US20140363467A1 - Avian induced pluripotent stem cells and their use - Google Patents

Avian induced pluripotent stem cells and their use Download PDF

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US20140363467A1
US20140363467A1 US14/100,809 US201314100809A US2014363467A1 US 20140363467 A1 US20140363467 A1 US 20140363467A1 US 201314100809 A US201314100809 A US 201314100809A US 2014363467 A1 US2014363467 A1 US 2014363467A1
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Steven L. Stice
Franklin West
Yangqing Lu
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University of Georgia Research Foundation Inc UGARF
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Definitions

  • the present invention relates to the production of avian induced pluripotent stem cells from non-pluripotent somatic cells, including embryonic fibroblasts and adult somatic cells.
  • avian including quail or chicken somatic cells are reprogrammed into a state closely resembling embryonic stem cells including the expression of key stem cell markers alkaline phosphatase, etc. by transfecting/transducing the non-stem cells with genes (preferably using a non-integrating vector as otherwise described herein or alternatively an integrating vector, such a lentiviral vector, retroviral vector or inducible lentiviral vector, among others) which express at least nanog, Lin28 and cMyc.
  • the transfected/transduced vectors express nanog, Lin28, cMyc, Oct 4 (POU5F1 or PouV), SOX2 and KLF4.
  • the induced stem cells which are produced contribute to all 3 germ layers, the trophectoderm and in certain aspects, the gonad in chimeric offspring.
  • the experiments which are presented herein evidence that the induced quail and chicken pluripotent stem cells according to the present invention are bona fide stem cells that exist in a pluripotent state where they can be differentiated or turned into any cell type in the body or even a chimeric cell or mature bird.
  • the present invention represents the first time that avian iPSCs were demonstrated to be capable of contributing to chimeric offspring, an essential component in their use as a biotechnology tool, including the preparation of vaccines.
  • avian induced pluripotent germ cells (aiPGCs) are produced from somatic cells using the methods which are described herein. These avian induced pluripotent germ cells (aiGSCs) exhibit behavior in developing embryo and offspring.
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • avian ESC and primordial germ cell (PGC) lines have been established (11, 12), they have not been used in gene targeting studies mostly likely because they have not been clonally isolated nor are they highly proliferative in extended cultures. Additionally, many of these avian lines have not been shown to robustly undergo directed in vitro differentiation into multiple lineages with phenotypic characteristic similar to mammalian pluripotent cells. Given some of the issues that plague the development of avian ESCs, the probability that avian iPSCs could be generated and show diverse differentiation potential in vitro and in vivo would seem unlikely. Generating non-mammal iPSCs faces other challenges and difficulties. Intuitively species specific or at least related reprogramming factors may be required for proper reprogramming and little is known of the required reprogramming factors in phylogenetically diverse species.
  • Avian embryonic models have a long history of providing critical new insights into developmental biology including organ function (13, 14), disease progression (e.g. Pompe disease) (15), eye disorders (16, 17) and many others (18, 19).
  • the advantage that avian species have is their relative size and ease of access to the embryo for manipulation.
  • Cells and tissues including whole sections of the spinal column can be transplanted into the avian embryo and can be monitored in real time during development (20). This is not possible in mammalian species.
  • the quail-chicken chimera is an attractive and widely used model for developmental patterning and cell fate studies given that cells can be readily tracked in this model (21, 22).
  • the quail also has a short generation interval (3-4 generation per year)(23), facilitating genetic selection studies and experiments requiring multiple generational observations (24). Coupling a robust and clonal feeder free iPSC lines and derived committed cell lines or tissues with these model systems offers new opportunities to manipulate and study developmental process both in vitro and in vivo.
  • the present invention also relates to germ cell development wherein pluripotent germ cells are induced from non-pluripotent somatic cells, including embryonic fibroblast cells and adult somatic cells. Germ cells are critically important as the vehicle that passes genetic information from one generation to the next. Correct development of these cells is essential and perturbation in their development often leads to reproductive failure and disease. Despite the importance of germ cells, little is known about the mechanisms underlying the acquisition and maintenance of the germ cell character. Using a reprogramming strategy, the present invention provides a method which demonstrates that over-expression of ectopic transcription factors in embryonic fibroblast and other somatic cells can lead to the generation of avian (chicken) induced-PGCs (ciPGCs).
  • avian chicken
  • induced-PGCs avian induced-PGCs
  • ciPGCs express pluripotent markers POU5F1, SSEA1 and the germ cell defining proteins CVH and DAZL, closely resembling in vivo sourced PGCs instead of embryonic stem cells (ESCs). Moreover, it was shown that CXCR4 expressing ciPGCs of the present invention were capable of migrating to the embryonic gonad after injection into the vasculature of stage 15 embryos, indicating the acquisition of a germ cell fate in these cells. Direct availability of ciPGCs in vitro are useful to facilitate the study of germ cell development as well as providing a potential strategy for conservation of important genetics of agricultural and endangered birds using somatic cells.
  • PSC pluripotent stem cell
  • ESC embryonic
  • iPSC induced pluripotent stem cells
  • PGCs are likely more representative of a na ⁇ ve in vivo germ line transmissible cell type than the undifferentiated PSCs, thus their response to a germ cell toxin or other perturbations may be more representative of how PGCs and eventually GCs behave in the developing embryo and offspring.
  • PGC studies are difficult in mammals since mouse PGCs, when cultured in vitro, do not maintain a PGC phenotype and will revert back to a cell type that is equivalent and resembles ESC [13-15].
  • PGCs have significant potential for the development of transgenic animals on unique backgrounds or conservation of valuable genetics as in the case of endangered birds. Over 1300 avian species, or 13% of the total population, are threatened by extinction, and an additional 880 species are threatened [19]. Although various conservation measures such as habitat protection and captive reproduction are a solution used to protect and repopulate endangered birds, the overall number of species is still on the decline and some of them are extinct [20]. A PSC to GC approach offers an additional theoretical strategy for the conservation of genetic diversity and repopulation of the endangered avian species and studies using unique adult genotypes [21].
  • the present invention demonstrated that quail iPSCs could be generated from somatic cells using human pluripotency transcription factors and revealed that the regulatory mechanisms of pluripotency are conserved across species. Therefore, it was viewed that it might be possible to reprogram chicken somatic cells using similar factors and chicken and other avian germ cell culture conditions to derive chicken or other avian germ cells [18,23].
  • the inventors have successfully generated PSCs by over expression of human transcription factors in chicken somatic cells.
  • the PSCs express typical stem cell markers and were capable differentiating into all 3 germ layers.
  • these induced chicken PSCs when propagated in medium used to expand PGCs expressed important germ cell genes such as CVH and DAZL.
  • FIG. 1 shows the derivation of quail induced pluripotent stem cells (qiPSCs) from quail embryonic fibroblast cells (QEFs).
  • QEFs prior to addition of reprogramming factors (A).
  • Incomplete reprogrammed QEFs maintain a fibroblast-like morphology at day 6 post-transduction (B), while qiPSC colonies at day 17 showed defined borders (C) and at the single cell level a high nuclear to cytoplasm ratio, clear cell boarders and prominent nucleoli (D, E).
  • qiPSCs were positive for Alkaline Phosphotase (AP; F) and Periodic Acid Schiff's (PAS) staining (G). 5 out of 6 human pluripotent stem cell factors were integrated and expressed in qiPSCs (H).
  • AP Alkaline Phosphotase
  • PAS Periodic Acid Schiff's
  • FIG. 2 shows qiPSCs demonstrate rapid proliferation, high levels of telomerase activity and clonal expansion after genetic manipulation.
  • B 8 days post transduction a subpopulation qiPSC expressed green fluorescent protein (GFP).
  • C, D, E After 9 days post-fluorescence activated cell sorting (FACS), clonally expanded GFP+ qiPSCs generated colonies (F, G) that have maintained GFP expression long term (H).
  • FACS fluorescence activated cell sorting
  • FIG. 3 shows qiPSCs express pluripotent genes. Immunocytochemistry demonstrated that QEFs were negative for POU5F1 (A) and SOX2 (B), while qiPSCs were POU5F1 (D) and SOX2 (E) positive. Scale bars are 50 ⁇ m.
  • FIG. 4 shows that qiPSCs generate EBs that form all 3 germ layers.
  • Compact EBs were formed after culture for 6 days (A). EBs were replated for further differentiation for 2 days (B). Ectoderm (TUJ1, PAX6), endoderm (Vimentin) and mesoderm (Brachyury) genes were expressed in EBs (C). Immunocytochemistry demonstrated that EB derived cells were positive for ectoderm (TUJ1, D), endoderm (SOX17, E) and mesoderm ( ⁇ SMA, F) proteins. Scale bars are 50 ⁇ m.
  • FIG. 5 shows directed differentiation of qiPSCs to 3 neural lineages.
  • qiPSCs were subjected to a 3 step neural differentiation process, with cells first cultured in neural derivation medium for 12 days, then in proliferation medium for 7 days, followed by continual maintenance in differentiation medium. Neurite extensions could be found after culture in differentiation medium for 48 days.
  • Neuron-like cells expressing Hu C/D+ (A) and MAP2 (B) were present after 10 days of differentiation and astrocytes (C) and oligodendrocyte (D) like cells after 23 and 39 days of differentiation respectively.
  • Scale bars in E, F and G are 50 ⁇ m.
  • Scale bars in H are 100 ⁇ m.
  • FIG. 6 shows chimeric chicken embryos derived from qiPSC.
  • GFP+ qiPSCs were incorporated in brain (A, ectoderm), eye (B, ectoderm), trachea/lung (C, endoderm), heart (D, mesoderm) and yolksac (F, extraembryonic tissue) of quail-chicken chimeric embryos.
  • PCR results demonstrated that various tissues were positive for the hPOU5F1 trasngene (E, G).
  • FIG. 7 also shows chimeric chickens derived from qiPSC
  • FIG. 8 shows the transduction efficiency in QEF.
  • QEFs were transduced with lentiviral eGFP gene constructs. Flow cytometry was carried out 48 hours after transduction. Utilizing GeneJammer, at 10 MOI 14.6% of QEFs were transduced (A) and at 20 MOI 25.3% of QEFs were transduced and (B) expressed GFP. While using TransDux, at 10 MOI 31.4% of QEFs were transduced (C) and at 20 MOI 40.5% QEFs were transduced and (D) expressed GFP.
  • FIG. 9 shows the injection of qiPSC into stage X chicken embryo.
  • qiPSCs To inject qiPSCs into chicken embryos, a single window was drilled into the shell of stage X White Leghorn chicken egg (A). qiPSC were then injected into the subgerminal cavity with a micropipette (B). Windows were sealed with hot glue and injected eggs were then transferred to incubators (C).
  • FIG. 10 shows the derivation of ciPSCs.
  • Chicken embryonic fibroblasts CEFs
  • A human stem cell factor
  • MEF feeder MEF feeder
  • Colonies of reprogrammed CEFs were observed at day 5 (B) and an enlarged view of the typical ciPSC-like colony demonstrating stem cell morphology (C).
  • the reprogrammed cells were around 50% confluent at day 7 after transduction and subjected to serial passages (D) and finally plated on Matrigel coated plates (E).
  • the ciPSCs were positive for Alkaline Phosphotase (F) and Periodic Acid Schiff's Staining (G) were also positive for pluripotent markers SSEA1 (H) and germ cell marker DDX4 (I) in immunostaining.
  • FIG. 11 shows pluripotent genes expression in ciPSCs.
  • PCR showed that human stem cell genes hNANOG, hLIN28 and hC-MYC were incorporated in ciPSCs (A).
  • Quantitative PCR showed that the chicken endogenous gene cPOUV, cSOX2, cC-MYC, Telomerase reverse transcriptase (cTERT) and chicken VASA Homologue (cVH) were significantly upregulated in ciPSC BA3 and BA4 line over the CEF (B, C, D, E, F).
  • FIG. 12 shows ciPSCs differentiated into cells from all 3 germ layer in vitro.
  • ciPSC were plated in Aggrewell plates for 24 hours and the aggregates were then harvested and cultured in suspension for 6 days (A).
  • RT-PCR revealed that the ciPSCs differentiaed into all 3 germ layers.
  • FIG. 13 shows the screening for NDV-resistant ciPSC
  • the NDV virus screening was initiated at 50 MOI using GFP-NDV, with GFP indicating successful infection.
  • An infection efficiency of >99% was seen in either CEFs (E) or ciPSCs (M).
  • CEFs were used as control cell line for virus screening (A) and they grew to confluence in the mock infection dish (B, F) while mostly died in the infected dish 48 hours after infection (C, G). The infected CEFs could not recover from the infection 9 days after infection.
  • ciPSCs plated on Matrigel dish (I) were used for NDV screening and they grew to confluence in the mock infection dish (J, N) while mostly died in the infected dish 48 hours after infection (K, O). 9 days after infection, ciPSC survived the NDV infection formed colonies and some of the cell were clear of the NDV virus.
  • FIG. 14 shows the established ciPSC line demonstrates significant resistance to NDV.
  • E infected CEF
  • F ciPSC I0
  • G ciPSC 112
  • H ciPSC 114
  • A, B, C, D mock infection control CEF
  • FIG. 15 shows that more pluripotent ciPSCs were derived by a 2nd round of reprogramming.
  • the ciPSCs are positive for OCT4, SOX2, NANOG and SSEA1 after a 2 nd round of reprogramming ( FIG. 6A-D ).
  • the SSEA1+ciPSCs was enriched by MACS sorting and additional selection by manual picking yielded a population of ciPSCs >82% positive for SSEA1 ( FIG. 15E-G ).
  • FIG. 16 shows the generation of chicken induced pluripotent stem cells (ciPSCs) from CEFs.
  • ciPSCs chicken induced pluripotent stem cells
  • A CEFs prior to addition of reprogramming factors
  • B GFP expressed 72 hours post-transfection
  • C Transfection efficiency comparison between different transfection reagents
  • D Lipofectamine showed highest transfection efficiency.
  • FIG. 17 shows the typical ciPSC colony formed in culture.
  • A Reprogrammed CEFs showed defined borders at day 20 post-transfection
  • B Typical ciPSC colonies were present in culture.
  • C colony formation number counted and comparison between 4 different transfection reagents. *P ⁇ 0.05 between 4 groups using different transfection reagents.
  • FIG. 18 shows that the rate of doubling the population of ciPSCs was only slightly greater than the doubling time for the somatic cell from the ciPSCs were derived.
  • FIG. 19 shows that ciPSCs injected into gonads evidenced migration consistent with formation of gametes.
