EP1981968A2 - Cellules souches et germinales embryonnaires de primates non humains, leurs méthodes d'utilisation et leurs méthodes de fabrication - Google Patents

Cellules souches et germinales embryonnaires de primates non humains, leurs méthodes d'utilisation et leurs méthodes de fabrication

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Publication number
EP1981968A2
EP1981968A2 EP07718078A EP07718078A EP1981968A2 EP 1981968 A2 EP1981968 A2 EP 1981968A2 EP 07718078 A EP07718078 A EP 07718078A EP 07718078 A EP07718078 A EP 07718078A EP 1981968 A2 EP1981968 A2 EP 1981968A2
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European Patent Office
Prior art keywords
cells
cell
human
embryos
pgcs
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EP07718078A
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German (de)
English (en)
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EP1981968A4 (fr
Inventor
Gerald P. Schatten
Calvin Simerly
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Magee-Womens Health Corp
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Magee-Womens Health Corp
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Publication of EP1981968A2 publication Critical patent/EP1981968A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
    • 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
    • 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
    • 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
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8776—Primate embryos
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/106—Primate

Definitions

  • the present invention relates to the study and development of non-human primate stem cells.
  • hES cells human embryonic stem cells'
  • the goals of the research described herein are: (i) To understand the fundamental biology of pluripotent stem cells during development and differentiation by investigating the NIH-approved hES cell lines and as additional models, deriving and studying nonhuman primate ES cells (nonhuman primate embryonic and germ cell lines) and; (ii) To determine the transplantation potentials of ES cells by non-invasively imaging autografts, allografts, and xenografts into primates and mice, with special attention devoted to focus on the safety of differentiated stem cell transplantation of nhp-ESCs derived from NT embryos, as well as immune tolerance.
  • the present invention provides a composition comprised of a chimeric primate embryo derived from non-human ES cells plus a fertilized primate embryo.
  • the present invention further provides a method of generating a chimeric primate embryo, which comprises reaggregating nhp ES cells with biopsied fertilized primate embryos.
  • the present invention provides a method of determining the differentiation status of an embryonic cell.
  • this method comprises the steps of determining the cell transcriptional pattern of the embyronic cell; comparing this embryonic cell transcriptional pattern to various prototype transcriptional patterns, where each of these prototype transcriptional patterns are derived from an embryonic cell of a specific embryonic cell differentiation status; determining which of these prototype transcriptional patterns most closely resembles the embryonic cell transcriptional pattern; and finally assigning the specific embryonic cell differentiation status corresponding to the prototype transcriptional pattern most closely matching the embryonic cell transcriptional pattern, to the embryonic cell.
  • the embryonic cell transcriptional pattern is determined by hybridizing labeled RNA, isolated from single colonies of cells, to microarray chips.
  • these microarray chips display genomic DNA fragments originating from a species selected from the group consisting of mouse, human and Rhesus monkey.
  • these microarray chips are selected from the group consisting of the Affymetrix ® hg-u133+2 chip, Affymetrix ® GeneChip ® Rhesus Macaque Genome Array and Affymetrix ® mg-u74Av2 chip.
  • the specific embryonic cell differentiation status determined by the method of the present invention can be the inner cell mast, the epiblast, the mesoderm, endoderm, ectoderm, lateral plate mesoderm, gut and neuroectoderm, extraembryonic mesoderm, amniotic ectoderm and visceral endoderm.
  • the present invention provides the composition of an isolated nhp pluripotential EG cell, methods for deriving this cell and methods for the use of this cell in the treatment and/or prevention of various diseases.
  • a method for deriving nhp EG cells wherein non-human primates are mated to establish pregnancy; the pregnancy is terminated at between 28 and 45 days and an nhp fetus obtained; gonads are isolated from this fetus and placed on plates of feeder cells; the plates are cultured, then fixed and stained for TNAP 1 a marker for PGCs; the PGCs are detected by TNAP staining; the PGCs placed into culture, supplemented with LIF, bFGF and forskolin; the culture is fed daily; EG cells are isolated by picking, then plated singly; and the EG colonies resulting are then tested for specific EG-expressed markers.
  • the present invention also provides for the composition that is a differentiated cell derived from an nhp EG cell. Further, the present invention provides for the composition that is the embryoid body derived from an nhp EG cell. The present invention also provides for a method of administering a composition comprised of the differentiated cell derived from an nhp EG cell, in order to treat, prevent and/or alleviate the occurrence or negative effects of disease.
  • this disease may be Alzheimer's, Parkinson's, muscular dystrophy, diabetes, stroke, and/or cardiovascular disease such as congestive heart failure, ischemic heart disease, cardiomyopathy, hypertension, coronary artery disease, high blood pressure, arrhythmia, and thrombogenicity.
  • this disease may be Alzheimer's, Parkinson's, muscular dystrophy, diabetes, stroke, and/or cardiovascular disease such as congestive heart failure, ischemic heart disease, cardiomyopathy, hypertension, coronary artery disease, high blood pressure, arrhythmia, and thrombogenicity.
  • FIG. 1 Multidimensional scaling plot of relationships between pluripotent cell types of mouse and human origin.
  • Mouse single inner cell mass (mlCM) isolated from 3 E3.5 blastocysts; epiblast manually dissected from 3 E3.5 blastocysts;
  • Figure Z Bisulfite methylation analysis of the HI9 DMR in three hESC lines. Each line represents a unique strand of DNA. Circles represent positions and methylation of individual CpGs as follows: filled, methylated; open, unmethylated. The first and last CpG sites are numbered relative to the H19 transcriptional start site.
  • PO1 Primate Core B has now isolated of epiblasts from day 15 nhp embryos. These isolated samples can be placed into culture of frozen in OCT for laser capture experiments, directly dissected and processed for RNA analysis, or even cultured for epiblast-derived embryonic stem cells.
  • IGF2 sample 073 DNA sequence is SEQ ID NO: 5.
  • IGF2 sample 052 DNA sequence is SEQ ID NO: 6.
  • IGF2 sample 022 DNA sequence is SEQ ID NO: 7.
  • hES cells human embryonic stem cells'
  • mice embryonic stem cell derived tissues
  • ESCs embryonic stem cell derived tissues
  • cell based transplantation therapies are likely to consist of allogeneic grafts and the most relevant endpoints for assessment will be comparable allogeneic grafts performed within a relevant species.
  • mouse ESCs Because of the extensive evolutionary distance between rodents and humans, and already evident differences between mouse and primate ESCs, there are limits to the use of mouse ESCs to demonstrate safety and efficacy of stem cell-based transplants. Nevertheless, mice (particularly immunodeficient strains) can have great utility in demonstrating the pluripotency of primate ESCs through the development of teratomas induced by transplanting ESCs to ectopic sites.
  • pluripotency is surrogates for a more thorough assessment of differentiative capacity and long-term stability of the differentiated state of primate ESC derived cells, their pluripotency, epigenetic status and stability of their differentiated state.
  • This can only be ultimately achieved through even more fundamental assessment of the developmental capacity of pluripotent stem cells as a formal assessment of chimeric studies in embryos, in which contribution and function of ESC derived cells in all primary germ layers and their differentiated derivatives can be definitively assessed.
  • the present research relies heavily on a non-human primate model as the most relevant model for assessing the therapeutic potential of pluripotent stem cells.
  • Rhesus monkeys While a number of such species are germane options, several considerations point to Rhesus monkeys: their high evolutionary relatedness to humans; the scope and availability of existing breeding stocks (with SNPs valuable to the epigenetic assessments and donor vs. host identification in chimeric tissues); and progression of the Macaque genome project, with imminent production of a Rhesus Genechip. In studying a non-human primate model, a focus on a definitive assessment of pluripotency, capacity for functional differentiation, and epigenetic status in ESCs and EGCs will be undertaken.
  • pluripotency includes the characteristic of immortality and the plasticity to develop into any of the body's cell lineages (perhaps excluding trophectoderm descendents like some extraembryonic and placental tissues). Immortality includes both cellular immortality in vitro and our potentially immortal reproductive cycle in vivo. Fertilized eggs, embryonic cells, inner cell mass cells, embryonic stem cells, embryonic germ cells, primordial germ cells, and embryonic carcinoma cells are endowed with pluripotency. That is, they have the potential to develop into any cell type in vitro and, under special situations, to contribute to any cell lineage (including, at times, the germ cells) when aggregated chimeric embryos are transferred to properly staged surrogates.
  • Pluripotency is best described operationally (e.g., by ectopic transplantation or chimera formation), since only a few of the molecular and cellular constituents have yet been identified.
  • These molecular markers of pluripotency are the subjects of intense international investigation, since regardless of their role in conferring pluripotency, these markers are presently the only keys for distinguishing pluripotent cells from those committed to specific lineages.
  • Molecular markers for pluripotency include Oct314, Nanog, Rex, Sox-2 (although Sox-2 continues to be expressed in early neuroectoderm).
  • teratoma formation is used as an in vivo demonstration of pluripotency, since hES cells differentiate into all three germ layers in SCID mice.
  • the progenitors of-the entire organism- are encompassed-in just three-lineages ⁇ the so called "primary germ layers," endoderm, ectoderm, and mesoderm.
  • mesoderm is important as an inducer of organotypic differentiation in the other two, and because it forms key tissues in its own right, for.
  • musculoskeletal system which emerges from dorsal components of primary mesoderm, and the blood stem cells, which emerge from the ventral-most aspect of the primary mesoderm
  • endoderm is important as the epithelial progenitor of the entire system of gut organs, including lungs, pancreas and liver
  • ectoderm is important as the epithelial progenitor of the brain and skin, thus the source of neurons and the supportive cells of the nervous system as well as the multipotent neural crest population.
  • Epigenesis comprises the special circumstances that enable both pluripotency and differentiation. Even with genetically identical twins or inbred mouse strains, the potential for alterations in genomic imprints, cytoplasmic organelle differences and variations in intracellular and extracellular environments are together capable of generating considerable phenotypic diversity.
  • Embryonic stem cell pluripotency is most convincingly demonstrated in reaggregated embryos in which the resultant offspring have ES cell contributions to all germ layers and tissues, including the germ line. This has only been achieved using mouse embryonic stem (mES) cells. Overwhelming ethical concerns obviously preclude attempts with human embryonic stem (hES) cells. This project responsibly bridges gaps in the scientific knowledge between mES and hES cells through the generation of chimeric primates with nonhuman primate ES (nhpES) cells - including nhpES cells derived from blastocysts generated after nuclear transfer (NTnhpES cells).
  • nhpES nonhuman primate ES
  • Aim I primate ES cell pluripotency during development
  • Aim II primate ES cell stability and utility after NT
  • Aim III genomic imprinting status after ART and nuclear transfer
  • Aim IV HESC and primate ES cell fate after transplantation, especially after SCNT
  • Aim I To dynamically image nhpES cell contributions in developing primate chimerae: 1.1. WiII nhpES cells contribute to chimeric blastocysts, fetuses and healthy offspring? 1.2.
  • ES cells contribute primarily to the inner cell mass in chimeric blastocysts, fetus and offspring?
  • Primate chimera are generated in four ways, and the fate of each chimera is followed in vitro during preimplantation development, determining the cellular contributions of the ES and tetraploids to the expanded blastocyst stage after differentially labeling ES or embryos with GFP-transgenes, as well as in utero during fetal development and in the offspring.
  • Aim H Are ES cells derived after nuclear transfer (NTnhpES cells) developmentally restricted? Two questions are posed: 2.1. Are NTnhpES cell lines stable?
  • Example I 1 To dynamically image nhpES cell contributions in developing primate chimerae.
  • nhpES cells contribute to chimeric blastocysts, fetuses and healthy offspring?
  • tetraploid embryos (4N)
  • will ES cells contribute primarily to the inner cell mass in chimeric blastocysts, fetus and offspring?
  • This question delves into nonhuman primate chimera generation by performing four sets of reaggregations: fertilized embryo ⁇ fertilized embryo; fertilized embryo ⁇ pluripotent nonhuman primate embryonic stem cells (nhpES cells); fertilized embryos ⁇ tetraploid embryo $c?+4N; and tetraploid embryo * ⁇ nhpES cell (4N + ES).
  • each chimera will be followed in vitro during preimplantation development, determining the cellular contributions of the ES cell and tetraploid to the expanded blastocyst stage after differentially labeling ES cells or embryos with GFP-transgenes. Fetal development will also be traced, as well as offspring potential of each chimera after embryo transfer to timed rhesus recipients using MRM noninvasive imaging. Finally, developmental normalcy of embryos, fetuses, placentae and the resultant offspring will be ascertained, along with contribution of nhpES to the offspring.
  • nhpES cells contribute to all three somatic cell lineages as well as to the germ-line in reconstructed chimeric embryos in ⁇ tero and after birth, and in vitro investigating pluripotency markers.
  • NT involves multiple manipulations and developmental events culminating in the somatic nucleus within the activated and enucleated oocyte.
  • steps include: a) 'enucleation' of meiotic metaphase-ll spindle and chromosome complex (SCC); b) somatic cell selection and preparation; c) nuclear transfer or intracytoplasmic nuclear injection (ICNI); d) wound healing and drug recovery from both spindle removal and nuclear introduction, as well as recovery; and e) oocyte activation.
  • SCC meiotic metaphase-ll spindle and chromosome complex
  • ICNI nuclear transfer or intracytoplasmic nuclear injection
  • e oocyte activation.
  • somatic cell preparation and selection has been investigated in several species (Wakayama et al., 1998, Nature 394:369-374; Wilmut, 2002b, Nature 419:583-587; Wilmut et al., 1997, Nature 385:810-813) and electrofusion ('Dolly') versus direct injection ('Honolulu' or ICNI) are being compared.
  • Wound healing after microinjection, cell fusion and 'enucleation' has not been investigated, yet cell sealing in other systems involves new membrane vesicle recruitment by microtubules (Togo et al., 1999, J. Cell Sci 112(Pat 5):719-731).
  • oocyte activation typically is initiated by the sperm and SCNT has succeeded with DMAP/ionomycin (Navara et al., 1994, Dev Biol 162:29-40), electrofusion (Wilmut et al., 1997, Nature 385:810-813), sperm factor (Perry, 2000, Dev Biol 217:386-393), ethanol (Ng et al., 2004, Development 131:2475-2484), and strontium chloride (Wakayama, 1997, Zygote 5:229-234).
  • the GFP-Oct4 imaging permits rapid and reliable selection of the most fully reprogrammed nuclei for later transfer.
  • Aim I it is proposed to produce chimera by aggregating dynamically labeled nhpES cells with either fertilized or tetraploid embryos and explore ES cell lineage contributions to the resultant chimeric blastocysts. Afterward, nhpES cell-derived chimera will be transferred to recipient rhesus females for noninvasive imaging by MRI during fetal development and the production of healthy offspring.
  • Chimeric mice have been derived after injecting GFP-transfected mouse ES cells into the blastocoel cavity of BALB/c mice. Following embryo transfer, 5/10 (50%) chimeric pups were successfully born as shown by coat color. The nhp embryos have been labeled with GFP insertion by lentivirus.
  • Aim I is designed to dynamically image nhpES cells' contributions in developing primate chimerae.
  • the rationale is that this aim will determine the developmental potentials of nhpES cells in reconstructed chimeric embryos, and investigate nhpES cell behavior and fate after transplantation by non-invasive MRI imaging.
  • Pluripotency in nhpES cells will be demonstrated by rhesus embryo reaggregations with nhpES cells, with the resultant embryos analyzed for ICM/TE distribution in vitro.
  • rhesus chimera produced with nhpES cells will be transferred to surrogate rhesus females for establishing pregnancies and to generate offspring.
  • nhpES cells will be produced with rhesus $ ⁇ f? embryos for embryo transfer to investigate fetal development in utero using MRI imaging and for offspring production.
  • CMRL + 10% FCS Hyclone Laboratories, Inc., Logan, UT
  • Buffalo rat liver cell monolayers BBL 1442; ATCC, Rockville, MD
  • the lentivirus is pseudotyped with vesicular stomatitis virus (VSVG) glycoprotein (Pfeifer et ai, 2002, Proc Natl Acad Sci USA 99:2140-2145) and is obtained in collaboration with Dr. Carlos Lois (MIT).
  • VSVG vesicular stomatitis virus
  • the pseudotyped VSVG lentiviral vector is derived as follows:
  • the viral vector backbone used in these experiments is based on a self-inactivating vector.
  • the long terminal repeat (LTR) is required for integration into the cell genome and for transcription of the viral RNA and is derived from HIV-1 with modifications: a deletion of the U3 enhancer plus a 1.2 KB insertion between U3 and R sequences of the 3' LTR and a deletion of U5 and insertion of human CMV enhancer/ promoter in the 5' LTR. This modification does not affect generation of the viral genome in the producer cell line, but results in self-inactivation of the lentivirus after transduction of the target cell.
  • the lentiviral genome is no longer capable of producing packageable viral genome.
  • the PSI sequence is required for packaging genomic RNA into the capsid.
  • transgene green fluorescent protein (GFP) gene
  • GFP green fluorescent protein
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • the envelope vector contains G glycoprotein gene from Vesicular Stomatitis Virus (VSV-G) as a pseudotyping envelope and allowing production of a high titer lentiviral vector with a significantly broadened host cell range.
  • VSV-G Vesicular Stomatitis Virus
  • the three plasmids (1: transgene expression vector contains GFP cDNA, 2: packaging vector contains gag, pol, rev and tat genes and 3: envelope vector contains VSVg gene are co-transfected into 293FT cells (Invitrogen, CA).
  • the 293FT cell is a cell line derived from human embryonic kidney cells that has been immortalized with the early region of adenovirus (EIA and E1B genes) and has been stably transfected with the early region of SV40 to increase transfectability.
  • helper plasmids contains deletions in LTR 1 psi sequence, rev responsive element and env, vif, vpu, vpr, and nef coding sequences.
  • the expression of these coding sequences is driven by a CMV promoter, and the polyadenylation signal is derived from SV40 virus.
  • VSVg vesicular stomatitis virus glycoprotein envelope gene is provided by cotransfection.
  • VSV coding sequence is driven by a CMV promoter, and the polyadenylation signal is derived from SV40 virus.
  • the three plasmids (1: transfer vector, 2: gag, poi, rev, tat encoding plasmid and 3: VSVg plasmid) are transfected into 293T cells. Due to the multiple deletions and mutations in the vector backbone and the packaging plasmids, it is highly unlikely to generate a replication competent virus by recombination (coding sequences for env, vpr, vpu, nef; rev responsive element sequence is absent; U3 enhancer from LTR is deleted).
  • 293T is a cell line derived from human embryonic kidney cells that has been immortalized with the early region of adenovirus (E1A and E1B genes) and has been stably transfected with the early region of SV40 to increase transfectability.
  • This cell line does not produce any known infectious agent and does not contain any of the genes that would complement the genetic defects of the viral vector system.
  • cell lines producing viruses will be grown exclusively in tissue culture incubators in the BL-2 approved facility at the Pittsburgh Development Center (PDC). Incubators will be appropriately labeled to indicate the type of recombinant virus being generated.
  • the viral supernatant is collected in a laminar flow hood and placed in ultracentrifugation tubes. The tubes are spun for 1.5 hours at 25000 rpm. In the laminar flow hood, ultracentrifugation tubes are removed from holders, and supernatant disposed of in an appropriate container. Viral pellets are resuspended in PBS and stored frozen at -80C. All procedures involving plasmid growth, DNA transfection into packaging cells, ultracentrifugation, and viral resuspension will be performed in dedicated BL2 facilities at the PDC.
  • the modified Lentil-GFP virus will be used, containing either CMV, Elongation factor 1 ⁇ , ubiquitin, chicken ⁇ -acting, or Oct-4 constitutively active promoters that can express as early as the 2- to 4-cell stages in NHP.
  • the concentrated vector solution is back- loaded into a 6-7 ⁇ m sterile microinjection needle (Homage, Inc., Charlottesville, VA) and a fixed amount deposited into the perivitelline space of pronuclear stage embryos ( ⁇ 8hrs post-insemination). After microinjection, the oocytes are returned to culture at 37 0 C in 5% CO 2 .
  • NHP-ESCs The ICM from ICSI-fertilized expanded blastocysts are isolated by immunosurgery following removal of the zona pellucida with 5mg/ml Pronase for 2-3 min. Anti-monkey whole serum antibody and guinea pig complement are prepared in 20 ⁇ l drops in 35 mm petri dishes overlaid with mineral oil and incubated at 37°C in 5% CO2.
  • Zona-free blastocysts are exposed to anti-monkey antiserum for 30 min at 37 0 C and washed 3 times with a washing media [1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's nutrient mixture F-12 supplemented with 0.1 mM 2-mercaptoethanol, 1% nonessential amino acid, and 15% fetal bovine serum].
  • the blastocysts are transferred to guinea pig complement and incubated for 15 min at 37 ⁇ C in 5% CO 2 .
  • the blastocysts are washed three times in washing media and incubated for 15 min at 37°C in 5% CO 2 .
  • the intact ICM is separated from lysed trophectoderm cells by pipetting, and plated on mitomycin C-treated mouse embryonic fibroblasts (MEF). MEF-ICM plates are incubated for 2 days to prevent detachment of the ICM in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's nutrient mixture F-12 supplemented with 0.1 mM 2- mercaptoethanol, 1% nonessential amino acid, 1000 U/ml leukemia inhibitory factor and 15% fetal bovine serum. After 7 to 14 days, expanded ICM are dissociated with a fine glass capillary. Colony pieces are transferred to new MEF for expansion.
  • DMEM Dulbecco's modified Eagle's medium
  • F-12 Ham's nutrient mixture F-12 supplemented with 0.1 mM 2- mercaptoethanol, 1% nonessential amino acid, 1000 U/ml leukemia inhibitory factor and 15% fetal bovine serum.
  • nhpESC cell lines should be derived, with 5 cell lines actively maintained for investigative work.
  • efficiency of early passage stage nhp-ES cells ⁇ 20
  • later passage stages >20
  • nhpESC Karyotype analysis of nhpESCs will be performed by the Cytogenetics Department at Magee Womens Hospital. NHP-ES cells are trypsinized and gently pipetted to give a single cell suspension. A small droplet of the cell suspension is dropped onto precleaned glass slide and placed in an oven at 55-65°C for 45 minutes. Slides are then incubated in 2 x SSC (150 mM NaCI, 15 mM trisodium citrate) at 60-65 0 C for 90 minutes followed by rinsing thoroughly in 0.9% w/v NaCI at room temperature.
  • 2 x SSC 150 mM NaCI, 15 mM trisodium citrate
  • Slides are stained in Trypsin-Giemsa (Bio/medical Specialties, Santa Monica CA) solution for 4-6 minutes before transfer to fresh buffer (Ix SSC; twice rinsed) and dried by compressed air. Slides are then mounted and viewed under 100X oil immersion using a Nikon E1000 upright microscope equipped with high numerical aperture objectives. Chromosome spreads are captured digitally using an ORCA Cooled CCD camera (Hamamatsu, New Jersey).
  • GFP-reporters for ES cell tracking are proposed as the best and longest lasting reliable marker.
  • Two different approaches will be followed towards the generation of transgenic non-human primate embryonic stem cells using GFP plasmids or retroviral vectors containing GFP.
  • the first involves GFP-lentiviral transfection of rhesus zygotes for the production of GFP-expressing embryos that are cultured to the expanded blastocyst stage prior to the derivation of NHP-ES cells, as described in 1.1.
