iPSC induction

Abstract

A method for inducing pluripotency in somatic cells from non-human domestic animals or livestock includes culturing neural stem cells (NSCs) in the presence of vectors expressing one or more reprogramming factors, resulting in canine, porcine, and bovine iPSCs with distinct genetic marker profiles.

Description

[Technical Field]

[0001] The present invention relates to the production of induced pluripotent stem cells in domesticated and farm animals. [Background technology]

[0002] The generation of induced pluripotent stem cells (iPSCs) from primary human and mouse cells has become established and routine in many laboratories. These cells can grow indefinitely in culture and differentiate into derivatives of the three germ layers, making them of great scientific, medical, and economic importance.

[0003] Pluripotency, in reference to stem cells, refers to the ability to generate cells of all three somatic cell lineages: mesoderm, endoderm, and ectoderm. Pluripotent stem cells can therefore serve as progenitors for all cell types in the adult body. This definition should not be confused with multipotency, which refers to the ability of stem cells to generate daughter cells of limited somatic cell types.

[0004] In humans, it has been shown that iPSCs can be generated by somatic cell treatment using only Oct4 and Nanog (WO 2010 / 111,409).

[0005] Much effort has been made to generate similar iPSCs from large domestic and livestock animals, such as horses, dogs, cats, pigs, sheep, and cattle, with the hope that these cells will provide similar benefits in animal research and for the veterinary industry.

[0006] To produce iPSCs from these species, retroviral or lentiviral vector integration has been used (see, e.g., WO 2016 / 204,298 and Koh and Piedrahita, 2014, "From ES-like cells to induced pluripotent stem cells: A historical perspective in domestic animals," Theriogenology 81:103-111). These methods involve the integration of vector sequences into the host genome, which can cause several problems, including the generation of unpredictable mutations, uncontrolled silencing of exogenous factors, uncontrolled expression of residual transgenes, and strong immunogenicity (Okita et al., 2007, "Generation of germline-competent induced pluripotent stem cells," Nature 448:313-317; Zhao et al., 2011, "Immunogenicity of induced pluripotent stem cells," Nature 474:212-251). Attempts to overcome these problems have been made to generate iPSCs from domestic animals and livestock using non-integrating vectors. Unfortunately, however, non-integrating vectors have been shown to be significantly less effective in domestic animals and livestock, and so-called iPSC clones that can be advanced for analysis are very rare and difficult to maintain (see, e.g., Tsukamoto et al., 2018).Furthermore, these clones were confirmed to exhibit undesirable phenotypes (Congras et al., 2016. “Non-integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine-induced pluripotent-like stem cells” Scientific Reports 6:27059; Chow., 2017. “Safety and immune regulatory properties of canine-induced pluripotent stem cell-derived mesenchymal stem cells” Stem Cell Research 25:221-232).

[0007] Fibroblasts are the most commonly used somatic cell starting material for iPSC preparation; this is common in humans and non-human animals. This is because the cells can be obtained from easily accessible tissues using minimally invasive methods, and these primary cells can be sufficiently expanded in culture before senescence. Other somatic cell starting materials that require more invasive derivation (establishment) are usually avoided due to adverse effects on the subject. In the case of autoimmune diseases, certain somatic cell starting materials, such as cells from the brain, are generally prohibited due to the risk of death associated with the derivation procedure.

[0008] Therefore, there is a need for improved methods for producing iPSCs from domestic and livestock animals that avoid both the drawbacks of using integrating vectors and the inefficiencies of current methods that use non-integrating vectors. Summary of the Invention [Problem to be solved by the invention]

[0009] It is therefore an object of the present invention to provide an efficient and effective method for inducing pluripotency in somatic cells from domestic and farm animals. In specific embodiments, the present invention aims to provide alternative, preferably improved, methods for the derivation of iPSCs, particularly in dogs and pigs, as well as iPSCs themselves. [Means for solving the problem]

[0010] The present invention provides a method for inducing pluripotency, comprising culturing neural stem cells (NSCs) in the presence of a vector expressing one or more reprogramming factors, wherein the NSCs are derived from domestic animals or livestock.