  • FIG. 20 shows the viPSTM vector kit lentiviral vector which expresses human Oct4 (POU5F1, Sox2, Nanog, Klf4, c-Myc, and Lin28 under control of the human elongation factor-1 alpha promoter (EF1 ⁇ ).
  • EF1 ⁇ human elongation factor-1 alpha promoter
  • FIG. 21 shows that reprogramming of CEF into cPSC requires 5 factors and cKO medium for stable propagation.
  • Chicken embryonic fibroblasts were transduced with human stem cell factors and replated in cKSR medium and cKO medium formed cPSC colonies in 7 days (A), but cKO medium was required to establish stable cPSC lines.
  • Incorporation of reprogramming factors varied in the different lines (B), but only the 5-factor line was immortal (>40 passages).
  • the established cPSCs were positive for AP and PAS (C).
  • the five factor cells were positive for pluripotent markers POU5F1, SOX2 and SSEA1 (D).
  • FIG. 22 shows the reactivation of endogenous pluripotent genes in cPSCs.
  • Quantitative PCR showed that the endogenous pluripotent genes PouV, Sox2, Nanog, Lin28 and Tert were highly up-regulated, while c-Myc was down regulated in cPSCs. This indicates a significant difference relative to CEFs: # P ⁇ 0.01.
  • A RT-PCR revealed that endogenous c-Myc and Klf4 were expressed in the parent CEF cells (B).
  • FIG. 23 shows that the cPSCs express germ cell markers.
  • Quantitative PCR by using PGCs as positive control and CEF as negative control revealed that the germ cell related genes Cvh, Dazl and c-Kit were up regulated in cPSCs compared to CEFs. This indicates a significant difference relative to CEFs: #P ⁇ 0.01.
  • A Immunocytochemistry demonstrated that germ cell related markers EMA1, DAZL and CVH were highly positive at the protein level
  • B Flow cytometry confirmed that 65.0% of the ciPSCs are positive for EMA1, 92.0% for DAZL and 93.4% for CVH (C).
  • Scale bar B 50 ⁇ m.
  • FIG. 24 shows that iPGCs are capable of differentiation into all 3 germ layers.
  • iPGCs formed compact EBs after suspension culture for 6 days in differentiation medium (A).
  • Expression of mesoderm (Me), ectoderm (Ec) and endoderm (En) genes were detected in the EBs using RT-PCR (B).
  • B After replating of EBs and differentiation for an additional 3 days, cells positive for endoderm (SOX17), mesoderm ( ⁇ SMA) and ectoderm (Hu C/D and SOX1) were detected by immunocytochemistry (C).
  • FIG. 25 shows that iPGCs are capable of migrating to the embryonic gonad.
  • Immunocytochemistry showed that CEFs were CXCR4 negative while both PGCs and iPGCs were positive (A). Flow cytometry confirmed that 51% of the ciPSCs were CXCR4 positive.
  • B Cells labeled with PKH26 (red) were injected into vasculature system of stage 15 chicken embryos and the embryonic gonads were isolated 6 days after injection. No PKH26 positive CEFs were found in the embryonic gonad, while significant migration was found in embryos injected with PGCs and iPGCs (C). A high-magnification view of the boxed area in FIG. 5C showed significant cells present in the embryonic gonad injected with iPGCs ( FIG. 5D ). Scale bar A: 50 ⁇ m; C: 400 ⁇ m.
  • the present invention relates to a method of inducing embryonic and/or mature avian, in particular, quail or chicken, somatic cells, including fibroblasts, to a pluripotent stem cell state (expressing the pluripotent stem cell markers Nanog, PouV (Oct 4) and preferably also SSEA1) comprising transfecting or transducing (often transfecting) said somatic cells (which can be from any number of avian species, especially including quail and chickens, preferably avian fibroblast cells, more preferably avian embryonic fibroblast cells) with at least three reprogramming genes consisting of Nanog, Lin28 and c-Myc and optionally, at least one additional reprogramming gene selected from the group consisting of Oct 4 (POU5F1 or PouV), SOX2 and KLF4 such that the somatic cell, after reprogramming, exhibits characteristics of an avian induced pluripotent stem cell (aiPSC).
  • aiPSC avian induced
  • the present invention relates to a method of inducing embryonic and/or mature avian, in particular, chicken or quail, among other somatic cells, including fibroblasts, to a pluripotent germ cell state (expressing the pluripotent germ markers cell markers Nanog, PouV (Oct 4) and SSEA1, as well as the germ cell markers DAZL, CVH, EMA1 and CXCR4 as well as C-KIT)) comprising transfecting or transducing (often transfecting) said somatic cells (which can be from any number of avian species, especially including chicken and quail, preferably avian fibroblast cells, more preferably avian embryonic fibroblast cells) with at least four reprogramming genes consisting of Pou5F1 (PouV or Oct4), Nanog, SOX2 and Lin28 and optionally, c-Myc such that the somatic cell, after reprogramming, exhibits characteristics of an avian induced pluripotent germ cell (aiPSC
  • At least one reprogramming vector population expressing the reprogramming genes is used to reprogram the cells, preferably more than one reprogramming vector population is used, preferably as many reprogramming vector populations as there are reprogramming genes to be transfected or transduced are used such that each reprogramming vector population comprises no more than one reprogramming gene (e.g., Nanog, Lin28, c-Myc, Oct4 (POU5F1 or PouV), SOX2, KLF4, depending on whether the desired pluripotent cell to be produced is a pluripotent stem cell or a pluripotent germ cell).
  • reprogramming vector population e.g., Nanog, Lin28, c-Myc, Oct4 (POU5F1 or PouV), SOX2, KLF4, depending on whether the desired pluripotent cell to be produced is a pluripotent stem cell or a pluripotent germ cell.
  • the reprogramming vector is a non-integrating or integrating vector preferably selected from the group consisting of a circle DNA vector (episomal DNA), a lentiviral vector, an inducible lentiviral vector or a retroviral vector, preferably, a circle DNA vector or a lentiviral vector, and each vector preferably comprises only one reprogramming gene per vector).
  • the somatic cells are transfected/transduced with the reprogramming vectors and reprogrammed under conditions wherein said reprogramming genes optionally integrate with the genome of the somatic cells, thus producing avian induced pluripotent stem cells (aiPSCs) according to the present invention.
  • aiPSCs avian induced pluripotent stem cells
  • the somatic cells are thus converted into an induced pluripotent stem cell (iPSC) state, which closely resembles embryonic stem cells (as evidenced by their expressed biomarkers, as well as their differentiation capacity and other physical and functional characteristics), or the somatic cells are converted into inducted pluripotent germ cells (iPGCs), which closely resemble naturally occuring pluripotent germ cells.
  • iPSC induced pluripotent stem cell
  • the induced pluripotent cells may be expanded/propagated in nutrient medium (e.g., stem cell expansion medium, germ cell explanation medium or related enriched culture medium), optionally stored (e.g.
  • cryopreservation used in differentiation processes to produce differentiated cells such as neural cells or muscle cells or gamete cells as in the case of aiPGCs or to genetically manipulate phenotypic changes in an avian population, preferably including chickens.
  • the avian e.g. chicken or quail
  • iPSCs induced pluripotent stem cells
  • iPGCs pluripotent germ cells
  • Genes can be introduced into either the somatic cells which are used to derive iPSCs or iPGCs or directly into iPSCs or iPGCs which have been produced. These cells can then be utilized in a variety of differentiation methods to form differentiated cells such as neural cells or other differentiated cells or to form chimeras (e.g.
  • iPSCs genetically manipulated iPSCs or iPGCs into embryos
  • iPGCs these cells can be used also to differentiate into gametes, which can be used to produce or generate offspring with the modified phenotype.
  • the phenotypes could include, but are not limited to, animals that exhibit improved production and disease resistance (in particular, Avian flu and/or Newcastle disease) or the development of animals to study specific diseases and/or injury.
  • Another aspect of the invention relates to chimeric embryos which are produced by injecting aiPSCs or aiPGCs into avian embryos (generally contained within eggs) in an effective amount, and allowing the chimeric embryo to grow to maturity.
  • the resulting matured embryos exhibit phenotypic characteristics of the wild-type or native embryo as well as those of the aiPSCs or aiPGCs.
  • chimeric embryos and/or mature chimeric chickens are produced by injecting an effective population of aiPSCs or aiPGCs (generally from about 1,000 to 50,000 or more, preferably about 8500-15,000, about 10,000 cells) into an avian, preferably chicken embryo in an egg shell anytime during stage X to XV, preferably at stage X soon after incubation begins (preferably within the first few hours of incubation), by providing an opening in the shell and injecting the cells (preferably, those cells exhibiting disease resistance) into the subgerminal cavity of an avian, preferably chicken egg.
  • aiPSCs or aiPGCs generally from about 1,000 to 50,000 or more, preferably about 8500-15,000, about 10,000 cells
  • the chimeric embryo produced by injecting the aiPSCs/aiPGCs into the embryo is allowed to grow and mature, thus producing a chimeric embryo soon after injection (generally, between two and tens days after injection, depending upon the stage X-XV when the aiPSC/aiPGC is injected into the embryo, exhibiting at least in part, the characteristics of the injected aiPSCs/aiPGCs.
  • the resulting chimeric embryo may be grown to maturity producing chimeric adults exhibiting characteristics of the aiPSCs/aiPGCs, including resistance to disease, other desired characteristics or they may be used in vaccine production or research.
  • the aiPSCs/aiPGCs can be used to produce a chimeric germline cell, by introducing the aiPSCs/aiPGCs into a germline cell population, thus producing a chimeric germline cell, which can mature into gametes such that any selected trait which is introduced into the aiPSCs/aiPGCs, including disease resistance, may be transmitted to offspring through the gamete.
  • the aiPSCs/aiPGCs (which may advantageously exhibit advantageous selected traits such as disease resistance) are injected into the reproductive organs, including the testes or ovaries of an adult bird and in the environment of the testes or ovaries, differentiate into sperm cells or ova.
  • aiPSCs/aiPGCs are selected for disease resistance (Cellular Adaptive Resistance or CAR) by exposing the na ⁇ ve cells (i.e., those cells not exposed to a disease agent), generally about 10 ⁇ 10 9 or more cells) to a disease agent for which resistance as a trait is desired (e.g. Newcastle disease, Avian influenza) in a growth medium for a period sufficient to induce cell death in a large majority of cells (often approaching 100% of the na ⁇ ve cells), generally at least about 48 hours to about 72 hours, preferably at least about 72 hours where almost all cells (generally, greater than 99%, and often close to 100% of the na ⁇ ve cells) die from infection.
  • CAR Cellular Adaptive Resistance
  • the resistant cells which are isolated may be reprogrammed pursuant to methods according to the present invention to increase the number of aiPSCS/aiPGCs in the resistant cell population.
  • species specific cell lines offer potential for species specific vaccines
  • these resistant cells may be particularly useful for providing a better host for species specific vaccines, especially where vaccines could be generated from a specific genetic or tissue specific genetic or epigenetic background that is shown to be a better host for the production of vaccines, for example, an animal infected with a virus, but cells from it demonstrate resistance to cell death.
  • the present invention is directed to a cell viability assay for identifying cells which are viable and resistant after exposure to a disease agent as described above.
  • a measurement of metabolic activity of the cells exposed to disease agent after an appropriate time e.g.
  • viable resistant aiPSCs/aiPGCs demonstrate metabolic activity which is at least about 10 times, at least about 20 times, at least about 25 times, at least about 30 times and at least about 35-40 times the metabolic activity of na ⁇ ve cells after exposure to ND virus which are not resistant.
  • a method referred to as the aiPSC/aiPGC virus exposure survival and recovery assay is used to determine the resistance of cells in a sample.
  • a sample of resistant aiPSCs or resistant aiPGCs which have been exposed previously to a disease agent (e.g. ND virus) is plated, exposed to the disease agent and then measured to determine viability.
  • Resistant cells evidence higher cell numbers at earlier points in time and a more rapid increase in cell numbers over time at later points in time evidencing a faster recovery rate in comparison to na ⁇ ve cells which have not been exposed to the disease agent.
  • resistant aiPSCs/aiPGCs demonstrated a higher survival than na ⁇ ve aiPSCs/aiPGCs at early (about 12-14 fold or more higher than na ⁇ ve aiPSCs/aiPGCs) and later (20 to 26 fold or more high than na ⁇ ve aiPSCs/aiPGCs) time points, evidencing a resistant population of aiPSCs/aiPGCs which exhibits increased survivability and rapid recovery of resistant cell lines.
  • the present invention is directed to a method for producing avian induced pluripotent stem cells comprising providing a population of somatic cells, preferably quail, chicken, turkey, duck or geese somatic cells (including fibroblast cells, especially embryonic fibroblast cells), to be reprogrammed to pluripotent stem cells (PSCs) or pluripotent germ cells (PGCs), transfecting or transducing these somatic cells with at least one population of reprogramming vectors (preferably more than one reprogramming vector) comprising reprogrammaing genes selected from at least Nanog, Lin28 and c-Myc and optionally one or more of Oct4 (POU5F1 or PouV), SOX2 and KLF4 in the case of PSCs, or Oct4 (Pou5F1 or PouV), SOX2, Nanog and LIN28, and optionally c-MYC in the case of PGCs, allowing said reprogramming genes to reprogram said somatic cells into pluripotent
  • somatic cells
  • the present invention is directed to embodiments related to one or more of the following specific embodiments of the invention, among others.
  • a population of avian induced pluripotent stem cells reprogrammed from avian somatic cells said somatic cells being reprogrammed with at least the three reprogramming genes Nanog, LIN28 and c-MYC and optionally, at least one reprogramming gene selected from the group consisting of OCT4, SOX2 and KLF4.
  • somatic cells are embryonic somatic cells, preferably, embryonic fibroblast cells.
  • the population of cells above wherein the somatic cells are adult somatic cells.
  • the population of cells above wherein the somatic cells are adult fibroblast cells.
  • the population of cells above wherein the reprogramming genes are avian or mammalian genes each of which has a sequence homology of at least about 50% of the identical gene of the avian species from which the somatic cells are reprogrammed.
  • the population of cells above wherein the reprogramming genes are chicken, mouse, human, pig or cow genes.
  • the population of cells above wherein the avian induced pluripotent cell is a quail, chicken, turkey, duck or goose pluripotent cell.
  • the chimeric bird described above which is a quail, chicken, turkey, duck or goose, often a chicken or turkey, most often a chicken.
  • the chimeric bird described above which exhibits resistance to a disease agent, including Newcastle disease virus or avian influenza virus.