  • D The second method involves either direct lentiviral transfection or Lipofectamine transfection of previously derived ES cells. Lipofectamine transfection of nhpESC is performed according to (Valuer et a/., 2004, Stem Cells 22:2-11).
  • Rhesus 8-cell embryos are prepared and a small clump of transfected ES cells introduced into the depression wells. After all reaggregations are completed, the plate is gently rotated to bring the fertilized embryos in close contact with the ES cells before returning to culture. All plates are checked the following day for cellular reaggregation and subsequent embryonic development. Collected embryos are kept at 37°C in a 5% CO 2 until tetraploid formation or chimera aggregation as described above.
  • Noninvasive in utero imaging by high definition ultrasound (U/S) and MRI, and Amniocentesis are performed at defined post-implantation intervals to document attainment of normal in utero parameters, i.e., 40-44 days gestation, a differentiated tissue mass showing body axis, limb-buds, the developing brain, and liver; at 60-65 days gestation, the developing brain and many internal organs are apparent (e.g., heart, liver, spleen, intestines, bladder, etc.). The placenta is also clearly detected. Specific measures of mean gestational sac size, yolk sac diameter, greatest length, and embryonic heart rates are collected to approximate the gestational age of the conceptus.
  • MRI magnetic resonance imaging
  • Quantitative imaging methods such as diffusion tensor imaging, provide information about developmental changes at the cellular and microstructure level at near-cellular resolution. Any tissue morphological changes, progression of disease states, and biochemical changes will be visualized with MR microscopy.
  • mtDNA mitochondrial DNA
  • nested PCR followed by automated sequencing of portions of the mtDNA displacement loop (D-loop) region will be used to screen polymorphism among animals within the Pittsburgh rhesus colony. Specifically, we will amplify the hypervariable regions (hv1 and hv2) of the D- loop of the mitochondrial genome from blood and or tissue samples (skin biopsy). The amplimers will then be sequenced using standard automated sequencing methods (Hopgood et al., 1992, Biotechniques 13:82-92).
  • Sequences will be analyzed for unique polymorphisms using a sequence analysis package such as the freely available ClustalW (www.ebi.ac.uk/clustalw).
  • Real-Time PCR primers and probes will be designed based upon each animal's unique polymorphisms. Multiplexed Real-Time PCR will be performed on DNA samples obtained from chimeric offspring using primers specific to each parent of origin. Overall levels of chimerism will be determined as a percentage of the mtDNA present from each parent of origin as determined by the multiplexed Real-Time PCR.
  • NTnhpESCs incorporate Left histology of pregnancies, urogenital chemistry nhpESC adhere, form with ICM organ, ascertained cells (from & fetus, then junctions, preference; tissues by Part V, A urine-soaked placental epiblasts viable & GFP-Oct4 Especially with U/S & diapers). cultures, collected compact. expression germs cells MRI, onAnalyzed in day 15.
  • Chimeras will be culture, their development monitored, and their normalcy confirmed by cytogenetic analysis prior to initiation of El' into recipient females, SCID mice, or isolation of ES cells. We have already demonstrated that embryo reaggregation can result in viable rhesus offspring. We expect that chimeras created from transgene blastomeres will retain gene expression throughout preimplantation development and we are hopeful that GFP- transgene presence and expression will be maintained in the majority of fetal and newborn tissues.
  • 1.2.A Production of rhesus fertilized and GFP-expressing transgenic embryos is described 1.1. A and 1.1. B. 1.2.B. Production of tetraploid intraspecific NHP will be performed by a modified technique of (Nagy et al., 2003, Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • two-cell embryos grown in vitro are placed into an electrode fusion chamber (500 ⁇ m gap; GSS-250, BLS 1 Hungary) in fusion media consisting of 0.25 M sorbitol, 0.10 M mM calcium acetate, 0.10 M magnesium acetate, and 0.5mg/ ml BSA (Fatty-acid Free; Sigma), pH 7.2 (265 mOsm).
  • fusion media consisting of 0.25 M sorbitol, 0.10 M mM calcium acetate, 0.10 M magnesium acetate, and 0.5mg/ ml BSA (Fatty-acid Free; Sigma), pH 7.2 (265 mOsm).
  • an alignment pulse of 2 V is applied to correctly orient the 2-cell embryos relative to the electric field before applying the fusion current (60 V; 35 ⁇ s) to fuse the two blastomeres.
  • fusion current 60 V; 35 ⁇ s
  • nhpES cell segregation largely to the inner cell mass while the tetraploid cells will largely be restricted to the mural and polar trophectoderm in successful chimera.
  • some ES cells may contribute to the trophectoderm (Xu 1 2002) and we plan to estimate this contribution in the fully expanded blastocyst.
  • the nhpES cells are expected to contribute to all three tissue lineages (endoderm, ectoderm, and mesoderm) as well as to germ cells. All chimeric concepti will be monitored for defects in cranial, somite, and limb development after ET by high definition ultrasound and MRI analysis.
  • GFP transgene infected embryos will be examined by a brief exposure to attenuated epifluorescent illumination to confirm the extent of mosaic expression using appropriate fluorescein filters. All confirmed fluorescent embryos will be cultured separately or transferred to proper stage females for pregnancy establishment and noninvasive imaging. Control embryos will be exposed to similar levels of fluorescent light to monitor any long-term effects of mercury light exposure on developmental progression and pregnancy establishment.
  • the integration of the GFP transgene into various tissues will be determined by PCR analysis as described in 1.1. L We estimate that 50-60 chimeric blastocyst will be needed for %S x 4N tetraploid chimera allocation to the ICM or trophectoderm following in vitro culture.
  • 4N embryo formation by electrofusion may be complicated by low efficiency or embryo lysis.
  • An alternative method to produce 4N rhesus embryos is incubating embryos at the late 2-cell stage (38-44 hrs post ICSI) in 20 ⁇ M cytochalasin B, a reversible microfilament inhibitor that blocks cytokinesis without interrupting karyokinesis, for 12 hours. After inhibiting one round of cell division, the cytochalasin is removed by washing in culture medium and continuing development in CMRL. Control embryos will be run in parallel using similar DMSO (the cytochalasin solvent) concentrations, which never exceed 0.5%. Based on preliminary evidence, we do not anticipate difficulties in generating tetraploid + fertilized chimeric rhesus embryos or culture in vitro. We have also demonstrated the feasibility of producing live transgenic and reaggregated NHPs.
  • Example I are nhpES cells derived after NT developmentally restricted? Rationale: SCNT may overcome immune rejection problems since the major histocompatibility antigens (MHCs) are nuclear transcripts.
  • MHCs major histocompatibility antigens
  • Our recent findings suggest that meiotic spindle extraction during SCNT in primates depletes the oocyte of vital microtubule motors preventing normal development.
  • primate SCNT embryos display aneuploidy, techniques have been adopted from the successful report of stem cell derivation from cloned human blastocyst and methods are now being optimized in nonhuman primates in collaboration with newly recruited scientists from Seoul National University (Eul-Soon Park, MS), MizMedi Hospital and Hangyang University (Jong-Hyuk Park, PhD).
  • ES cells will be isolated from SCNT-derived blastocysts and stable cell lines produced. These ES cells will be investigated in reaggregation experiments with fertilized and tetraploid embryos for cellular lineage contributions during blastocyst formation. Later, NTnhpES cell- derived chimeric embryos will be transferred for pregnancy and offspring production.
  • this second aim focuses on deriving ES cells from blastocysts generated by NT and addresses two important goals.
  • NT will provide important advances to the generation of normal embryos for the production of genetically identical nonhuman primates.
  • the production of euploid embryos will facilitate deriving NT ES cells that can be used for chimera production, attempts that, if successful, will quickly advance therapeutic cloning technology.
  • This latter question explores whether NTnhpES cells contribute to chimeric blastocysts, fetuses, and offspring when reaggregated with fertilized embryos (NTnhpES+$ ⁇ J) or with tetraploid embryos (NTnhpES+4N).
  • NTnhpES embryonic stem cells from blastocysts generated by nuclear transfer
  • enucleation by squeezing out the SCC instead of pipette aspiration a) enucleation by squeezing out the SCC instead of pipette aspiration; b) enucleation of pre-metaphase-ll SCC, i.e. at telophase-l or prometaphase-ll just after first polar body extrusion, accomplished by live imaging of the meiotic spindle with orientation independent polarization optics.
  • enucleation Before enucleation, cumulus cells are removed mechanically by pipetting in TALP-Hepes containing 0.3% BSA and 1 mg/ml hyaluronidase. Oocyte aspiration is performed as described in Part V at 27- 28 hrs post-hCG and maturing oocytes that have not elicited the first polar body selected for nuclear transfer experiments. Oocytes are cultured in CMRL medium at 37°C in 5% CO 2 and observed every 30 minutes for signs of first polar elicitation, confirmed by dynamic imaging of the SCC in unfertilized NHP oocytes using polarization optics (SpindleViewTM; CRI, Cambridge, MA).
  • the forming second meiotic spindle is seen as a bright, birefringent bipolar - structure against the dark cytoplasm.
  • 'Squish' enucleation of oocytes is performed as follows: pre-metaphase-11 stage oocytes are placed into TALP-Hepes media containing 0.3% BSA and 7.5 ⁇ g/ml cytochalasin B overlaid by mineral oil. The zona pellucida is partially dissected with a fine glass needle to make a slit near the forming first polar body. Next, the first polar body and underlying cytoplasm containing the SCC is extruded by gently squeezing them with the same needle. Enucleation is confirmed by visualizing the karyoplast stained with Hoechst 33342 (Sigma.) under attenuated UV illumination to confirm removal of the SCC.
  • 2.1.B Preparation of somatic donor cells for SCNT.
  • a small pieces of skin from the same female selected for oocyte - donation is collected from a punch biopsy near the shoulder blade, washed three times in Dulbecco's phosphate buffered saline (DPBS, Life Technologies), and then digested in 0.25% (v/v) trypsin-EDTA (Life Technologies) solution for 1 h at 37°C.
  • DPBS Dulbecco's phosphate buffered saline
  • trypsin-EDTA Life Technologies
  • the pellet After washing three times in Ca 2+ - and Mg 2+ -free DPBS by centrifugation at 43 x g for 2 min, the pellet is resuspended in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% FBS, 1 mM glutamine, 25 mM NaHCO 3 and 1% nonessential amino acid solution and the cells are plated in 75 mm culture flask. The cells are subcultured 2-4 times every 2-3 days before cryopreserving at -196 0 C until needed. Prior to nuclear transfer experiments, thawed cells are grown to confluency by culturing for 2-3 days in the DMEM. Individual cells are collected by trypsinization as above for 30 sec, washed by low speed centrifugation, and used within two hours for SCNT.
  • DMEM Dulbecco's modified Eagle's medium
  • Enucleated oocytes are washed 3 times TALP-Hepes containing 0.3% BSA and placed in a 50 ⁇ l drop of TALP-Hepes containing 0.3% BSA in a micromanipulation chamber containing donor cells.
  • Donor cells are aspirated into a microinjection pipette (internal diameter, 20-25 ⁇ m; Humagen, Inc) and introduced through the same slit in the zona pellucida made during enucleation step. The cell is wedged between the zona and the cytoplast membrane to facilitate close membrane contact.
  • Couplets are equilibrated in fusion medium [0.28 M mannitol (Sigma Co.), 0.5 mM HEPES, and 0.05% fatty acid-free BSA with 0.1 mM magnesium chloride] at room temperature in a chamber consisting of two stainless steel electrodes 3.3mm apart (BTX, San Diego, USA) prior to electrical fusion using two DC pluses of 1.78kV/cm for 15 ⁇ s each, delivered by BTX Electro-cell Manipulator 2001. Couplets are rinsed in TALP-Hepes, and then incubated for 2 hrs in G1.2 (Vitrolife, Inc, Englewood, CO) at 37°; 5% CO 2 . Electrofusion is confirmed by inverted HMC optics within 30 min.
  • fusion medium 0.28 M mannitol (Sigma Co.), 0.5 mM HEPES, and 0.05% fatty acid-free BSA with 0.1 mM magnesium chloride
  • reconstructed oocytes are activated in 5 ⁇ M ionomycin (Sigma) for 4 min in TALP-Hepes. After rinsing, the constructs are placed in G1.2 medium containing 2 mM 6-DMAP (Sigma) and incubated at 37°, 5% CO 2 5% O 2 and 90% N2 for 4 hrs.
  • Activated SCNT Constructs Activated oocytes are washed with TALP-Hepes and cultured in 20 ⁇ 1 drops of G 1.2 medium at 37° , 5% CO 2 , 5% O 2 and 90% N 2 for 48 hrs. On the third day of culture, cleaving embryos are either selected for transfer to timed female recipients or transferred to the hmSOFaa (Vitrolife, Inc) media and cultured for another 7 days until the blastocyst stage for derivation of NT- nhp-ES cells as described below.
  • NT-ES cells using anti-monkey IgG serum and guinea pig complement will be performed as described in 1.1.
  • D. 2.1.G. NT blastocyst imaging for pluripotency markers The zona pellucida in SCNT blastocysts will be removed with 0.5% pronase and the blastocyst recovered for 30 min in hmSOFaa medium.
  • the embryos After attaching zona-free blastocyst to polylysine-coated cover slips in serum-free hmSOFaa, the embryos are fixed in 2% formaldehyde in hmSOFaa for lhr or in absolute methanol (-20 0 C; 20 min) diluted directly into the well. Methanol addition is performed while watching under the dissecting scope to ensure blastocysts are not displaced. After fixation, the embryos are rinsed overnight in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 detergent (PBS-TX) to permeabilize the blastocyst.
  • PBS phosphate-buffered saline
  • PBS-TX Triton X-100 detergent
  • Primary antibody staining for formaldehyde fixed blastocysts includes SSEA-1 and SSEA-3 or-4 (1:5 dilution; Developmental Hybridoma Bank, Iowa City, Iowa) as well as fluorescein-labeled Tra 1-60 or Tra 1-81 (1:10; Santa Cruz Biotechnology, Inc, Santa Cruz, CA).
  • Anti-Oct-4 antibody (Santa Cruz Biotechnology) is used 1:5 on methanol fixed blastocyst. All primary antibodies are applied for lhr at 37 ⁇ C.
  • Cytogenetic analysis of cultured NT-ES cells by FISH analysis is in described 1.1. E and the Imaging Part IV. Cytogenetic analysis is performed once every six months to ensure genetic stability within the isolated NT-ES cells.
  • testingpluripotent markers in derived NT-ES cells will be performed on methanol or formaldehyde fixed colonies after transfer to gelatin-coated cover slips for 24 hours, lmmunocytochemistry will be performed using appropriate molecular markers for inner cell mass cells [Oct 4; SSEA-4, Tra 1-60, Tra 1-81] and trophectoderm lineages (SSEA-1) as described in 2.1.G.
  • blastocysts are incubated in guinea pig complement (Sigma) diluted 1:10 in CMRL medium, containing 20mg/ml propid urn iodide (Pl) and incubated for 10-15 min at 37°C. This activates the complement cascade rendering the trophectoderm cells permeable to Pl.
  • blastocysts are washed in TALP-Hepes and transferred to hmSOFaa medium containing 5 ⁇ M Hoechst and cultured for at least 30 min at 37°C before being mounted on microscope slides in a small drop of water-based antifade solution (Slow-Fade, Molecular Probes, Eugene, OR) underneath a cover slip.
  • Blastocysts are examined by epifluorescence as describe in Part IV.
  • the TE nuclei, labeled with both Pl and Hoechst are pink; the ICM nuclei labeled with Hoechst are blue.
  • TdT terminal deoxy nucleotidyl transferase
  • TUNEL mediated dUTP nick-end labeling
  • Zona-free blastocysts are first fixed in 2% formaldehyde (pH 7.4) for 30 min, rinsed in PBS 1 then permeabilized in PBS with 0.1% Triton X-100 and 0.1% NaCitrate solution at 4° C for 2 min. The broken DNA ends of the embryonic cells are labeled with TdT and fluorescein-dUTP for 60 min at 37° C. The blastocysts are counter-stained with 1 ⁇ g/ml Hoechst to visualize total DNA. The blastocysts are then mounted onto glass slides using Vectashield.
  • two coverglass spacers (170 ⁇ m height, i.e. >130-150 ⁇ m rhesus embryo diameter) are placed beneath the cover slip alongside the droplet of Vectashield.
  • Mouse blastocysts pretreated with DNase I (5 U / 50 ⁇ l PBS, Roche Diagnostic Corp., Indianapolis, IN) will be used as positive controls for the TUNEL assay and blastocysts in which terminal TdT transferase is omitted will serve as negative controls.
  • NT- blastocyst we will record the staining profile of the inner cell mass cells as well as the polar and mural trophectoderm. We will also determine the ratio of each pluripotent marker by comparing the staining profile of each probe to the total number of cells labeled by the DNA stains. Estimates of the number of aneuploid cells observed in each blastocyst will also be determined. For derived NT-ES cells, colony morphology as well as the staining characteristics of each pluripotent marker will be recorded. NT-ES cells that demonstrate Oct 4 nuclear staining and plasma membrane SSEA-4 but are negative for SSEA-1 will be considered undifferentiated.
  • NT-ES cell reaggregations with fertilized or tetraploid embryos will produce viable chimeric blastocysts in vitro.
  • NT-ES cells will contribute predominately to the ICM in either fertilized or tetraploid embryos by either cell injection or zona-free co-culture.
  • various PCR methodologies including nested PCR 1 AS-PCR, RT-PCR and real time PCR will be employed as described in 1.1. L.
  • NT-ES cell chimera will implant following embryo transfer and demonstrate axis development as imaged dynamically by high resolution U/S and MRI imaging.
  • NT-ES cell-derived chimera rates may be slightly lower compared with the viability of non-manipulated controls or even £ ⁇ f? + $ ⁇ 5 chimera. Therefore, we predict that we will need to produce 120 chimeras by aggregating $ ⁇ $ or 4N embryos with NT-ES cells.
  • Chimera development will demonstrate normal blastocyst formation as analyzed by total cell proliferation, intercellular interactions, compaction, and cavitation.
  • Example I 3 What is the level of DNA methylation in embryos, fetuses, placentae, and offspring after ART, NT, or natural coatings?
  • nhp embryos after 1VF or ICSI will be provided by Part V as detailed in section 1.1.G. Since the reported demethylation of the paternal genome in mouse and bovine zygotes happens within 4 hours of sperm incorporation, we will initially analyze 1VF and ICSI-derived oocytes at 2, 6, 12, and 24 hr post insemination. Baseline methylation of the paternal genome will be established by probing mature ejaculated sperm. We will also collect 2-to-4- cell, 8-to-16-cell, morula and expanded blastocyst stage for both IVF and ICSI-derived embryos for determining DNA methylation patterns.
  • SCNT nhp embryos will be produced as described in Part 1.
  • NT-nhp embryos will be harvested at the 2-to-4-cell, 8-to-16-cell, morula and expanded blastocyst stages to determine DNA methylation patterns.
  • . 3.1.D Production of NHP post-implantation tissues. Isolation of early post-implantation embryos (epiblast cells; Day 14-18) and embryonic germ cells (day 25-30) will be performed after punctomies as described in Part V, section 2.1.E, and Projects Il and III, respectively. To determine methylation levels in newborn and adult somatic tissues, we will examine methylation patterns in fibroblast cells isolated from newborn offspring and in adults, as described in 2.1.B.
  • 3.1.E Detection of Global Methylation Patterns: After fixation in absolute methanol for 15 min or 4% paraformaldehyde (Polysciences) for 24 hrs. Fixed cells will be permeabilized with PBS containing 1% Triton X-100 for 40 rains and then treated with 3N HC1 for 20 min to denature the DNA. After washing in PBS, the fixed cells are blocked in PBS containing 3 mg/ml BSA and 150 mM glycine to reduce non-specific antibody binding.
  • Igf2/H19 and DIM/GU2 gene pairs are selected because of their well-characterized DNA methylation regions (DMRs), and the tendency for H 19 to undergo relaxation of paternal repression as a consequence of in vitro embryo culture (Mann et al., 2004, Development 131:3727-3735) as well as in cultured hESCs.
  • RNA Isolation The methods described for mouse embryos will be employed (Szabo et a/., 1995, Genes Dev 9:3097- 3108). Embryos are mechanically stripped of all somatic cells (primarily cumulus cells) in the presence of 2mg/ml hyaluronidase and washed extensively in TALP-Hepes. The zona pellucida is removed using a brief Incubation in 5 mg/ml Pronase (Sigma) and the embryo washed. RNA is isolated from single embryos by placing individual embryos into 10 ⁇ l aliquots of RNAzol followed by snap freezing and storage in LN2 until assayed.
  • a cDNA is generated by preparing a reaction containing: 5mM MgC12, 1 x RT buffer, 1mM each dNTP, lunit/ ⁇ l ribonuclease inhibitor, 15unit/ ⁇ g AMV RT, 0.5 ⁇ g random hexamers, and nuclease free water to a 20 ⁇ 1 final volume. This reaction is added to the RNA and incubated at room temperature for 10 min and then 42°C for 15 min. Finally the sample is heated to 95°C for 5 minutes, and immediately placed at 4°C for PCR. PCR primers are designed that span polymorphisms Identified in the rhesus colony at the Pittsburgh Development Center.
  • SNuPE is performed in a reaction mix containing 10 ng of the PCR product, 1 ⁇ M of the SNuPE primer 10 mM Tris-HC1 (pH 8.3), 50 mM KCI, 2 mM MgC12, 0.75 units of AmpliTaq DNA polymerase, and 2 ⁇ Ci of [32P]dNTP.
  • One cycle is run; 95°C for 30 sec, 42°C for 30 sec and 72°C for 1 min. Bands are separated by electrophoresis and imaged using a phosphor imager.
  • the objective of these experiments will be to assess the methylation status in 5 preimplantation stage Rhesus embryos, specifically at the blastocyst stage.
  • the selection of this stage for analysis reflects the minimum requirement of ⁇ 100 cells for the DNA methylation analysis by the bisulfite method, which involves conversion of unmethylated cytosines to uracils, which are detected as thymines upon DNA sequencing.
  • This method has been recently refined 0 so that it is possible to assess methylation in single preimplantation mouse embryos (8 cell stage or later), and this is the approach that will be applied for this purpose (Millar et a/., 2002, Methods 27:108-113; Wamecke et a/., 1998, Genomics 51:182-190).
  • DNA will be isolated from embryos essentially as described previously for mouse embryos (Millar et a/., 2002, Methods 27:108-113; Warnecke et a/., 1998, Genomics 51 :182-190). Embryos in the quantities identified above will be mechanically stripped of any adhering maternal cells and concentrated in 2-5 ⁇ 1 of PBS. Embryos are then resuspended in 18 ⁇ l of the following solution (2 ⁇ g Escherichia coli
  • PCR of Bisulfite-Treated DNA We will choose primers that span differentially methylated regions and which contain informative polymorphisms. Nested PCR will be used to amplify low quantities of DNA. PCR products will be subcloned into plasmids, transfected into chemically competent E. coli and selectively grown on agar plates containing antibiotic.
  • PCR bias one allele being preferentially amplified over the other
  • PCR bias can be controlled by first testing the primers on DNA with known ratios of fully methylated and unmethylated DNA. Only primers that demonstrate no bias will be employed.
