phosphotransferase, wherein said differentiated cells are selected by administering G418.
- any mammalian host may be employed, i.e., including adults, embryos, fetuses and embryoid bodies.
- the pluripotent cells of the invention may be implanted into an immune comprised host, such as a SCID mouse.
- an immune comprised host such as a SCID mouse.
- the host environment need not necessarily be completely an in vivo environment. For instance, an embryoid body may be itself maintained in vitro depending on the stage of development sought and the extent of differentiation possible.
- replacement cells and/or tissues may be purified by chemical selection in vitro or in vivo.
- a preferred method employs two separate selectable markers, for instance, neomycin and hygromycin, operably linked to promoters that are specifically expressed in an overlapping group of tissues. For instance, by expressing neomycin from a promoter specifically expressed in bone and splenocytes, and by also expressing hygromycin from a promoter specifically expressed in bone and cartilage, one can achieve a stronger selection for bone cells via an overlapping selection mechanism. In such a system, although the promoters are not cell-specific, the combined selection for both markers results in cell- specificity.
- replacement cells may be purified away from surrounding host cells and tissues, for instance, using immunopurification targeting a cell-specific, species-specific cell surface protein.
- Antigens employed for purification of the desired cells may also be expressed via one or more exogenous gene construct(s) that are transfected into said primordial cells.
- exogenous genes may be preferentially expressed in the cells or tissues of interest via cell-specific or tissue-specific promoters.
- the CD4 antigen can be preferentially expressed in pancreatic beta cells by operably linking the gene for the CD4 antigen to an insulin promoter. Because the CD4 antigen is not generally expressed in pancreatic, the seeded donor cells may be purified away from surrounding host cells by immunopurification using anti-CD4 antibodies, or via another purification process that targets the CD4 protein.
- the present invention also encompasses the replacement cells and/or tissues produced by the methods disclosed herein, and methods of using the same to treat patients in need of replacement cells and tissues, i.e., via transplantation.
- the invention contemplates the production of cardiac tissue, pancreatic islet tissues, hematopoietic cells, immune cells, in vivo, in vitro, or ex vivo.
- cardiac, renal, pancreatic, immune, liver, lung cells or tissues will be generated in a bioreactor that contains pluripotent cells, e.g., inner cell mass cells or optionally, lineage detective pluripotent cells, substituents necessary for the production of a desired cell lineage, and optionally containing a lineage or tissue specific promoter operably linked to a selectable marker DNA.
- pluripotent cells e.g., inner cell mass cells or optionally, lineage detective pluripotent cells, substituents necessary for the production of a desired cell lineage, and optionally containing a lineage or tissue specific promoter operably linked to a selectable marker DNA.
- human tissues will be generated in such bioreactor by using human pluripotent or lineage-deficient pluripotent cells optionally containing a lineage or tissue promoter operably linked to a selectable marker and culturing under appropriate conditions for formation of the desired tissue type.
- the bioreactor will allow for infusion of necessary nutrients and substituents for tissue growth and development and ideally for formation of vasculature. As discussed, this may be achieved by addition of helper cells, e.g., endothelial or stromal cells or membranes or substituents desired therefrom.
- a DNA that encodes a desired polypeptide e.g., a therapeutic polypeptide, such as a hormone, growth factor, etc.
- a desired polypeptide e.g., a therapeutic polypeptide, such as a hormone, growth factor, etc.
- EXAMPLE 1 A study is ongoing wherein primate, parthenogenetically derived cells (Cyno-1 ) are surgically implanted or injected into fetal lambs at different time points and different ages. Because the sheep does not develop self recognition until 60 to 85 days, there should be a difference in the stability and development of cells implanted prior to self recognition development and afterwards.
- EXAMPLE 2 A study is underway to determine whether cell mixtures encourage differentiation of pluripotent cells either in vivo or in vitro. Briefly, Cyno-1 and other pluripotent cells will be mixed with fetal bud cells and placed under the kidney capsule of a SCID mouse in order to recover islet cells. Also, pluripotent cells may be mixed with pancreatic duct cells with or without Matrigel in order to aggregate cells into three dimensional structures, and examined both in vivo and in vitro for the development of islet cells.