  • the bird described above which is a quail, chicken, turkey, duck or goose, often a chicken or turkey, most often a chicken.
  • the bird described above which exhibits resistance to a disease agent, including Newcastle disease virus or avian influenza virus.
  • a method comprising reprogramming an avian embryonic or adult somatic cell to an avian induced pluripotent stem cell (aiPSC) or an avian induced pluripotent germ cell (aiPGC), the method (in the case of aiPSCs) comprising transfecting or transducing the somatic cell with the three reprogramming genes Nanog, Lin28 and c-Myc and optionally, at least one reprogramming gene selected from the group consisting of Oct 4 (Pou5F1/PouV), SOX2 and KLF4, or (in the case of aiPGCs) with the four reprogramming genes Oct 4 (Pou5F1/PouV), SOX2, Nanog, LIN28 and optionally c-MYC, allowing the genes to reprogram the somatic cells into pluripotent stem cells or pluripotent germ cells and optionally, isolating and/or expanding the stem or germ cells, wherein each of the reprogramming genes is an avian or mamm
  • avian somatic cell is reprogrammed with the reprogramming genes Nanog, LIN28, c-MYC, Oct4, SOX2 and KLF4 to produce avian induced pluripotent stem cells (aiPSCs).
  • avian somatic cell is reprogrammed with the reprogramming genes Oct4, SOX2, Nanog, LIN28 and c-MYC to produce avian induced pluripotrent germ cells (aiPGCs).
  • reprogramming genes are human, chicken, mouse, pig or cow genes.
  • somatic cells are embryonic somatic cells, often embryonic fibroblast cells or adult somatic cells, including adult fibroblast cells.
  • somatic cells are reprogrammed by transfecting or transducing the cells with at least one vector comprising the reprogramming genes.
  • the vector is a viral vector and the reprogramming genes integrate into the genome of the somatic cell to produce the induced pluripotent stem cell.
  • the vector is a non-viral vector and the reprogramming genes are non-integrated with the genome of the somatic cells to produce the induced pluripotent stem cell.
  • the avian induced pluripotent cell is a quail, chicken, turkey, duck or goose pluripotent cell, often a chicken pluripotent cell.
  • a method of genetically manipulating avian species, especially chickens comprising selecting and introducing genes into somatic cells to be reprogrammed into avian iPSCs or aiPGCs or directly into avian aiPSCs or aiPGCs or to produce genetically manipulated aiPSCs or aiPGCs, introducing the genetically manipulated avian aiPSCs cells or aiPGCs into avian embryos to form chimeric embryos; and generating offspring with the chimeric embryos, wherein said offspring exhibit a phenotype consistent with the introduced genes.
  • the embryo is a chicken, turkey or duck embryo, often a chicken embryo.
  • aiPSC avian induced pluripotent stem cell
  • aiGSC avian induced pluripotent germ cell
  • a method of instilling disease resistance into a population of avian induced pluripotent stem cells or avian induced pluripotent germ cells from disease resistant somatic cells comprising producing disease resistant somatic cells by exposing somatic cells to a disease agent for a period of time sufficient to produce resistance to the disease agent in the cells, more than 99% of the population of which succumb to the disease agent, isolating the resistant somatic cells, propagating the cells to provide a population of resistant somatic cells and producing a population of disease resistant avian induced pluripotent stem cells or avian induced pluripotent germ cells from the disease resistant somatic cells according to any of the methods described above.
  • resistant aiPGCs are allowed to mature into gametes or are differentiated into gametes which may be used to reproduce disease resistant birds.
  • a method of producing a chimeric avian gamete comprising administering into a germline cell an avian induced pluripotent stem cell or an avian induced pluripotent germ cell described above to produce a chimeric germline cell, introducing the chimeric germline cell into the testes or ovaries of an adult bird and isolated a chimeric gamete after a period sufficient to produce the chimeric gamete from the germline in the adult bird.
  • a method of producing an avian gamete cell comprising introducing an aiPGC into the testes or ovaries of an adult bird and isolating the gamete after a period sufficient to produce the gamete from the aiPGC in the adult bird.
  • a method of producing immunogenic material for a vaccine comprising providing a population of aiPSCs or aiPGCs resistant to a disease agent described above, growing the disease agent in the population of aiPSCs or aiPGCs and removing the disease agent from the population of aiPSCs or aiPGCs after said growing step.
  • the disease agent is influenza virus or Newcastle disease virus.
  • a vaccine produced with immunogenic material prepared according to the method described above.
  • aiPSCs according to the present invention exhibit a robust clonal rate of 20% (i.e. a single cell will produce a colony of cells 20% of the time). This unique feature of aiPSCs according to the present invention allows these cells to be particularly useful in studies that require multiple clonal isolation, such as complex genetic manipulation or virus screening, among others.
  • the present invention is directed to a method comprising reprogramming an avian embryonic or adult somatic cell to an avian induced pluripotent germ cell (aiPGC), the method comprising transfecting or transducing the somatic cell with the four reprogramming genes Oct4 (Pou5F1 or PouV), SOX2, Nanog and Lin28 and optionally, c-Myc, allowing the genes to reprogram the somatic cells into pluripotent germ cells (which are believed to pass through a transitory pluripotent stem cell which could not be isolated on their way to a germ cell state) and optionally, isolating and/or expanding the germ cells, wherein each of the reprogramming genes is an avian or mammalian gene which exhibits at least about 50% sequence homology with the identical gene from the species from which the somatic cells are reprogrammed.
  • aiPGC avian induced pluripotent germ cell
  • the present invention is directed to a population of avian cells consisting essentially of avian induced pluripotent germ cells having the following characteristics similar to or distinguishable from other related cells):
  • Standard techniques for growing cells, separating cells, and where relevant, cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art.
  • a number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth. Enzymol.
  • patient is used throughout the specification within context to describe an animal, generally an animal which is a member of an avian species, especially, for example, a quail, a chicken, a turkey or other commercially relavent species which is exposed to methods and/or compositions which are described herein (including vectors and/or cells) according to the present invention.
  • an animal generally an animal which is a member of an avian species, especially, for example, a quail, a chicken, a turkey or other commercially relavent species which is exposed to methods and/or compositions which are described herein (including vectors and/or cells) according to the present invention.
  • avian species especially, for example, a quail, a chicken, a turkey or other commercially relavent species which is exposed to methods and/or compositions which are described herein (including vectors and/or cells) according to the present invention.
  • patient refers to that specific animal, including numerous avian species, especially quail, chicken, turkeys, ducks and gees
  • transfect refers to a process of introducing nucleic acids into cells pursuant to the present invention.
  • transfecting is used notably for introducing non-viral DNA (generally plasmids, although naked DNA, including supercoiled naked DNA and RNA including modified mRNA and MicroRNA may also be used) into eukaryotic cells, but the term may also refer to other methods and cell types, although other terms may also be used.
  • Transfection or transduction of animal cells typically involves opening transient pores or “holes” in the cell membrane, to allow the uptake of material into the cells to be transfected.
  • Transfection is often carried out using an agent such as calcium phosphate or other agent to assist transfection into the target cell, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit cargo within the cell.
  • Transduction is a term which describes the process by which foreign DNA is introduced or transferred from one bacteriuim to another by a virus. This term also refers to the process whereby foreign DNA is introduced into another cell via a viral vector, as are preferred aspects of the present invention.
  • Transduction does not require cell-to-cell contact (which occurs in conjugation), and it is DNAase resistant, but it often is benefitted by the inclusion of a transduction factor such as GeneJammerTM or TransDuxTM transduction reagents.
  • Transduction is a relatively common tool used by those of skill to stably introduce or integrate a foreign gene into a host cell's genome.
  • somatic cell is used to describe any cell which forms the body of a multicellular organism that is other than a gamete, germ cell, gametocytes or undifferentiated stem cell.
  • gametes are cells that are involved in sexual reproduction
  • germ cells are gamete precursory cells
  • stem cells are cells that can divide (mitosis) and differentiate into a variety of cell types.
  • somatic cells make up all of the internal organs, skin, feathers, bones, blood, and connective tissue. Somatic cells are diploid.
  • any avian somatic cell may be used to induce pluripotent stem cells, but preferred somatic cells for use in the present invention include those cells which may be readily propagated, especially including fibroblast cells, including adult fibroblast cells.
  • Embryonic fibroblast cells are preferred for use in the present invention although stomach cells, liver cells, keratinocytes, amniotic cells, blood cells, adipose cells, neural cells, melanocytes, among numerous others, may also be used.
  • germ cell is used to describe a biological cell that is a pluripotent precursor cell that eventually matures into gametes of an organism that engages in sexual reproduction.
  • the germ cells originate near the gut of an embryo and migrate to the developing gonads of the embryo. There, in the gonads, they undergo cell division, which is of two types, mitosis and meiosis and these cells further differentiate into mature gametes (eggs or sperm).
  • the germ cell lines are established by signals controlled by genes in the zygotes of the animal.
  • avian induced pluripotent stem cells are derived from avian somatic cells by reprogramming the somatic cells with at least three reprogramming genes including NANOG LIN28 and c-MYC, and optionally one or more of Oct4 (POU5F1, PouV) SOX2 and KLF4 using transfection and/or transduction of the reprogramming genes in certain instances (when higher efficiency is desired) to preferably integrate the genes into the genome of the somatic cells.
  • Avian induced pluripotent stem cells include both adult and embryonic induced pluripotent stem cells, often produced from adult fibroblast cells or embryonic fibroblast cells.
  • avian induced pluripotent stem cells are adult and embryonic cells from various types of somatic cells, preferably fibroblast cells, often embryonic fibroblast cells Any cells of avian origin that are capable of producing progeny that are derivatives of all three germinal layers are included, regardless of whether they were derived from adult somatic cells, embryonic somatic tissue (e.g., embryonic fibroblasts), fetal, or other sources.
  • the aiPSCs are preferably not derived from a malignant source.
  • aiPSCs according to the present invention exhibit a robust clonal rate of 20% (i.e. a single cell will produce a colony of cells 20% of the time). This unique feature of aiPSCs according to the present invention allows these cells to be particularly useful in studies that require multiple clonal isolation, such as complex genetic manipulation or virus screening, among others.
  • aiPSC cultures are described as “undifferentiated” when a substantial proportion of the stem cells and their derivatives in the population display morphological characteristics of undifferentiated cells, clearly distinguishing them from differentiated cells of embryo or adult origin. Undifferentiated aiPSCs are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli. It is understood that colonies of undifferentiated cells in the population may often be surrounded by neighboring cells that are differentiated. “Na ⁇ ve” aiPSCs refer to aiPSCs that have not been exposed to a disease agent (such as Newcastle disease virus or avian flu virus).
  • a disease agent such as Newcastle disease virus or avian flu virus
  • Resistant aiPSCs refer to aiPSCs that have been exposed to a disease agent and are viable after such exposure compared to non-resistant aiPSCs, which exhibit substantially reduced metabolic activity compared to resistant aiPSCs in the virus exposure cell viability assay as otherwise described herein.
  • aiPSCs are distinguishable over avian embryonic pluripotent stem cells (ESCs) in the following ways:
  • a desirable phenotype of propagated aiPSCs is the high potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues.
  • Pluripotency of pluripotent stem cells which may be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers.
  • SCID severe combined immunodeficient
  • pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers, using RT-PCR as otherwise described herein.
  • Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a “normal karyotype,” which means that the cells are euploid, wherein all chromosomes are present and not noticeably altered.
  • the propagated aiPSCs may be re-exposed to the reprogramming vectors (DNA integrating or non-integrating transient expression) and selected again for pluripotent traits as provided above, especially including positive expression of the biomarker SSEA1.
  • avian induced pluripotent germ cells are derived from avian somatic cells by reprogramming the somatic cells with the reprogramming genes Oct4 (Pou5F1 or PouV), SOX2, NANOG and LIN28 and optionally c-MYC, using transfection and/or transduction of the reprogramming genes in certain instances (when higher efficiency is desired) to preferably integrate the genes into the genome of the somatic cells.
  • Avian induced pluripotent germ cells (aiPGCs) may be produced from both adult and embryonic somatic cells, often from adult or embryonic fibroblast cells, more often embryonic fibroblast cells.
  • avian induced pluripotent germ cells are aiPGCs produced from various types of somatic cells, preferably fibroblast cells. Any cells of avian origin that are capable of producing gametes are included, regardless of whether they were derived from adult somatic cells, embryonic somatic tissue (e.g., embryonic fibroblasts), fetal, or other sources.
  • the aiPGCs are preferably not derived from a malignant source.
  • chimera refers to cells and/or embryos in which aiPSCs according to the present invention have been introduced.
  • aiPSCs may be injected into avian embryos to instill those embryos with certain favorable characteristics which may introduced into the aiPSCs, including resistance to disease agents such as Newcastle disease virus, avian influenza virus or other traits.
  • aiPSCs may be introduced into an embryo preferably at embryonic Stage X to XV (i.e., from the point that an egg is freshly laid (about 20 hours uterine age) to about 6-8 hours of that time, although embryos of greater maturity may also be manipulated to produce a chimeric offspring.
  • a germline chimera may be produced (introduction of the aipSCS into germlines cells to produce germline chimeras) so that the germ cells may mature into gametes (ova or sperm) and any selected trait which is incorporated into the aiPSC may be transmitted to the offspring.
  • aiPSCs may be injected directly into the testes of a chicken, such that in that environment
  • avian embryonic stem cell refers to embryonic pluripotent cells, of avian origin, which are isolated from the blastocyst stage embryo.
  • Embryonic stem cell refers to a stem cell from an avian species and are used for comparison purposes to the avian induced pluripotent stem cells of the present invention.
  • Many of the physical and functional characteristics of aESCs are identical to aiPSCs, although there are some difference of functional significance.
  • reprogramming refers to a process pursuant to the present invention whereby avian somatic cells are supplied, tranfected or transduced with a population of reprogramming vectors comprising the reprogramming genes Nanog, LIN28 and c-MYC and optionally one or more of Oct4 (Pou5F1 or PouV), SOX2 and KLF4 in the case of aiPSCs or Oct 4 (Pou5F1), SOX2, Nanog and LIN28, and optionally C-MYC, and reprogrammed such that each of the genes which are transfected or transduced into the somatic cells is incorporated into cell and preferably integrated with the somatic cell genome, whereby the reprogrammed genes instill characteristics in the somatic cells to produce a population of avian induced pluripotent stem cells (aiPSCs) or avian induced pluripotent germ cells (aiPSCs).
  • aiPSCs avian induced pluripotent stem cells
  • the term reprogramming is used generically herein to describe a process by which a somatic cell is induced to become a pluripotent stem cell (PSC) or pluripotent germ cell (PGC) pursuant to the present invention.