  • Example 1 Dynamic imaging of ES cell fates after transplantation.
  • NHPs e.g. subcutaneous, testicular, intramuscular or kidney capsule.
  • Table Il Introduction
  • Female Autograft SCNT in which the cumulus donor cell and oocytes are from the transplanted female; Fetal Fibroblast Autograft: Amniotic cell NT transplanted to born NHP; Male and/or Female Autograft: Somatic fibroblast NT into unrelated oocyte, transplant to nuclear donor.
  • SPIO Labeling of pluripotent NT-nhpES cells for in vivo MRI Imaging is described in Part IV.
  • SPIO particles obtained commercially (Miltenyi Biotec Inc., Auburn, CA). For transfection, SPIO particles are pre-mixed with the transfection agent Lipofectamine 2000 for ⁇ 20 minutes before adding to the culture media. Previously, we determined that 6 ⁇ l/ml of Lipofectamine provides effective coverage of the SPIO particles (see Part IV).
  • nhp- ESC and NT-ES cells ( ⁇ 10 5 -10 7 cells) will be harvested (Project Vl) labeled while in suspension at 37 0 C in a humidified 5 % CO 2 atmosphere. At the end of the incubation period, the ES cells will be washed twice to remove excess agents prior to transplantation as described next.
  • 4.1.D. NHP Stem cell transplantation Pluripotent nhpES cells are harvested by cell scraping followed by treatment in 0.25% trypsin-EDTA for 5 minutes to break up cell colonies. Individual cells along with small colonies will be washed twice in sterile PBS and the final concentration between 0.05-5 X 10 6 in 0.4 ml. NHPs are anesthetized (Project V) and the entire volume of the stem cells transferred subcutaneously, intramuscularly, or to the kidney or testicular capsules, as described in Part V.
  • 4.1.F. MRI Imaging is performed as described in the Part IV. Image frequency will be once every two weeks for the first two months and thereafter on a monthly basis providing no rejection is observed.
  • 4.1.G. Analysis of teratomas or biopsied material Teratoma formation, as determined both by palpation and imaging, will result in tumor excision (as in Part V) and subsequent fixation in 4% paraformaldehyde (EM grade; Polysciences,). After embedding in paraffin, sections will be processed for histology. Typically, 8- ⁇ m sections are cut and stained with hematoxylin for identification of tissue types (endoderm, ectoderm and mesoderm).
  • tissue markers Neurodethelial growth factor (Tuj1:neuron-specific beta-Ill tubulin; MF20: anti-myosin, mesoderm; alpha-fetoprotein: endoderm marker as in Imaging Part IV. Images of sections are collected by conventional or confocal microscopy.
  • NT-nhpES cells are immunotolerated when transplanted to the female from which the very NT blastocyst was initially derived (i.e. both donor nucleus and oocyte).
  • NT- nhpES cells will form teratomas demonstrating all three germ layers (endoderm, ectoderm and mesoderm) as confirmed by specific marker antibodies (see 4.1.G) for each tissue type and high resolution EM analysis of tissue sections.
  • endoderm, ectoderm and mesoderm as confirmed by specific marker antibodies (see 4.1.G) for each tissue type and high resolution EM analysis of tissue sections.
  • 4.2.A Derivation and GFP transgene insertion of pluripotent nhpES and NT- nhpES cells as in 4.1. A-B. 4.2. B. Differentiation of ES or NT-ES cells. We will prepare two differentiated stem cells populations for transplantation:
  • Pluripotent ES stem cells are cultured under conditions that promoted differentiation into neural progenitor (NP) cells.
  • NP neural progenitor
  • Two protocols were employed that produced 80-90 % NP cells, one, an adherent protocol that required a graded reduction of mouse feeder cells and the second, a feeder free protocol that required a specific period of culture as embryoid bodies ⁇ NP cells cultured by these methods retained markers of progenitor cells for more than 2 months. These cultures could be further driven to neuronal lineages by media conditioned by glial cells.
  • CD34+-sorted control and gene-targeted NHP EB cells will be re-cultured on OP9 bone marrow stromal layers, or NOD-/scid fetal thymic organ cultures (FTOC)(Robin et a/., 1999, Br J Haematol 104: 809-19) in the presence of Kit-Ligand, Flt-3 Ligand, Thrombopoietin, IL-2, IL-15, and IL-7 for the generation of B and NK (OP9 system) (McCune, 1991, Curr Opin Immunol 3:224-28; Vugmeyster et a/., 1998, Proc Nat'l Acad Sci 95: 12492-97) or T lymphoid cells (FTOC).
  • FTOC NOD-/scid fetal thymic organ cultures
  • NHP Stem cell transplantation Differentiated hematopoetic SC, nhpESCs and/or NT-nhpESCs are analyzed as in 4.1.D and Part V.
  • Imaging is performed as described in the Imaging Part IV. Image frequency will be once every two weeks for the first two months and thereafter on a monthly basis providing no rejection is observed.
  • the bioinformatics strategy is designed to identify a set of robust molecular markers that characterizes each stage and tissue lineage of the early mammalian conceptus. This analysis provides the basis for inducing stem cell development along specific tissue lineage pathways, and it establishes benchmarks for judging the normalcy and stability of differentiated states achieved by in vitro differentiation.
  • pluripotent stem cells It is also important to define the epigenetic status of pluripotent stem cells and their differentiated progeny. This will be accomplished through an analysis of the allele-specific transcriptional activity of imprinted genes in pluripotent stem cells and in non-human primate embryonic tissues using single nucleotide polymorphisms. See Examples Il 2 and 5.
  • ES cell pluripotency and its maintenance are important issues with both fundamental and practical implications.
  • directing hESC differentiation requires knowing what developmental stage they represent.
  • Cultured mES cells spontaneously differentiate to cells with characteristics of ectoderm, mesoderm or endoderm, suggesting that they reflect the potentiality of the epiblast, rather than the ICM (inner cell mass).
  • hES cells can differentiate to trophectoderm cells (Xu et al., 2002, Nat Biotechnol 20:1261-1264), unlike mouse ES cells, suggesting that they could have the potentiality of the earlier, morula stage.
  • the resulting human and mouse gene expression data sets contained processed expression values for 54,675 transcripts (Affymetrix ® hg-u133+2 chip, Human genome) and 12,488 transcripts (Affymetrix ® mg-u74Av2 chip, Murine Genome U74v2 Set) respectively.
  • the expression sets were analyzed to assess the significance of differential gene expression between ESC and ICM groups (3 arrays in each).
  • the moderated t-statistic of Smyth (2004, Statistical Applications in Genetics and Molecular Biology 3(1) Article 3) was applied to both human and mouse expression sets to assess the significance of differential expression between ESC and ICM groups for each transcript present.
  • 127 possessed (one or more) orthologs that were labeled significant on the mouse expression data as well.
  • the final list includes 132 orthologous transcript pairs that display significant differential regulation between ESC and ICM groups on both mouse and human data.
  • the orthologous pairs include 127 human transcripts and 98 mouse transcripts (some appearing more than once, as multiple probe-set associations were preserved when assigning mouse orthologs to human gene probe-sets).
  • An interesting initial observation is that 28 of the 132 pairs display contrasting differential regulation (e.g. a transcript up-regulated in human ESC, with ortholog down-regulated in mouse ESC).
  • This gap compels us to turn to primate embryos a) as a relevant model to complement the mouse system as a means of defining the embryonic stage that hESCs most resemble, and b) for establishing transcriptional marker genes as benchmarks for characterizing in vitro hESC differentiation, again, in comparison with comparable mouse tissues.
  • Figure 1 was generated by normalizing human and mouse microarray data using 6161 uniquely orthologous probe-sets from Affymetrix hg-u133+2
  • Example Il Assessment of the epigenetic status of hESCs and their differentiated progeny
  • hES cells The epigenetic status of hES cells is relatively stable at low and medium passages ( ⁇ p50), but that it may be susceptible to perturbation at higher passage in the case of specific gene(s) and cell lines.
  • imprinted genes that showed detectable expression of the 'silent' allele were those in which the paternal allele is typically silent (i.e., maternally expressed imprinted genes H19, SLC22A18 and NESP55).
  • the concurrence of derepression of paternally silent genes in human hES cells observed here and mouse embryos mutant for Eed shows that hES cells provide a system for analyzing this interesting phenomenon.
  • Bisulfite sequencing studies will be performed to evaluate the methylation status of key imprinting control regions.
  • the bisulfite treatment protocol has now been optimized so that we are obtaining high (>95%) cytosine conversion rates, but still retaining one or two unconverted non-CpG associated cytosines to aid with the identification of unique clones.
  • Control studies have been carried out as detailed in the experimental procedures in order to preclude any PCR or cloning bias in these assays. Primers and conditions for analysis of the H19 DMR have been identified and selected for the absence of any bias.
  • Example Il 4 Determine the transcriptional identity of early embryonic cells and their in utero progeny
  • the pluripotency of embryonic stem cells commends their use in derivation of differentiated progeny with clinical potential.
  • the genuine process of differentiation takes place in mammals in an intrauterine environment, aided by a three-dimensional structure and numerous factors that are acquired from the intraembryonic microenvironment during the course of normal development.
  • the hypothesis that underlies the proposed use of pluripotent stem cells as exogenous sources of cells for transplantation therapies is that such cells and composite tissues will be comparable in function and stability of their differentiated state to in vivo generated tissues. While it can be argued that the successful development of mouse ESCs in chimeric circumstances proves the normality of ESC- derived tissues, the numerous differences that are emerging between mouse and primate ESCs renders this case less than compelling.
  • the first priority will be to validate a set of markers for the range of phenotypes expected for in vitro differentiation of hESCs.
  • Robust markers of inner cell mass and epiblast will be identified, as well as definitive endoderm, ectoderm and , mesoderm, plus extraembryonic mesoderm, amniotic ectoderm and visceral (extraembryonic) endoderm.
  • markers that define them as robust they need to be unambiguously associated with the bona fide, native tissue as it develops in vivo, consistently expressed by genetically different individuals within a species, expressed across a broad time range (i.e., a day or more), rather than transiently.
  • Expression will be detected in the first instance by transcriptional profiling using DNA microarrays.
  • Candidate markers will then be further analyzed by a set of progressively more quantitative and qualitative methods, including RT-PCR to detect expression in cell populations, real time PCR, to assess absolute levels of mRNA, and in situ hybridization to determine the cellular distribution of transcripts.
  • Marker genes can be defined as those genes whose expression is correlated with an observed biological phenotype or difference between discrete conditions.
  • Microarray experiments involve the capture of thousands of gene expression measurements for each experimental condition/tissue analyzed. The result is thousands of expression pairs, the difference between which may, in principle, be used to identify genes responsible for the biological phenomenon, or difference between conditions, described by comparison of any two array experiments. Many methods exist with which to identify the genes responsible for the differences in comparisons between two microarray experiments. Linear fold-change thresholds have recently been superseded by variable fold-change thresholds that incorporate the knowledge that genes have differing propensities to change according to their position on the recorded range of expression intensity.
  • the transform facilitates the application of sophisticated machine learning techniques, such as the data description method of Tax and Duin (2004, Machine Learning 54(1):3097-3108).
  • machine learning techniques such as the data description method of Tax and Duin (2004, Machine Learning 54(1):3097-3108).
  • This approach readily detected standard, known markers of pluripotency (Oct4, Nanog, FGF4, Nodal, Cripto and Lefty), among numerous others whose role has yet to be defined.
  • Our objective in this aim is to define 10 or more robust marker genes for each of the tissues to be analyzed.
  • Our first objective will be to generate transcriptional profiles for the inner cell mass, epiblast, and the primary germ layers and their earliest derivatives (definitive mesoderm, endoderm and ectoderm, as well as lateral plate mesoderm, gut and neuroectoderm) by laser capture microdissection of day 15-16 post-fertilization embryos.
  • Such early stage embryos are exceptionally rare in humans, but can be straightforwardly obtained in the Rhesus monkey (Enders, 2002, Plancenta 23:236-238; Enders et al., 1986, Am J Anat 177:161-185).
  • Affymetrix hg-u133+2 GeneChips Human Genome U133 Plus 2.0 Array, Affymetrix, Inc., Santa Clara, CA
  • Rhesus cDNAs Choismar et al., 2002, Biotechniques 33:516-518, 520, 522; Wang et al., 2004, Nat Genet 36:687-693
  • Rhesus chips from Affymetrix (GeneChip® Rhesus Macaque Genome Array).
  • the next objective will be to generate transcriptional profiles for morula and inner cell masses of three or more Rhesus embryos.
  • Epiblast cells from three or more gastrulation stage (day 13-14) Rhesus embryo will be obtained as described above.
  • the transcriptional profiles obtained for these pluripotent embryonic cell types will be compared to transcriptional profiles of triplicate samples of three or more Rhesus embryonic stem cell lines.
  • hES cell culture hES cells are maintained on a mouse embryonic fibroblast feeder layer in KO-DMEM (Gibco ® , Invitrogen, Inc., Carlsbad CA) supplemented with 10% Serum Replacer (Gibco ® ) and 4ng/m! basicFGF (R&D), or in feeder free conditions (Xu et al., 2002, Nat Biotechnol 20:1261- 1264).
  • KO-DMEM Gibco ® , Invitrogen, Inc., Carlsbad CA
  • Serum Replacer Gibco ®
  • R&D basicFGF
  • EBs Embryoid bodies
  • CDM composition is IMDMEM/F12 1:1 , 1X lipid concentrate, 15 ⁇ g/ml transferrin, insulin 7 ⁇ g/ml, monothioglycerol 450 ⁇ M, BSA 5mg/l or PVA 0.1% according to (Johansson and Wiles, 1995, id).
  • concentration range of factors added is as follows: FGF-2 (0-50 ng/ml), BMP (0-4ng/ml), LiCI (0-20 ⁇ M), Activin (0- 4ng/ml).
  • the cDNA of the genes of interest will be synthesized by RT- PCR 1 purified from agarose gel and cloned in TOPO ® vector (Invitrogen, Carlsbad, CA).
  • TOPO ® vector Invitrogen, Carlsbad, CA.
  • the protocol for probe labeling and whole mount in situ hybridization from (Streit and Stern, 2001, Methods 23:339-344) will be used on hES cells and on EBs fixed in methanol and kept at -20 0 C.
  • proteins are extracted with Trizol ® using a cocktail of proteinase inhibitors. After separating by SDS PAGE, proteins are transferred onto nitrocellulose membrane by western blotting.
  • Membranes are incubated with primary antibody and horseradish peroxidase conjugated secondary antibody is used for detection by chemiluminescent substrate.
  • EBs are dissociated by exposure for ⁇ 30 min to Cell Dissociation Buffer (Invitrogen, Carlsbad CA) and further mechanical dissociation.
  • GFP fluorescent cells are detected and quantified on a Becton Dickinson FACScalibur (Becton Dickinson, Franklin Lakes, NJ), or when preparative amounts are needed, by cell sorting on a MoFIo FACS sorter (Cytomation, Inc., Fort Collins, CO).
  • Transcriptional profiling For transcriptional profiting, either 1) the standard amplification protocol for Affymetrix arrays (Affymetrix, Inc., Santa Clara, CA) is used with large populations of cells (10 5 to 10 7 ) to generate labeled cRNA probes, starting from a total RNA amount of ⁇ 1 ⁇ g; or 2) the amplification method described for single cell transcriptional profiling (Tietjen et al., 2003, Neuron, 38: 161-175) is used with small numbers of cells (10-1000) to generate sufficient cDNA for profiling. Affymetrix hg-u133+2 GeneChips are hybridized according to Affymetrix methodology.
  • MAS v ⁇ .O will be used to assess technical/experimental quality control and RMA will be used to background correct, normalize and determine the absolute expression values of all individual elements (genes) on each chip.
  • RMA is preferred to MAS v5.0 for the latter stage as published results
  • a host of state-of-the-art marker identification methods will be employed when analyzing array data for differential expression.
  • Bayesian statistics (Smyth, 2003, Statistical Applications in Genetics and Molecular Biology 3(1), Article 3), variable fold-change methods (Quackenbush, 2002, Nat. Genet. 32 Suppl. 496-501) and the in- house method of Trotter et al. (2004, id.).
  • Computational machine learning methods will be applied to microarray data using project microarray data.
  • novel methods for marker gene identification will be applied to project data.
  • Data visualization and functional clustering will be performed on project microarray data, using techniques such as multi-dimensional scaling (see Example Il 1) and hierarchical clustering, available via third party software packages such as dChip (Harvard University, Cambridge MA). Visual analysis is a great aid in discerning consistency in biological repetitions and in identifying clusters of genes that behave differently from the norm.
  • Lysis is subsequently performed at 65°C for 1 min.
  • First-strand cDNA synthesis is then initiated by adding 50 U of MMLV and 0.5U of AMV reverse transcriptases (Invitrogen, Carlsbad CA) followed by incubation at 37 ⁇ C for 15 min. Samples are heat inactivated at 65°C for 10 min, and poly(A) is added to the first-strand cDNA product by-adding an equal volume of 200 mM potassium cacodylate (pH 7.2), 4 mM CaCI 2 , 0.4 mM DTI, 200 ⁇ M dATP containing 10 U of terminal transferase (Roche Diagnostics, Basel, Switzerland) at 37°C for 15 min.
  • Samples are heat inactivated at 65°C for 10 min, and the contents of each sample is brought to 100 ⁇ l with a solution made of 1X PCR buffer Il (Applied Biosystems, Foster City, CA), 2.5 mM MgCI 2 , 100 ⁇ g/ml bovine serum albumin, 0.05%Triton X-100 and containing 1 mM of dATP, dCTP, dGTP, dTTP, 10 U of AmpliTaq polymerase (Applied Biosystems), and 5 ⁇ g of the PCR primer AL1.
  • the AL1 sequence is 5'-ATT GGA TCC AGG CCG CTC TGG ACA AAA TAT GAA TTC (T)24-3' (SEQ ID NO: 8).
  • PCR amplification is then performed according to the following schedule: 94°C 1 min, 42°C 2 min, and 72°C6 min with 10 s extension per cycle for 25 cycles. An additional 5 U of Taq polymerase is added before performing 25 more cycles of PCR without the 10 s extension per cycle. In this manner, 10-20 ⁇ g of PCR-amplified cDNA can be synthesized from RNA of individual or small numbers of cells. Five microliter aliquots of each single-cell cDNA are checked for the presence of ubiquitous and cell type- specific markers by Southern blot hybridization.
  • the objective of this step is to verify that sequences relevant to the tissue identity (e.g., Oct4, Nanog, Cripto in ESCs; Sox1, NeuroDI, nestin in neuroectoderm) are present in the amplification products, and that irrelevant sequences (e.g., epidermal cytokeratins) are absent. It is anticipated that the majority but not all of the amplified samples will qualify for further analysis on the basis of this criterion.
  • tissue identity e.g., Oct4, Nanog, Cripto in ESCs; Sox1, NeuroDI, nestin in neuroectoderm
  • irrelevant sequences e.g., epidermal cytokeratins
  • Tissue-tissue Comparisons in Human and Rhesus Data from Affymetrix arrays will be processed using default parameters of the RMA model, as described above (Irizarry et al., 2003, id.). In order to directly compare human and Rhesus data captured on human arrays; a set of Affymetrix hg- u 133+2 GeneChip probe-sets that respond to both human and Rhesus expression signal must be identified. Information regarding human-Rhesus probe equivalence is already at our disposal (Rob Norgren, University of Wyoming Medical Center, Omaha, NE ⁇ and the macaque GeneChip is available from Affymetrix. Further correspondence may be identified by a control study of pooled fetal or adult Rhesus tissues to establish a subset of human GeneChip array probes that can be reliably detected in both human and Rhesus samples.
  • the normalized array data for that subset will be visualized in 2-13-dimensions and distance analysis will be performed on array profiles corresponding to the different tissue groups present.
  • MDS multi-dimensional scaling
  • the distance between any two expression profiles is estimated using the Pearson correlation co-efficient subtracted from unity, to provide a bounded distance in the region (0,2).
  • the distance between two groups of profiles e.g. those that describe two types of tissue, is calculated using the average linkage (the mean of all pairwise distances (linkages) between members of the two groups concerned).
  • the standard error of the average linkage distance between two groups may be used to evaluate the significance of difference between two or more average linkage distances. Distance analysis of this nature will provide the basis for definition of relationships between the various pluripotent cell types that characterize early mammalian development, including hESCs.
  • Example Il 5 Compare the epigenetic status of hESCs and their in vitro differentiated progeny with the in utero epigenetic status of non-human primate lineages
  • the potential use of hES cells in cell-replacement therapies prompts a genuine concern about whether their in vitro differentiated progeny would possess the same differentiative stability as cell types arising in the course of normal development.
  • Derivation and culture of mouse ES cells can perturb the expression and methylation status of imprinted genes (Dean et al., 1998, Development 125:2273-2282).
  • mouse embryos can undergo perturbation of imprinted gene expression as a consequence of exposure to certain culture environments (Doherty et al., 2000, Biol Repred. 62:1526- 1535).
  • imprinted gene expression is to be examined at various times of cell culture (i.e., low, medium and high passage), both in ESCs and in differentiated cells derived from them.
  • Example Il 2 strictly monoallelic expression of all three paternally expressed genes was observed, both at moderate passage (50-60) and at high passage (75-100) numbers, implying the maintenance of normal genomic imprinting at this stage, thus appearing to refute the 'imprint perturbation 1 hypothesis.
  • monoallelic expression of the maternally expressed H19 gene was observed in moderate passage H9 cells, 'relaxation' of strict monoallelic expression of this gene was observed at high passages (>75) in one subline, and low levels of 'silent' allele expression in other maternally expressed genes, thus appearing to support the 'imprint perturbation * hypothesis.
  • H19 is similar to the other imprinted human genes that have been studied in hESCs in being predominantly monoallelic (H 19 being strictly monoallelic at low passages). This suggests that the loss of H19 repression observed in hESCs could occur by a distinct mechanism from the loss of H19 repression in cultured mouse embryos (Mann et al., 2004).
  • the epigenetic status of imprinted genes will be determined in undifferentiated ESC (both human and non-human primates) and their in vitro differentiated progeny. This will provide the background for an assessment of their expression during in vivo development in conceptuses of non-human primates.
  • Candidate maternally expressed genes include MEG3/GTL2, CDKN1C/P57kip, p73, COPG2, ASCL2, KCNQ1DN, TSSC3, ZNF215, HTR2A, UBE3A, ATP10C and ElonginA3. It is important to point out that the objective of this research is not to characterize the expression status of all imprinted human genes, or even a majority of them. Rather, it seeks to identify the overall pattern of imprinted gene activity as a function of pluripotency vs. differentiative state, or as a function of culture history. Then the focus will be on defining the underlying mechanisms of whatever pattern is observed. The priority in selection of which of these candidate genes to analyze will be determined by the body of information available for the mouse embryo, the depth of equivalent information about their pattern of imprinting in the human, and the identification of polymorphisms in available hESC lines.
  • the effort to expand the number of informative genes by examining additional hES cell lines available will be aided by undertaking an analysis of the imprinted status of key imprinted genes in existing hESCs, including DNA and RNA extracts of numerous existing hESC lines listed by the NIH Stem Cell Registry (NIH, Bethesda MD). This will enable a search for additional SNPs that can define allele-specific transcription in other hESC lines. Having established the set of informative imprinted genes, the effect of passage number will be examined as the variable most likely to influence the status of imprinted gene expression. .