[0011] The present invention also provides a method for inducing pluripotency, comprising culturing somatic cells of low relative potency in the presence of a non-integrating vector expressing one or more reprogramming factors, wherein the cells are derived from domestic animals or livestock.

[0012] Induced pluripotent stem cells (iPSCs) produced by the methods of the present invention also constitute part of the present invention. The present invention also provides iPSCs that exhibit a characteristic marker profile. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the expression of NANOG and REX1 in canine iPSCs. [Figure 2] FIG. 2 shows the expression of NANOG and REX1 in porcine iPSCs. [Figure 3] Figure 3 shows a heat map of pluripotent stem cell marker expression. [Figure 4] Figure 4 shows a heat map of somatic marker expression. [Figure 5]FIG. 5 shows the SSEA-3 and SSEA-4 marker profiles in iPSCs of the present invention. [Figure 6] Figure 6 shows the difference in iPSC induction efficiency when starting from porcine neural stem cells compared to porcine fibroblasts. [Figure 7] FIG. 7 shows that Oct4 alone cannot induce reprogramming of porcine neural stem cells into iPSCs. [Figure 8] Figure 8 confirms the gene expression patterns in pig and dog iPSCs that were found to be differentially expressed in the RNAseq study. [Figure 9] FIG. 9 shows the derivation of bovine NSCs and their subsequent reprogramming into iPSCs. DETAILED DESCRIPTION OF THE INVENTION

[0014] Accordingly, the present invention provides a method for inducing pluripotency, comprising culturing neural stem cells (NSCs) in the presence of a vector expressing one or more reprogramming factors, wherein the NSCs are derived from domestic animals or livestock.

[0015] The livestock and / or domestic animals are non-human; preferably selected from dogs, cats, cows, sheep, pigs, goats, horses, chickens, guinea pigs, donkeys, deer, ducks, geese, camels, llamas, alpacas, turkeys, rabbits and hamsters.

[0016] Somatic NSCs are more preferably derived from canine, bovine, ovine, porcine and equine sources; in specific embodiments, they are derived from canine, porcine, bovine and equine sources, and in the specific examples below, from canine, porcine and equine sources.

[0017] The present invention allows for efficient and proven pluripotency of iPSCs derived from domestic animals and livestock. The efficiency of reprogramming using NSCs as the starting material for pluripotency induction is surprisingly and advantageously higher than that using fibroblasts as the starting material. This increased efficiency is evident from the fact that thousands of iPSC clones can be generated when starting from NSCs, compared to only a few iPSC clones generated when starting from fibroblasts.

[0018] As shown in more detail in the Examples below, iPSCs from dogs, pigs and cattle have been successfully derived and maintained in culture according to the present invention.

[0019] The maintenance of self-renewal capacity during expansion is an advantage of the iPSCs of the present invention. It has been observed in the Examples that canine, porcine, and / or bovine-derived iPSCs maintain their morphology (forming smooth-edged colonies) and the ability to differentiate into derivatives of all three germ layers over successive passages. After at least 40 cell culture passages, preferably at least 50 passages, preferably at least 100 passages, more preferably at least 200 passages, or even more preferably at least 1000 passages, iPSCs that maintain the ability to differentiate into derivatives of the three germ layers are available.

[0020] The vector is preferably a non-integrating vector.The non-integrating vector is preferably selected from adenovirus vector, adeno-associated virus vector, respirovirus vector, integrase-deficient retrolentivirus vector, poxvirus vector, episomal vector, plasmid vector and artificial chromosome vector.The advantage of the obtained iPSC is that there is no unnecessary and potentially confusing integrated genetic material in the descendants of iPSC.Preferably, the non-integrating vector is Sendai virus.