  • the reprogramming genes integrate into the genome of the somatic cells, but integration is not required to induce the somatic cells to a pluripotent stem cell state or pluripotent germ cell state, although such an approach is often efficient.
  • reprogramming genes are non-integrating and instead activate the endogenous pluripotency genes, resulting in alternative favorable characteristics (including the tendency to have much lower incidence of mutation and tumor formation).
  • reprogramming genes refers to the genes which are transfected or transduced into the avian somatic cells, are preferably integrated into the somatic cell genome and as a consequence of the expression of the induced polynucleotides reprogram or induce the somatic cells to a pluripotent stem cell state or a pluripotent germ cell state which the art refers to as an induced pluripotent stem cell or avian induced pluripotent stem cell (aiPSC) or induced plurioptent germ cell or avian induced pluripotent germ cell (aiPGC).
  • aiPSC induced plurioptent germ cell or avian induced pluripotent germ cell
  • these genes include the following six genes, the first three of which are necessary and the last three of which are optionally used.
  • Nanog, LIN28 and c-MYC are required to produce avian induced pluripotent stem cells according to the present invention and additional genes which are optional, including one or more of POU5F1 (PouV), SOX2 and KLF4. It is noted that POU5F1 and PouV are the human and chicken homologues of Oct4 (octamer-binding transcription factor 4). It is an unexpected result that reprogramming of avian (preferably including chicken) somatic cells into aiPSCs may be induced using only the three genes Nanog, LIN28 and c-MYC as described.
  • the reprogramming genes include four genes, namely, OCT4 (Pou5F1 or PouV), SOX2, Nanog and Lin28 and optionally, a fifth reprogramming gene, C-MYC.
  • OCT4 Pou5F1 or PouV
  • SOX2 SOX2
  • Nanog Nanog
  • Lin28 optionally, a fifth reprogramming gene, C-MYC.
  • C-MYC a fifth reprogramming gene
  • the genes which are employed to reprogram the avian somatic cells pursuant to the present invention have a sequence homology identity which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% up to 100% of the genes from the species from which the somatic cells to be induced to pluripotent cells are obtained and are generally from mammalian or avian species.
  • reprogramming vector is used to describe a transfection or transduction vector which may be used to reprogram a somatic cell to produce aiPSCs or aiPGCs according to the present invention.
  • Preferred reprogramming vectors for use in the present invention may be integrating or non-integrating and include viral vectors, especially integrating lentiviral vectors (see, Yu, et al., Science, 38, 1917-1920, 2007; Stadtfeld, et al., Curr.
  • Non-integrating vectors which may be preferred in certain aspects of the invention, such as adenoviral vectors (Zhou, et al., Stem Cells, 27, 2667-2674, 2009 and Stadtfeld, et al., Science, 322, 949-953, 2008), plasmid vectors (Okita, et al., Science, 322, 949-953, 2008 and Si-Tayeb, et al., BMC Dev,.
  • excisable vectors including transposon (Woltjen, et al., Nature, 458, 766-770, 2009) and loxP-lentiviral vectors (Somers, et al., Stem Cells, 28, 1728-1740, 2010) and DNA free vectors, including Sendai virus vectors (Fusaki, et al., Proc. Jpn Acad.
  • Vectors used in the present invention are well known in the art and may be purchased, readily constructed and manipulated to incorporate reprogramming genes using standing genetic engineering methods.
  • vectors are prepared using methods which are generally known in the art.
  • a number of reprogramming vectors are available commercially, from establishments including the viPSTM Vector Kit (integrating), developed by ArunA Biomedical Inc., Athens, Ga., and made available commercially by Thermo Fisher Scientific, Inc., Waltham Massachussets, USA or Minicircle DNATM (non-integrating episomal DNA vectors) available from System Biosciences, LLC, Mountainview Calif., USA), the PiggyBacTM Mouse 4-in-1 Transposon Vector (non-integrating), also available from System Biosciences, LLC.
  • Non-integrating modified mRNAs of the reprogramming genes e.g., Stealth ExpressTM exogenous gene expression system, available from Allele Biotechnology, San Diego, Calif., USA
  • reprogramming genes e.g., Stealth ExpressTM exogenous gene expression system, available from Allele Biotechnology, San Diego, Calif., USA
  • reprogramming genes required for reprogramming e.g., LIN28, c-Myc.
  • the viPSTM Vector Kit for reprogramming fibroblasts (or other differentiated somatic cells) into induced pluripotent stem cells was developed by Thermo Fisher Scientific, Inc. in collaboration with ArunA Biomedical, Inc.
  • Six transcription factors/genes (Lin28, c-Myc, Klf4, Nanog, Sox2 and Oct4 (Pou5f1)) have been cloned into a lentiviral vector system to create a resource for producing induced pluripotent stem (iPS) cells allowing for the generation of patient- and disease-specific cells.
  • iPS induced pluripotent stem
  • Ectopic expression of these factors has been shown to create pluripotent cells which resemble embryonic stem (ESCs) cells.
  • the set of six factors is available in high-titer virus, ready for transduction.
  • vectors each of which expresses a single reprogramming gene are preferably used in the present invention. These vectors are commercially available from Thermo Fisher Scientific, Inc. or Systems Biosciences, LLC, as well as other sources and alternatively, may be readily prepared by inserting the relevant DNA sequence into the vector, the sequence of which genes are available online at Genebank.
  • the viPSTM vector kit lentiviral vectors express human Oct4 (POU5F1), Sox2, Nanog, Klf4, c-Myc, and/or Lin28 under control of the human elongation factor-1 alpha promoter (EF1 ⁇ ) or alternatively a CMV, SV40, UBC, PGK and/or CAGG promoter.
  • Vectors are generated by PCR amplifying each pluripotency factor open reading frame (ORF) followed by direct cloning downstream of the EF1 ⁇ promoter. Endonuclease restriction sites placed within the PCR primers are used to facilitate cloning. A Kozak consensus sequence may also be included to ensure efficient translation.
  • Minicircles are episomal DNA vectors that are produced as circular expression cassettes devoid of any bacterial plasmid DNA backbone. Their smaller molecular size enables more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days. They are non-integrating.
  • Non-integrating or footprint free transfection of reprogramming genes using mRNA (transposon vectors or cDNA expression vectors (minicircles) or even non-integrating virus such as Sendi virus or adenovirus, described above, may often be used to generate aiPSCs or aiPGCs according to the present invention because these systems are less likely to induce a mutation (and possibly a tumor in the resulting chimeric bird) because the reprogramming genes do not integrate into the somatic cell genome using these methods of transfection/transduction.
  • the gene cannot be turned on later (as with integrated genes) and induce tumors, which may be preferably in certain instances.
  • the integrating vectors described above lentivirus, retrovirus, inducible retrovirus and additionally, adenoassociate virus AAV vectors
  • the cDNAs used for PCR are obtained from the Human Pluripotency Tool KitTM (Catalogue number PPK4919), which consists of: Oct4 (Pou5f1) (NM — 002701, Incyte clone ID:LIFESEQ259583); Sox2 (NM — 003106. IMAGE clone ID:2823424); Nanog (NM — 024865.
  • Klf4 NM — 004235, IMAGE clone ID: 5111134
  • c-Myc NM — 002467, IMAGE clone ID: 2985844
  • Lin28 NM — 024674, IMAGE clone ID: 841184.
  • alternative reprogramming genes from other mammalian and/or avian species, including chickens, these are obtained by nucleotide synthesis of the gene sequences for each of the identified reprogramming genes. Examples include the following genes listed in the table below:
  • One of ordinary skill can identify the various reprogramming genes from a variety of avian and/or mammalian species with the requisite homology, synthesize the cDNA's in each instance and incorporate the cDNA's into reprogramming vectors as otherwise described herein to transfect/transduce avian somatic cells and provide aiPSCs according to the present invention.
  • the preferred lentiviral vector used in the present invention also contains a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and the pput/cPPT (CTS, DNA flap) sequence.
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • CTS pput/cPPT
  • the pVIPS lentiviral vector is self-inactivating (SIN).
  • iPSCs induced pluripotent stem cells
  • iPGCs pluripotent germ cells
  • treat refers to any action providing or imparting a benefit to a subject at risk for or afflicted by a disease state, condition or deficiency which may be improved using cellular compositions according to the present invention.
  • Treating a condition includes improving the condition through lessening or suppression of at least one symptom, delay in progression of the effects of the disease state or condition, including the prevention or delay in the onset of effects of the disease state or condition, etc,.
  • Treatment encompasses both prophylactic and therapeutic treatment and especially includes instilling resistance to disease agents such as Newcastle disease virus or avian (bird) influenza.
  • the term “differentiation” is used to describe a process wherein an unspecialized (“uncommitted”) or less specialized cell acquires the features of a more specialized cell such as, for example, a neural cell, a muscle cell, a cardiomyocite or other somatic cell.
  • the term “differentiated” includes the process wherein a pluripotent stem cell according to the present invention becomes a more specialized intermediate cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell.
  • “De-differentiation” refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell.
  • the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
  • aiPSCs may be used to differentiate into a large number of somatic cells, in the same manner that ESCs may be differentiated.
  • aiPGCs these may be used to produce gamete cells after migrating to gonads in an embryo, or alternatively, through differentiation processes to gamete cells.
  • cell growth medium As used herein, the terms “cell growth medium”, “cell propagating medium”, “cell transfecting medium”, “cell reprogramming medium” and “differentiation medium” are all used to describe a cellular growth medium in which (depending upon the additional components used) the somatic cells, aiPSCs or aiPGCs are grown and/or propagated, transfected and/or reprogrammed or instilled with resistance to a disease agent (e.g. Newcastle disease virus and/or avian influenza). Specific examples of these are presented in the examples section which follows.
  • a disease agent e.g. Newcastle disease virus and/or avian influenza
  • the somatic cells are generally maintained and/or propagated in a minimum essential medium such as a basic cellular medium (basic salt solution) which includes one or more components such as ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (where indicated and not excluded), and other agents well known in the art and as otherwise described herein.
  • a useful cell growth medium is Dulbecco's modified Eagle's medium (DMEM) with greater than 0.1% bovine serum albumen (Sigma), 5% CO 2 at 37° C.
  • a minimum essential medium with additional components such as the preferred medium Dulbecco's modified Eagle's medium (DMEM) high glucose (Hyclone) with 10% fetal bovine serum (Hyclone), 4 mM L-glutamine (Gibco) and 50 U/ml penicllin and 50 mg/mL streptomycin (Gibco), 4 ng/ml of basic FGF in 5% CO 2 at 37° C.
  • DMEM Dulbecco's modified Eagle's medium
  • Hyclone high glucose
  • Hyclone fetal bovine serum
  • 4 ng/ml of basic FGF in 5% CO 2 at 37° C. may be used.
  • cells can be expanded in 10% knockout DMEM conditioned by exposure to buffalo rat liver cells for 24 hours, 7.5% fetal bovine serum, 2.5% chicken serum, 1% glutamax, 2% GS nucleoside supplement, 1% antibiotic, 2-mecaptoethanol, 4 ng/ml of basic FGF and 6 ng/ml of stem cell factor.
  • Alternative media may also be used but may not be as effective as the above described media.
  • Exemplary preferred media for maintaining or expanding somatic cells is a cell expansion media which is based upon Dulbecco's Modified Eagle's Medium (DMEM) High Glucose (*2): General Range 50%-95%, preferred range of about 80%-90%; more preferably 89% KO-DMEM is used; however minimal essential medium (MEM) is sufficient and these media are further supplemented with Fetal Bovine. Serum (FBS) in the range of 0%-30%, with a preferred range of about 5%-20%. In most instances, about 10% FBS is used to supplement the somatic maintenance/expansion medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM minimal essential medium
  • L-glutamine within the general range of about 0.1-15 mM and preferred range of about 1-8 mM; most most about 4 mM L-glutamine is included; and penicillin/streptomycin: within a general range of about 0%-10%, a preferred range of about 0.5%-5% and a more preferred amount of about 1%.
  • penicillin/streptomycin within a general range of about 0%-10%, a preferred range of about 0.5%-5% and a more preferred amount of about 1%.
  • Reprogramming of the somatic cells to aiPSCs or aiPGCs should be done in an enriched culture medium (“reprogramming medium”).
  • a minimum medium is (Dulbecco's modified Eagle medium (DMEM)/F12 (Gibco) or other similar minimum essential medium.
  • DMEM Dulbecco's modified Eagle medium
  • F12 Gibco
  • a protein source is required to be added to the medium to assist in the reprogramming of the somatic cells, but the amount of protein required can range from 0.1% to 20%. Additional components such as knockout serum replacer (KSR) may also be preferably included.
  • KSR knockout serum replacer
  • bFGF at effective concentrations is added as well.
  • a preferred reprogramming medium is [DMEM/F12 (Gibco), supplemented with 20% knockout serum replacer (KSR; Gibco), 2 mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 50 U/mL penicillin/50 mg/mL streptomycin (Gibco), 0.1 mM b-mercaptoethanol (Sigma-Aldrich), and 10 ng/mL basic fibroblast growth factor (bFGF; Sigma-Aldrich and R&D Systems)].
  • growth factor rich medium which can be used as a reprogramming medium includes a medium such as mTeSR1 medium (from Stemcell Technologies), but the reprogramming media, to be maximally effective must contain some protein source in effective amounts. Media may be pre-conditioned (e.g. 1 day in MEF (inactivated mouse embryonic fibroblasts) before use.
  • MEF inactivated mouse embryonic fibroblasts
  • An alternative reprogramming medium for production of aiPGCs is cKO medium (KO-DMEM, Invitrogen) containing 4 ng/ml bFGF, 7.5% defined FBS, 2.5% chicken serum (Sigma), 1 ⁇ Pen/Strep, 1 ⁇ GlutaMAX (GIBCO), 1 ⁇ GS nucleoside supplement (Millipore) and 0.1 mM b-mercaptoethanol, with 10% of the KO-DMEM preconditioned in BRL (Buffalo rat liver) cells (ATCC) for 3 days before use.