  • Allele-specific gene expression in hESCs and their differentiated progeny For analysis of imprinted gene expression in hESCs and their differentiated progeny, similar procedures involving RFLP and DNA sequence polymorphisms will be used as for the data presented in Examples Il 1 and 2. Briefly, in order to assess parent-specific expression of imprinted genes, informative polymorphisms between the two parental alleles of an imprinted gene will first be identified DNA is extracted from the hES cells, and the polymerase chain reaction (PCR) is used to amplify a fragment containing the putative polymorphism. Many of these polymorphisms are situated within a restriction site. In these cases, the PCR fragment is then purified and digested with the appropriate enzyme.
  • PCR polymerase chain reaction
  • Digested fragments are run on a gel (either agarose or polyacrylamide). Informative polymorphisms yield two bands after digestion, one reflecting the cut and the other the uncut fragment.
  • a control cell line MRC- 5 (fetal lung fibroblast) which was informative for the H19 polymorphism will be used. This line revealed mono-allelic expression as expected, and is used as a control in all restriction digests for H19, for example.
  • all PCR products are sequenced. For genes in which the informative polymorphism does not occupy a restriction site, identity of the hES cell transcript is determined by DNA sequencing of the PCR product.
  • RNA sample 2.5 U/ ⁇ l reverse transcriptase (RT; Superscript H, Invitrogen, Carlsbad CA) is added and the reaction continued at 42°C for 15 min. The reaction is stopped by heating to 99 0 C for 5 min, and finally held at 65 0 C. Controls without RT are done for each RNA sample to ensure that DNA is not amplified. Two microliters of cDNA are then added to a polymerase chain reaction (PCR) mix (20 mM Tris-HCI pH 8.4, 50 mM KCI, 1.5 mM MgCI 2 , 200 mM dNTP, 0.5 mM lower and upper primers, 2.5 U/Rxn Taq DNA polymerase (Invitrogen).
  • PCR polymerase chain reaction
  • reaction conditions are as follows: 94°C for 30 s, 42°C for 30 s, 72 0 C for 2 min (40 cycles), 72°C for 10 min followed by a 4°C soak for H 19, lgf-2 and Snrpn, and 94 0 C for 45 s, 56°C for 45 s and 72°C for 1.5 min (40 cycles), 72 for 10 min followed by a 4°C soak for other genes.
  • PCR products are purified with Prep-Agene DNA purification Kit (BioRad, Hercules CA).
  • PCR product Approximately 10 ng of PCR product is added to the SNuPE reaction (20 mM Tris-HCI pH 8.4, 50 mM KCI, 1.5 mM MgCI 2 , 2 ⁇ Ci of the specific [32PJdNTP 1 I mM SNuPE primer, 0.75 U Taq DNA polymerase) and the reaction proceeds as follows: 4°C for 30 s, 42°C for 30 s, 72°C for 1 min. Electrophoresis through 12% polyacrylamide (BioRad) gels allows SNuPE reaction visualization by autoradiography and quantification with a Phosphorlmager. Specific [32P]dNTPs added to each reaction are as appropriate to the polymorphisms in the parental alleles, as determined by DNA sequencing of imprinted Rhesus gene coding sequences.
  • the first hypothesis to be tested is that the gametic imprint controlling imprinted expression of the H19 gene, the H19 DMR, progressively loses its methylation during hESC culture, thus accounting for the onset of 'silent' (presumptively paternal) allele transcription at high passage.
  • the H19 DMR is still represented by both methylated and unmethylated sequences at a frequency not significantly different from 50% (X-square test, P > 0.05; see Examples Il 1 and 2), affirming that the normal gametic imprinting of this gene is still present at this stage.
  • the further analysis in each case will involve an assessment of DNA methylation at any promoters known to depend for their methylation on the methylation status of the gametic imprints (H19 on the paternally methylated state of the H19 DMR;Gtl2 on the maternally unmethylated state of the IG DMR; Snrpn/Snurf on the status of the SNRPN DMR; and KCNQ1OT1 on the maternally methylated KV DMR.
  • methylation status of each DMR and their relevant sub-domains will clarify whether inappropriate methlyation per se can be attributed a role in disregulation (if any) of the respective imprinted gene.
  • a parallel analysis using nhpESCs will serve to generalize the findings, and importantly, to establish the foundation for perturbation of regulatory mechanisms and assessment of its developmental consequences.
  • any hESC or nhpESC sublines in which there is evident disregulation of imprinted genes will be considered particularly valuable resources for evaluating the consequences of such disregulation, especially if such (biallelic) imprinted gene expression patterns are stable throughout subsequent passages and during differentiation (see Preliminary Studies). Further analysis of these lines will be conducted as described below. In the event that biallelic transcription correlates with altered DNA methylation, either of the DMR or of gene-specific promoters, this will encourage further experimental approaches involving perturbation of the methylation status of the cells.
  • DNA methyl transferase inhibitors such as azacytidine, procainamide or zebularine
  • azacytidine DNA methyl transferase inhibitors
  • procainamide DNA methyl transferase inhibitors
  • zebularine DNA methyl transferase inhibitors
  • hESCs or nhpESCs will be grown at exponential phase, then exposed to normal ESC medium containing the methylation inhibitor 5- azacytidine.
  • an initial concentration of 1-5 ⁇ M 5-azacytidine for 2 days will be used to determine the extent and trajectory of inhibition. Treatments will be performed in triplicate, analyzing imprinted gene expression and changes in DMR and specific promoter methylation. Studies on mouse ES cells have indicated that once methylation is lost or removed, the cells remain devoid of imprints.
  • nhpESCs The developmental consequences of any perturbations in nhpESC imprinted gene expression status, whether spontaneous or experimentally induced, will be accessed by transplantation to chimeras and analysis of developmental outcomes, including both imprinted status and global transcriptional identity.
  • the first task will be to characterize any sublines of nhpESCs that have undergone alteration in their pattern of imprinted gene expression status. This will be accomplished in parallel with the analysis on hESCs. Once nhpESC lines or sublines have been identified that have characteristic perturbations in imprinted gene expression, these will be archived. The further analysis of their fate involves generating chimeras by blastocyst injection or morula aggregation.
  • chimeras Once chimeras have been generated in vitro, they will be transferred to the Rhesus uterus for development to early postimplantation stages. Embryos will be recovered at E15-20 for subsequent analysis of contribution to various germ layers and lineages, and for transcriptional profiling using the markers as criteria for normality of particular tissue lineages.
  • the characterization of physical integration and transcriptional complexity of the ESC-derived component of chimeric tissues will be accomplished by marking the donor nhpESCs with green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • This gain-of-function approach has been developed extensively using lipofection to introduce vectors containing the non-toxic hrGFP gene (Stratagene, La JoIIa CA) in a pTP6 vector containing a drug selection marker under IRES regulation into hESCs (Valuer et a/., 2004, Stem Cells 22:2-11).
  • This vector confers ubiquitous, stable GFP expression on hESCs and their differentiated progeny, without loss of pluripotency. Therefore, it is an optimal means of distinguishing the donor (nhpESC-derived) cells from the recipient embryo descendant cells.
  • resultant chimeric embryos will be cryosectioned (as described above, laser capture methods), using immunohistochemistry with complementary (i.e., red) fluorescent secondary antibodies to identify particular tissues. Alternate sections will thus be available for analysis of transcriptional status using laser capture microdissection, with GFP fluorescence as the identifying characteristic of donor-derived progenitor cells.
  • sorting of fluorescent cells using FACS could be used to capture donor nhpESC-derived differentiated cells after dissociation of chimeric embryos using Cell Dissociation Buffer (Invitrogen, Carlsbad CA).
  • the pattern of incorporation and transcriptional fidelity will provide a means of assessing definitively the pluripotent status of nhpESCs, an undertaking that cannot be accomplished using hESCs, owing to practical and ethical constraints against the use of human cells and animal embryos for this purpose.
  • DNA methylation analysis The protocol we use for bisulphite-based methylation analysis was adapted from (Olek et al., 1996, Nucleic Acids Res 24:5064-5066). hESCs are treated overnight with 1mg/ml proteinase K (Qiagen, Crawley, UK), and the following day DNA is extracted using the Genomic DNA Extraction Kit (Promega, Southampton, UK). 2 ⁇ g of DNA is digested overnight with a restriction enzyme that does not cut within the fragment of interest, for example for H19 DMR analysis Apal is used. DNA samples are serially diluted and denatured by boiling, then freshly made NaOH is added to a final concentration of 0.4 M.
  • DNA is embedded in 2.5% LMP agarose (Cambrex, Wokingham, UK) to form beads at DNA concentrations of 100, 10 and 1ng/bead.
  • Beads are treated (in the dark) with 5 M sodium bisulphite (Sigma, G ⁇ llingham, UK)) 110 itiM hydroxyquinone (Sigma) on ice for 30 minutes, incubated at 50 0 C for 3.5 hours and washed thoroughly with TE and water.
  • 5 M sodium bisulphite Sigma, G ⁇ llingham, UK
  • 110 itiM hydroxyquinone Sigma
  • First-round PCR is run in 100 ⁇ l in the presence of 2 U BlO-X-ACT Taq polymerase (Bioline, London, UK), 1 x manufacturer's buffer, 3 mM MgCk, 400 ⁇ M dNTPs and 1 ⁇ M primers.
  • Second round PCR is run in 25 ⁇ l in the presence of 0.8 U BIO-X-ACT Taq, 1 x buffer, 2.5 mM MgCI 2 , 400 ⁇ M dNTPs and 1 ⁇ M primers.
  • PCR conditions for both rounds are five cycles at 94°C for 1 minute, 5O 0 C for 2 minutes and 72°C for 3 minutes, followed by 30 cycles of 94 0 C for 30 seconds, 50 0 C for 2 minutes and 72°C for 90 seconds.
  • PCR products are subcloned using the TOPO TA cloning kit (Invitrogen, Paisley, UK) and sequencing.
  • Our standard for successful bisulphite conversion is 95% converted non-CpG associated cytosine residues; a minimal level (5%) of non-converted CpGs facilitates the identification of uniquely cloned (as distinct from repetitively cloned) sequences.
  • At least 2 independent bisulphite treatments are performed, with analysis of between 10 to 15 unique clones per treatment.
  • hrGFP-expressing nhpESC lines will be generated as described for hESC lines by (Valuer et al., 2004, Stem Cells 22:2-11) using Lipofection of the hrGFP pTP6 vector with either puromycin or neomycin as drug selection markers.
  • the in vitro pluripotency of such lines will first be determined by generating embryoid bodies, assessing the diversity of cell types using RT-PCR with markers specific to the primary germ layer derivatives, and with transcriptional profiling, using the markers established previously.
  • the analysis of embryos will initially be accomplished by sensitive, viable imaging of the recipient embryos, and then again after recovery at E15 to E20.
  • the assessment of chimeras will include an overall inspection of integration of nhpESC-descendant cells into the recipient embryo tissues, as defined by uniform expression of tissue-specific markers using immunohistochemistry (e.g., Sox 17 for definitive endoderm; Sox 1 for neuroectoderm, and MyoD for definitive mesoderm), using GFP fluorescence to identify donor cell descendants.
  • Teratoma imaging will be performed both with and without Gd-DTPA enhancement, magnetic resonance microscopy, and post-implantation visualization of SPIO-labeled stem cells, as well as optical and electron microscopy. In vivo MRI of SPIO- labeled HESCs implanted in the mouse brain, arid ⁇ 10,000 implanted cells are detectable.
  • Receptor-mediated endocytosis and lipofectamine SPIO labeling procedures are presented above. Because of likely variability between ESC lines and individual embryos, at least two independent nhpESC lines will be examined for each aberrantly expressed imprinted gene, or experimental perturbation, and triplicate embryos for each line.
  • ES cells embryonic stem cells
  • EG cells embryonic germ cells
  • PPCs primordial germ cells
  • Differentiated cells produced from pluripotent stem cells could potentially be used to treat a wide variety of human diseases including Alzheimer's, Parkinson's, diabetes, stroke and heart disease.
  • major questions about the growth, normal differentiation, stability of genomic imprints and potential for tumor formation of stem cells need to be addressed before they can be used clinically.
  • Technical and ethical barriers preclude many of these questions being addressed directly using human ES or EG cells. Therefore, many of these questions will need to be studied in pre-clinical animal models. Differences in the growth characteristics, marker expression .and gene imprinting status of murine and human ES and EG cells suggest that mice might not represent the most appropriate model for all pre-clinical studies.
  • ES and EG cells in primate species closely related to humans could help fill gaps in knowledge concerning the utility and safety of cell-based therapies.
  • ES cells have been developed from non-human primates (nhp)
  • primate EG ceils and to compare their growth, differentiation capacity and marker expression with existing primate ES cells and with human ES and EG cells.
  • Primate ES and EG cell lines can then be used to analyze questions that cannot currently be addressed using human stem cells. These include whether such stem cells can differentiate normally into all cell lineages in the embryo, whether stem cells will form tumors upon transplantation and whether altered genomic imprints exist and if they present a significant barrier to stem cell transplantation.
  • Such analysis of non-human primate ES and EG cell lines will fill the gaps in our knowledge of the usefulness and safety of pluripotent stem cells in cell-based therapies.
  • Pluripotent stem cells have two important properties. First, they are immortal and can be grown indefinitely in the laboratory. Second, they can be differentiated into all the cell types present in the animal body. In mammals three types of pluripotent stem cells have been identified and isolated into culture (reviewed in Donovan and Gearhart, Nature 414:92-97, 2001; Thomson and Odorico, Trends in Biotechnology 18:53-57, 2000). Embryonal carcinoma (EC) cells are the pluripotent stem cells of testicular cancers and are derived from primordial germ cells (PGCs) in the fetal gonad (Stevens, 1967).
  • PPCs primordial germ cells
  • Embryonic stem (ES) cells are derived from the inner cell mass of the pre-implantation embryo (Evans and Kaufman, Nature 292:154- 156, 1981; Martin, Proc Nat Acad Sci USA 78:7634-7638, 1981).
  • Embryonic germ (EG) cells are derived from cultured PGCs isolated from the fetal gonad (Resnick et al., Nature 359:550-551, 1992; Matsui et al., Cell 70:841- 847, 1992).
  • EC, ES and EG cells of murine origin all share certain markers and properties but also differ in certain important respects (Donovan and Gearhart, 2001; Pera et al., J Cell Sci 113:5-10, 2000; Thomson and Odorico, 2000).
  • EC cells typically are aneuploid whereas ES and EG cells are karyotypically normal and also maintain a normal karyotype in culture.
  • ES and EG cells are karyotypically normal and also maintain a normal karyotype in culture.
  • EC cells when EC cells are injected into the blastocyst cavity of the pre-implantation embryo they can contribute to the somatic cell lineages but they do not contribute to the germline (reviewed in Donovan and Gearhart, 2001). In contrast both ES and EG cells form good germline chimeras.
  • ES cells The ability of murine ES cells to maintain a normal karyotype and to enter the germline in chimeric animals allowed the manipulation of the mouse germline in a way that was unimaginable before their isolation.
  • EG cells Like ES cells, EG cells also are able to form germline chimeras and have been used to manipulate gene expression in the germline (Labosky et al., Development 120:3197-3204, 1994; Stewart et al., Developmental Biology 161:626-628, 1994).
  • hES human ES
  • hEG human EG
  • hEG cells may have all the same properties as hES cells in terms of disease treatment. Indeed, hEG cell-derived cells were the first karyotypically-normal human pluripotent stem cell to be used in any animal model to show proof of principle of the use of such cells to treat human disease (Kerr, Llado, et al., J Ne ⁇ rosci 23:5131-5140, 2003). Second, other than ES cells, EG cell lines represent the only other karyotypically-normal pluripotent stem cell type. Because of their shared pluripotency but distinct origin, comparisons of ES and EG cells could provide an enormous tool to study the mechanisms regulating developmental potency.
  • hEG and nhpEG cells will reflect fundamental similarities in their cellular programs, potentially providing critical insights into mechanisms regulating developmental potency.
  • a fundamental understanding of developmental potency will advance our knowledge of many areas of biology including embryonic development, gametogenesis, pluripotent stem cell self-renewal, aduit stem cell potential and nuclear reprogramming and could have widespread implications for disease treatment.
  • derivation of hEG cell lines is eligible for Federal funding. Therefore, if EG cell lines prove to be as useful as ES cell lines for cell transplantation they have the distinct advantage that new cell lines could be derived with Federal support.
  • testicular tumors are the most common form of tumor in young men. Studying the mechanisms by which PGCs form EG cells in different species could provide new information about mammalian gametogenesis and the etiology of testicular cancer.
  • ES or EG cells can be used for human disease therapy.
  • the first problem to be overcome is to develop improved conditions for growth of hES and hEG cells. Although some improvements in conditions for growing ES and EG cells have been made, further improvements are likely to be necessary if such stem cells are to be used in cell-based therapy (Richards et at., Nature Biotech 20:933-936, 2002; Xu et al., Nature Biotech 19:971-974, 2001). A major problem is that we know so little about these stem cell populations in terms of molecules that are essential for their growth. These molecules are likely to include growth factors, growth factor receptors, cell adhesion molecules, gap junction proteins, cell-cell adhesion molecules etc.
  • the growth properties of the small numbers of existing hES and hEG lines may not fully represent the conditions that will be used to grow all potential human pluripotent stem cell lines.
  • One powerful method for understanding critical growth mechanisms is to compare cells derived from different • species. For example, major advances in understanding the fundamental and evolutionarily-conserved mechanisms that regulate the cell division cycle came from studies in a variety of species including yeast, flies, worms, toads, mice and humans. Similar comparative studies of pluripotent stem cell populations from different mammalian species could provide crucial information about the factors regulating both the growth and differentiation of human ES and EG cells that would be difficult, if not impossible, to determine by other mechanisms.
  • nhpES provide important information on stem cell behavior that is distinct from that obtained from murine cells (Kawasaki et al., Proc Nat Acad Sci USA 99:1580-1585, 2002; Sone et al., Circulation 107:2085-2088, 2003).
  • the development of non- human primate EG cell lines could provide an important new reagent for understanding stem cell growth and differentiation in species more closely related to humans.
  • the second major problem to be addressed concerns the ability to differentiate pluripotent stem cells into stable, fully functional differentiated derivatives.
  • One major underlying safety issue to be addressed is whether transplanted cells, either stem cells or their derivatives, will behave appropriately when transplanted into host animals.
  • murine ES and EG cells introduced into embryos produced chimeras, that showed skeletal and other growth abnormalities (Dean et al., Development 125:2273- 2282, 1998; Durcova-Hills et al., Differentiation 68:220-226, 2001). Therefore an important question is whether ES and EG cells are capable of forming stable, fully-functional differentiated cells such as neurons, astrocytes, myoblasts, cardiomyocytes, etc.
  • pluripotent stem cells are provided with the appropriate temporal and spatial signals to differentiate into the full spectrum of cells present in the body.
  • ethical considerations prevent this experiment from being conducted with human ES and EG cells.
  • the production of nhpES and nhpEG chimeras could reveal potential drawbacks of primate pluripotent stem cells that would be difficult or impossible to uncover by other means.
  • Example III 2 Regard the ability of NHP ES and EG cells to differentiate in these assays.
  • ES and EG cells can form tumors when placed in the appropriate environment. Therefore, another important question is whether ES or EG cells or their derivatives transplanted into host animals will form tumors. This question is also addressed in Example III 2.
  • Example 111 1 describes experiments that are designed to develop methodology for analyzing imprinting in non-human primates, and in Example III 3 to test the effect of culture conditions on imprinting status of NHP ES and EG cells. This data will allow one to test the consequences of impaired genomic imprints on stem cell behavior, which is determined in Example III 2.
  • Example III 1 markers will be identified for non-human primate PGCs. This will be a pre-requisite for unequivocally identifying PGCs in vitro and for unequivocally following their growth and differentiation in culture. The choice of markers are based on previous studies and those of others that have identified markers of mammalian PGCs. The first part of this analysis involves histochemical and immunocytochemical staining of PGCs in vivo in the context of the developing gonad. To this end were isolated gonads from a single non-human primate fetus and sections of those gonads were cut to stain with antibodies to known mammalian PGC markers. An analysis of marker expression in the human gonad was performed, and it was found that human PGCs express both SSEA-1 and SSEA-4.
  • SSEA-1 and SSEA-4 are markers of mouse and human PGCs these data suggest that they will serve as good markers of non-human primate PGCs also and support the idea that one will be able to develop markers of PGCs in monkeys. In addition will be determined at what stage of development PGCs could be isolated from nhp fetuses, culture conditions developed for growing nhp PGCs in vitro.
  • Nonhuman primate fetuses have been isolated at different stages of development. It was determined that at day 30 post fertilization fetuses were morphologically equivalent to 10.5 days post coitus (dpc) mouse embryos. Next were isolated gonads from these fetuses, which were dissociated onto irradiated feeder layers of STO cells. After 2 days of culture the plates were fixed and stained for TNAP, the classical marker of PGCs in many mammalian embryos and fetuses. Many TNAP+ cells were detected and these cells had alt the morphological features of PGCs.
  • dpc days post coitus
  • nhp primary embryo fibroblasts that can serve as feeder cells for stem cell populations as well as providing a source of nuclei for nhp nuclear transfer.
  • the imprinting status of genes in nhp PGCs will also be analyzed using PCR based techniques.
  • Example III 2 the fate of either nhp EG cells or nhp ES cells will be followed in chimeras by making use of cells expressing green fluorescent protein (GFP) markers.
  • GFP green fluorescent protein
  • GFPNeor GFP/Neomycin resistance gene fusion protein
  • the Oct4 gene is expressed in the totipotent blastomeres of the pre-implantation embryo, the inner cell mass and in primordial germ cells.
  • a genomic clone (GOF18) contains all the regulatory elements required to recapitulate endogenous Oct4 expression and was used to drive expression of GFP. This construct was transfected into hES cells and allowed one to follow the fate of undifferentiated ES cells in culture.
  • Example III 3 will be analyzed the imprinting status of EG cell lines derived from non-human primates.
  • reagents were developed for analyzing the imprinting status of genes in nonhuman primates. It was decided to look for informative polymorphisms between both Rhesus and Cynomolgus monkeys.
  • the genes analyzed included the Peg1 and Peg3 genes, Igf2, Snrpn and H 19. These genes were chosen because previous studies in humans had shown that they represented imprinted loci, that is, they encoded genes that were differentially expressed depending on the parent of origin. Additionally, both examples were chosen of genes that were maternally imprinted and ones that were paternally imprinted.
  • Primers were designed to analyze these genes in non-human primate samples based on similar studies in humans. Genomic DNA was isolated from both Rhesus and Cynomolgus monkeys and products were amplified for Pegl, Peg3 and Igf2 but not for Snrpn or H19.
  • Example III Define the characteristics of PGC development in Non- Human Primates
  • DAZ Deleted in Azoospermia
  • DAZ Deleted in Azoospermia
  • targeted mutation of a Daz-like (Dazl) gene in the mouse revealed an essential role for the murine Dazl gene in germ cell development (see (Cooke, Reviews of Reproduction 4:5-10, 1999; Fox and Reijo Pera, Molecular and Cellular Endocrinology 184:41-49, 2001) for reviews).