[0021] The reprogramming factors expressed by the vector are preferably selected from two or more or all of Oct4, Sox2, cMyc, and Klf4. Somatic cells are preferably cultured with vectors expressing all of the reprogramming factors; in the following examples, all factors were used for canine and porcine iPSCs. Optionally, a smaller number of factors may be used in combination; for example, Oct4 may be used in combination with Sox2 and / or cMyc, or may be used in combination with KLF4 in particular. Preferably, at least Oct4 and cMyc are present. In any case, when factors are used in combination, each reprogramming factor may be expressed on the same vector or on a different vector. In a particularly preferred embodiment, somatic cells are cultured with a three-vector preparation, where the first vector expresses the polycistronic Klf4-Oct3 / 4-Sox2, the second vector expresses cMyc, and the third vector expresses Klf4. This provides a ratio of factors suitable for inducing pluripotent cells.

[0022] iPSCs produced by the methods of the present invention are preferably cultured in knockout serum replacement (KOSR) medium.

[0023] After successful induction of pluripotency by the present invention, iPSCs benefit from reduced levels of viral vectors in successive rounds of passaging, which is a major benefit of using non-integrating vectors. 6 cells, suitably at least 10 8 cells, or preferably at least 10 10 Preferably, the iPSC population reaches a purity whereby the total number of iPSCs (including cells) is less than 1%, preferably less than 0.1%, or more preferably less than 0.01% of the original vector concentration. The original vector concentration may be defined as the concentration of vector present in the iPSC population at passage 1 in cell culture. In certain embodiments of the present invention, a substantially vector-free iPSC population is obtained.

[0024] Previously, the art has not been able to derive and reliably maintain iPSCs from the animal species of the present invention. It has now been discovered that iPSCs can be advantageously derived and maintained using a media supplement. Preferably, the iPSC growth media contains a gp130 agonist.

[0025] Preferably, the gp130 agonist is leukemia inhibitory factor (LIF). Alternatively, the gp130 signaling pathway can be stimulated using other available and known agonists, including IL-6, cardiotrophin 1 (CT-1), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), and IL-11. Separately, the iPSC growth medium preferably contains an FGF receptor agonist. Preferably, the FGF receptor agonist is basic fibroblast growth factor (bFGF). Again, other agonists are known and commercially available. A preferred medium contains both a gp130 agonist and an FGF receptor agonist; this combination was successfully used in the examples below.

[0026] Optionally, iPSCs are cultured in a growth medium containing a GSK3 inhibitor. Preferably, the GSK3 inhibitor is selected from insulin, SB216763, SB415286, azakenpaullone, AR-A0144, bis-7-azaindolylmaleimide, BIO, CHIR-98014, CHIR-99021, TWS119, A1070722, TDZD8, and AZD1080. Preferably, the GSK3 inhibitor is CHIR-99021. Good results have been obtained using GSK3 inhibitors on both porcine and canine iPSCs, particularly porcine iPSCs.

[0027] It is further advantageous to maintain iPSCs on a layer of feeder cells, typically an adherent layer of somatic feeder cells, preferably non-human feeder cells. In the examples, iPSCs are cultured in the presence of a feeder layer of irradiated mouse embryonic fibroblast feeders (MEFs).

[0028] The present invention also provides a method for inducing pluripotency, comprising culturing somatic cells in the presence of a non-integrating vector expressing one or more reprogramming factors, wherein the cells are derived from domestic animals or livestock.

[0029] The livestock and / or domestic animals are non-human and are preferably selected from dogs, cats, cows, sheep, pigs, goats, horses, chickens, guinea pigs, donkeys, ducks, geese, camels, llamas, alpacas, turkeys, rabbits and hamsters. Very suitable animals are dogs, cows, sheep, pigs and horses.

[0030] Preferably, the livestock is a pig, cow, sheep or horse (ie a porcine, bovine, ovine or equine).

[0031] Preferably, the domestic animal is a dog (ie, a canine).