  • BRL Breast Reprogramming medium
  • a preferred media which is employed is a KO-DMEM (knockout dulbecco's minimal essential medium): within a general range of about 50%-90%, a preferred range of about 60%-80%, and a most preferred amount of about 75-80% (especially 75.8%), which includes Basic Fibroblast Growth Factor (bFGF) in a general range of about 0-100 ng/mL, a preferred range of about 2-50 ng/mL; and a more preferred amount of about 4 ng/mL, Fetal Bovine Serum (FBS) within a general range of about 5%-30%, a preferred range of about 6%-20%; and a most preferred amount of about 7.5% of the final medium, Chicken Serum in a general range of 0%-30%, preferred range of about 1%-10% and a most preferred amount of about 2.5%, Penicillin/Streptomycin: General Range 0%-10%, Preferred Range 0.5%-5%; Most Preferred 1%; G
  • KO-DMEM preconditioned in buffalo rat liver (BRL) cells or equivalent such as chicken embryonic fibroblast is further added to the medium at a range of about 0%-30%, a preferred range of about 5%-20%, and a most preferred amount of about 10% of the final volume of the media used.
  • BBL buffalo rat liver
  • Induced pluripotent cells are generated and propagated in tissue culture plates.
  • Cells can be in suspension or adherent with or without any additional extracellular matrix or feeder layer.
  • a matrix like Matrigel or other is used and is adherent to the matrix.
  • Most optimal is use of a mouse embryonic fibroblast feeder layer, although the cells may be grown feeder free.
  • Cells are propagated to the desired cell number and express Avian pluripotent markers as otherwise described herein. At a minimum, cells should display a high nucleus to cytoplasmic ratio and large nucleoli. Cells should also express alkaline phosphatase and be positive for the periodic acid shift assay. At a higher level of pluripotency of stem cells, the cells should be positive for the pluripotency markers Sox2, Nanog and PouV, possess high levels of telomerase whereas somatic cells are largely negative or low expressers of these genes and markers. Optimally the pluripotent stem cells express the cell surface marker SSEA1.
  • Differentiation media comprise at least a minimum essential medium plus one or more optional components such as growth factors, including fibroblast growth factor (FGF), ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (where indicated and not excluded), Activin A, transferrin, beta mercaptoethanol, and other agents well known in the art and as otherwise described herein.
  • FGF fibroblast growth factor
  • Preferred media includes basal cell media which contains between 1% and 20% (preferably, about 2-10%) fetal calf serum, or for defined medium (preferred) an absence of fetal calf serum and KSR, and optionally including bovine serum albumin (about 1-5%, preferably about 2%).
  • Preferred differentiation medium is defined and is serum free.
  • agents which optionally may be added to differentiation medium according to the present invention include, for example, nicotinamide, members of TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, Activin A, nodal, serum albumin, members of the fibroblast growth factor (FGF) family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II, LR-IGF), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, betacellulin, ITS, noggin, neurite growth factor, nod
  • suitable media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM) with high glucose (Hyclone) with 10% fetal bovine serum (Hyclone), 4 mM L-glutamine (Gibco) and 50 U/ml penicillin and 50 mg/ml streptomycin, DMEM/F12 (Gibco), supplemented with 20% knockout serum replacement KSR (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 50 U/ml penicillin/50 mg/,ml streptomycin (Giobco), 0.1 mM b-mercaptoethanol (Sigma-Aldrich) and 10 ng/mL basic fibroblast growth factor (bFGF; Sigma Aldrich and R&D systems) as well as the following five (5) media, which utilized Matrigel as the substrate:
  • DMEM Dulbecco
  • KSR medium DMEM/F12 (Gibco), supplemented with 20% knockout serum replacement (KSR; Gibco), 2 mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 50 U/mL penicillin/50 mg/mL streptomycin (Gibco), 0.1 mM b mercaptoethanol (Sigma-Aldrich), and 10 ng/mL basic fibroblast growth factor (bFGF; Sigma-Aldrich and R&D Systems)];
  • TGF ⁇ 1/LIF medium DMEM/F12, supplemented with 20% KSR, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/mL penicillin/50 mg/mL streptomycin, 0.1 mM b mercaptoethanol, 0.12 ng/ml TGF ⁇ 1 (Pepro Tech), 1000 unites/ml LIF (Millipore).
  • LIF/Wnt3a medium DMEM/F12, supplemented with 20% KSR, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/mL penicillin/50 mg/mL streptomycin, 0.1 mM b mercaptoethanol, 100 ng/ml Wnt3a (R&D Systems), 1000 unites/ml LIF.
  • 2i/LIF medium DMEM/F12 supplemented with N2 (Gibco) and mix 1:1 with Neurobasal medium (Gibco) supplemented with B27 (Gibco), 1 mM L-glutamine, 0.8 ⁇ M PD0325901 (Sigma), 3 ⁇ M CHIR99021 (Selleckchem), 20 ng/ml LIF.
  • TGF ⁇ 1/activin A/nodal medium DMEM/F12, supplemented with 20% KSR, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/mL penicillin/50 mg/mL streptomycin, 0.1 mM b mercaptoethanol, 0.12 ng/ml TGF ⁇ 1 (Pepro Tech), 10 ng/ml Activin A (R& D Systems), 50 ng/ml mouse recombinant nodal (R & D systems).
  • DMEM/F12 50:50 which contains about 2% proalbumin (albumin; Millipore/Serologicals), 1 ⁇ Pen/Strep, 1 ⁇ NEAA, 1 ⁇ Trace Elements A,B, C (Mediatech), Ascorbic Acid (10-100 ng/ml, about 25-65 ng/ml, about 50 ng/ml), about 0.1 mM (0.025-0.5 mM) ⁇ -Mercaptoethanol (Gibco), about 2-10 ng/ml, about 5-9 ng/ml, about 8 ng/ml bFGF (Sigma), with additional components added depending upon the cells to which the aiPSCs are to be differentiated.
  • proalbumin albumin; Millipore/Serologicals
  • Pen/Strep 1 ⁇ Pen/Strep
  • NEAA 1 ⁇ Trace Elements A,B, C (Mediatech)
  • Ascorbic Acid 10-100 ng/ml, about 25-65 ng/ml, about 50 ng/
  • Each of the above-media may also be used to provide aiPSGCs, however, the efficiency of production and relative purity are not as significant as occurs with the use of the preferred media, described in detail hereinabove.
  • Various media which are useful in the present invention include commercially available media available from and can be supplemented with commercially available components, available from Invitrogen Corp. (GIBCO), Cell Applications, Inc. and Biological Industries, Beth HaEmek, Israel, among numerous other commercial sources, including Calbiochem.
  • at least one differentiation agent such as fibroblast growth factor (FGF), LR-IGF (an analogue of insulin-like growth factor), Heregulin and optionally, VEGF (preferably all three in effective amounts) is added to the cell media in which a PSC is cultured and differentiated into a mature differentiated cell line.
  • FGF fibroblast growth factor
  • LR-IGF an analogue of insulin-like growth factor
  • Heregulin optionally, VEGF (preferably all three in effective amounts) is added to the cell media in which a PSC is cultured and differentiated into a mature differentiated cell line.
  • VEGF preferably all three in effective amounts
  • Cell differentiation medium is essentially synonymous with basal cell medium but is used within the context of a differentiation process and includes cell differentiation agents to differentiate cells into other cells.
  • Growth/stabilizing medium is a basal cell medium which is used either before or after a reprogramming or differentiation step in order to stabilize a cell line for further use.
  • Growth/stabilizing media refers to media in which a pluripotent or other cell line is grown or cultured prior to differentiation.
  • the various cell media which are used may include essentially similar components of a basal cell medium, but are used within different contexts and may include slightly different components in order to effect the intended result of the use of the medium.
  • the inclusion of a protein in an effective amount is highly important.
  • pluripotent stem and germ cells may be cultured (preferably) on a layer of feeder cells that support the pluripotent stem cells in various ways which are described in the art.
  • pluripotent stem cells may also be cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stem cells.
  • the growth of pluripotent stem and germ cells in feeder-free culture without differentiation is often supported using a medium conditioned by culturing previously with another cell type.
  • the growth of pluripotent stem cells in feeder-free culture without differentiation may be supported using a chemically defined medium.
  • 6,642,048 discloses media that support the growth of pluripotent stem (pPS) cells in feeder-free culture, and cell lines useful for production of such media, which can be readily adapted for use in the present invention.
  • pPS pluripotent stem
  • US20070010011 discloses a chemically defined culture medium for the maintenance of pluripotent stem cells, also adaptable for use in the present invention.
  • the cells especially including the aiPSCs and aiPGCs may be grown on a cellular support or matrix, as adherent monolayers, rather than as embryoid bodies or in suspension.
  • Matrigel as a cellular support is preferred.
  • Cellular supports preferably comprise at least one reprogramming or substrate protein.
  • reprogramming protein or “substrate protein” is used to describe a protein, including a matrix protein, which is used to grow and/or propagate cells and/or to promote reprogramming of a somatic cell into a pluripotent stem cell.
  • Reprogramming proteins which may be used in the present invention include, for example, an extracellular matrix protein, which is a protein found in the extracellular matrix, such as laminin, tenascin, thrombospondin, and mixtures thereof, which exhibit growth promoting and contain domains with homology to epidermal growth factor (EGF) and exhibit growth promoting and differentiation activity.
  • Other reprogramming proteins which may be used in the present invention include for example, collagen, fibronectin, vibronectin, polylysine, polyornithine and mixtures thereof.
  • gels and other materials such as methylcellulose of other gels which contain effective concentrations of one or more of these reprogramming proteins may also be used.
  • Exemplary reprogramming proteins or materials which include proteins which may be used to reprogram cells include, for example, BD Cell-TakTM Cell and Tissue Adhesive, BDTM FIBROGEN Human Recombinant Collagen I, BDTM FIBROGEN Human Recombinant Collagen III, BD MatrigelTM Basement Membrane Matrix, BD MatrigelTM Basement Membrane Matrix High Concentration (HC), Growth Factor Reduced (GFR) BD MatrigelTM, BDTM PuraMatrixTM Peptide Hydrogel, Collagen I, Collagen I High Concentration (HC), Collagen II (Bovine), Collagen III, Collagen IV, Collagen V, and Collagen VI, among others.
  • the preferred material for use in the present invention includes MatrigelTM.
  • BD MatrigelTM Basement Membrane Matrix Another composition/material which contains one or more proteins for use in reprogramming is BD MatrigelTM Basement Membrane Matrix. This is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins. Its major component is laminin, followed by collagen IV, heparan sulfate, proteoglycans, entactin and nidogen.
  • EHS Engelbreth-Holm-Swarm
  • the induced pluripotent stem and germ cells are generated and propagated in tissue culture plates.
  • Cells can be in suspension or adherent with or without any additional extracellular matrix (e.g. Matrigel or other matrix protein as described above) or feeder layer.
  • a matrix like Matrigel or other as described above is used and is adherent to the matrix.
  • use is made (in all instances, but preferably during reprogramming and/or propagation) of a mouse embryonic fibroblast feeder layer.
  • the pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.
  • Cells are reprogrammed and propagated to the desired cell number and express avian pluripotent biomarkers. At a minimum, cells should display a high nucleus to cytoplasmic ratio and large nucleoli. Cells should also express alkaline phosphatase and be positive for the periodic acid shift assay. At a higher level of pluripotency, aiPSCs should be positive for the pluripotency markers Sox2, Nanog and PouV, possess high levels of telomerase whereas somatic cells are largely negative or low expressers of these genes and markers. Optimally pluripotent cells express the cell surface marker SSEA1. In the case of aiPGCs, the cells are generally positive for the biomarkers OCT4 (PouV), AP, PAS, SSEA1, EMA1, Nanog, DAZL, CVH, CXCR4 and C-KIT.
  • aiPSCs and aiPGCs which are produced according to the present invention can be selected for certain in vitro traits such as expression of an added exogenous gene (transgene) or loss of a endogenous trait (knockout) which may be considered for the production of transgenic animals or a new trait such as resistance to cell death when exposed to a virus such as Newcastle virus as described in detail herein.
  • transgene an added exogenous gene
  • knockout loss of a endogenous trait
  • a method of instilling resistance to a disease agent such as Newcastle disease virus or avian influenza represent an additional embodiment according to the present invention.
  • the propagated reprogrammed cells can be re-exposed (reprogrammed) to reprogramming gene expression (DNA integrating or non integrating transient expression) and selected again for pluripotent traits as described above, including optimally SSEA1 expression.
  • the term “activate” refers to an increase in expression or upregulation of a marker such as or an upregulation of the activity of a marker associated with aiPSC or aiPGC, a chimeric cell or a differentiated cell including a neuron, muscle cell or a related cell as otherwise described herein (e.g., nanog, SSEA1, etc.).
  • the term “deactivate” generally refers to a decrease in expression or down regulation of the activity of a marker associated with a cell.
  • the term “isolated” when referring to a cell, cell line, cell culture or population of cells refers to being substantially separated from the natural source of the cells such that the cell, cell line, cell culture, or population of cells are capable of being cultured in vitro.
  • the term “isolating” is used to refer to the physical selection of one or more cells out of a group of two or more cells, wherein the cells are selected based on cell morphology and/or the expression of various markers.
  • magnetic and florescence cell sorting have been found to significantly improve upon the ability to isolate fully reprogrammed selectively. Isolated cells typically express higher levels of pluripotency markers and behave more like pluripotent stem cells with respect to expandability.
  • the term “express” refers to the transcription of a polynucleotide or translation of a polypeptide (including a marker) in or on the surface of a cell, such that levels of the molecule are measurably higher in or on a cell that expresses the molecule than they are in a cell that does not express the molecule.
  • Methods to measure the expression of a molecule are well known to those of ordinary skill in the art, and include without limitation, Northern blotting, RT-PCT, in situ hybridization, Western blotting, and immunostaining.
  • the term “markers” or “biomarkers” describe nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest.
  • differential expression means an increased level for a positive marker and a decreased level for a negative marker.
  • the detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
  • the term “contacting” or “exposing” is intended to include incubating the compound and the cell together in vitro (e.g., adding the vector or compound to cells in culture).
  • the step of contacting the cell with reprogramming or differentiation medium and one or more growth proteins or other components such as reprogramming vectors as otherwise described herein can be conducted in any suitable manner.
  • the cells may be treated in adherent culture as an adherent layer, as embryoid bodies or in suspension culture, although the use of adherent layers may be preferred because they provide an efficient process oftentimes providing reprogramming to a target cell population of high relative purity (e.g. at least 50%, 60%, 70-75%, 80% or more). It is understood that the cells contacted with the reprogramming or differentiation agent may be further treated with other cell differentiation environments to stabilize the cells, or to differentiate the cells further.
  • the term “differentiation agent” refers to any compound or molecule that induces a cell such as an aiPSC or aiPGC or other cell to partially or terminally differentiate.
  • the term “differentiation agent” as used herein includes within its scope a natural or synthetic molecule or molecules which exhibit(s) similar biological activity.