  • azl Daz-like gene in the mouse revealed an essential role for the murine Dazl gene in germ cell development (see (Cooke, Reviews of Reproduction 4:5-10, 1999; Fox and Reijo Pera, Molecular and Cellular Endocrinology 184:41-49, 2001) for reviews).
  • several germline markers are widely conserved within vertebrate and non-vertebrate species.
  • the Vasa gene was originally identified in Drosophila as a gene involved in early germline development (reviewed in Raz, Genome Biol 1:REVIEWS 10
  • Vasa The murine Vasa gene, mVasa, is involved in several aspects of germline development and marks PGCs from 11.5 dpc. onwards in the mouse embryo (Toyooka et al., Mech Dev 93:139-149, 2000). Vasa is also a germline marker in the Zebrafish and Chicken suggesting that this gene function has been conserved through evolution (Knaut et al., Current Biol 12:454-466, 2002; Krovel and Olsen, Mech Dev 116:141-150, 2002; Olsen et al., Mech Dev 66:95-105, 1997; Tsunekawa et al., Development 127:2741-2750, 2000).
  • One of the goals of this project is to determine the safety and efficacy of pluripotent stem cells for transplantation in humans.
  • these safety issues will be tested in a closely-related primate species.
  • a major issue concerning stem cell safety is whether the imprinting status of imprinted genes is normal and, if not, whether that affects their ability to stably differentiate into specialized cells types.
  • Genomic imprinting involves epigenetic modification of DNA in the germline that leads to preferential expression of a specific parental allele (monoallelic expression) in the offspring.
  • An example of imprinting involves the gene for insulin-like growth factor Il (lgf-2) encoded on human chromosome 11.
  • the lgf-2 gene is active during early embryogenesis and is only expressed from the paternal allele (Vu and Hoffman, Nature 371:714-717, 1994).
  • H19 another imprinted gene also encoded on human chromosome 11 is expressed only from the maternal allele (Zhang and Tycko, Nature Genetics 1:40-44, 1992). Loss of imprinting of these genes producing biallelic expression has been observed in Wilm's tumor and cancer development, suggesting that altered imprinting could lead to developmental abnormalities. In both mice and humans, mutations that affect genomic imprinting can cause severe, sometimes lethal, conditions (Greally, Molec Biotech 11:159-173, 1999).
  • Imprinting of genes is associated with changes in DNA methylation and may also involve changes such as histone modification (reviewed in Jaenisch and Bird, 2003). Such monoallelic expression is thought to play an important role in regulating embryonic and fetal growth (Reik et al., Novartis Foundation Symposium 237:19-31, 2001). Consistent with this idea, androgenetic and gynogenetic embryos in which both alleles are derived from a single parent, show significant developmental defects (McGrath and Solter, CeW 37:179- 183, 1984; Surani and Barton.
  • mice suggest that there are differences in imprinting status between ES cells and EG cells while studies in human EG lines suggest that there are not.
  • a problem with the studies utilizing human samples is that the investigators had no access to parental DNA and were therefore unable to definitively address this question. So a major question is whether the mouse data accurately represents the situation in human pluripotent stem cells. This question could be addressed easily in nhps where it is possible to study stem cells and the parents from which they were derived.
  • a second major question is whether there are major differences in gene imprinting status between ES cells and EG cells and whether those imprints are normal. Again this question can only be addressed definitively if the imprinting status of the parental cell of origin is ascertained.
  • EG cells are derived from PGCs during the fetal period. It is during this fetal period that genomic imprints are erased and then later re-established (reviewed in Arney et al., lntl J Developmental Biol 45:533-540, 2001; Davis et al., Human Molecular Genetics 9:2885-2894, 2000; Surani, 2001). Consequently, determining whether EG cells have appropriate imprints requires examining imprinting status in PGCs. Therefore, methods will be developed for studying genomic imprints in nhps and that technology used to begin to analyze imprinting in nhp PGCs.
  • Biomarkers will be defined that can be used to unequivocally identify PGCs in vitro and to follow their fate. This approach is the same one that was employed previously to develop biomarkers and culture conditions for mouse PGCs (Donovan et al., 1986). Previously, PGCs were isolated from mouse, rat, porcine, bovine and human embryos and successfully placed into culture on feeder layers of irradiated STO cells (Donovan et al., 1986; Shamblott et al., 1998). Non-human primate PGCs should , behave in the same way as their counterparts from the other mammalian species. In order to be able to identify PGCs in culture it is first necessary to determine what markers are expressed by PGCs in vivo.
  • TNAP - tissue non-specific alkaline phosphatase a marker of murine and human PGCs (Donovan et al., 1986; Shamblott et al., 1998); mVasa - The murine homolog of the Drosophila vasa gene which is expressed in PGCs from 10.5 dpc (Toyooka et al., 2000); Oct4 - The POU domain transcription factor that is a marker of PGCs in all mammals tested (Yeom et al., Deve/opmenM22:881-894, 1996);
  • SSEA-1 Stage-specific embryonic antigen-1 - a carbohydrate differentiation antigen expressed on PGCs, EC cells, ES cells and EG cells of human and murine origin.
  • EMAI a differentiation antigen expressed on murine PGCs from 11.5 dpc.
  • CXCR4 A seven-transmembrane G-protein coupled receptor involved in PGC migration in species as diverse as Zebrafish and mice.
  • GCNA-1 - germ cell nuclear antigen-l (Enders and May, Developmental Biol 163:331-340, 1994); lntegrin Beta-1 - a cell surface receptor for extracellular matrix proteins (Anderson et al., Deve/op/ ⁇ enM 26(8): 1655-1664, 1999);
  • PG cells will enter the genital ridge (gonad anlagen) at around 32 days post fertilization. Histochemical staining for TNAP will be carried out as described previously (Donovan et al., 1986).
  • the reagents recognizing PGC antigens include rabbit polyclonal antisera and mouse monoclonal antibodies, all of which have been described previously. As controls, either pre-immune rabbit sera, isotype-matched control monoclonal antibodies or pre-incubate antibodies with excess antigen will be used.
  • Primary antibodies will be detected with rhodamine- or fluorescein-conjugate secondary antibodies and staining observed using a Nikon microscope equipped with fluorescence optics. In controls, the secondary antibody will be omitted. Histochemical and immunocytochemical staining of gonads will allow the pattern of marker expression by PGCs to be determined.
  • PGCs will be isolated into culture.
  • techniques will be employed that were developed previously for the culture of PGCs from murine and human embryos and which have also been used successfully to culture bovine and porcine PGCs (Dolci et al., Nature 352:809-811, 1991; Donovan et at, 1986; Resnick et at., 1992).
  • Gonads will be isolated from non-human primate fetuses at or before 35 days of gestation.
  • Gonads are dissociated with trypsin/EDTA, and the resulting cell suspensions either centrifuged onto glass slides or plated onto feeder layers of irradiated STO cells in 96-well plates. Cytospin preparations will be air dried and stained for TNAP to determine the percentage of PGCs in the cell suspension.
  • Cultured PGCs will be fed with complete medium (High Glucose formulation of DMEM supplemented with 15% fetal bovine serum) and LIF and bFGF. Twenty-four hours after isolation, one plate will be fixed and stained for TNAP activity using a standard protocol. TNAP positive cells will be visualized under a microscope. Cultures will also be fixed arid stained for other PGC markers using standard protocols.
  • cultures will be fixed with 2% para-formaldehyde in PBS or 1:19 Acetic Acid:Ethanol, washed three times in PBS, and incubated with antibody for 30 minutes at room temperature. Following antibody incubation, the cells are incubated with secondary antibody conjugated to either horseradish peroxidase or rhodamine isothiocyanite and observed under a microscope.
  • the degree of PGC proliferation in culture will be assessed by incubating cultures with bromodeoxyuridine (BrdU). Detection of PGCs incorporating BrdU will be carried out using anti-BrdU antibodies and TNAP staining as previously described (Dolci et al., 1991). Incorporation of BrdU will indicate that PGCs are in S-phase of the cell division cycle and will allow one to assess the suitability of the culture conditions for PGC growth.
  • Imprinting analysis will be carried out as follows: briefly, expression from either the paternal or maternal allele will be examined by RT-PCR together with restriction enzyme digestion and DNA sequence analysis. To identify instructive polymorphisms between parental alleles, genomic DNA will be amplified using PCR primers designed for a variety of genes. These genes have been shown to be imprinted in mouse and humans and include IGF2, PEG1, PEG3, SNRPN, IPW and KCNQIOTI (also called LIT 1) which represent paternally expressed genes and H 19, SLC22A18 (also known as TSSC5) and NESP55 which represent maternally expressed genes.
  • IGF2, PEG1, PEG3, SNRPN, IPW and KCNQIOTI also called LIT 1
  • SLC22A18 also known as TSSC5
  • NESP55 which represent maternally expressed genes.
  • PCR primers will be used that have been used successfully to amplify the appropriate regions of the human genes, since non-human primates share 98% DNA sequence homology with humans.
  • the list of genes provided below may however not reveal identical polymorphisms between humans and non-human primates, due to evolutionary divergence. In which case the primers will be used to amplify the appropriate region and sequence to search for new polymorphisms (see Alternative Approaches section- Examples III 1 and 3).
  • the PCR primers used to analyze DNA polymorphism will be:
  • DNA and cDNA create bands of about 310bp. Digestion with AfIIII cuts to 260bp.
  • the frequent codon is cgc which can change to cac if polymorphic. No restriction site so just have to sequence and observe the peak at the polymorphic site.
  • the surrounding sequence is: cagctcttcaatgaccGcctgtccctcgcca
  • DNA and cDNA create bands of about 310bp. Digestion with AfIIII cuts to 260bp.
  • the frequent codon is cgc which can change to cac if polymorphic. No restriction site so just have to sequence and observe the peak at the polymorphic site.
  • the surrounding sequence is: cagctcttcaatgaccGcctgtccctcgcca
  • Forward primer aaccaggctccatctactctttg (SEQ ID NO: 17)
  • Backward primer tcttgcaggatacatctcattcta (SEQ ID NO: 18)
  • DNA - about 1100bp band reduced to about IOOObp when cut.
  • cDNA - about 220bp band reduced to about 150bp when cut.
  • DNA- is a 1550 bp band.
  • cDNA- is a 868 bp band.
  • Polymorphism is C to T KCNQ1OT1- silenced on maternal chromosome .
  • DNA and cDNA create bands of about 268 bp.
  • Polymorphism is G to A -SLC22A18 DNA - silenced on paternal chromosome Onyango, P. et al. Pro ⁇ . Natl. Acad. Sci. U.S.A. 99, 10599-10604
  • gaggaggctgctccactcgctg (SEQ ID NO: 26)
  • DNA- is a 315 bp band.
  • Polymorphism is G to A .
  • cDNA- is a 231 bp band. Polymorphism is G to A
  • DNA- is a 233 bp band.
  • Polymorphism is T to C
  • DNA- is a 1141 bp band.
  • Polymorphism is T to C.
  • PGCs will be examined for parental allele- specific expression using RT-PCR as previously described. Briefly, PGCs will be isolated at different stages of nhp development by fluorescence-
  • FACS 96 activated cell sorting
  • Markers will have been developed for nhp PGCs. Some of these markers will likely be recognized by antibodies that can be used to sort PGCs by the techniques described above. Since one of the markers to be analyzed, SSEA-1, is expressed on both mouse and human PGCs it seems likely that anti-SSEA-1 antibodies could be used for cell separation of nhp PGCs as they have for mouse PGCs.
  • RNA isolated from PGCs will be converted into cDNA, and then analyzed by sequencing of RT-PCR products.
  • Preliminary Data Previously, pools of 2500 mouse PGCs have been sorted for molecular biology analysis, so the numbers of cells that need to be isolated will not likely be prohibitive (see Preliminary Data). PGCs from nhp will be isolated at different stages of development both before and after their entry into the genital ridge. It is at these times that imprinting marks are thought to be erased from PGCs in mouse embryos. Preliminary Data demonstrates the ability to accurately stage nhp embryos and fetuses and that stages have been identified at which one can isolate PGCs.
  • nhp PGCs 97 mammalian systems. Other genes could be used in the future to analyze these questions and include: IPW, KCNQ10T1, SLC22A18 and NESP55. Determination of the imprinting status of several genes in nhp PGCs could also provide the essential baseline for analysis of imprinting in non-human primate embryos and ESCs 1 including those derived from ART (IVF and ICSI).
  • STO cell feeder layers may not support the growth of non-human primate PGCs. Again, this is a. formal possibility but seems unlikely given the successful culture of human PGCs on STO cells. Nevertheless, in the course of isolating PGCs from non-human primate embryos, stromal cells lines have also been developed from the gonad region (see Preliminary Studies). Immortalized cell lines will be tested for
  • Example III Derive nhp EG cells and compare growth and developmental potential of primate ES and EG cells
  • EG cells of non-human primate origin should have many of the same properties as their murine and human counterparts (Thomson, 1998; Thomson and Marshall, Current Topics in Developmental Biol 38:133-165, 1998; Thomson and Odorico, 2000). These properties include cell cycle progression, response to sub-culturing, developmental potential and potential for tumor formation.
  • EC, ES and EG cells of murine origin have been described as being pluripotent based on their ability to give rise to a wide variety of differentiated derivatives.
  • Three widely-used assays have been used to test developmental potency; embryoid body formation in suspension culture in vitro, chimera formation following introduction into a donor blastocyst and teratoma formation in nude or histocompatible mice.
  • EG cell lines developed from primates should, like their murine counterparts, be pluripotent.
  • human EG cells have many features of pluripotent stem cells, ethical considerations preclude some of the in vivo testing of these cell lines that have been carried out with murine ES and EG cells. In fact it is illegal to introduce human ES or EG cells into human embryos. This fact effectively prevents a strict comparison of human ES and EG cells with their murine counterparts and, for all intents and purposes, also prevents one from making the conclusion that the human cells are truly pluripotent. There are no such ethical considerations surrounding the use of non-human primate EG cell lines, providing an important rationale for the development of nhp EG cells. To test developmental potency of the cells which will be developed, three distinct and commonly used assays will be used: embryoid
  • Objective 1 Develop nhp EG cells from nhp PGCs.
  • monkey EG cells will be derived from PGCs.
  • monkey PGCs can be cultured in vitro; the Preliminary Data suggests that this will be straightforward.
  • PGCs will be isolated from embryos and fetuses of non- human primates and placed into culture as described above. Monkeys will be mated following hormonal stimulation of the female (Part V). Pregnancy will be followed by sonography (Part IV) and terminated between 28 and 45 days post coitus. In this experiment, the cultures will be supplemented with both LIP and bFGF, factors which are required for the formation of EG cells in mouse and human embryos (Matsui et al., 1992; Resnick et at, 1992; Shamblott et al., 1998).
  • Cultures will also be supplemented with forskolin, a cAMP agonist, and one of the most potent PGC mitogens.
  • PGC cultures will be fed on a daily basis and observed by Hoffman modulation optics to look for the appearance of colonies of EG cells. After 5-7 days, the cultures will be trypsinized and re-plated onto 24 well plates pre-plated with irradiated feeder cells. Each well of a 96 well plate will be transferred onto a single well of a 24 well plate. The cultures will continue to be fed daily for another 5-7 days, at which time colonies of EG cells should be apparent.
  • EG cell colonies will be isolated by trypsinization and manual isolation (picking), then transferred into a solution of trypsin/EDTA for 5 minutes at 37°C. Following trypsinization, individual colonies are dissociated manually with a micropipette and diluted in medium containing fetal bovine serum to inhibit trypsin action. The single cell suspension is then plated onto preformed feeder layers of STO cells. Cultures will be fed again on a daily basis and colonies of EG cells should be visible within 3-5 days after plating. EG colonies will be expanded and frozen down. Expanded colonies of EG cells will be tested for expression of markers expressed on EG cells or murine and human origin such as: TNAP, SSEA-1 and Oct4 (see above for details) and:
  • SSEA-3 stage-specific embryonic antigen-3, a carbohydrate differentiation antigen expressed by human EC cells and nhp and human ES cells (Thomson, 1998)
  • SSEA-4 stage-specific embryonic antigen-4, a carbohydrate differentiation antigen expressed by human EC cells and nhp and human ES cells (Thomson, 1998)
  • TRA-1-60 a marker of human EC cells and nhp and human ES cells (Henderson et al., Stem Cells 20:329-337, 2002)
  • TRA-1-81 a marker of human EC cells and nhp and human ES cells (Henderson et al., 2002)
  • Staining will be carried out using standard immunocytochemical and histochemical techniques currently used in the lab (Resnick et al., 1992). Controls will include the use of pre-immune sera, isotype-matched control antibodies and omission of the secondary antibody. Telomerase activity will be measured using a commercially available TRAP assay according to the manufacturer's protocol. Human and murine ES and EG cell lines will be used as positive controls and commercially available somatic cells will be used as negative controls. To further characterize primate EG cells, karyotype analysis will also be done using standard protocols. Once nhp EG cell lines have been established, karyotype analysis will be carried out.
  • Objective 2 Compare the growth characteristics and markers of human and primate ES and EG cells. To compare the growth properties of non-human primate EG cells with the existing non-human primate ES cells or human ES and EG cells a standard analysis of population growth, cell cycle kinetics, programmed cell death and efficiency of sub-culture will be done. To compare marker expression on non-human primate EG cells, standard immunocytochemical assays will be used. All of these assays will be carried out using many standard techniques.
  • EG cells will be grown in feeder-dependent culture and cell number will be determined by counting dissociated cells in a hemacytometer. Because the feeder cells are irradiated and are quiescent, use may also be made of fluorometric assays for determining cell number. In addition, plating efficiency will be determined using similar techniques. Cell number will be determined in cultures before and after plating and the efficiency of cell plating determined.
  • Flow cytometric analysis of propidium iodide-labeled cells will be used to analyze cell cycle kinetics. Briefly, EG and ES will be separated from feeder cells by trypsinization at room temperature followed by unit gravity sedimentation. ES and EG cell colonies will be harvested and dissociated into single cells by trypsinization. Single cell suspensions will be fixed in ethanol, labeled with propidium iodide (Pl), and analyzed by flow cytometry using standard techniques in use in the lab (Lincoln et al., Nature Genetics 30:446-449, 2002).
  • Pl propidium iodide
  • cells will be pulse labeled with BrdU, fix cells as above, and then stained with fluorescein-conjugated anti-BrdU antibody.
  • BrdU fluorescein-conjugated anti-BrdU antibody.
  • BrdU fluorescein-conjugated anti-BrdU antibody.
  • BrdU fluorescein-conjugated anti-BrdU antibody.
  • Pl-labeled cells By comparing the BrdU- labeled and Pl-labeled cells over time one can determine the rate at which BrdU-labeled cells transit through the cell cycle.
  • To determine the rate of apoptosis one can examine the population of sub-G1 cells in the Pl-stained population or carry out specific staining for markers of apoptosis such as Annexin V. Such assays are currently in use (Lincoln et al., 2002).
  • immunocytochemistry will be done using antibody reagents recognizing stage-specific embryonic antigens (SSEAs) and other lineage-specific antigens. These include the Trafalgar (TRA) antigens (Draper et al., J Anatomy 200:249-258, 2002). Immunocytochemistry protocols will follow those routinely in use. Briefly, cultures will be fixed with 2% paraformaldehyde in PBS and then incubated with primary antibodies to SSEAs or other antibody reagents. Following incubation with the primary
  • EG cells To assay the developmental potential of EG cells, they will be differentiated into embryoid bodies and those structures then allowed to differentiate further. Embryoid bodies will be generated from EG cell lines using well- established techniques (Shamblott et al., Proc Nat Acad Sci USA 98:113- 118, 2001). Briefly, EG cell lines will be cultured on feeder layers and then passaged onto tissue culture dishes without feeder cells. After 3 days of feeder— independent culture the EG cells will be dislodged from the tissue culture dish and plated into non-tissue culture dishes. These cultures will be allowed to grow for several days in suspension until they form so-called cystic embryoid bodies in which the balls of cells begin to develop cavities.
  • embryoid bodies will be harvested after 2 days of suspension culture and be plated back onto tissue culture dishes and allowed to differentiate further. By plating embryoid bodies onto gelatin-coated dishes one will enrich for endoderm derivatives.
  • histological, molecular and immunocytochemical analyses will be done using standard techniques. In brief, the presence of differentiated cells will be examined initially simply by careful microscopic examination of growing cultures. Differentiated cells should have, in many cases, distinct morphologies from the un-differentiated stem cells. Next, cultures will be fixed with 2% paraformaldehyde and stained with antibodies to lineage specific antigens. For example, antibodies
  • intermediate filament proteins can be used to distinguish between cells of neuronal, glial and muscle origin. Antibodies to the intermediate filament protein neurofilament will specifically stain neurons, whereas antibodies to glial fibrillary acidic protein or desmin will stain glial cells and muscle cells respectively. Similarly, differentiation of EG cells into differentiated derivatives will be examined using RT-PCR analysis as described by Shamblott et al. (Shamblott et al., 2001). For example, differentiation into endoderm will be assayed by looking for expression of classical endoderm markers such as alpha fetoprotein, GATA4, and hepatic nuclear factor 3 ⁇ .
  • classical endoderm markers such as alpha fetoprotein, GATA4, and hepatic nuclear factor 3 ⁇ .
  • Production of muscle cells will be assayed by looking for expression of muscle-specific genes such as MyoD, Myf5 and Myf6 (Shamblott et al., 2001).
  • Neuronal cell lineages, indicative of ectoderm differentiation will be identified by looking for expression of neuronal markers such as nestin, neurofilament and Tau protein (Shamblott et al., 2001).
  • Cardiomyocytes will be identified using RT-PCR and antibodies specific for recognition of cardiac differentiation such as cardiac muscle troponin I, desmin, atrial natriuretic peptide, anti-sarcomeric ⁇ actinin and anti-nebulin. These analyses will allow the spectrum of the developmental potential of nhpEGCs to be determined. Noncontracting EBs or other cell types will be used as controls. The ability of primate EG cells to differentiate into different derivatives will be directly compared in side by side experiments with the ability of nhpES cells to form differentiated derivatives.
  • nhpEG cell lines will be introduced into pre- implantation rhesus embryos as described in Part 1. Briefly, 4- to 8-cell embryos will be derived from hormone-stimulated rhesus macaques and collected by laparoscopy (as described in Core B). Approximately 10-15 EG cells will be introduced into each embryo as described in Part 1 and the embryos cultured briefly prior to embryo transfer into staged recipients (Core B): Females will be monitored until day 30 at which time the fetuses will be harvested for analyses (Core B).
  • nhpESCs and nhpEGCs will be transfected with GFP-expressing constructs. This will allow noninvasive imaging of developing chimera embryos in vitro, as well as determination of EG and ES in chimeric pregnancies after amniocentesis. Such cells can be imaged in developing chimeras by high definition ultrasound (U/S) and MRI. This will allow one to follow the fate of groups of nhpEG cells in chimeric embryos and fetuses.
  • U/S high definition ultrasound
  • Rhesus 8-cell embryos are prepared as above and a small clump of labeled or transfected nhpESCs or nhpEGCs are introduced into the well. After all reaggregations are completed, the plate is gently rotated to bring the fertilized embryos in close contact with the stem cells before returning to culture. All plates are checked the following day for chimera formation and embryonic development. Embryo transfer of nhpESCs + rhesus $c?chimeras is described in the Primate Animal Core.