[0032] Preferably, the somatic cells are NSCs.

[0033] The reprogramming factors expressed by the vector are preferably as described elsewhere herein, for example, selected from Oct4, Sox2, cMyc, and Klf4.

[0034] Also, the non-integrating vector is preferably as described elsewhere herein, and the non-integrating vector is Sendai virus.

[0035] Again, the medium is preferably as described elsewhere herein. Accordingly, iPSCs are preferably cultured in knockout serum replacement (KOSR) medium, and the iPSC growth medium preferably contains a gp130 agonist, preferably LIF, and more preferably contains an FGF receptor agonist. Optionally, iPSCs are cultured in a growth medium containing a GSK3 inhibitor. Preferably, the GSK3 inhibitor is selected from insulin, SB216763, SB415286, azakenpaullone, AR-A0144, bis-7-azaindolylmaleimide, BIO, CHIR-98014, CHIR-99021, TWS119, A1070722, TDZD8, and AZD1080. Preferably, the GSK3 inhibitor is CHIR-99021.

[0036] Also, as described elsewhere herein, iPSCs are preferably cultured in the presence of a feeder cell layer.

[0037] The methods of the present invention, as described above and below, are capable of producing thousands of successful iPSC clones. The observed high transduction efficiency is significantly advantageous and therefore provides an improved method for producing iPSCs from somatic cells derived from livestock and domestic animals. Based on technical methods, it was estimated that iPSC derivation would be approximately 1,000 times more efficient using the methods of the present invention.

[0038] Therefore, the present invention also provides iPSCs themselves obtained by the methods described above and below. Preferably, the iPSCs are positive for the pluripotency markers NANOG, REX1, SSEA-3, and SSEA-4.

[0039] In a preferred embodiment, the present invention provides iPSCs derived from livestock or domestic animals, wherein the iPSCs are positive for the pluripotency markers NANOG, REX1, SSEA-3, and SSEA-4. Preferably, the iPSCs are provided as isolated cells.

[0040] The iPSCs of the present invention are preferably characterized by high levels of expression of SSEA-3 and SSEA-4. In the cell populations of the present invention, preferably 50% or more of the cells express SSEA-3 and 50% or more of the cells express SSEA-4. These populations generally contain tens of thousands or hundreds of thousands or millions of cells, suitably at least 10, at least 10, or at least 10 cells. More preferably, 60% or more of the cells are SSEA-4 positive and 60% or more of the cells are SSEA-3 positive. In embodiments described in more detail below, more than 60% of the iPSCs are positive for SSEA-4 expression, and SSEA-4 expression of the iPSCs is high. + Over 50% of the population was SSEA-3 + It was also.

[0041] Preferably, the iPSCs of the present invention are positive for one or more, two or more, three or more or all of GLDN, PTK2B, LOC110260197, ANGPT1, LY96, NYAP2, THBS2, ULK4, CRSP3, CHST8, SKOR1, KCNMB2, LMNA, HTRA1, PHLDA1, FGF1 and GASK1B expression.

[0042] More preferably, the iPSCs are positive for one or more, two or more, three or more or all of LMNA, HTRA1, PHLDA1, FGF1 and GASK1B expression, indeed most preferably the iPSCs express all of these gene markers.

[0043] In the iPSC population of the present invention, preferably 50% or more of the cells express LMNA, more preferably 60% or more of the cells express LMNA, more preferably 70% or more of the cells express LMNA, more preferably 80% or more of the cells express LMNA, more preferably 90% or more of the cells express LMNA, and most preferably 95% or more of the cells express LMNA.

[0044] In the iPSC population of the present invention, preferably 50% or more of the cells express HTRA1, more preferably 60% or more of the cells express HTRA1, more preferably 70% or more of the cells express HTRA1, more preferably 80% or more of the cells express HTRA1, more preferably 90% or more of the cells express HTRA1, and most preferably 95% or more of the cells express HTRA1.