- Tissue-engineered renal units were created from cells cloned from adult bovine fibroblasts. Urine-like fluid production and long-term viability were demonstrated after transplantation back into the nuclear donor animal despite expressing a different mtDNA haplotype. Examination of the explanted devices revealed formation of organized glomeruli and tubule-like structures. Immunohistochemical and RT-PCR analysis confirmed the expression of specific renal mRNA and proteins exclusively in the cloned constructs, whereas the relative expression of CD4 and CD8 in allogeneic control units support the conclusion that there was no rejection response to the cloned cells following either primary or secondary (DTH test) challenge.
- DTH test primary or secondary
- Renal cells were isolated by enzymatic digestion using 0.1% collagenase/dispase, and passaged until the desired number of cells were obtained.
- In vitro immunocytochemistry confirmed expression of renal specific proteins, including synaptopodin (a protein produced by podocytes), aquaporin 1 (AQP1 , a protein produced by proximal tubules and the descending limb of the loop of Henle), aquaporin 2 (AQP2, a protein produced by collecting ducts), Tamm-Horsfall protein (a protein produced by the ascending limb of the loop of Henle), and factor VIII (a protein produced by endothelial cells).
- synaptopodin and AQP1 & 2 expressing cells exhibited circular and linear patterns in two-dimensional culture, respectively.
- the retrieved implants demonstrated extensive vascularization, and glomeruli and tubule-like structures.
- the later were lined with cuboid epithelial cells with large, spherical and pale-stained nuclei (Fig 3A), whereas the glomeruli-like structures exhibited a variety of cell types with abundant red blood cells (Fig. 3B).
- Immunohistochemical analysis confirmed expression of renal specific proteins, including AQP1 , AQP2, synaptopodin, and factor VIII (Fig. 4).
- Antibodies for AQP1 , AQP2, and synaptopdin identified tubular, collecting tubule, and glomerular segments within the constructs, respectively.
- RT-PCR analysis confirmed the transcription of AQP1 , AQP2, synaptopodin, and Tamm-Horsfall genes exclusively in the cloned group (Fig. 4).
- Cultured and cloned cells also expressed high protein levels of AQP1 , AQP2, synaptopodin, and Tamm- Horsfall protein as determined by Western blot analysis. Expression intensity of CD4 and CD8, markers for inflammation and rejection, were also significantly higher in the control vs cloned group (Fig.4).
- m-DNA polymorphisms have been shown to define what have been referred to as maternally transmitted minor histocompatibility antigens (miHA) in mice (K. Fischer Lindahl, E. Hermel, B.E. Loveland, & C.R.Wang, Ann. Rev. Immunol, 9, 351 (1991).).
- the cloned skeletal muscle cell explants showed spatially oriented tissue bundles with elongated multinuclear muscle fibers (Fig.5D,G). Immunohistochemical analysis using sarcomeric tropomyosin antibodies identified skeletal muscle fibers within the implanted constructs (Fig.5F). In contrast to the cloned implants, the allogeneic, control cell implants failed to form muscle bundles, and showed an increased number of inflammatory cells, fibrosis, and necrotic debris consistent with acute rejection (Fig. 5H,I). [0056] Histological examination revealed extensive vascularization throughout the implants, as well as the presence of multinucleated giant cells surrounding the remaining polymer (polyglycolic acid)(PGA) fibers.
- PGA polyglycolic acid
- Poly(glycolic acid) is one of the most widely used synthetic polymers in tissue engineering.
- PGA polymers are attractive due to their biodegradability and biocompatibility, and have been used in experimental and clinical settings for decades as scaffolds for delivering cells and drugs, absorbable suture materials and prosthetic implants.
- the scaffolds are immune acceptable, the PGA construct is known to stimulate a characteristic pattern of inflammation and fibrovascular in growth similar to that observed in the cloned constructs of the present study.
- RT-PCR and Western blot analyses Semi-quantitative RT-PCR and Western blot analysis confirmed the expression of specific mRNA and proteins in the retrieved tissues despite the presence of allogeneic mitochondria.
- Desmin expression was significantly greater in the cloned versus control tissue sections (85 ⁇ 1 and 68 ⁇ 4 vs. 30 ⁇ 2 and 16 ⁇ 2 at 6 weeks for the skeletal and cardiac implants, respectively, PO.001 ; and 80 ⁇ 3 and 121+24 vs. 53 ⁇ 2 and 52 ⁇ 8 at 12 weeks for the constructs generated from the skeletal and cardiac cells, P ⁇ 0.05).