  • Another approach for marking cells and following their fate in chimeras is possible.
  • EF1 ⁇ Elongation Factor 1 ⁇
  • That construct will be modified to express GFP- and RFP-tagged proteins.
  • a reporter gene will be used a GFP-tagged deletion fragment of the Cyclin B1 gene, termed GFP- ⁇ Cyclin B1.
  • This construct accurately reports native Cyclin B1 localization and, importantly, unlike the full-length Cyclin B1, it's overexpression does not interfere with cell cycle progression. Because of cell cycle-dependent changes in Cyclin B1 localization, this construct can also be used to accurately monitor cell cycle progression in living stem cells. Therefore, one should be able to not only follow stem cells in chimeras but also ascertain whether they are dividing. Two methods of chimera formation
  • biopsied embryos (obtained in Part V) will be microinjected with 8-10 GFP-labeled nhpESCs and nhpEGCs into the space vacated by blastomere withdrawal and the chimera's returned to BRL co- culture for embryonic development.
  • a second method will employ re- aggregation of fertilized embryos with small colonies (10-15 cells) of nhpESCs and nhpEGCs carrying GFP transgene reporters (Part IV), and then counterstained with 5 ⁇ M propidium iodide (Molecular Probes, Eugene, OR) to label the DNA.
  • Fixed samples will be optically sectioned using a Leica TCS-SP2 laser scanning confocal microscopy as described in Imaging Part IV.
  • cultured blastocysts will be transferred to staged recipient females. Females will be monitored until day 30 at which time the fetuses will be harvested for analysis or the animals allowed to proceed to term (Part V). Analysis of chimera formation will also be carried out using genetic markers such as isoenzymes and mitochondrial DNA (mtDNA) polymorphisms (Dyke et al., Progress in Clinical and Biol Research 344:363-574, 1990; Hewitson et al., 2002; Holmes et al., Alcoholism: Clinical and Exptl Research 10:623- 630, 1986; Khan, Genetica 73:25-36, 1987).
  • mtDNA mitochondrial DNA
  • determining mtDNA polymorphisms in the Rhesus Colony will be accomplished by analyzing the D-loop of the rhesus monkey mtDNA genome through nested PCR and automated sequencing (Core B). Specifically, the hypervariable regions of the D-loop of the mitochondrial genome will be amplified from blood and or tissue samples. These products will then be sequenced using direct sequencing methods for mtDNA according to Hopgood et al. (Hopgood et al., Biotechniques 13:82-92, 1992). Unique polymorphisms will allow one to design specific primers for PCR amplification, a technique known as Allele Specific-PCR (AS-PCR).
  • AS-PCR Allele Specific-PCR
  • nhpESCs and nhpEGCs are expected to contribute to all three tissue lineages (endoderm, ectoderm and
  • 106 mesoderm as well as the germ cells. Contribution to the germline can be monitored in the fetus by examining the fetal gonads and looking for donor- derived germ cells in the gonad. Isolation and culture of germ cells from fetuses could also be used to determine whether TNAP+ PGCs also express the GFP-tagged Cyclin B1 protein. All chimeric concept! will be monitored for defects in cranial, somite and limb development after ET by high definition ultrasound and MRI analysis. GFP transgene infected embryos will be examined by a brief exposure to attenuated epifluorescent illumination to confirm the extent of mosaic expression using appropriate fluorescein filters.
  • nhpES and nhpEG cells Contribution of nhpES and nhpEG cells to the germline of animals after birth could be determined by biopsy of one of the gonads or, at a later stage, by mating. It is also expected that male stem cell lines introduced into female blastocysts will convert the embryo into a phenotypic and functional male as occurs in male/female murine chimeras. In this is the case, all of the offspring wilt be donor derived.
  • Objective 5 Determine the ability of primate ES and EG cells to form teratomas in nude mice.
  • EG cell lines will be injected intraperitoneally or intramuscularly into nude or SCID mice again using standard protocols (Thomson, 1998 #2). Briefly, 10x106 cells will be injected intramuscularly or intraperitoneally into nude or SCID mice and allowed to grow for approximately 60 days by which time palpable tumors should be present. Animals will be sacrificed and tumors removed for histology. Histological analysis will be carried out using standard techniques such as hematoxylin and eosin staining.
  • nhp ES and EG cell lines will prove to be an excellent reagent for genome-wide analyses of gene expression by pluripotent stem cells.
  • chimeras formed between rhesus nhpEGCs or nhpESCs and rhesus QS + QS embryos to form normal blastocysts with proper ICM and trophectoderm lineages in vitro. It is anticipated that rhesus ES and EG cells will contribute exclusively to the ICM when reaggregated by either blastocyst injection or zona-free co-culture.
  • Pregnancy in rhesus will be monitored by high definition ultrasound or MRI analysis. Amniocentesis and CVS will be collected.
  • paired control and chimeric concepti will be compared for defects in cranial, smite and limb development after embryo transfer or xenotransplantation of chimera into mice.
  • the ability of chimera to form all three germ layers (ectoderm, endoderm and mesoderm) will be examined, noting deficiencies in their morphology and proper development. It is expected that xenotransplantation into SCID mice will prove to be a more useful tool in determining tissue development extent in nhpES and nhpEG cells QS because early concepti recovery is possible. Effect of disaggregation and reaggregation on the viability of newly created chimeras will be compared with the viability of non-manipulated controls. Chimeras will be placed in culture, their development monitored to the
  • blastocyst stage 108 blastocyst stage, and their normalcy confirmed by cytogenetic analysis prior to initiation of embryo transfer into recipient females, SCID mice or isolation of embryonic stem cells. Embryo development will be evaluated by total cell proliferation, intercellular interactions, compaction, cavitation and blastocyst formation. It has already been demonstrated that embryo re-aggregation can result in viable rhesus offspring, as shown by the birth of Tetra (see Part 1). One expects that chimeras created from transgenic blastomeres will retain gene expression throughout preimplantation development.
  • Example III Analyze the role of imprinting status in primate ES and EG cell lines on the stability of differentiation and tumor formation
  • genomic imprints are erased and then later re-established (reviewed in Arney et al., 2001; Davis et al., 2000; Surani, 2001).
  • Some studies examining genomic imprinting in murine EG cells found that the cells had abnormal genomic imprints and biallelic gene expression, perhaps reflecting their derivation during the process of imprint erasure (Tada et al., Devt Genes and Evol 207:551-561, 1998). Consistent with these observations, some chimeras made from EG cells showed
  • non-human primate stem cells Because such stem cells can be derived from matings of identified parents, embryos (and subsequently stem cells) can be derived which carry easily-followed imprinting marks. EG cell lines derived from informative coatings can be differentiated and their differentiated progeny transplanted into suitable hosts. In this way, it will be possible to test whether pluripotent stem cells, and cells derived from them, obtained from non-human primates will behave correctly in transplant situations and whether differential genomic imprinting will have a significant impact on transplantation success.
  • Example III 1 techniques will have been developed for imprinting analysis in non-human primates.
  • the imprinting status of genes will be analyzed and compared in early embryos and derived ES cells and in PGCs and derived EG cells.
  • the data on imprinting status of genes in the early period of development will be carried out in Part 1. Briefly, expression from either the paternal or maternal allele will be examined by RT-PCR together with restriction enzyme digestion and DNA sequence analysis. To identify instructive polymorphisms between parental alleles, genomic DNA will be amplified using PCR primers designed for a variety of genes.
  • PCR primers will be used that have been used successfully to amplify the appropriate regions of the human genes since non-human primates share extensive DNA sequence homology with humans.
  • the PCR primers used to analyze DNA polymorphisms are described above (see Example III 1).
  • RNA isolated from cell lines will be converted into cDNA, and then analyzed by sequencing of RT-PCR products. This analysis will be carried out on newly-derived EG cell lines and on existing non-human primate ES cell lines for which parental information is known.
  • nhp ES and EG cells will be grown under different conditions and the effect of these different conditions on the expression of imprinted genes will be tested.
  • hES and hEG cells are grown on feeder layers
  • nhpES cells 112 or mitotically-inactive cells.
  • the nhpES cells are grown in these conditions also.
  • Some studies report the growth of hES cells on MatrigelTM, an extracellular matrix extract of sarcoma cells. Therefore, one of the conditions tested will be whether growth on MatrigelTM affects the imprinting status of nhpEG and nhpES cells.
  • Another method commonly used to culture hES cells is to grow cells in the presence of serum-replacer rather than serum. Therefore, the effect of serum replacer on imprinting stability will be tested. Briefly, nhpES and nhpEG cells will be plated onto MEFs in serum or serum replacer or onto MatrigelTM and cultured for at least two " passages.
  • hESCs could be maintained in a pluripotent state for repeated passages in the chemically-defined medium of Johansson and Wiles (MoI Cell Biol 15(1):141-151, 1995) in the presence of recombinant (R and D systems) or transgenically expressed Nodal; this condition thus represents the minimal culture circumstances known to be compatible with maintenance of pluripotency, thus establishing a basal culture condition in which maintenance of ESC imprinting might be perturbed.
  • Cells will be harvested and RNA isolated using standard methods in use in the lab. The cDNA will be constructed also using standard methods.
  • Amplification of regions containing informative polymorphisms will be carried out using the methods described above (see Example III 1) and the PCR products sequenced.
  • the sequence of the amplified fragments will be compared with that of the genomic sequences derived from the DNA of the parental samples to determine whether there is mono- or bi-allelic expression from that locus. This process will be repeated for as many genes as informative polymorphisms can be found.
  • experiments will also be carried out in which cells are treated with Azacytidine or Trichostatin, agents which would be expected to affect the expression of imprinted genes and which can cause reactivation of silenced alleles.
  • Example III 2 the differentiation potential and tumor potential of nhp ES and EG cell lines will have been determined. In this specific aim will be described.
  • the imprinting status of a number of imprinted genes and the two sets of data correlated will be determined and compared with the degree of differentiation observed in the assays of developmental potency.
  • the imprinting status of the battery of paternally- and maternally-imprinted genes in nhpES and nhpEG cell lines will be determined and compared with the degree of differentiation observed in the assays of developmental potency.
  • For tumor assays will be determined the size of tumors, timing of tumor development from the time of inoculation, degree of differentiation into different lineages and percent of undifferentiated stem cells in tumors. These parameters will be determined using lineage specific markers as described in Example MI 2.
  • For in vitro assays will be carried out similar analyses of the ability of nhpES and nhpEG cells to differentiate into different cell lineages.
  • Another potential concern is the risk of not finding informative polymorphisms in the imprinted genes selected for analysis. However, it seems highly likely that there will be polymorphisms to identify each of the candidate imprinted genes, because there are over 200 animals within the PDC primate colony that can be screened in this way. Another approach will be to develop stem cell lines
  • Part III are designed to address fundamental questions concerning the safety and utility of pluripotent stem cells such as ES and EG cells.
  • the development of pluripotent stem cell populations from primate species closely related to humans should accelerate efforts to develop cell- based therapy for the treatment of human disease.
  • data will be generated concerning the development of the germline in primates. This data could significantly improve the understanding of gametogenesis in primates, including humans, and how that process can go wrong.
  • Parts I, Il and III above will all use the 4.7 T, and 11.7 T micro-imaging system and 4.7 T MRI system, and monkey and mouse model systems.
  • the tissues of interest will be teratomas, kidneys, testes, subcutaneous
  • the MRI methods used will be 3D anatomical, Gd-contrast enhancement, MR microscopy and SPIO-labeled stem cells.
  • Mouse Preparations will begin after the mice have been transferred to the NMR Center at least 24 hours prior to imaging. Anesthesia is needed to immobilize the animal during the MRI scan and to alleviate stress to the animal.
  • the mouse will be anesthetized with isoflurane in air, intubated, and then connected to a mechanical ventilator (150 strokes/minute, 250 ⁇ l/stroke) delivering 1.25 % isoflurane in an oxygen/nitrous oxide mixture (70 %: 30 %).
  • the mouse will be positioned in the bore of the 11.7 T microimaging system. Mouse physiology, including EKG 1 and pulse oximetry will be monitored in situ throughout the experiments. The animal's temperature will be maintained at 37°C using a temperature-regulated collet surrounding the mouse. An imaging session will last a maximum of 3 hours. From previous experience, anesthetized animals can be routinely maintained in the vertical bore 11.7 T instrument for up to 8 hours and then safely recover the animal.
  • Heart rate, body temperature, and blood oxygenation will be monitored continuously to insure an adequate level of anesthesia; these measurements will be performed in situ in the magnet bore during imaging.
  • a typical imaging session to last a maximum of two hours.
  • the laboratories containing the monkeys will be quarantined and restricted area signs will be posted. The only personnel allowed in the magnet area during
  • the study will be the outside personnel from PDC accompanying the primate, the NMR Center's licensed veterinarian technician (LVT) and the MRl operator. All personnel will be required to wear gowns, mask, face shield, gloves, and shoe covers.
  • the monkey After imaging, the monkey will be recovered from the anesthesia and then transported back to the Development Center while under sedation. After the completion of each imaging session, the LVT cleans the magnet bore and other surfaces that have been in contact with the primate with Quatricide/TB. A bite and scratch kit will accompany the primate for each study. Many additional details concerning the preparation, handling, and care of the primates during these procedures are outlined in the Part V.
  • Mouse - Mouse data will be acquired in the 11.7 T micro-imaging system. Either a 25 mm birdcage RF coil or a laboratory-built surface coil will be used for imaging localized regions of the mouse at high sensitivity. Motional image artifacts wili be suppressed by triggering all data acquisitions at the same place in the respiratory cycle. Multiple, contiguous, two-dimensional (2D) slices (12) will be obtained along all three axes (axial, coronal, and sagittal) through the abdomen or ES cell implantation site.
  • 2D two-dimensional
  • a T1 or T2-weighted spin-echo pulse sequence with 256x256 or 256x128 image points will be used, and a resolution of approximately 80 micrometers and a 1-0.3 mm slice thickness.
  • the acquisition time for a package of slices will be approximately 20 minutes.
  • a rapid three-dimensional (3D) T2-weighted RARE (Rapid-Acquired-Relaxation- Enhanced) imaging sequence will be used.
  • the RARE sequence works well at 11.7 T and will be used to acquire a 3D datasets in about an hour with 256x128x128 image points.
  • mice/year will be imaged for using MRI. It will take approximately 1.5 hours/mouse to acquire the data, and approximately 2-4 mice will be imaged per day. The number of mice and scan times will be adjusted as needed.
  • Monkey - The monkey data will be acquired in the 4.7 T MRI system. This magnet has a bore size of just over 20 cm with the gradient and a volume RF coil in place. From past experience, in this system primates that are approximately ⁇ 8.5 kg can be imaged. Several different RF coils will be used for these studies, including a birdcage volume coil, a laboratory-built hemi-cylindrical birdcage RF coil, and assorted surface coils.
  • RF coil technology will be refined to optimally-image the tissues of interest.
  • Multiple, contiguous, two- dimensional (2D) slices (12-24) will be obtained along all three axes (axial, coronal, and sagittal) through the abdomen or ES cell implantation site.
  • 2D imaging a T1, T2, or T2*-weighted or diffusion-weighted spin-echo, gradient-echo, RARE, or EPI (Echo-Planar Imaging) pulse sequences will be used with a resolution of approximately 100-300 micrometers in plane, and a 2 mm slice thickness.
  • mice and monkeys with large teratomas ⁇ 1 cm
  • high- resolution 3D volumetric images of these cell masses will be acquired after they have been excised and fixed. This will allow the digitally 'sectioning' the
  • Data from all MR imaging sessions will be loaded into the software program Amira (TGS Inc., San Diego, CA). Using this software, the volumes will be edited to display the regions of interest. Other parameters will be adjusted as needed such as image intensity threshold, contrast, and image opacity in the case of volume rendered data.
  • image intensity threshold contrast
  • image opacity image opacity in the case of volume rendered data.
  • images of teratomas both in vivo and excised
  • the outer boundaries of the teratomas will be digitally segmented to examine the 3D structure. Teratoma boundaries will be traced using a combination of coronal, horizontal, and sagittal orientations. Starting from a user-defined seed point, boundaries are delineated using an AMIRA algorithm that selects contiguous voxels falling below a user-defined threshold intensity.
  • the software will detect contiguous voxels falling within a user-specified range of intensities and seed point.
  • the automated segmentation is then checked by eye slice-by-slice, and minor corrections can be applied using a stylus on a digital tablet (Wacom Technology Inc., Vancouver, WA).
  • AMIRA the pixel-to-pixel borders will then be smoothed in 3D to obtain the most accurate representation of
  • a component of the imaging experiments will be to refine methods for labeling stem cells using MRI contrast agents for in vivo MRI.
  • the undifferentiated cell types that will be used are mES cell 129/Sev (mouse), mES cell w95 (mouse), nhpES cell r366 (nonhuman primates), nhpES cell PDC-1 (nonhuman primate), hES cell H-1 (human) and hES cell HSF-6 (human).
  • cationic lipids e.g. LipofectamineTM
  • the SPIO agents will be obtained from commercial sources (Miltenyi Biotec Inc., Auburn, CA). These agents consist of an iron-oxide crystal (-10 nm diameter) coated with polysaccharide resulting in a ⁇ 50 nm diameter particle.
  • Lipofectamine will be premixed with the SPIO agents for ⁇ 20 minutes before adding to the culture media. 6 ⁇ l/ml of Lipofectamine provides effective coverage of the SPIO particles.
  • Table IV displays a tentative list of the cell surface carbohydrates expressed on stem cells that will be targeted with mAb.
  • SPIO agents that are conjugated to streptavidin (Miltenyi Biotec Inc., Auburn, CA).
  • Biotinylated primary mAbs against the molecular targets molecules (Table 4) will be obtained commercially (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Pre-saturation of btotin on cells will minimize non-specific labeling.
  • cells will be harvested and labeled while in suspension.
  • the incubation time and the SPlO particle concentration in the culture media will be systematically varied.
  • a population of cells ( ⁇ 10 7 cells) will be subdivided into multiple sample populations (6-10).
  • each sample population will be incubated with various concentrations of SPIO agent (in equally-spaced concentration increments) at a fixed incubation time ( ⁇ 3 hours).
  • each sample population will be incubated for a different time period, ranging from
  • each sample population will be washed 2-times in culture medium to remove excess agents.
  • the total number of cells will be re-counted using a hemocytometer in each sample population.
  • the total number of dead cells will be estimated using the Trypan Blue exclusion assay.
  • the effective intracellular concentration of SPIO particles will be estimated in the remaining living cells in each sample population.
  • the SPIO particles will be uniformly suspended in a known volume ( ⁇ 50 ⁇ l) of agarose at the bottom of a small glass capillary tube.
  • the longitudinal relaxation time (T2) of the capillary tube contents will be measured using a 20 MHz relaxometer (Bruker Instruments Inc., Billerica, MA).
  • a control measurement will be made of cells in agarose that have not been incubated From the measured T 2 -values one can estimate the effective SPIO concentration per cell.
  • the T 2 relaxation time is related to agent concentration according to the relation
  • 1/T 2 1/T 2 ' + R 2 [M] (1)
  • T 2 ' is the measured relaxation time in the non-incubated cell sample
  • R 2 is the relaxivity of the SPIO particles
  • [M] is the net SPIO concentration of the cells in the agarose.
  • the constant R 2 is a measure of the agent's effectiveness at relaxing spins; R 2 will be measured for our SPIO particles in solution in a separate experiment. From Eq. 1 , [M] will be calculated, which is the only (unknown) quantity that will vary with incubation conditions. Dividing [M] by the total number of cells in each of the agarose gel samples will yield the effective SPIO concentration per cell or the cellular uptake of SPIO. Using this information several plots will be generated summarizing the results:
  • the quantity "labeling efficiency” is defined as the effective concentration per cell divided by the survival fraction (SF) of the cells.
  • the SF is defined as
  • SF (No-N d ead)/No (2) where N 0 is the total number of cells prior to incubation, and N is the number of dead cells after incubation (determined by the Trypan Blue exclusion assay).
  • N 0 is the total number of cells prior to incubation
  • N is the number of dead cells after incubation (determined by the Trypan Blue exclusion assay).
  • the plot of labeling efficiency versus incubation time will exhibit a maximum.
  • the incubation time at this maximum, or the "optimum" incubation time will be used for all subsequent labeling experiments for that specific cell type.
  • the comparable plot for the added SPIO concentration exhibits a maximum in the labeling efficiency
  • the appropriate optimum concentration will be used for subsequent experiments.
  • the other plots describing the effective cellular uptake will be used for our modeling studies.
  • ES cells will be probed for the positive ES cell markers: Oct-4, SSEA-3 and 4, Tra 1-80 and Tra 1-61, as well as the negative ES cell marker: SSEA-1.
  • Immunocytochemistry will be performed in one or more of the following manners. 1.) Culture dishes containing undifferentiated colonies will be fixed by addition of either 100% methanol -20 0 C for 15 min followed by a 15 min wash in PBS + 1 % Triton X- 100 (PBS-Tx, Sigma, St.
  • EM will be performed on labeled cells.
  • Cells will be washed, pelleted, and fixed in PBS containing 2% glutaraldehyde at room temperature for 30 minutes and held overnight at 4°C.
  • the cells will then be treated with 1 % OsO 4 in PBS for 10 minutes. All of the samples will be washed three times in H 2 O and dehydrated m an ascending series of ethanol.
  • Propylene oxide (PO) will be used as a transitional solvent.
  • the cells will be infiltrated overnight in a solution containing a 1: 1 mixture of PO and Epon-Araldite (EA).
  • the sample will be placed in plastic capsules containing EA and polymerized at 60 0 C for 48 hours.
  • Thin (0.1 ⁇ m) sections will be cut using a microtome and were placed on 200 mesh Cu grids.
  • the samples were stained with 1% aqueous uranyl acetate and Reynolds lead citrate. Sections will be imaged using a transmission electron microscope. SPIO particles typically appear as a large number of punctuate dark spots within the cell.
  • MRI Sensitivity of Labeled Cells A crucial issue when devising in vivo cellular MRI experiments is sensitivity limitations. Although MRI is a powerful tool for non-invasively mapping biological structures, it has a ubiquitous limitation in the available signal-to-noise ratio. A consequence of this limitation is that the ability to discriminate between tissues on the basis of differences in T 2 can be limited. Thus, when exogenous contrast agents are used to enhance T 2 differences between labeled and unlabeled cells, it is
  • a simple mathematical model will be constructed for evaluating the minimal intra-cellular agent concentration and cell concentration per voxel required to produce "satisfactory" image contrast.
  • the parameters describing MRI intensity and contrast are well characterized analytically.
  • the case of intracellular T 2 agents will be analyzed.
  • the results of this T 2 contrast model will describe the minimal intracellular agent concentration and cell density required for satisfactory contrast enhancement (e.g. contrast-to-noise ratio > 5).
  • the model will take the form of a general set of equations that express this minimal concentration in terms of a small number of parameters that can realistically be estimated for a given experimental subject.