[0045] In the iPSC population of the present invention, preferably 50% or more of the cells express PHLDA1, more preferably 60% or more of the cells express PHLDA1, more preferably 70% or more of the cells express PHLDA1, more preferably 80% or more of the cells express PHLDA1, more preferably 90% or more of the cells express PHLDA1, and most preferably 95% or more of the cells express PHLDA1.

[0046] In the iPSC population of the present invention, preferably 50% or more of the cells express FGF1, more preferably 60% or more of the cells express FGF1, more preferably 70% or more of the cells express FGF1, more preferably 80% or more of the cells express FGF1, more preferably 90% or more of the cells express FGF1, and most preferably 95% or more of the cells express FGF1.

[0047] In the iPSC population of the present invention, preferably 50% or more of the cells express GASK1B, more preferably 60% or more of the cells express GASK1B, more preferably 70% or more of the cells express GASK1B, more preferably 80% or more of the cells express GASK1B, more preferably 90% or more of the cells express GASK1B, and most preferably 95% or more of the cells express GASK1B.

[0048] In the iPSC population of the present invention, preferably 50% or more of the cells express LMNA, HTRA1, PHLDA1, FGF1 and GASK1B, more preferably 60% or more of the cells express LMNA, HTRA1, PHLDA1, FGF1 and GASK1B, more preferably 70% or more of the cells express LMNA, HTRA1, PHLDA1, FGF1 and GASK1B, more preferably 80% or more of the cells express LMNA, HTRA1, PHLDA1, FGF1 and GASK1B, more preferably 90% or more of the cells express LMNA, HTRA1, PHLDA1, FGF1 and GASK1B, and most preferably 95% or more of the cells express LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.

[0049] The maintenance of specific marker expression during expansion is an advantage of the iPSCs of the present invention. It has been observed in the examples that canine, bovine, and / or porcine iPSCs maintain their morphology (forming smooth-edged colonies) and the ability to differentiate into derivatives of all three germ layers over successive passages. iPSCs are available that maintain the expression of LMNA, HTRA1, PHLDA1, FGF1, and GASK1B even after at least 10 cell culture passages, preferably at least 20 passages, preferably at least 50 passages, more preferably at least 100 passages, or even more preferably at least 1000 passages.

[0050] In an embodiment of the present invention, the iPSCs are derived from dogs, pigs, cattle, horses, or sheep. Preferably, the iPSCs are dog, cattle, or pig iPSCs. Preferably, the iPSCs are dog or pig iPSCs.

[0051] In certain embodiments of the invention, described in more detail below, more than 60% of canine iPSCs and more than 80% of porcine iPSCs are positive for SSEA-4 expression, and the SSEA-4 + Of the iPSC population, over 55% of the canine iPSCs were SSEA-3 + and more than 50% of porcine iPSCs were also SSEA-3+ Canine and porcine iPSCs were also positive for Rex1 and Nanog.

[0052] The present invention also provides for the use of iPSCs in medical / veterinary treatment. Preferably, the treatment is an allogeneic cell-based treatment. This has the advantage that the somatic cell starting material for iPSC production does not need to be derived from the recipient of the treatment. [Example]

[0053] The present invention will now be described in further and more particular detail with reference to the accompanying drawings, in which:

[0054] The DNA, RNA and amino acid sequences are as follows: SEQ ID NO: 1 is the porcine LMNA forward primer DNA sequence; SEQ ID NO:2 is the porcine LMNA reverse primer DNA sequence; SEQ ID NO:3 is the canine LMNA forward primer DNA sequence; SEQ ID NO: 4 is the canine LMNA reverse primer DNA sequence; SEQ ID NO: 5 is the porcine HTRA1 forward primer DNA sequence; SEQ ID NO: 6 is the porcine HTRA1 reverse primer DNA sequence; SEQ ID NO: 7 is the canine HTRA1 forward primer DNA sequence; SEQ ID NO: 8 is the canine HTRA1 reverse primer DNA sequence; SEQ ID NO: 9 is the porcine FGF1 forward primer DNA sequence; SEQ ID NO: 10 is the porcine FGF1 reverse primer DNA sequence; SEQ ID NO: 11 is the canine FGF1 forward primer DNA sequence; SEQ ID NO: 12 is the canine FGF1 reverse primer DNA sequence; SEQ ID NO: 13 is the porcine GASK1B forward primer DNA sequence; SEQ ID NO: 14 is the porcine GASK1B reverse primer DNA sequence; SEQ ID NO: 15 is the canine GASK1B forward primer DNA sequence; SEQ ID NO: 16 is the canine GASK1B reverse primer DNA sequence; SEQ ID NO: 17 is the porcine PHLDA1 forward primer DNA sequence; SEQ ID NO: 18 is the porcine PHLDA1 reverse primer DNA sequence; SEQ ID NO: 19 is the canine PHLDA1 forward primer DNA sequence; and SEQ ID NO: 20 is the canine PHLDA1 reverse primer DNA sequence.

[0055] Example 1 - Derivation of primary canine neural stem cells Neural stem cells (NSCs) were harvested from the brain of a 6-year-old dog.

[0056] A large sandwich box was cleaned and placed in a Class II cabinet that had been sprayed with 70% industrial methyl alcohol and allowed to air dry. The UV light was turned on and the box was left for 20 minutes. Two separate 10 cm 2 Tissue culture dishes were recoated with iMatrix Laminin 511 and stored at 4°C overnight.

[0057] Upon receipt, the dog brains were placed in a sterile sandwich box containing calcium- and magnesium-free phosphate-buffered saline (PBS). The brains were cut in half using a scalpel to separate the two cerebral lobes. The brain region containing the subventricular zone (covering the lateral ventricles of the forebrain) was isolated.

[0058] The excised subventricular zone was cut into small pieces and transferred to a 50 ml tube containing 10 ml of Accutase. The tube was incubated at 37°C for 10 minutes with intermittent shaking. Cells were then separated from the tissue using a pipette. 20 ml of PBS was added to the tube, allowing large pieces of tissue to sink to the bottom, and the supernatant was removed and placed in a new tube. The Accutase step was then repeated with the tube containing the larger pieces of tissue.

[0059] The new tubes containing the supernatant were centrifuged at 1800 rpm for 4 minutes. The supernatant in these tubes was removed, resuspended in 10 ml of PBS, and passed through a 70 μm cell strainer. Then, two 10 cm 2 The cells were seeded on laminin-coated dishes (each containing 20 ml RHB-A medium + 10 ng / ml huEGF + 10 ng / ml HuFGF + penicillin, dihydrostreptomycin, and primocin).

[0060] The culture medium was changed every 1-2 days until the cultures reached approximately 70% confluence (approximately 9-14 days). 2 The cells were divided into two laminin-coated flasks.

[0061] The morphology of NSCs was assessed microscopically; cells appeared to grow as single cells, but as they became more confluent, they began to appear as networks with thin dendrites.

[0062] Before day 20 of culture, NSCs were cryopreserved in vials according to standard laboratory practice.

[0063] Example 2 - Derivation of primary porcine neural stem cells Neural stem cells (NSCs) were harvested from the brains of 1-day-old piglets.

[0064] A large sandwich box was cleaned and placed in a Class II cabinet that had been sprayed with 70% industrial methyl alcohol and allowed to air dry. The UV light was turned on and the box was left for 20 minutes. Two separate 10 cm 2 Tissue culture dishes were recoated with iMatrix Laminin 511 and stored at 4°C overnight.

[0065] Upon receipt, the pig brains were placed in a sterile sandwich box containing calcium- and magnesium-free phosphate-buffered saline (PBS). The brains were cut in half using a scalpel to separate the two lobes. The brain region containing the subventricular zone (covering the lateral ventricles of the forebrain) was isolated.