  • an effective amount of a differentiation agent is that amount which, in combination with other components, in a differentiation medium for an appropriate period of time (including sequential times when different differentiation agents are exposed to cells to be differentiated) will produce the differentiated cells desired.
  • the present invention is broadly applicable given that the avian species are a widely used developmental research and agricultural species, but gene targeting studies have been limited given an absence of robust pluripotent stem cells that can serve as a vector for these changes. Birds supply about 25-30% of the animal protein consumed in the world. Recent publications in Science and PLoS One have highlighted a number of gene based strategies (siRNA, DNA and recombinant protein) to prevent the spread of Avian Influenza (4-6). Genetically distinct disease resistant birds would in theory not require vaccination and offer the people of underdeveloped and developed countries alike animals that require reduced veterinary care and increased food source safety. However, these significant advances have several short comings that can be potentially overcome using avian induced pluripotent stem cells including:
  • avian stem cells that can be genetically manipulated, clonally isolated, easily expanded over numerous passages and can form chimeric animals, can overcome these major challenges and provide an economically advantageous approach to advantageous phenotypic modification of the animal.
  • aiPGCs may be used to produce gametes for fertilization efforts for birds for agricultural production or in instances where a species is endangered.
  • the use of these cells in promoting agricultural efficiency and in enhancing a population of endangered bird species or otherwise producing particular birds where such a need arises.
  • these cells may be used to do further genetic modification to the cells to incorporate genes for knockout models or for incorporating genes for animals studies in drug development.
  • Avian embryonic models have a long history of providing critical new insights into developmental biology including organ function (13, 14), disease progression (e.g. Pompe disease) (15), eye disorders (16, 17) and many others (18, 19).
  • the advantage that avian species have is their relative size and ease of access to the embryo for manipulation.
  • Cells and tissues including whole sections of the spinal column can be transplanted into the avian embryo and can be monitored in real time during development (20). This is not possible in mammalian species.
  • the quail-chicken chimera is an attractive and widely used model for developmental patterning and cell fate studies given that cells can be readily tracked in this model (21, 22).
  • the quail also has a short generation interval (3-4 generation per year)(23), facilitating genetic selection studies and experiments requiring multiple generational observations (24). Coupling a robust and clonal feeder free iPSC lines and derived committed cell lines or tissues with these model systems offers new opportunities to manipulate and study developmental process both in vitro and in vivo.
  • cell-based vaccine production could more easily meet “surge capacity needs” because iPSCs could be frozen and stored in advance of an epidemic or developed rapidly in response to an epidemic.
  • Cell-based vaccine production dramatically reduces the possibility for contamination and promises to be more reliable, flexible, and expandable than egg-based methods.
  • cell-based vaccine production utilizes avian iPSC cell lines that are capable of hosting a growing virus in bioprocessing cell culture vessels. The virus is introduced into the cells where it multiplies to produce a large amount of virus per iPSC. Generally the cells' outer walls are removed, harvested, purified, and inactivated. A vaccine can be produced in a matter of weeks. While other cell lines are capable of generating a vaccine.
  • the avian iPSC will produce high concentrations and thus be more economical and faster than mammalian cell lines.
  • qiPSCs The generation of qiPSCs was initiated by testing the lentiviral transduction efficiency of isolated (QEF) ( FIG. 1A ) with an eGFP reporter construct using both GeneJammer and TransDux transduction reagents. A 20 MOI transduction with Transdux resulted in the highest efficiency with 40.5% (GFP) positive cells (S. FIG. 1 ). QEFs were then transduced with the six human pluripotency genes hPOU5F1, hNANOG, hSOX2, hLIN28, hC-MYC and hKLF4 driven by the elongation factor 1-alpha (EF1- ⁇ ) promoter with each construct in individual lentiviral vectors. After 24 hrs, cells were replated on feeder cells in stem cell expansion medium.
  • FIG. 1B Colonies were observed beginning 6 days after transduction with irregular shaped borders and fibroblast-like cell morphology. These initial colonies failed to proliferate and expand indicating that these colonies were not fully reprogrammed. Potential qiPSCs were observed around 17 days after transduction and grew as compact colonies ( FIG. 1C ). The compact colonies were mechanically picked and initially replated on feeder plates in stem cell expansion medium. However, replated cells failed to proliferate and appeared apoptotic. Additional colonies were collected and replated on matrigel coated plates in mTeSR1 stem cell medium. This system supported the growth and expansion of colonies and subsequent qiPSC expansion was performed using this system.
  • qiPSC colonies were highly refractive and at the single cell level showed clear cell boarders, high nuclear to cytoplasm ratio and prominent nucleoli ( FIG. 1D , 1 E).
  • qiPSCs were strongly positive for alkaline phosphatase (AP) and periodic acid Schiff staining (PAS; FIG. 1F , 1 G).
  • AP alkaline phosphatase
  • PAS periodic acid Schiff staining
  • PCR and RT-PCR using human specific primers revealed that 5 out of 6 pluripotent stem cell factors, hPOU5F, hSOX2, hNANOG, hLIN28 and hC-MYC, were integrated and expressed in qiPSCs, while hKLF4 was not present ( FIG. 1H ).
  • qiPSCs are Highly Proliferative, Express Pluripotent Marks and are Capable of Clonal Expansion after Genetic Manipulation
  • telomere activity revealed a significant (P ⁇ 0.01) increase of >11 fold from 8.4 total product generated (TPG) in QEFs to 95.3 TPG in qiPSCs ( FIG. 2B ).
  • qiPSCs were plated in AggreWell plates for 24 hrs and then transferred to suspension culture in mTeSR1 medium for differentiation. Six days of suspension culture resulted in round and compact EBs from qiPSCs ( FIG. 4A ). EBs were collected for RNA isolation and RT-PCR or replated for additional differentiation for 2 days in stem cell expansion medium without bFGF—the removal of which will enable differentiation ( FIG. 4B ).
  • Results of RT-PCR showed expression of TUJ1 (ectoderm), PAX6 (ectoderm), Vimentin (endoderm) and Brychyury (mesoderm) in EBs, but not in qiPSCs or QEF cells ( FIG. 4C ).
  • Immunocytochemistry showed cells positive for TUJ1 (ectoderm, FIG. 4D ), SOX17 (endoderm, FIG. 4E ) and alpha smooth muscle actin ( ⁇ SMA, mesoderm, FIG. 4F ) in plated EBs.
  • qiPSCs Differentiate In Vitro Into Neuronal, Astrocytes and Oligodendrocytes
  • qiPSCs were subjected to a 3 step neural differentiation process.
  • Cells were initially cultured in neural derivation medium for 12 days, proliferation medium for 7 days and differentiation medium continuously. Immunostaining showed that these cells were positive for neural proteins Hu C/D+ and MAP2+ ( FIG. 5A , 5 B) after 10 days of differentiation. A significant number of neurite extensions were observed after differentiation.
  • Differentiated qiPSCs were found to be positive for the astrocyte and oligodendrocyte associated proteins GFAP and 04 after 23 and 39 days of differentiation, respectively, in neural differentiation medium ( FIG. 5C , 5 D).
  • GFP+ qiPSCs at passage 26 were injected into the subgerminal cavity of stage X embryos ( FIG. 10 ). Embryos were incubated for 14 or 19 days and were then dissected to determine GFP+ qiPSC incorporation into embryos. GFP+ qiPSCs were incorporated in brain ( FIG. 6A ), eye ( FIG. 6B ), trachea/lung ( FIG. 6C ), heart ( FIG. 6D ) and yolk sac ( FIG. 6F ) tissues. PCR was performed for the human POU5F1 gene used to reprogram QEFs into iPSCs to further determine qiPSC contribution in chimeric animals.
  • qiPSCs were present in tissues from the ectoderm (brain, eye and skin), endoderm (intestine, liver and lung), mesoderm (muscle and heart), extraembryonic tissue (yolk sac) and the gonad ( FIG. 6E and FIG. 9 ).
  • PCR products from qiPSCs from the yolk sac and skin were sequenced to validate that PCR primers were solely expanding the human POU5F1 sequence.
  • Results showed that brain, liver and gonad samples from two individuals were positive for the human POU5F1 reprogramming gene ( FIG. 7C ).
  • the presence of feather chimerism with early passage cells and incorporation of qiPSCs into tissue with later passages indicate that these cells retain pluripotent characteristics following long term culture and are still capable of contributing to multiple lineages following prolonged culture.
  • Induced pluripotent stem cells have been generated from numerous mammalian species (1, 2, 5-10), but never before in a non-mammalian species.
  • iPSCs the first non-mammalian iPSCs and paradoxically these avian iPSC were generated using human reprogramming factors.
  • a constitutively expressed GFP marker was introduced into qiPSCs and subclones were selected and expanded based on expression of GFP in a feeder free culture system. Therefore these qiPSC are amenable to future gene targeting, and exhibit a proliferative potential never before reported for any avian pluripotent cell lines (11, 25-27).
  • the qiPSCs show morphology consistent with previously established pluripotent stem cells at the single cell level.
  • the qiPSCs are highly positive for the stem cell markers AP, PAS, POU5F1 and SOX2 that have been previously used to characterize avian ESCs and PGCs (26, 28-31). These iPSCs are highly proliferative with a doubling time of 16.6 hr, similar to iPSCs from mouse (1) and pig (32), and have undergone over 50 passages.
  • qiPSCs generated all three germ layer cells after spontaneous EB differentiation and then using a mammalian neural directed in vitro differentiation process we generated astrocytes, oligodendrocytes and neuronal cells.
  • qiPSC contributed to fetal tissues from all 3 germ layers and extraembryonic tissues and ultimately contributed to tissues in live offspring.
  • avian ESC and PGC have generated chimeric offspring (29, 33)
  • the qiPSC differ because for the first time an avian stem cell exhibits the robust in vitro proliferative and clonal attributes needed for future gene targeted birds.
  • qiPSCs were found to significantly contribute to the brain and eye tissue when injected into stage X chicken embryos in an undifferentiated state. Upon proper signaling in vitro, qiPSCs could differentiate into a neural progenitor (TUJ1+) and all 3 lineages of neural cells: neuronal (Hu C/D+ and MAP2+), astrocyte (GFAP+) and oligodentrocyte (O4+) in vitro.
  • TUJ1+ neural progenitor
  • GFAP+ astrocyte
  • O4+ oligodentrocyte
  • qiPSCs overcome impediments inherent to avian ESC and PGC.
  • Previously quail or chicken PGCs (31, 33) and ESCs (29) contributed to chimeras when injected into embryos immediately after collection from the donor embryo or after only a few passages.
  • qiPSC still efficiently incorporated into tissues from all three germ layer in chimeric embryos at passages 26 and 45. This will further enable complex genetic manipulations like homologous recombination, multiple gene introductions, drug selection and other strategies that may require extended culture. This significantly increases the value of these cells for future developmental studies.
  • the present invention is directed to avian iPSCs which will greatly facilitate the insertion of genetic reporters and gene targeting.
  • Future studies generating cells with gene specific and multiple promoters, inducers and conditional expression systems in avian iPSCs is likely feasible; thus enhancing the research communities' capabilities when it comes to investigating cell migration and contribution in developing embryos in ova (50). Since these qiPSC and ciPSC were capable of clonal expansion after genetic modification, targeting genes of interest is potentially possible, which would facilitate research on gene function and signaling pathways underlying the development process in chimeric embryo.
  • avian iPSC derivatives such as neural cells should compliment mammalian cell transplant models for regenerative medicine (51). In total, this unique source of avian iPSC and derivative cells will provide biologist with multiple opportunities to enhance and expedite developmentally related discoveries.
  • QEFs were isolated from day 11 embryos and cultured in fibroblast medium (DMEM high glucose (Hyclone) with 10% FBS (Hyclone), 4 mM L-Glutamine (Gibco) and 50 U/ml penicillin and 50 ⁇ g/ml streptomycin (Gibco)) in 5% CO 2 at 37° C. Cells were split using 0.05% trypsin (Gibco) upon confluency. For transduction, a total of 150,000 QEF cells were plated in one well of a 12-well plate.
  • QEFs were trypsinized 24 hrs after transduction and passaged onto inactivated feeder cells in embryonic stem cell expansion medium (Dulbecco's modified Eagle medium (DMEM)/F12 (Gibco), supplemented with 20% knockout serum replacement (KSR; Gibco), 2 mM L-glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 50 U/ml penicillin/50 ⁇ g/ml streptomycin (Gibco), 0.1 mM ⁇ mercaptoethanol (Sigma-Aldrich) and 10 ng/ml bFGF (Sigma-Aldrich and R&D Systems).
  • DMEM embryonic stem cell expansion medium
  • KSR knockout serum replacement
  • 2 mM L-glutamine Gibco
  • 0.1 mM non-essential amino acids Gibco
  • 50 U/ml penicillin/50 ⁇ g/ml streptomycin Gibco
  • qiPSC was manually harvested and plated on Matrigel (BD Biosciences; diluted 1:100 in DMEM/F12) coated dishes in mTeSR1 (Stemcell Technologies) medium. qiPSCs were mechanically dissociated using glass Pasteur pipette every 4 to 5 days. For clonal expansion, qiPSCs were transduced with GFP viral vector and single cells were FACS sorted into individual wells of a 96-well plate.
  • AP staining was carried out with VECTOR Red Alkaline Phosphatase Substrate Kit (Vector Laboratories) according to the manufacturer's instructions. PAS staining was performed by 4% fixation for 5 min. PAS (Sigma-Aldrich) was added to the plate and incubated at room temperature (RT) for 5 min, followed by PBS washes. Schiff's reagent (Sigma-Aldrich) was added and incubated at RT for 15 min, followed PBS washes and then observation.
  • VECTOR Red Alkaline Phosphatase Substrate Kit Vector Laboratories
  • Embryoid bodies were formed by plating 2.0 ⁇ 10 6 qiPSCs in mTeSR1 medium and 0.1 mM Y-27632 ROCK inhibitor (Calbiochem) in AggreWell plate (Stemcell Technologies). After 24 hrs, aggregates were harvested and maintained in mTeSR1 medium for 7 days. Differentiation was assessed by RT-PCR using the primers in S. Table 2.
  • qiPSCs were sequentially cultured in neural derivation medium (DMEM/F12 supplemented with 200 mM L-Glut, 4 ng/ml bFGF and 1 ⁇ N2) for 12 days, proliferation medium (AB Medium supplement with 200 mM L-Glut, 1 ⁇ ANS and 20 ng/mL bFGF) for 7 days and then in differentiation (AB Medium supplement with 200 mM L-Glut, 1 ⁇ ANS and 10 ng/mL LIF) mediums continuously.