- the expression intensities of troponin I in the cloned and control cardiac muscle explants was 68 ⁇ 4 and 16 ⁇ 2 at 6 weeks (P ⁇ 0.001), respectively, and 94 ⁇ 7 and 54 ⁇ 12 at 12 weeks (P ⁇ 0.05).
- the most straight-forward approach to resolve the question of minor antigen involvement is the identification of potential antigens by nucleotide sequencing of the mtDNA genomes of the clone and fibroblast nuclear donor.
- the contiguous segments of mtDNA that encode 13 mitochondrial proteins and tRNA's were amplified by PCR from total cell DNA in five overlapping segments. These amplicons were directly sequenced on one strand with a panel of sequencing primers spaced at * 500 bp intervals.
- the resulting nucleotide sequences (13,210 bp) revealed only nine nucleotide substitutions (Table 3). One substitution was in the tRNA-Gly segment and five substitutions were synonymous.
- the identification of two amino acid substitutions that distinguish the clone and the nuclear donor confirm that a maximum of only two minor histocompatibility antigen peptides could be defined by this donor:recipient transplant combination.
- first- and second-set clone devices functionally survived for the duration of these experiments without significant increases in infiltration of second-set devices by CD4+ and CD8+ T lymphocytes.
- cloned cardiac and skeletal tissues remained viable >3 months after second-set transplantation (comparable to in vitro control specimens).
- Multiple, viable, myosin- and troponin l-containing cells were observed throughout the tissue constructs, consistent with functionally active protein synthesis and expression.
- human and primate embryonic stem (ES) cells have been successfully differentiated in vitro into derivatives of all three germ layers, including beating cardiac muscle cells, smooth muscle, and insulin- and dopamine-producing cells, among others (Itskovitz-Eldor, J et al. Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol. Med 5, 88-95 (2000).; Schuldiner, M. Yanuka, O., Itskovitz-Eldor, J, Melton, D.A. & Benvenisty, N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad.
- Mitochondrial DNA analyses Mitochondrial DNA products ranging in size from 3 to 3.8kb were amplified by PCRs using Advantage-GC Genomic Polymerase (Clontech, Palo Alto, CA) and total genomic DNA templates from the clone and nuclear donor. The regions of the mitochondria that were amplified included all of the protein-coding sequences and the intervening tRNAs.
- PCR products were electrophoresed in 1 % SeaPlaque GTG agarose (Rockland, ME), extracted from the gels with the use of QIAquick Gel Extraction Kits (Qiagen, Valencia, CA), and sequenced by the Molecular Biology Core Facility (Mayo Clinic, Rochester, MN) with a series of primers located approximately 500 base intervals. Sequences have been submitted to Genbank with Bankit #'s 456319 and 456325.
- Bovine (Bos taurus) oocytes were obtained from abattoir-derived ovaries (from multiple animals) as previously described (Lanza, R.P. et al. Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2, 79-90 (2000).). Oocytes were mechanically enucleated at 18-22 hours post maturation, and complete enucleation of the metaphase plate confirmed with b/sBenzimide (Hoechst 33342) dye under fluorescence microscopy. A suspension of actively dividing cells was prepared immediately prior to nuclear transfer.
- the cell suspension was centrifuged at 800 x g and 5 ⁇ l of the resulting cell pellet used for the donor cells. A single cell was selected and transferred into the perivitelline space of the enucleated oocyte. Fusion of the cell-oocyte complexes was accomplished by applying a single pulse of 2.4 kV/cm for 15 ⁇ sec. Nuclear transfer embryos were activated as previously described by Presicce et al (Presicce, G.A. & Yang, X. Parthenogenetic Development of Bovine Oocytes Matured In Vitro for 24 Hr and Activated by Ethanol and Cycloheximide. Molecular Reproduction and Development 38, 380-385 (1994)) with slight modifications.
- Cardiac muscle and skeletal muscle tissue from 5 to 6 week-old cloned and natural (control) fetuses were retrieved.
- Cardiac and skeletal cells were isolated by the explant technique using DMEM supplemented with either 15% heat-inactivated fetal calf serum (FCS) (HyClone Laboratories, Inc., Logan, Utah), plus the following: 1% penicillin-streptomycin, 1% L-glutamine, 1% nonessential amino acids, and 1 % P-mercaptoethanol.
- FCS heat-inactivated fetal calf serum
- the cells were incubated in a humidified atmosphere chamber containing 5% CO2 and maintained at 37°C.