  • Example IV Light and Electron Microscopy Techniques. lmmunocytochemistry will be preformed in one or more of the following 5 manners: i.) Culture dishes containing undifferentiated colonies will be fixed by addition of either 100% methanol -20" C for 15 min followed by a 15 min wash in PBS + 1 % Triton X-100 (PBS-Tx 1 Sigma, St. Louis MO) for 15 min, or it) 2% paraformaldehyde in PBS for 40 minutes followed by quenching in PBS + 150 mM glycine for 30 minutes subsequently washed out with PBS-
  • Live Cell Brightfield Video Microscopy Cells are plated on a glass coverslip coated with either 0.1% gelatin or 1 ng/ml laminin and polyornithine. Coverslips are mounted in a closed perfusion chamber (Warner Instruments,
  • Metamorph Universal Imaging
  • Live cells were imaged by real-time- spinning disk confocal microscopy (Perkin Elmer Ultraview LCI equipped with a Krypton-Argon ion laser) to minimize photobleaching and to discriminate nuclei movement in multilayered ES cell colonies.
  • GFP-H2B transfected cell colonies in Matek glass bottomed 35 mm dishes were mounted in a chamber (Warner Instruments) perfused with humidified CO2.
  • the Nikon TE2000E inverted microscope was enclosed in a LIS Systems temperature control chamber to eliminate thermal fluctuations and maintain focus for long-term experiments.
  • Cells were imaged with 2Ox NA 0.45 plan fluor objectives, or 4Ox and 6Ox planapo 1.4 NA objectives.
  • Image stacks are further processed with Volocity (Improvision) to produce 3-D reconstructions, multispectral movies of XY, XZ, and YZ cross-sections: Live GFP-H2B labeled stem cells have been observed for > 11 days continuously. More than 20,000 images (60GB) in 4 dimensions can be acquired with no harmful effect on cell movement or mitotic progression.
  • GFP-H2B transfected cells were pattern photobleached with 488 argon ion laser light using a Leica TCS SP2 scanning laser confocal microscope using the 63x 1.4NA planapo lens with the aperture set to 3 urn (2x airy disk). Cells were imaged with low intensity light (4-8% of bleaching intensity) for 10 images, photobleached during 30-60 frames, for 40-9Os and then imaged for and additional 20-100 frames at low intensity. Intensity profiles were calculated from submaximal rectangular areas selected to avoid edge effects and movement artifacts. Fluorescence photobleaching will be used to provide fiducial marks for studies of chromatin dynamics and subunit exchange. Photobleaching artifacts are possible due to stray light and photodamage. Photobleaching artifacts were evaluated by
  • First beached zones provide a histone marker to aid in studies of speed and direction of motion by conventional methods (using Metamorph kymographs and related analysis, Molecular Devices Corp., Sunnyvale, CA).
  • the GFP-H2B FLIP and FRAP experiments are expected to agree on the presence or absence of a diffusible histone component. Quantitative evaluation of these experiments will include the dependence on Oct-4 expression, detected by immunocytochemistry after completion of the experiment. FLIP may be an artifact of overexpression and so the dependence on total GFP expression in the cells will be evaluated. GFP- H2B and GFP-H3 will be extracted with 0.5% Tx-100 and analyzed for %
  • Dynamic imaging of cell tracer-labeled chimeric embryosjzy a spinning microlens array confocal microscopy will be performed as follows: Successful reaggregations will be transferred to glass bottom 35 mm dishes (MatTek, Ashland, MA), mounted on a warming plate (Warner Inst, Holliston, MA), and the CO 2 regulated with a home built acrylic chamber and humidifier. Oil immersion objectives are warmed with a temperature controller (Bioptechs, Butler, PA): An automated Nikon 2000E inverted microscope is integrated with the Perkins Elmer Ultraview LCI spinning disk confocal microscope to record time-lapse image stacks at 488, 514 and 647 nm excitation.
  • Image stacks are further processed with Volocity (Improvision) to produce 3-D reconstructions and multispectral movies of XY, XZ, and YZ cross-sections.
  • Chimera constructs will be recorded beginning 24 hr after reaggregation and continue each 24 hr until development ceases or attains expanded blastocyst.
  • chimeric embryos will be fixed in 2% formaldehyde overnight, which preserves the GFP staining profile, and then counterstained with 1 ⁇ M Toto-3 (Molecular Probes, Eugene, OR) to label the DNA.
  • a mouse GFP antibody (1:50; Molecular Probes) may also be employed to immunostain fixed embryos to confirm GFP protein expression in selected embryos.
  • Alexa-568 antimouse IgG secondary antibody is applied to detect GFP blastomeres in these fixed samples. Trophectoderm and inner cell mass cells will be stained according to published protocols. All static samples will be optically sectioned using a Leica TCS-SP2 laser scanning confocal microscope.
  • Quantitative image analysis Confocal image stacks were acquired with the Leica TCS SP2 or the Perkin Elmer Ultraview LCI confocal microscope under standardized conditions. Pseudocolor representation of time sequences can be prepared in Photoshop. Quantitative image analysis will be performed with a combination of Volocity, a 3D software for voxel quantification (size, intensity and distribution) and 3-D display. More sophisticated image quantitation of shape parameters is provided by Metamorph. Image slices were selected, background subtracted and further processed with Metamorph (Universal Imaging). Nuclear masks were prepared from DNA images derived from Toto-3 staining.
  • a 1-bit mask was prepared by thresholding the nuclear image, which was then used to select nuclei from the oct-4 and lamin images by image multiplication and normalization.
  • Each nuclear object in the field was identified and analyzed by the Image Morphology Tool to tabulate the size, circularity, and intensity of nuclei. Individual nuclei and their associated data were sorted manually for quality and degree of differentiation. Statistical differences will be determined with Student T tests.
  • Negative stain electron microscopy Pellets of pluripotent HSF-6 cells are fixed in 2.5% Glutaraldehyde overnight. Cells are then given three 15 minute washes in PBS, and then incubated in 1% OsO 4 (Osmium) with Potassium Ferricyanide for an hour at 4°C. After that the sample is once again given three 15 minute washes in PBS, and then dehydrated in 30% EtOH (15 min), then 50% EtOH (15 min), then 70% EtOH (15 min), then 90 %EtOH (15 min), then three times in 100% EtOH (15 min each). The sample is then given two
  • Determining mtDNA polymorphisms in rhesus colony is accomplished by analyzing the D-loop of the rhesus monkey mtDNA genome through nested
  • the hypervariable regions of the D- loop of the mitochondrial genome will be analyzed from blood and/or tissue
  • Follicle stimulation regimen Ovarian stimulation of female rhesus monkeys exhibiting regular menstrual cycles is induced with exogenous gonadotrophins (VandeVoort, 1989, J In Vitro Fertil Embryo Transfer 6:85- 91; Zelinski-Wooten, 1995, Hum Reprod 51:433-440). Beginning at menses, females are down-regulated by daily subcutaneous injections of a GnRH antagonist (Antide; Ares Serono, Aubonne, Switzerland; 0.5 mg/kg body weight) for 6 days during which recombinant human FSH (r-hFSH; Ares Serono or Organon Inc., West Orange, NJ; 30 IU, i.m.) is administered twice daily.
  • GnRH antagonist Antide; Ares Serono, Aubonne, Switzerland; 0.5 mg/kg body weight
  • r-hFSH Ares Serono or Organon Inc., West Orange, NJ; 30 IU, i.m.
  • r-hFSH + r-hLH r-hLH
  • Ares Serono 30 IU each, i.m., twice daily
  • Ultrasonography is performed on day 7 of the stimulation to confirm adequate follicular response.
  • an i.m. Injection of 1000 IU r-hCG (Serono, Randolph, MA) is administered for the induction of ovulation.
  • Follicular aspiration by laparoscopy Follicular aspiration is performed approximately 27 h post-hCG via laparoscopy.
  • Stimulated females are anesthetized with an intramuscular dose of ketamine (10 mg/kg), intubated with a cuffed endotracheal tube, fitted with a 22 gauge angiocath in the radial or saphenous vein, sterilely prepped for surgery, and maintained on isoflurane anesthesia to prepare for them for laparoscopy.
  • Oocytes are aspirated from follicles using a needle suction device lined with Teflon tubing. Briefly, a 10 mm trocar is placed through the abdominal wall and a laparoscope is introduced. The ovaries are visualized via a camera attached to the inserted laparoscope. Two small skin incisions facilitate the insertion
  • Collection and evaluation of rhesus oocytes The contents of each collection tube are diluted in TALP-Hepes supplemented with 2 mg/ml hyaluronidase to facilitate removal of the cumulus cells (Hewitson et al., 1998, Hum Repro 13:3449-3455). Oocytes are rinsed and then transferred to pm-equilibrated TALP medium containing 3 mg/ml BSA (TALP). Metaphase ll-arrested oocytes, exhibiting expanded cumulus cells, a distinct perivitelline space, and first polar body, are maintained in TALP for up to 8 h before fertilization.
  • Immature oocytes are matured in TALP plus hormones for up to 24 h (Bavister, 1983, Biol Reprod. 28:983-999; Boatman, 1987, "In Vitro Growth of Non-Human Primate Pre- and Peri-implantation Embryos,” Plenum Press; Morgan, 1991, Biol Reprod 45:89-93).
  • sperm suspensions are incubated at 37 ⁇ C under 5% CO 2 in air for 6 h at which point 1mM caffeine and 1mM dibutyryl cyclic
  • dbcAMP 137 adenosine monophosphate
  • ICSI intracytoplasmic sperm injection
  • IVF in vitro fertilization
  • ICSI intracytoplasmic sperm injection
  • microinjection needles 10 ⁇ m
  • microinjection needles O.D. 6-7 ⁇ m and LD. 4-5 ⁇ m
  • Humagen, Inc., Charlottesville, VA are mounted on a Nikon TE300 inverted fluorescent microscope equipped with Hoffman modulation contrast (HMC) optics.
  • the manipulation pipettes are controlled by hydraulic Narishigi manipulators attached to a Hamilton
  • ICSI can also be used as a method to produce androgenotes (see below). Fertilization is scored between 5-12 hrs post-insemination by confirming extrusion of the second polar body and by the presence of two pronuclei in the cytoplasm. Zygotes are cultured in fresh CO 2 -equilibrated TALP medium until the 2-cell stage.
  • 2-cell embryos are co-cultured in CMRL + 10 % FCS (Hyclone Laboratories, Inc., Logan, UT) on Buffalo rat liver cell monolayers (BRL 1442; ATCC, Rockville, MD) seeded in 100 ⁇ l drops overlaid with oil. Two-cell embryos will be used
  • Embryo Sexing Simultaneous FISH will be performed for several known chromosome sequences in order to determine embryo sex.
  • Blastomere biopsy from 8-cell embryos ( ⁇ 60 hours post ICSI) are based on the methods of Handyside et al. (Handyside et al., 1990, Nature. 344(6268):768-70). Each embryo is immobilized by suction with a flame-polished holding pipette held in one micromanipulator.
  • the second micromanipulator with a double holder controls a drilling pipette (internal diameter 10 ⁇ m) containing acid Tyrode's solution (pH 2.4) and a sampling pipette (internal diameter 30 pm) containing buffered medium.
  • the drilling pipette is placed in close contact with the zona pellucida and a hole made with a controlled stream of acid Tyrode's solution. Immediately, the zona is penetrated and this pipette removed, then the sampling pipette is pushed through the hole. One blastomere is then removed by gentle suction. In all cases, an interphase nucleus is observed in the isolated blastomere.
  • the single blastomere is transferred to .1% sodium citrate at 30 0 C for 20 min. After a brief immersion in cold methanol/acetic acid (3:1 ratio), the fixed cells are transferred to a clean slide and dried on a warming plate (Clyde, 2001; Vollmer, 2000). Fixed blastomeres are then sexed using a premixed cocktail of centromeric probes for chromosomes 18, X and Y (MultiVysions; Vysis) according to the techniques of Clyde et al. (Clyde et al., 2001). Slides are treated for 30 min at 37°C in RNAase and placed into 0.01 M HCL containing 0.005% Pepsin Slides for 5 min.
  • Example V Techniques related to Pregnancies
  • Embryo transfer for cleavage stage embryos Surgical embryo transfers are performed on day 2, 3 or 4 following ovulation by transferring two 8-cell to morula stage embryos into the oviduct of the recipient. All transfers are performed via laparoscopy whereby the oviduct is cannulated using a Cook embryo transfer catheter, preloaded with the embryos in TALP-Hepes medium. Embryos are expelled from the catheter in about 0.05 ml of medium while the catheter is withdrawn. The catheter is flushed with medium into a Petri dish following removal from the female to ensure that the embryos are successfully transferred. Control and manipulated cleavage stage embryos will be obtained from the various questions addressed in Part 1 for embryo transfer.
  • Natural Mating is necessary for the establishment of control pregnancies, staged pregnancies for fetal gonad collections and the collection of in vivo
  • Pregnancy confirmation Pregnancy is visually confirmed by transabdominal ultrasound on day 16-30 post-transfer. During ultrasound, mean gestational sac size, yolk sac diameter, greatest length, and embryonic heart rate measurements are collected to approximate the gestational age of the conceptus. These are compared to similar measurements made from IVF and natural pregnancies (Tarantal, 1988, Am J Primat 15:309-323). Ultrasound is performed monthly, to determine developmental normalcy.
  • a blood sample is also collected from all ET recipients between days 16-20 post ET and is sent to UC Davis for analysis of monkey chorionic gonadotropin levels (mCG); (Shimizu et al., 2001, Am J Primatol 54:57-62) to determine if an early pregnancy was possibly established, despite not being sustained.
  • Fetectomies for Epiblast and PG Cell Isolations Fetuses will be harvested at between days 14-18 for epiblast and hypoblast isolations and days 22 and 30-day-old (prior to and after germ cell migration) for PG cell isolation using the following procedure. After a 12-hour fast, monkeys are sedated with
  • 141 ketamine (10 mg/kg IM) and taken to the surgical prep area.
  • the animal's hair is clipped from the ventral abdomen, the abdomen is cleaned with betadyne solution, a cuffed endotracheal tube is placed in the trachea, and an angiocath is placed in a radial or saphenous vein to provide fluid therapy throughout the surgical procedure.
  • Maintenance anesthesia is then provided by isoflurane gas (0.5 - 1.5%) vaporized in 100% oxygen delivered via the endotracheal tube, a sterile prep of the abdomen is performed using betadyne scrub, and the surgical field is draped using sterile drapes.
  • a midline abdominal skin incision is made from the umbilicus to the pubis, the linea alba is located and elevated, and the abdomen is entered using sharp dissection along the linea.
  • the uterus is then exteriorized and surrounded with sterile laparotomy pads soaked in warm saline.
  • a longitudinal incision is then made in the serosa along the body of the uterus. This incision is gently deepened until the amniotic sac is barely visible. Mild blunt dissection is then utilized to tease the amniotic sac away from its uterine connections and the fetus is harvested within an intact amniotic sac.
  • the uterus is then closed using absorbable suture in a Gushing over a Lembert pattern.
  • the linea alba is then closed using 2-0 vicryl in a simple interrupted pattern.
  • the SQ tissue is closed (if necessary) using 3-0 vicryl in a simple continuous pattern, and the skin is closed using 4-0 vicryl in a subcuticular pattern.
  • Excised tissues will be used in Part Vl for the derivation of epiblast and PG cells for use in Part Il and Part III, respectively.
  • Ultrasound (U/S) imaging of macaque fetuses will be performed using a Sonoline Antares high definition system with 2D/grayscale, M-mode, color M-mode, and color Doppler capabilities.
  • the average gestational age of a rhesus-macaque is 165 days.
  • the gestational sac (GS) and crown-rump length (CRL) are evaluated within the first trimester, beginning at gestational day (GD) ⁇ 14-18 days.
  • the yolk sac, embryo proper, and developing heart rate can be visualized as early as -GD 21-25 which confirms pregnancy.
  • ⁇ GD 31-33 the embryo displays isolated movements of the cranium, and begins to show flexion and extensions of the extremities.
  • ⁇ GD 46-47 whole body activity is observed.
  • ⁇ GD 50-60 the amnion and chorion fuse.
  • Other body measurements recorded by U/S from GD 60 until birth include, i. biparietal diameter, ii. occipitofrontal diameter; iii. head area, iv. head circumference, v. abdominal area, vi. abdominal circumference, and vii. femur length (Tarantal, 1988, Am J Primat 15:309-323; Tarantal, 1990, J Med Primatol 19:47-58).
  • the AVS is a digital multiplex recorder and broadcast server currently configured for approximately 7 days of digital video to be recorded. It currently supports 16 video camera signals and 5 remote vet staff can simultaneously connect to the video server. The high- resolution video cameras generate quality video pictures during daylight hours and overnight. This allows animals to remain undisturbed, but still be monitored, overnight when most pregnant females go into labor. When an animal appears to be going into labor, the appropriate staff is notified so that they can assist the delivery and/or survival of the neonate.
  • the AVS can also be used to broadcast, record or archive any surgery or procedure, which can be used a vet-teaching tool.
  • Amniocentesis will be performed on pregnant females between days 55-70 of gestation.
  • ketamine is administered intramuscularly at a dose of 10 mg/kg.
  • the animal is delivered to the veterinary procedure room and placed in a supine position on the examination table.
  • the skin of the abdomen is then shaved with an electric trimmer and prepped thoroughly with betadyne scrub followed by an alcohol rinse.
  • the abdomen is covered with sterile gel and imaged with an ultrasound probe covered with a sterile sleeve.
  • the uterus is located within the abdomen it is scanned closely to determine the position of the fetus, umbilical cord and placenta.
  • a sterile 20-22 gauge spinal needle is
  • anesthesia is then provided by isoflurane gas (0.5 - l.5%) vaporized in 100% oxygen delivered via the endotracheal tube, a sterile prep of the abdomen is performed using betadyne scrub, and the surgical field is draped using sterile drapes.
  • isoflurane gas 0.5 - l.5%
  • a sterile prep of the abdomen is performed using betadyne scrub, and the surgical field is draped using sterile drapes.
  • a midline abdominal skin incision is made from the umbilicus to the pubis, the linea alba is located and elevated, and the abdomen is entered using sharp dissection along the linea.
  • the uterus is then exteriorized and surrounded with sterile laparotomy pads soaked in warm saline.
  • a longitudinal incision is then made along the serosa of the body of the uterus avoiding the placental discs.
  • the tissue of the amniotic sac is identified and elevated using forceps and then fine surgical scissors are used to incise the sac.
  • the fetus is grasped through the incision in the amniotic sac and the fetus is delivered. Blunt dissection is used to remove the placental discs and the uterus is then closed using absorbable suture in
  • the umbilical cord is clamped immediately after caesarian section and cord blood and placental tissue collected for either mitochondrial DNA analysis or transgene expression as described in Part I.
  • the umbilical vein is cannulated with a 22 gauge butterfly catheter prior to placental detachment as described by (Pafumi et al., 2002, Gynecol Obstet Invest 54:73-77).
  • the catheter is fitted with an empty 10 ml syringe and blood is gently aspirated and then immediately transferred to blood tubes containing heparin.
  • Multiple 5 mm x 5 mm x 5 mm cubes of placenta will also collected and analyzed immediately or snap frozen in liquid nitrogen and stored at -80 0 C for future analysis. If pregnancies spontaneously abort,
  • nhpES cells Transplantation of nhpES cells into adult rhesus macaques: MHC haplotyping of primates used for transplantation studies will be performed. Based on rodent studies, nhpES cells will be transplanted subcutaneously, into the parenchyma of the testes and intramuscularly. If cells are rapidly rejected, cells may also be transfered under the capsule of the kidney, which is considered a more immunologically-privileged site and is easy to image by ultrasound and MRI.
  • the recipient animal Prior to administration of subcutaneous, intramuscular, and intratesticular ES cells, the recipient animal will be anesthetized with an intramuscular dose of ketamine (10 mg/kg) and the hair over the administration site will be clipped and the skin will be prepared with betadyne scrub followed by an alcohol rinse to maintain sterility. Subcutaneous administration will be performed under the skin between the shoulder blades to reduce the chance of the animal traumatizing the administration area. Intramuscular administration will be performed in the large muscle groups such as the quadriceps or biceps by incising the skin located over the target muscle and injecting the ES cells superficially into the belly of the muscle. Post-intramuscular injection, the skin over the injection site is closed with 4-0 vicryl in a simple interrupted pattern.
  • Testicular administration is performed by grasping one testicle firmly, moving it caudally and laterally away from the other testicle, puffing the skin of the scrotum tightly over the isolated testicle, and injecting the ES cells into the parenchyma of the organ. Renal subcapsular transplant of ES cells will be done via laparoscopy. Briefly, once the animal is insufflated with CO 2 via a gas port inserted just cranial to the umbilicus, two small skin incisions facilitate the insertion of 5 mm trocars bilaterally. One kidney is stabilized using a fine curved forceps inserted through one trocar and a small incision is made in the renal capsule using scissors inserted through the other trocar. The capsulotomy is then closed using 4-0 vicryl in a simple interrupted pattern. The renal capsule is then gently lifted with the fine curved forceps and the ES cells are deposited beneath the capsule via polyethylene tubing inserted through one of the
  • trocars Each site listed above will be injected with 0.5 to 5 x 10 6 ES cells.
  • nhpES cells are derived by Parts Vl and IV.
  • the ES cell administration sites of the subcutaneous, intramuscular, and intratesticular inoculated animals will be monitored daily for evidence of inflammation, infection, or teratoma growth. Once per week, these animals will be anesthetized with an intramuscular dose of ketamine (10 mg/kg) and the ES cell administration site will be palpated for evidence of teratoma growth. Any growth will be measured with Vernier calipers and recorded in the animal's clinical record. Teratoma growth will also be evaluated monthly using MRI imaging as described in Part IV. Subcutaneous, intramuscular, and intratesticular teratomas will be excised when they grow to 1.0 cm in diameter.
  • the administration sites will be monitored closely for evidence of inflammation and necrosis that is often associated with rejection.
  • the animal Prior to the excision of a teratoma, the animal will be sedated with an intramuscular dose of ketamine (10 mg/kg) and the affected area will be shaved of hair and prepped with betadyne scrub. The animal will then be intubated with a cuffed endotracheal tub, a radial or saphenous vein catheter will be placed, and general anesthesia will be induced and then maintained with 0.5-2.0% isoflurane gas. The skin over the subcutaneous, intramuscular, and testicular teratoma will be then be infiltrated with 1.0% lidocaine to alleviate post-surgical incisional pain.
  • Subcutaneous teratomas will be excised by making an elliptical incision through the skin and subcutaneous tissue surrounding the abnormal growth and removing the affected area plus an additional 1 /2-centimeter border of unaffected skin. The remaining subcutaneous tissue will be closed using 3-0 vicryl in a simple continuous pattern and the overlying skin will be closed using 4-0 vicryl in a subcuticular pattern. Muscle teratomas will be removed by incising the skin over the affected muscle, locating the teratoma within the affected muscle belly, removing the affected muscle tissue using sharp dissection, and closing the muscle belly using 2-0 prolene with interrupted horizontal mattress sutures. The subcutaneous tissue and skin over the muscle belly will be closed as described above. Testicular teratomas will be removed by performing a
  • Example V Techniques to Study and Insure Health of Nonhuman Primate Primate NICU and nursery. Infants born under all projects enter an intensive care nursery immediately after either caesarian section or vaginal delivery and are reared under established guidelines (Ruppenthal and Sackett, 1992, Research Protocol and Technician's Manual: A Guide to the Care, Feeding, and Evaluation of Infant Monkeys, Seattle: Infant Primate Reasearch Laboratory). The nursery is staffed 24 hours a day by a well-trained group of animal care technicians, veterinary technicians, and behaviorists. Infants are intensively monitored for various health (temperature, hydration, respiratory and nutritional status) and developmental parameters to reduce morbidity and mortality and to document progress for later analysis and comparison to other infants undergoing similar rearing experience.