  • neural derivation medium DMEM/F12 supplemented with 200 mM L-Glut, 4 ng/ml bFGF and 1 ⁇ N2
  • proliferation medium AB Medium supplement with 200 mM L-Glut, 1 ⁇ ANS and 20 ng/mL bFGF
  • differentiation AB Medium supplement with 200 mM L-Glut, 1 ⁇ ANS and 10 ng/mL LIF
  • Stage X white leghorn chicken embryos were used to produce chimeras.
  • Egg shells were removed by Dremel rotary tool to make injection window ( FIG. 9A ).
  • qiPSCs were introduced into the subgerminal cavity using a glass micropipette ( FIG. 9B ) with pressure controlled microinjector (Parker Automation).
  • the window was sealed by hot glue ( FIG. 9C ) after injection and eggs were incubated at 37° C.
  • iPS cell lines were derived by using all 6 factors of OCT4, SOX2, NANOG, LIN28, KLF4 and CMYC.
  • OCT4 the first time demonstrating OCT4 is dispensable in iPSC derivation.
  • these ciPSC could be cultured on feeder free system, on plates coated with Matrigel or directly on plate without any matrix. This feature would reduce the cost of cell culture and facilitate the cell screening or vaccine production where large amount of culture are required.
  • CEFs Black australorp chickens embryonic fibroblast cells
  • DMEM media Hyclone
  • FBS Hyclone
  • 4 mM L-Glutamine Gibco
  • 50 U/ml penicillin and 50 ⁇ g/ml streptomycin Gibco
  • Cells were subcultured using 0.05% trypsin (Gibco) upon confluency.
  • CEF cells were plated in one well in a 12-well plate. After 24 hrs CEFs underwent lintiviral transduction utilizing the viPS kit (Thermo Scientific) with viruses containing the human stem cell genes POU5F1, NANOG, SOX2, LIN28, KLF4 and C-MYC under the promoter of human elongation factor-1 alpha (EF1 ⁇ ). Transduction was carried out at a multiplicity of infection (MOI) of 10 in the present of 1 ⁇ TransDux (System Biosciences).
  • MOI multiplicity of infection
  • the transduced CEFs were replated onto inactivated mouse embryonic fibroblast (MEF), fed with conditioned embryonic stem cell (ESC) expansion media (Dulbecco's modified Eagle medium (DMEM)/F12 (Gibco), supplemented with 20% knockout serum replacement (KSR; Gibco), 2 mM L-glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 50 U/ml penicillin/50 ⁇ g/ml streptomycin (Gibco), 0.1 mM ⁇ mercaptoethanol (Sigma-Aldrich) and 10 ng/ml FGF-2 (Sigma-Aldrich and R&D Systems).
  • DMEM conditioned embryonic stem cell
  • KSR knockout serum replacement
  • 2 mM L-glutamine Gibco
  • 0.1 mM non-essential amino acids Gibco
  • 50 U/ml penicillin/50 ⁇ g/ml streptomycin Gibco
  • Conditioning of the medium was done by incubating the medium on MEF for 24 hours. Upon 50% confluent, the tranduced CEFs were dissociated using 0.05% tripsin and replated on fresh MEF plates. Attrition of incorrectly reprogrammed cells was performed by serial tripsin passages until the majority of the coloies were iPSC-like cells. At the end of the attrition, the ciPSCs were manually passaged onto tissue culture plate coated with Matrigel in ESC medium. The stable ciPSC cells were culture on plate coated with Matrigel or without any matrix and passaged every 3-4 days using accutase.
  • the primary antibody used in this experiment were SSEA1 (1:20; Developmental Studies Hybridoma Bank), DDX4 (1:500, Abcam). Secondary antibodies were Alexa Fluor (Invitrogen) diluted at 1:1000.
  • Embryoid bodies were formed follow the same protocol as in quail iPSC as described in the experiments described above.
  • the EBs were harvested on day 7 and RNA was isolated to for RT-PCR to check the expression of genes from different lineages.
  • CEFs were cultured in modified DMEM ( FIG. 10A ) were transduced with 6 human stem cell factors (hOCT4, hSOX2, hNANOG, hLIN28, hKLF4 and hCMYC) constructed in lentiviral vectors.
  • the transduced CEFs were replated onto inactivated MEF plate 24 hours after transduction and feed with conditioned ESC medium. Obvious colonies were observed 5 days after transduction ( FIG. 10B ).
  • the ciPSC-like cells demonstrated typical stem cell characteristics as large nuclear and prominent nucleoli ( FIG. 10C ).
  • a pure population of ciPSCs were derived by serial passages 50% confluence ( FIG.
  • PCR using human specific primers revealed that, of the 6 human transcription factors that we used to derive the ciPSC, only hNANOG, hLIN28 and hC-MYC were incorporated in ciPSC BA3 and BA4 cell lines ( FIG. 11A ).
  • the quantitative PCR demonstrated that the endogenous OCT4, SOX2 were highly up regulated in ciPSCs comparing to the CEFs ( FIG. 11-C ), indicating the activation of the endogenous stem cell regulatory network.
  • the chicken C-MYC was expressed at a similar level in CEFs and ciPSC.
  • telomerase reverse transcriptase cTERT
  • ciPSC ciPSC
  • cVH chicken vasa homologue
  • ciPSCs Differentiation of ciPSCs was performed through embryoid body formation using AggreWell system. Around 2.0 ⁇ 10 ⁇ 6 cells were plated in one well in the Aggrewell plate thus yielded 1,000 cells per aggregate. The aggregates were cultured in ESC medium for 24 hours and then transfer to a 100 mm plate for suspension culture in ESC medium without bFGF for 7 days. Compact EBs were formed at the end of the culture ( FIG. 12A ) and RNA was isolated from the EBs. The results of RT-PCR showed the gene expression of ectoderm (NESTIN, TUJ1), mesoderm (GLUT1, LPL, PPAR ⁇ ) and endoderm (HNF1 ⁇ , HNF4 ⁇ ) was found in the EBs derived cells.
  • ectoderm NESTIN, TUJ1
  • mesoderm GLUT1, LPL, PPAR ⁇
  • HNF1 ⁇ , HNF4 ⁇ endoderm
  • CEF and ciPSCs were plated in Matrigel coated 6-well plate at a density of 1 ⁇ 10 ⁇ 6 cells per well.
  • NDV infection was performed 24 hours after plating at an MOI of 50.
  • the medium from the culture is removed and washed with PBS once before infection.
  • the virus in ESC medium containing 1 KSR for ciPSCs or in DMEM medium containing 1% FBS for CEFs Add the infection solution to cells and incubate at 39° C. in 5% CO2 for 1 hour.
  • Cell viability was assayed 48 hours after the NDV infection. The survived cells were subjected to serial rounds of virus challenging with or without passaging.
  • the transduction protocol used in the 2 nd round reprogramming is similar to the 1 st round.
  • ciPSCs are plated on Matrigel coated plate in ESC medium.
  • Cells were tranduced with 6 human transcription factors (POU5F1, NANOG, SOX2, LIN28, KLF4 and C-MYC) constructed in lentiviral vectors at an MOI of 10 in the present of 1 ⁇ TransDux (System Biosciences).
  • Transduction medium was removed 24 hours after transduction and fresh ESC medium was added and change every other day.
  • the reprogrammed ciPSCs was maintained and expanded in feeder free system and purification of the more-pluripotent ciPSCs were performed by MACS sorting using magnetic beads conjugated with anti-SSEA1 antibody (Miltenyi Biotech). The purified cells were cultured on MEF feeder for better maintaining of the pluripotency.
  • NDV used in this study was genetically modified and labeled with GFP thus the infection could be tracked by fluorescence from the infected cells.
  • Result of flow cytometry showed an infection efficiency of >99% in either CEFs ( FIG. 13E ) or ciPSCs (M).
  • CEFs and ciPSCs There was significantly cell death in CEFs and ciPSCs 48 hours after the first round of NDV infection ( FIGS. 13C , 13 G and 13 K, 13 O).
  • the survived ciPSCs recovered from the infection and formed obvious colonies around 9 days after the infection, with significant number of cells negative for GFP ( FIG. 13L , 13 P), while the CEFs remained infected and slowly proliferate or even stopped growing (FIG. 13 D, 13 H).
  • the ciPSCs resistant to NDV were subjected to 2 nd of reprogramming by transduction of human stem cell factors constructed in lentiviral vectors and transduced were expanded on feeder free culture system.
  • Cells positive for OCT4, SOX2, NANOG and SSEA1 were found 7 days after transduction ( FIG. 6A ), this pluripotent state was not seen in the 1 st round of reprogramming.
  • the stable SSEA1+ ciPSCs were finally plated on MEF layer for a better maintaining of the pluripotency.
  • the benefits of non integrating systems are the following:
  • the molecular basis of reprogramming has been revealed by exogenous expression of combinations of transcription factors described in prior examples for quail and chicken, described above. Induction of reprogramming was carried by these factors is mostly carried out by co-infection with retroviral or lentiviral vectors.
  • the main problems of the retrovirus-based method are oncogenicity and mutagenesis. Chimeric mice derived from iPSCs as well as their offspring developed tumors, probably because of reactivation of the proviral cMyc oncogene.
  • CEFs Chicken embryonic fibroblast (CEFs) from black australorp chickens were isolated from day-11 embryos and cultured in fibroblast medium [Dulbecco's modified Eagle's medium (DMEM) high glucose (Hyclone) with 10% fetal bovine serum (Hyclone), 4 mM Lglutamine (Gibco), and 50 U/mL penicillin and 50 mg/ml streptomycin (Gibco) in 5% CO 2 at 37° C. Cells were trypsinized and passaged using 0.05% trypsin (Gibco) upon reaching confluence. For transfection, a total of 1 ⁇ 106 CEF cells were plated in a 35 dish.
  • DMEM Dulbecco's modified Eagle's medium
  • Hyclone high glucose
  • Hyclone fetal bovine serum
  • Gibco 4 mM Lglutamine
  • Gibco penicillin and 50 mg/ml streptomycin
  • CEF cells were transfected with Minicircle DNA (System Biosciences) containing the four reprogramming factors POU5F1, SOX2, LIN28, NANOG and the green fluorescent protein (GFP) reporter gene all driven by the cytomegalovirus promoter using four types of transfection reagents including lipofectamine (Invitrogen), Xfect (Clontech), purefection (System Biosciences) and genejammer (Agilent Technologies).
  • 5 ⁇ g of minicircle DNA was diluted in 250 ul DMEM/F12 and mixed with transfection reagent per transfection reagent manufacturer instructions. The mixture was incubated at room temperature for 20 min. The mixture was added to the CEF drop wise.
  • the transfection reagent mixture was removed and replaced with fresh medium.
  • the CEF cells were transfected a total of 3 times in this manner every other day over a 5 day period in.
  • the CEFs were trypsinized and plated onto inactivated mouse embryonic fibroblast feeder cells in 20% KSR stem cell medium [DMEM/F12 (Gibco), supplemented with 20% knockout serum replacement (KSR; Gibco), 2 mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 50 U/mL penicillin/50 mg/mL streptomycin (Gibco), 0.1 mM b mercaptoethanol (Sigma-Aldrich), and 10 ng/mL basic fibroblast growth factor (bFGF; Sigma-Aldrich and R&D Systems)].
  • KSR knockout serum replacement
  • bco nonessential amino acids
  • bFGF basic fibroblast growth factor
  • ciPSCs were maintained on feeders and were mechanically dissociated using a glass pasteur pipette or passaged using 0.05% trypsin every 4-5 days. After 10 passages on feeder, the colonies were picked up and dissociated in 0.05% trypsin into single cells, and ciPSCs were directly passaged into feeder free conditions on Matrigel (BD Biosciences; diluted 1:100 in DMEM/F12) coated dishes in 20% KSR plus 10 ng/ml bFGF.
  • ciPS cells were plated on 6-well plates at day 1 with 1 ⁇ 10 5 cells per well.
  • Population doubling time was determined using an exponential regression curve fitting (website at doubling-time.com/compute.php).
  • telomere activity of CEFs, ciPSCs, WA09 human embryonic stem cells (hESCs) and Hela cells (positive control) were determined using TRAPeze XL Telomerase Detection Kit (Millipore) following the manufacturer's instructions. Statistical analysis was done utilizing ANOVA and Tukey pair-wise comparisons between each population with p-values ⁇ 0.05 being considered significant.
  • Emryoid bodies were formed by plating 3.6 ⁇ 10 6 ciPSCs in 20% KSR medium and 0.1 mM Y-27623 ROCK inhibitor (Calbiochem) in an AggreWell plate (Stemcell Technologies). After 24 hours, cell aggregates were harvested and cultured in differentiation medium [DMEM/F12 (Gibco), supplemented with 20% fetal bovine serum (FBS; Hyclone), 2 mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 50 U/mL penicillin/50 mg/mL streptomycin (Gibco), 0.1 mM b mercaptoethanol (Sigma-Aldrich)] for 10 days. Differentiation was assessed by RT-PCR using the primers in Table 1 to assess the differentiation by immunostaining, EBs were replated in 4 well chamber slides and differentiate further for 4 additional days in differentiation medium.
  • Group 1 20% KSR medium DMEM/F12 (Gibco), supplemented with 20% knockout serum replacement (KSR; Gibco), 2 mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 50 U/mL penicillin/50 mg/mL streptomycin (Gibco), 0.1 mM b mercaptoethanol (Sigma-Aldrich), and 10 ng/mL basic fibroblast growth factor (bFGF; Sigma-Aldrich and R&D Systems)]; Group 2 TGF ⁇ 1/LIF medium: DMEM/F12, supplemented with 20% KSR, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/mL penicillin/50 mg/mL streptomycin, 0.1 mM b mercaptoethanol, 0.12 ng/ml TGF ⁇ 1 (Pepro Tech), 1000 unites/ml LIF (Millipore).
  • KSR knock
  • Group 3 LIF/Wnt3a medium DMEM/F12, supplemented with 20% KSR, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/mL penicillin/50 mg/mL streptomycin, 0.1 mM b mercaptoethanol, 100 ng/ml Wnt3a (R&D Systems), 1000 unites/ml LIF.
  • Group 4 2i/LIF medium DMEM/F12 supplemented with N2 (Gibco) and mix 1:1 with Neurobasal medium (Gibco) supplemented with B27 (Gibco), 1 mM L-glutamine, 0.8 ⁇ M PD0325901 (Sigma), 3 ⁇ M CHIR99021 (Selleckchem), 20 ng/ml LIF.