- Cells were passed by 1 :3 or 1 :4 every 3 to 4 days. Both muscle cell types were expanded separately until desired cell numbers were obtained. The cells were trypsinized, collected, washed and counted for seeding. Cells were seeded in 1 x 2 cm PGA polymer scaffolds with 5x10 7 cells. Vials of frozen donor cells were thawed, cultured, passaged and further propagated prior to seeding the second-set scaffolds.
- Implantation The cell-polymer constructs were implanted into the flank subcutaneous tissue of the same steer from which the cells were cloned; skeletal muscle and cardiac muscle constructs derived from a single fetus (and thus a single oocyte) were used for implantation. Fourteen constructs (8 first- set and 6 second-set) for each cell type were implanted. Control group constructs, with cells isolated from a single allogeneic fetus, were implanted on the contralateral side. The implanted constructs were retrieved at 6 weeks (first-set) and 12 weeks (second-set) after implantation. [0070] Histological and immunohistochemical analyses.
- hematoxylin and eosin were performed using specific antibodies in order to identify the cell types in retrieved tissues with cryostat and paraffin sections.
- Monoclonal sarcomeric tropomyosin (Sigma, St. Louis, MO) and troponin I (Chemicon, Temecula, CA) antibodies were used to detect skeletal and cardiac muscle fibers, respectively.
- Monoclonal CD4 and CD8 (Serotec, Raleigh, NC) antibodies were used to identify T cells for immune rejection. Specimens were routinely processed for immunostaining.
- Pretreatment for high-temperature antigen unmasking pretreatment with 0.1% trypsin was performed using a commercially available kit according to the manufacturer's recommendations (T-8128; Sigma).
- Antigen-specific primary antibodies were applied to the deparaffinized and hydrated tissue sections. Negative controls were treated with non-immune serum instead of the primary antibody. Positive controls consisted of normal cardiac and skeletal tissue. After washing with phosphate buffered saline, the tissue sections were incubated with a biotinylated secondary antibody and washed again. A peroxidase reagent (DAB) was added. Upon substrate addition, the sites of antibody deposition were visualized by a brown precipitate. Counterstaining was performed with Gill's hematoxylin. For determining the degree of immunoreaction, the immune cells were counted under 5 high power fields per section (HPF, x200) using computerized histomorphometrics (Biolmaging Analyses Software).
- RNA isolation, cDNA synthesis Fresh retrieved tissue implants were harvested and frozen immediately in liquid nitrogen. The tissue was homogenized in RNAzol reagent at 4°C using a tissue homogenizer. RNA was isolated according to the manufacturers protocol (Tel-Test). Complementary DNA was synthesized from 2 ug RNA using the Superscript! I reverse transcriptase (GIBCO) and random hexamers as primers.
- PCR For PCR amplification 1 ml of cDNA with 1 U Taq DNA polymerase (Roche), 200mM dNTP and 10 pM of each primer were used in a final volume of 30 ml.
- Myosin for skeletal muscle tissue was amplified from cDNA with primers 5'-TGAATTCAAGGAGGCGTTTCT-3' and 5'- CAGGGCTTCCACTTCTTCTTC-3'.
- Troponin T for cardiac tissue was done with primer 5'-AAGCGCATGGAGAAGGACCTC-3' and 5'- GGATGTAGCCGCCGAAGTG-3'.
- PCR products were visualized with agarose gel electrophoresis and ethidium bromide staining.
- CD4 and CD8 Monoclonal CD4 and CD8 (Serotec, Raleigh, NC) antibodies were used as markers for inflammation and rejection. Subsequently membranes were incubated with secondary antibodies for 30 minutes. The signal was visualized using the ECL system (NEN, Boston, MA). [0074] Statistical analysis. Data are presented as mean ⁇ SEM and compared using the two-tailed Student's t test. Differences were considered significant at P ⁇ 0.05.
- beating myocardial cells may be obtained by culturing ICM produced by parthenogenesis (activation of rabbit oocyte) or an endothelial cell monolayer.
- Endothelial cells, or other cells that induce myocardial differentiation can be isolated from spontaneous mutates of myocardial development from such cultures. Isolation may be effected by labeling with Dll-labeled LDC that is specifically taken up by vascular endothelial cells.
- Endothelial cells that induce differentiation are propagated in vitro, cryopreserved and used in screening assays to induce myocardial differentiation, or to produce myocardial cells for research or therapy.
- EXAMPLE 6 Production of Myocardial Tissues in Bioreactor
- the invention further contemplates the production of artificial organs and tissues in vitro by use of three-dimensional bioreactor.