  • the PDC has outfitted the NICU and nursery with state-of-the-art medical equipment.
  • the major pieces of equipment include an Ohmeda 7800 pediatric ventilator, a Phillips V24E modular patient monitor with ECG, respiratory, SPO2, expired CO2, and noninvasive blood pressure readouts and, and three infant incubators.
  • infants remain in intensive care until they can maintain physiological homeostasis, are capable of self-feeding, and are otherwise healthy. As they mature, they are moved to a traditional animal holding room containing other nursery graduates but are continually monitored to assess developmental parameters. During this time, all infants are housed singly. This is done for several reasons: (i) infant development (social, emotional, cognitive, growth, etc.) is assessed on a scheduled basis under closely controlled conditions so that all receive identical stimulation and experience.
  • Mother- rearing influences behavioral trajectories and behavior. Some mothers are overly protective and restrictive, others more relaxed and permissive. Some are more abusive than others. All influence behavioral development of offspring. Mothers may also reject their infants, necessitating nursery care of a subset of infants, confounding their histories; (ii) if mother-reared but then the infants are separated for assessments, the mothers react violently to separation, endangering infants. Also, repeated separations from their mothers negatively impacts emotional development of infants.
  • each infant receives 30 minutes a day of social play with one or more peers in large play cage. Their social development is also tested 3 times a week with other infants. This continues for up to a year, at which time, compatible animals are pair-caged.
  • Umbilical arterial lines may be necessary to constantly monitor arterial blood pressure, assess oxygenation and ventilation status through arterial blood gas measurements, and phlebotomy for laboratory assessment of blood glucose and electrolyte status.
  • Umbilical venous catheters can be used to supply necessary fluids (dextrose water solution initially followed by dextrose and electrolytes), antibiotics and eventually parenteral nutrition.
  • NH intraventricular hemorrhage
  • PVL periventricular leukomalacia
  • NH is strictly a lesion of the neonatal brain with inverse proportional risk and severity with decreasing gestational age.
  • the pathogenesis is complex and multifactorial, but generally results from the interaction of circulatory disturbances resulting in fluctuations in cerebral perfusion pressure and the preterm infants' ability to regulate cerebral blood flow. Severity is graded by the amount of blood filling the ventricular system of the brain.
  • PVL Very severe hemorrhages
  • NH periventricular white matter damage secondary to venous infarction
  • PVL white matter injury in the brain that can be focal, diffuse, and both diffuse and focal leading to permanent injury of the developing white matter tracts and eventually cerebral palsy, cognitive and behavioral disability. PVL can occur prior to birth, although the majority is related to postnatal aberrations in cerebral circulation that result from being premature and the need for mechanical ventilation. Both lesions, NH and PVL can also coexist in the premature brain. Cranial ultrasound can determine the extent and severity of NH as well as follow its course and assess for the development of posthemorrhagic hydrocephalus. Similarly, PVL and the
  • PVE periventricular echogenicity
  • serial cranial ultrasound assessments are generally performed at day 7 of life and sometimes earlier based on the clinical scenario and then at days 14 and 30 to assess for the presence of hydrocephalus and degree of white matter injury.
  • Magnetic resonance imaging is also adjuncts that can be employed to delineate the true extent of brain injury in cooperation with the Core A. This data is critical to correlate the future motor, cognitive, and behavioral assessments done on these infants. After the initial stabilization period, respiratory support will be weaned and parenteral nutrition started if appropriate all under the supervision of veterinary and neonatal support team.
  • Stabilized infants are assessed by a series of protocols (Ruppenthal and Sackett, 1992, id.) to determine developmental normalcy including physical, emotional, cognitive, immunological, and behavioral assessments that have been employed for several years and have proven to be of worth in support of research utilizing the species used by the PDC.
  • These protocols include measures such as: modified APGAR ratings, physical exams, weight gain/loss, reflex development, sleep/wakeful ness and activity cycles, vital signs, food intake, development of object permanence, recognition memory, social behavior development, learning, anthropometrical growth and development, emotion, immune system development, and reactivity to mild challenges on a scheduled basis with some age-based and others daily, weekly, or monthly.
  • Intakes are recorded each feeding and daily weights are taken to assess weight gain per calorie consumed.
  • Vital signs heart rate, respiration rate and temperature
  • Activity cycles are recorded on a scheduled basis 24/7.
  • infants are assessed for reactivity, clasping/grasping, righting, sucking, orientation and tracking of visual and auditory stimuli, and
  • the infant will be maintained in the vivarium and will be provided with appropriate clinical care fashioned to the animal's specific needs. If an infant's physical abnormalities are so severe that permanent intensive care is necessary, euthanasia will be elected. Euthanasia will be performed in accordance with the most recent recommendations of the AVMA Panel on Euthanasia.
  • infants are socialized daily in peer groups to insure behavioral normalcy. Should individuals with physical or emotional handicaps affect normal social development steps have been developed and utilized to overcome such deficits. Infants reared in social isolation for the first several months of life exhibit a majority of avoidance behavior and extreme negative reaction when placed in a social situation. By grouping them with younger, less threatening animals who do not overwhelm these subjects, the isolate-reared subjects behaviors are transformed into positive, exploratory, playful and affiliative behavioral interactors.
  • anesthesia for MRI Upon arrival at the Institute, the animal will be sedated with an intramuscular dose of ketamine (10 mg/kg) and atropine (0.04 mg/kg), removed from the squeeze cage, and taken to the NMR Center using the transfer box. Prior to the initiation of the imaging procedure, the animal will be intubated with a cuffed endotracheal tube and instrumented with a saphenous or radial vein catheter. As described previously by Benveniste et. al. (Benveniste et al., 2003, J Nucl Med 44:1522-1530), anesthesia will be maintained using intravenous propofol (Diprivan; AstraZeneca) at a dose of 120 -300 ⁇ g/kg/min.
  • Diprivan intravenous propofol
  • respiratory parameters will be maintained within acceptable limits by providing continuous mechanical ventilation.
  • a pulse oximeter will be utilized to monitor oxygen saturation and pulse rate, body temperature will be maintained with a water circulating heating pad and a warm air source blowing directly into the bore of the MRI system, and a mixture of 0.9% NaCl and 5% dextrose will be administered intravenously at maintenance rates throughout the imaging procedure.
  • Post-imaging care Post-imaging, the animal will be de-instrumented, recovered from anesthesia, placed back in the squeeze-cage of the van and transported back to its home cage at the PDC where it will be monitored closely for evidence of adverse reactions.
  • mice Each supplier of human embryonic stem cells prefers adifferent strain of mice for derivation of feeder cells. For those cells obtained from WiCeII 1 CF-1 mice will be used (Charles River, Wilmington MA). For cells from Bresagen, ICR mice will be used (Charles River) and for cells from ES Cell International, 129Sev mice will be used (Taconic, Germantown, NY). Female mice at eight weeks of age are injected I. P. with 5 I. U. Pregnant Mare's Serum Gonadotrophin (PMSG).
  • PMSG Pregnant Mare's Serum Gonadotrophin
  • mice This injection is followed 48 hr later with 5 I. U. human chorionic gonadotrophin (hCG) followed by mating overnight. The next morning females are checked for vaginal plugs to confm coitus and separated from the males. Pregnant mice are sacrificed on day 12.5 of gestation by C02 asphyxiation. The uterine horns are dissected and the fetuses removed and placed into Dulbecco's phosphate buffered saline (PBS 1 Invitrogen, Carlsbad, CA): To help ensure a more pure population of feeders the heads and internal viscera are removed from the fetuses and the remaining tissues are rinsed in several washes of PBS. The tissue is transferred to a 100 mm petri dish containing 2 ml of 0.05% TrypsinEDTA (Invitrogen, Carlsbad, CA). The
  • .tissue is minced, an additional 5 rnl of trypsin/EDTA added &d all 7 ml transferred to a 37" C incubator for 15 minutes.
  • Two volumes of mouse embryonic feeder cell isolation media (CF-1 media, 90% DMEM, 10% fetal calf serum, 0. ImM MEM nonessential amino acids, 1.0% Penicillin/Streptomycin) are added and placed in a 50 ml centrifuge tube to settle after fust breaking up cells with gentle pipetting. After 15 minutes entire contents of tube are split evenly into tissue culture flasks.
  • mouse embryonic feeder cells When mouse embryonic feeder cells reach confluency they may be frozen in the following manner. Cells will be passaged using 0.05% Trypsin/EDTA and transferred to a 50 ml conical tube, counted and centrifuged at 800x g for eight min. After centrifugation cells are resuspended in freezing media (30%
  • CF- 1 Media 60% defined fetal bovine serum (HyClone, Logan UT) and 10% DMSO (Sigma, St. Louis, MO)) and transferred to 1.5 ml cryovials (Nalgene, Rochester NY). Cryovials are placed in a cell freezer (Fisher Scientific, Pittsburgh, PA) in a -80 C freezer overnight before being placed in liquid nitrogen for long-term storage.
  • Mouse embryonic fibroblast cells are grown to confluence in tissue culture flasks and then mitotically inactivated by two-hour treatment with CF- 1 media containing 10 ⁇ g/ ml mitomycin C (Sigma, St. Louis, MO). Mitomycin C containing media is removed after two hours and the cells are washed three times with PBS before being harvested using 0.05% Trypsin/EDTA. Cells are transferred to a 50 ml conical tube, counted and centrifuged at 80Ox g for eight min.
  • centrifugation cells are resuspended in CF-1 media at a density of 7.5 x 104 cells/ml. These are transferred to culture dishes previously pretreated with 0.1% gelatin. Cells are plated at a density of -200K cells per well of a 6 well dish. Irradiation may be used as an alternative mechanism of inactivation. Cells will be irradiated with 3000 rad X-ray or gamma irradiation using a 2100 Cesium- source irradiator for seven minutes. Feeder plates prepared in either manner are used within two weeks of preparation.
  • Embryonic Stem Cell Culture and Passaging All cell lines are obtained under executed and approved material transfer agreements (MTA's) negotiated between the National Institutes of Health approved distributors and the Principal Investigators. Each cell line will be cultured according to the distributors provided methodology as described below. The culture media for each line is:
  • H1, H7, and H9 Cells are cultured in 80% DMEM/F12 (Invitrogen, Carlsbad, CA), 20% Knockout Serum Replacement (Invitrogen), 1mM L- glutamine (Invitrogen), 0.1 mM beta-mercaptoethanol (Sigma, St. Louis MO) 0.1 mM MEM non-essential amino acids (Invitrogen), and 4ng/ml basic human recombinant FGF (Invitrogen).
  • HSF-6 and HSF-1 (UCSF): These cell lines are cultured and propagated in 80% DMEM high glucose (Invitrogen), 20% Knockout serum replacement, 1mM L-glutamine, , 0.1 mM MEM non-essential amino acids, 0.1 mM (3- mercaptoethanol and 4ng/ml basic human recombinant FGF.
  • HES-3 and HES-4 (ES Cell International): These cells are cultured in 80% DMEM high glucose, 20% defined FBS (HyClone, Logan, UT) 0.1mM MEM non-essential Amino Acids, 0.5% Penicillin/Streptomycin, 2mM L-Glutamine, 1% Insulin-Transferrin-Selenium supplement (Invitrogen), and 0.1mM beta- mercaptoethanol.
  • Non-human Primate ES Cells (WiCeII and PDC): These cells are cultured in 80% DMEM high glucose, 20% defined PBS, 0.1mM MEM non-essential Amino Acids, 2mM L-Glutamine, and 0.1mM beta -mercaptoethanol.
  • the most consistent passaging technique for maintaining normal karyotype reportedly is manual passage as follows (Pera, 2004) A very fine glass needle is pulled over a flame or using a micropipette puller (Model P-87, Sutter Instruments, Novato CA). Individual colonies are sliced into small sections using the glass needle. One of two techniques will then be employed; either the sections displaying good compact morphology are detached using the glass needle followed by rinsing and pelleting as above or the differentiated sections of the colony can be scraped and rinsed away preceeding isolation of the good portions of the colony. This technique allows for the repeated passaging of embryonic stem cells with good morphology and is the recommended passaging technique of ES Cell International hESC lines (M. Pera, personal comm.).
  • Embryonic stem cells displaying an undifferentiated morphology are scraped as in passaging and transferred to a 15 ml conical tube. Each scraped well is then rinsed with 1 ml of the cell line specific media. This rinse is added to the 15 ml conical tube. The cells are pipetted gently to break up large clumps and pelleted at 20Ox g for 5 min. The supernatant is
  • Immunocytochemistry As immunocytochemistry probes the heterogeneity of colonies it will be used whenever possible to determine the undifferentiated state of the human ESCs (M. Pera, personal communication). The hESC's will be probed for the positive ESC markers: Oct-4, SSEA-4, Tra 1-80 and Tra 1-61, as well as the negative ESC marker: SSEA-1. Immunocytochemistry will be preformed in one or more of the following manners. 1.) Culture dishes containing undifferentiated colonies will be fixed by addition of either 100% methanol 20° C. for 15 min followed by a 15 min wash in PBS + 1 % Triton X-100 (PBS-Tx, Sigma, St.
  • 5OM TOTO-3 (Molecular Probes, Eugene OR) will be added for 20 min to label the nuclear DNA. Coverslips will be inverted onto slides and mounted in Vectashield anti-fade medium (Vector Labs, Burlinghame, NH) to prevent photobleaching (Navara et al., 2001).
  • RNAqueousTM-4PCR kit (Ambion, Austin TX) following manufacturer's instructions. Briefly, 100 ⁇ l of lysis/binding solution is added per 100-1000 cells and vortexed to lyse cells to homogeneity. An equal volume of 64% EtOH is added and gently mixed by inversion. This solution is added to the RNAqueous filter cartridge and centrifuged for one minute at 15,00Ox g.
  • the filter is washed sequentially through buffers one and 2/3. After washing the filter is centrifuged for 30 sec to remove final traces of wash solution.
  • the RNA is eluted from the filter by first placing in a fresh collection tube, adding 40 ⁇ l of preheated Elution solution (95°C) and centrifuging at maximum speed for 30 seconds. DNase I buffer and 1 ⁇ l DNase I is added to the eluate and the mixture incubated for 30 min at 37°C. The DNase is inactivated and the RNA removed to a new tube. To produce cDNA for PCR, 1 ⁇ g of total RNA is incubated for 10 minutes at 70 0 C quick spun and placed on ice.
  • the following reaction is prepared containing: 5mM MgCI2, 1 x RT buffer, 1mM each dNTP, 1 unit/ ⁇ l ribonuclease inhibitor, 15unit/ ⁇ g AMV RT 1 0.5 ⁇ g random hexamers, and nuclease free water to a 20 ⁇ l final volume.
  • This reaction is added to the RNA and incubated at room temperature for 10 min and then 42°C for 15 min. Finally the sample is heated to 95°C for 5 minutes, and immediately place at 4°C for PCR or long-term storage at -20 0 C.
  • Primers used for Oct-4 are forward cgaccatctgccgctttgag (SEQ ID NO: 33) and reverse ccccctgtcccccattccta (SEQ ID NO: 34).
  • Primers for Nanog are forward accttccaatgtggagcaac (SEQ ID NO: 35) and reverse gaatttggctggaactgcat (SEQ ID NO: 36).
  • mice Approximately 5 x 105 - 5 x 106 human embryonic stem cells (hES) or non-human primate embryonic stem cells (nhpES), are injected using a sterile 31 G needle into the testis of 8-12 week old NOD-SCID mice (Jackson Labs) using Institutional Animal Care and Use Committee approved protocols. Tumors will be grown until palpable or seven to eight weeks after injection. At the appropriate time mice will be euthanized by CO2 asphyxiation and tumors dissected, fixed in 4% formaldehyde, paraffin embedded, and examined histologically after hematoxylin and eosin staining. Formation of teratomas with cell lineages derived from ectoderm, mesoderm, and endoderm will be proof of the ES cell lines' pluripotency.
  • Example Vl Establishing And Maintaining A Resource Of Genetically And Epigenetically Characterized hESC, nhpESC, NTnhpESC and nhpEGC hES, nhpES, NTnhpES and nhpEG cells will be maintained, their imprinting status monitored at a group of imprinted loci, and these cells will be available to each project.
  • nhpES nhpES
  • NTnhpES nhpEG cells
  • nhpEG cells will be derived from specific matings as described in Parts I and III. Briefly, parents will be chosen based on analysis of their genomic DNA with primers specific for informative polymorphisms within imprinted genes. Matings will be established (see Part V for details)
  • nhpES 161 and embryos or fetuses harvested for derivation of nhpES, NTnhpES or nhpEG cell lines. Once established cell lines will be maintained using well- established protocols.
  • RNAqueousTM-4PCR kit (Ambion, Austin TX) following manufacturer's instructions. Briefly, 100 ⁇ l of lysis/binding solution is added per 100-1000 cells and vortexed to lyse cells to homogeneity. An equal volume of 64% EtOH is added and gently mixed by inversion. This solution is added to the RNAqueous filter cartridge and centrifuged for one minute at 15,00Ox g.
  • the filter is washed sequentially through buffers one and 2/3. After washing the filter is centrifuged for 30 sec to remove final traces of wash solution.
  • the RNA is eluted from the filter by first placing in a fresh collection tube, adding 40 ⁇ 1 of preheated Elution solution (95°C) and centrifuging at maximum speed for 30 seconds. DNase I buffer and 1 ⁇ l DNase I is added to the eluate and the mixture incubated for 30 min at 37°C. The DNase is inactivated and the RNA removed to a new tube. To produce cDNA for PCR, 1 ⁇ g of total RNA is incubated for 10 minutes at 70 0 C quick spun and placed on ice.
  • the following reaction is prepared containing: 5mM MgCI 2 , 1 x RT buffer, 1mM each dNTP, 1unit/ ⁇ l ribonuclease inhibitor, 15unit/ ⁇ g AMV RT, 0.5 ⁇ g random hexamers, and nuclease free water to a 20 ⁇ l final volume.
  • This reaction is added to the RNA and incubated at room temperature for 10. min and then 42°C for 15 min. Finally the sample is heated to 95°C for 5 minutes, and immediately place at 4 0 C for PCR or long- term storage at -20 0 C.
  • RNA will be isolated from nhpES, NTnhpES and nhpEG cells essentially as described above. RNA will be converted into cDNA and amplified using primers spanning informative polymorphisms. The primers to be utilized are: H 19 - silenced on paternal chromosome
  • DNA and cDNA create bands of about 300bp. Digestion with Apal cuts to 230bp. PEG1 - silenced on maternal chromosome
  • DNA and cDNA create bands of about 310bp. Digestion with AfIIII cuts to 260bp.
  • tcttgcaggatacatctcattcta (SEQ ID NO: 47) DNA - about 1100bp band, reduced to about IOOObp when cut. cDNA - about 220bp band, reduced to about 150bp when cut. Use BstUI restriction enzyme.
  • DNA- is a 1550 bp band.
  • cDNA- is a 868 bp band.
  • Polymorphism is C to T KCNQ1OT1- silenced on maternal chromosome Lee, MP. et al. Proc. Natl. Acad. Sci. U.S.A. 96, 5203-5208 (1999).
  • DNA and cDNA create bands of about 466 bp.
  • Polymorphism is C to T SNP2 in H7
  • DNA- is a 315 bp band.
  • Polymorphism is G to A
  • cDNA- is a 231 bp band. Polymorphism is G to A
  • DNA- is a 1141 bp band.
  • Polymorphism is T to C
  • PCR products will be sequenced. This analysis will be carried out on newly derived EG cell lines and on existing non-human primate ES cell lines for which parental information is known (see Part III). As data accumulates on improved hES or nhpES, NT-nhpES and nhpEG cell cultures, the effects of culture conditions on the stability of imprints will be examined. The sequence of the amplified fragments wili be compared with that of the genomic sequences derived from the DNA of the parental samples to determine whether there is mono- or bi-allelic expression from that locus. This process will be repeated for as many genes as informative polymorphisms are found.
  • Genomic sequence analysis would be used to search for polymorphisms within the coding regions of imprinted genes. Once those polymorphisms had been identified, matings from informative animals would be established. Rhesus monkeys and other macaques can be successfully mated and produce offspring that are viable and fertile. Such crosses could be used to generate embryos from which stem cell lines could be derived. Analysis of genomic imprinting in F1 hybrids derived from different species would follow the same principal as described above.

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Abstract

L'invention concerne une cellule souche embryonnaire (ES) pluripotentielle de primate non humain (nhp), pouvant être utilisée de différentes manières, notamment pour générer des embryons de primate chimériques. L'invention concerne également des méthodes destinées à déterminer l'état de différenciation d'une cellule embryonnaire, par comparaison de ses motifs de transcription avec ceux de cellules ES à des étapes de différenciation particulières. L'invention concerne également une cellule germinale embryonnaire (EG) de primate non humain pouvant être utilisée de différentes manières, notamment pour administrer une lignée cellulaire EG différenciée à un patient, afin de traiter un certain nombre de maladies. L'invention concerne également des méthodes de génération de cellules ES nhp et de chimères d'embryons de primates, ainsi que des méthodes de dérivation de cellules EG.
EP07718078A 2006-01-19 2007-01-19 Cellules souches et germinales embryonnaires de primates non humains, leurs méthodes d'utilisation et leurs méthodes de fabrication Withdrawn EP1981968A4 (fr)

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US75995006P 2006-01-19 2006-01-19
PCT/US2007/001280 WO2007084585A2 (fr) 2006-01-19 2007-01-19 Cellules souches et germinales embryonnaires de primates non humains, leurs méthodes d'utilisation et leurs méthodes de fabrication

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EP1981968A2 true EP1981968A2 (fr) 2008-10-22
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GB0215287D0 (en) * 2002-07-02 2002-08-14 Oxford Biomedica Ltd 5T4 antigen expression
US7332336B2 (en) * 2003-08-19 2008-02-19 Effector Cell Institute, Inc. Methods for inducing differentiation of pluripotent cells

Non-Patent Citations (4)

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Title
CALVIN SIMERLY ET AL: "Interspecies chimera between primate embryonic stem cells and mouse embryos: Monkey ESCs engraft into mouse embryos, but not post-implantation fetuses", STEM CELL RESEARCH, ELSEVIER, NL, vol. 7, no. 1, 10 March 2011 (2011-03-10), pages 28-40, XP028094073, ISSN: 1873-5061, DOI: 10.1016/J.SCR.2011.03.002 [retrieved on 2011-03-25] *
CHAN A ET AL: "Transgenic monkeys produced by retroviral gene transfer into mature oocytes" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, WASHINGTON, DC; US LNKD- DOI:10.1126/SCIENCE.291.5502.309, vol. 291, no. 5502, 12 January 2001 (2001-01-12), pages 309-312, XP002168955 ISSN: 0036-8075 *
CHAN ANTHONY WS: "Transgenic nonhuman primates for neurodegenerative diseases" REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY, XX, XX LNKD- DOI:10.1186/1477-7827-2-39, vol. 2, no. 1, 16 June 2004 (2004-06-16), page 39, XP021009405 ISSN: 1477-7827 *
See also references of WO2007084585A2 *

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WO2007084585A3 (fr) 2009-02-19
WO2007084585A2 (fr) 2007-07-26

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