  • TGF ⁇ 1/activin A/nodal medium DMEM/F12, supplemented with 20% KSR, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/mL penicillin/50 mg/mL streptomycin, 0.1 mM b mercaptoethanol, 0.12 ng/ml TGF ⁇ 1 (Pepro Tech), 10 ng/ml Activin A (R& D Systems), 50 ng/ml mouse recombinant nodal (R & D systems). All systems used Matrigel as the substrate.
  • PCR products were loaded into 2% agarose gels containing 0.6 ⁇ g/mL ethidium bromide and run in Tris-acetate-ethylenediaminetetraacetic acid buffer for 45 min.
  • the Alpha Innotech gel documentation station was used to observe PCR products.
  • Stage-X White Leghorn chicken embryos were used to produce chimeras. Small injection windows were drilled into injection egg shells using a Dremel rotary tool. ciPSCs from black australorp chickens were transduced with Turbo-GFP Lentiviral Vector (Thermo Scientific Open Biosystems) before injection according to the manufacturer's instructions. ciPSCs were injected into the subgerminal cavity using a glass micropipette with pressure controlled microinjector (Parker Automation). Each embryo was injected with 10,000 cells. The window was sealed by using a hot glue gun after injection and eggs were incubated at 37.8° C.
  • DNA was isolated from five different organs using DNeasy kit (Qiagen) following the manufacturer's instructions. PCR reactions were performed by initially denaturing cDNA at 95° C. for 3 min followed by 30 cycles of denaturing at 95° C. for 30 sec, annealing at 58° C. for 30 sec, polymerization at 72° C. for 30 sec and a final 10-min extension at 72° C. GFP primer used in PCR are listed in Table 2, above.
  • Sequencing verification of GFP gene was performed by extracting DNA from agarose gels after electrophoresis. DNA was extracted from the agarose gels using the QIAquick Gel Extraction Kit (Giagen) per manufacturer's instructions. Purified DNA was sent to the Georgia Genomic Facilities for sequencing. The resulted sequence was compared by Blast in the NCBI database.
  • Chicken embryonic fibroblasts isolated from day-11 black australorp chicken embryos were transduced with the minicircle vector containing the POU5F1, SOX2, NANOG and LIN28 reprogramming genes and the GFP reporter using four different transfection reagents: lipofectamine, Xfect, purefection and genejammer. The cells were transfected a total of 3 times every other day over a 5 day period. The GFP expression reached a peak at 72 hours post-transfection ( FIG. 16B ).
  • the lipofectamine transfected group showed the highest transfection efficiency with 16.4% population being GFP+, while transfection with genejammer, Xfect and purefection resulted in 2.3%, 2.1%, and 15.1% GFP+ cells respectively ( FIG. 16 . C, D).
  • the cells were trypsinized and plated onto inactivated feeder cells in 20% KSR stem cell medium.
  • ciPSC displayed morphological characteristics consistent with iPSCs including a colonial growth pattern with colonies forming highly refractive colonies with well defined boarders ( FIG. 17B ).
  • ciPSCs had a high nucleus to cytoplasm ratio and possessed large nucleoli indicative of a stem cell fate.
  • the chicken embryonic fibroblast (CEF) cells used in the transduction were isolated from day-11 Barred Rock (BR) embryos and cultured in fibroblast medium [Dulbecco's modified Eagle's medium (DMEM) high glucose (Hyclone) with 10% fetal bovine serum (Hyclone), 4 mM L-glutamine (Gibco) and 1 ⁇ Pen/strep (Gibco)] in 5% CO 2 at 37° C.
  • DMEM modified Eagle's medium
  • Hyclone high glucose
  • Hyclone fetal bovine serum
  • Pen/strep Gibco
  • CEFs were transduced utilizing the viPS kit (Thermo Scientific) with lentiviruses containing the human stem cell genes POU5F1, NANOG, SOX2, LIN28, KLF4, and C-MYC at an multiplicity of infection (MOI) of 10 and in the presence of 1 ⁇ TransDux (System Biosciences).
  • the transduced cells were replated at a ratio of 1:10 onto 1 ⁇ 100 mm plate pre-seeded with mitomycin inactivated mouse embryonic fibroblast (MEF) in cKSR medium [DMEM/F12 (Gibco), supplemented with 20% knockout serum replacement (KSR; Gibco), 2 mM L-glutamine (Gibco), 0.1 mM nonessential amino acids (Gibco), 1 ⁇ Pen/strep (Gibco), 0.1 mM ⁇ -mercaptoethanol (Sigma-Aldrich), and 10 ng/mL basic fibroblast growth factor (bFGF; R&D Systems).
  • AP staining was conducted by using VECTOR Red Alkaline Phosphatase Substrate Kit per manufacturer instructions. Briefly, culture medium was removed and then the cells were gently rinsed once with Tris-HCl buffer (pH8.3) and then incubated with the pre-mixed staining solution at room temperature for 20 min. At the end of incubation, the cells were gently rinsed with Tris-HCl twice. Then cells were overlaid with PBS and cells were imaged on the microscope.
  • Tris-HCl buffer pH8.3
  • PAS staining was conducted by using Periodic Acid Solution and Schiff's Reagent (Sigma) per manufacturer instructions. Briefly, cells were fixed in a culture dish with 4% paraformaldehyde (PFA) for 5 min and then washed with PBS 3 times. Periodic Acid Solution was then added into the plate and incubated at room temperature for 5 min. Then the cells were rinsed with PBS 3 times and Schiff's Reagent was added to the plate and incubated at room temperature for 15 min. The cells were gently rinsed with PBS 3 times and cells were imaged on the microscope.
  • Periodic Acid Solution and Schiff's Reagent
  • EBs were prepared in cKO medium without bFGF by using an AggreWell plate (Stemcell Technologies). A total of 2.4 ⁇ 10 6 chicken PSCs were plated in each well of the AggreWell plate, which is equivalent to 2,000 cells per microwell. After 24 h, the aggregates were harvested by gentle pippeting and then transferred to a petri-dish and continued to culture for 6 days. EBs were collected for RNA isolation and cDNA synthesis. PCR was performed to detect the differentiation using primers listed in Table 2. For immunocytochemistry assay, EBs were replated in 4-well chamber slide and cultured for 3 days. Then the cells were fixed in 4% PFA for use in immunostaining.
  • PKH26 (Sigma) according to the manufacturer's instruction.
  • White leghorn chicken embryos at stage 15 were used as a host for the injection.
  • a window 1-cm diameter was made on the blunt end above the air cell to expose the embryos.
  • a total of 1 ⁇ 10 4 cells were loaded into a micro glass needle and injected into the vasculature system of each embryo. The window was sealed by applying 2 layers of parafilm.
  • the injected embryos were incubated for 6 days and then euthanized to isolate the gonads under a stereomicroscope. Images of gonads were captured under an inverted microscope.
  • cPSCs chicken embryonic fibroblasts isolated from 5 different Barred Rock (BR) or 5 unique Black Australorp (BA) embryos were transduced with 6 human reprogramming factors (hPOU5F1, hSOX2, hNANOG, hLIN28, hKLF4 and hC-MYC). Each factor was individually packaged in a separate lentiviral vector. After 24 hours post-transduction, cells were replated on an inactivated MEF feeder layer. Putative chicken pluripotent stem cells (cPSCs) were cultured in cKSR medium following a previously developed protocol utilized to generate quail iPSC [24].
  • BR Barred Rock
  • BA Black Australorp
  • the cPSC colonies emerged as early as day 5 and were manually selected for propagation on day 7 after transduction ( FIG. 21A ). However, 8 of the 10 lines generated ceased to proliferate by passage 10. The remaining 2 lines demonstrated high levels of proliferation, but did not express the pluripotent markers POU5F1 or SSEA1 (data not shown) and were not utilized in further studies.
  • cPSC cultured in cKO are highly positive for alkaline phosphatase (AP) and periodic acid Shiff's staining (PAS, FIG. 21C ). Immunocytochemistry revealed that these cells are positive for POU5F1, SOX2 and SSEA1 ( FIG. 21D ) indicating pluripotency. Flow cytometry results showed that SSEA1 and POU5F1 positive cells represent 82.5% and 70.8%, respectively, of the whole population ( FIG. 21E ). To determine if cells cultured in cKO medium were less prone to spontaneous differentiation than cells in cKSR medium, we cultured the cKO medium-derived ciPSCs in cKSR medium. This resulted in a reduction of SSEA1 and POU5F1 positive cell down to 74.9% and 39.0%, respectively ( FIG. 21E ). Thus in the following experiments we used cKO as the preferred culture medium.
  • the chicken c-Myc was found to be down regulated in ciPSCs relative to CEF cells ( FIG. 22A ) and this finding was confirmed by RT-PCR.
  • This c-Myc result may be due to the fact that this was highly expressed in the parent cell line CEFs and not further increased when the exogenous gene was added ( FIG. 22B ).
  • the results of RT-PCR also revealed that Klf4 was highly expressed in CEF, comparable to that observed in cPSCs ( FIG. 22B ).
  • cPSCs were derived in a cKO medium that was optimized for germ cell maintenance and we, therefore, examined cPSCs for shared morphology and gene expression with cultured PGC.
  • cPSCs were positive for EMA1, DAZL and CVH at the protein level ( FIG. 23B ).
  • iPGCs are Capable of Differentiation into all 3 Germ Layers In Vitro
  • Chicken PGCs have previously demonstrated a high level of plasticity in vitro and are capable of generating cells from all 3 germ layers [26,27] in vitro.
  • iPGCs demonstrate the similar potential, these cells were subjected to in vitro embryoid body (EB) differentiation.
  • EB embryoid body
  • the recovered aggregates formed compact EBs after suspension culture for 6 days in differentiation medium ( FIG. 24A ).
  • RT-PCR analysis showed mesoderm (Glut, LPL and PPAR), ectoderm (TUJ1 and NESTIN) and endoderm (HNF1 and HNF4) genes were highly expressed in iPGC-derived EBs, but were negative in CEFs ( FIG. 24B ).
  • iPGCs To determine the migratory potential of these cells, we labeled the iPGCs, PGCs and CEFs with PKH26 and injected them into the vasculature system of stage 15 chicken embryos. All 10 embryos injected with PGCs and 13 out of 18 embryos injected with iPGCs exhibited migration of exogenous cells to the embryonic gonads. No CEFs were found in the gonads of the injected embryos (9 embryos) ( FIG. 25C ).
  • the inventors have demonstrated for the first time in the avian species the successful derivation of iPGCs from a somatic cell line by cellular reprogramming. These cells resemble PGCs in gene expression and protein profiles and are capable of migration to embryonic gonads after injection into stage 15 chicken embryos.
  • This advance represents the first step towards generating germ line chimeric individuals derived from somatic cells.
  • germ cells are responsible for passing genetics from one generation to the next, the capability to generate PGCs from somatic cells offers a potentially new strategy for the conservation of endangered birds.
  • This approach also provides scientists a new cell tool to gain insight into the biology of the germ cell development or for PGC studies such a developmental reproductive toxicology using the avian model.
  • iPGCs expressed a number of the key germ cell markers including CVH, DAZL and C-KIT, which are germ cell specific or highly enriched markers.
  • VASA or DDX4
  • DDX4 is the CVH homologue and is widely conserved and has been demonstrated to be a definitive germ cell marker in drosophila, xenopus , mice and human [33-36].
  • the chicken homologue Cvh has also been found to be specifically expressed in PGCs [37].
  • overexpression of Cvh has been demonstrated to drive chicken ESCs to a germ cell fate [38].
  • Dazl a member of the Daz gene family which encodes RNA-binding proteins, is also specifically expressed in germ cells and required for germ cell development in diverse organisms [39].
  • c-Kit is a key regulator of PGC development and binding of c-Kit to its ligand activates multiple downstream signaling events (such as MEK/MAPK) and promotes growth and survival of PGCs [43].
  • high expression of Cvh, Dazl and c-Kit in the iPGCs indicates that the ectopic expression of the transcription reprogramming factors not only induced the pluripotent network in CEFs, as indicated by the up-regulation of the endogenous genes.
  • Reprogramming also triggered the germ cell related signaling pathway and resulted in a germ cell fate in these somatic cells when placed in PGC culture conditions.
  • PGCs possess unique migratory properties in early embryonic development, and the signaling by chemokine receptor CXCR4 and its ligand SDF-1 was reported to be responsible for this migration in mammals as well as in avians [32].
  • CXCR4 was expressed in PGCs and iPGCs, but absent in CEFs.
  • PGCs and iPGCs injected into stage 15 chicken embryos resulted in significant migration to the embryonic gonads.
  • Chicken ESCs are negative for the germ cell associated CXCR4 and are unable to migrate to the putative gonad in a similar manner to PGCs [44].
  • the cells derived in this study resemble PGCs in gene transcription, protein cell morphology and in vivo characteristics (Table 1), and thus should be considered iPGCs.
  • bFGF has been demonstrated to play a key role in this process by activating the MEK/ERK cell signaling pathway and stimulates the proliferation of PGCs [45].
  • iPGCs in the present study were derived and maintained in cKO that contains bFGF. If bFGF is removed from cKO media iPGCs lose their 3-D colony morphology and are loosely attached to the feeder layer, but then become more adherent with some invading the feeder layer, a phenomena similar in the culture of chicken ES cells [46] and the conversion of PGCs into EGCs in the mouse [18].
  • cKSR medium used for human or chicken ESC [47] culture also contains bFGF, but failed to maintain the pluripotency in iPGCs as evidenced by a significant decrease of POU5F1 positive cells after culture in this medium ( FIG. 21E ). Additional factors in the cKO medium such as the those secreted by the BRL cells in the conditioned medium (cKO medium) are believed to be critical for the maintenance of avian pluripotent cells [46].
  • exogenous c-Myc gene may not be required to induce and maintain pluripotency in CEF-generated chicken iPGC, and donor CEF c-Myc levels may be sufficient.
  • exogenous c-Myc may be required in other cell types that are reprogrammed, perhaps in adult tissue that would have lower endogenous c-Myc expression. Therefore, results may vary depending on the original cell type that is reprogrammed.
  • Another tumor related gene Klf4 is a widely used reprogramming factor and when overexpressed can change prime state EpiESCs to the na ⁇ ve state [53].
  • the inventors have demonstrated for the first time the generation of chicken iPGC from somatic cells utilizing cellular reprogramming by ectopic expression of transcription factors, thereby laying the foundation for future demonstration of germ line transmission in chickens.
  • Availability of competent iPGC offers new strategies for endangered bird conservation and a new cell source for developmental biology and transgenic animal research.

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