- endothelial or other cells that induce differentiation into specific cell types e.g., myocardial cells
- myocardial cells may be added to three-dimensional bioreactors containing ICM, blastocyst, morula, or ES cells.
- endothelial cells that induce myocardial differentiation are trypsinized, and permitted to attach to polymer tubes or vessels that promote vascularization and the development of blood vessels. These tubes also allow media to perform and support endothelial attachment and cell viability.
- tissue culture media containing factors that promote the growth of helper cells, e.g., endothelial and which promote differentiation into a desired cell type, e.g., a desired cell type, e.g., myocardial cells.
- the media may comprise brain-derived growth . factor (BDNF), or vascular endothelial growth factor-A (VEGF-A), preferably isoform 165.
- BDNF brain-derived growth . factor
- VEGF-A vascular endothelial growth factor-A
- This approach will work with different endothelial cell types to give rise to different types of tissues.
- Such endothelial cells may be embryonic, fetal or adult and include those already identified.
- the invention further embraces the tissues generated using these three-dimensional bioreactors, which optionally may be transgenic.
- Such a three-dimensional culture system is depicted schematically in Figure 9.
- EXAMPLE 7 Production of Tissues in Bioreactor Using Stromal Cell Inducers
- the invention further embraces the combination of endothelial cells that induce differentiation with stromal (e.g., fibroblast) cell inducers.
- stromal e.g., fibroblast
- Such a system may be used with many different endothelial and stromal cell types in order to generate desired cells and three-dimensional tissues.
- the endothelial and stromal cells can be of the same tissue of origin and may be derived from different tissues, and may be of the same or different species as the ES, ICM, morula, or blastocyst cells that are co-cultured therewith.
- Such cells may be genetically modified and can be of embryonic, fetal or adult origin.
- Potential types of endothelial and stromal cells include by way of example kidney, liver, brain, heart, intestine, pancreas, stomach, eye, bone, skin, lung, etc.
- a co-culture according to the invention will comprise endothelial, stromal cell inducers on a membrane and undifferentiated cells, e.g., ICM, blastocyst, morula, or ES cells, preferably of human origin.
- undifferentiated cells e.g., ICM, blastocyst, morula, or ES cells
- such cells will be obtained by parthenogenic activation of human oocytes or by cross-species nuclear transfer, e.g., by transplantation of a human cell, nuclear or chromosomes into a rabbit oocyte, which is enucleated before, simultaneous or after transfer.
- the bioreactors in Figure 9 and 10 are intended to be exemplary as such bioreactors can take various forms in order to grow tissues in two or three dimensions. Bioreactors which are useful for producing tissues exhibiting desired morphology and tissue architecture are known in the art.
- Another embodiment of the invention includes the marking of human undifferentiated cells with marker genes that are expressed in differentiated progeny of such cells. Thereby genes which are turned on upon differentiation may be identified. For example, such cells may be produced by transfecting human donor cells with selectable marker genes, e.g., green fluorescent protein (GFP) DNA sequences linked to tissue specific promoter.
- GFP green fluorescent protein
- light up on cell differentiation will comprise those that are involved with and/or promote differentiation.
- the co-culture aspect of the invention includes the addition of cell surface molecules that facilitate differentiation of undifferentiated cells, e.g., which may be added as isolated proteins, DNA or RNAs, or as membrane extracts, e.g., membrane blebs derived from helper cells, e.g., endothelial stromal, and parenchyma! cells.
- cell surface molecules that facilitate differentiation of undifferentiated cells, e.g., which may be added as isolated proteins, DNA or RNAs, or as membrane extracts, e.g., membrane blebs derived from helper cells, e.g., endothelial stromal, and parenchyma! cells.
- the invention further embraces the use of helper cells (cells that induce differentiation) that are capable of cell division or which are arrested in their growth by various means, e.g., radiation, DNA damaging agents, viral invention, and others.
- the bioreactors and subject co-culture method may be used to provide the actual vasculature, i.e., perfusion of resulting tissue.
- the subject bioreactor co-culture system may be used to produce artificial and vascularized organs, e.g., artificial pancreas for treatment of diabetes.
- the bioreactor can take various forms, e.g., coated cylinders, tissue culture plates and dishes, comprising undifferentiated cells, helper cells and appropriate media to induce cell differentiation, e.g., of ICMS, blastocyst or morula cells.
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