[0066] The excised subventricular zone was cut into small pieces and transferred to a 50 ml tube containing 10 ml of Accutase. The tube was incubated at 37°C for 10 minutes with intermittent shaking. Cells were then separated from the tissue using a pipette. 20 ml of PBS was added to the tube, allowing large pieces of tissue to sink to the bottom, and the supernatant was removed and placed in a new tube. The Accutase step was then repeated with the tube containing the larger pieces of tissue.

[0067] The new tubes containing the supernatant were centrifuged at 1800 rpm for 4 minutes. The supernatant in these tubes was removed, resuspended in 10 ml of PBS, and passed through a 70 μm cell strainer. 2 The cells were seeded on laminin-coated dishes (each containing 20 ml RHB-A medium + 10 ng / ml huEGF + 10 ng / ml HuFGF + penicillin, dihydrostreptomycin, and primocin).

[0068] The culture medium was changed every 1-2 days until the cultures reached approximately 70% confluence (approximately 9-14 days). 2 The cells were divided into two laminin-coated flasks.

[0069] The morphology of NSCs was assessed microscopically throughout the culture period; cells appeared to grow as single cells, but as they became more confluent, they began to appear as networks with thin dendrites.

[0070] Before day 20 of culture, NSCs were cryopreserved in vials according to standard laboratory practice.

[0071] Example 3 - Derivation of primary bovine neural stem cells Neural stem cells (NSCs) were harvested from the brains of one-year-old and two-year-old cows (both chemically euthanized).

[0072] Two large sandwich boxes were cleaned and transferred to a Class II cabinet that had been sprayed with 70% industrial methyl alcohol and allowed to air dry. The UV light was turned on and the boxes were left there for 20 minutes. Four separate 10 cm 2 Tissue culture dishes were recoated with iMatrix Laminin 511 and stored at 4°C overnight.

[0073] Upon receipt, the bovine brains were placed in a sterile sandwich box containing calcium- and magnesium-free phosphate-buffered saline (PBS). The brains were cut in half using a scalpel to separate the two lobes. The brain region containing the subventricular zone (covering the lateral ventricles of the forebrain) was isolated.

[0074] The excised subventricular zone was cut into small pieces and transferred to a 50 ml tube containing 10 ml of Accutase. The tube was incubated at 37°C for 10 minutes with intermittent shaking. Cells were then separated from the tissue using a pipette. 20 ml of PBS was added to the tube, allowing large pieces of tissue to sink to the bottom, and the supernatant was removed and placed in a new tube. The Accutase step was then repeated with the tube containing the larger pieces of tissue.

[0075] The new tubes containing the supernatant were centrifuged at 1800 rpm for 4 minutes. The supernatant in these tubes was removed, resuspended in 10 ml of PBS, and passed through a 70 μm cell strainer. 2 The cells were seeded on laminin-coated dishes (each containing 20 ml RHB-A medium + 10 ng / ml bovine EGF + 10 ng / ml bovine FGF + penicillin, dihydrostreptomycin, and primocin).

[0076] The culture medium was changed every 1-2 days until the cultures reached approximately 70% confluence (approximately 9-14 days). 2 The cells were divided into two laminin-coated flasks.

[0077] NSC morphology was assessed microscopically throughout the culture period; cells appeared to form (1) dense colonies (epithelial cell-like) lacking processes, (2) elongated cells with a looser network of dendrites, and (3) small single cells that developed into a network with thin dendrites as they became more confluent.

[0078] Before day 20 of culture, NSCs were cryopreserved in vials according to standard laboratory practice.

[0079] Example 4 - Reprogramming of canine neural stem cells Canine neural stem cells (NSCs) were reprogrammed using the CytoTune 2.0 reprogramming kit. This kit uses a modified, non-transducing Sendai virus delivery system to introduce reprogramming vectors into primary cells, enabling the generation of iPSCs. The Sendai virus used in this kit is non-integrating and remains in the cytoplasm. Viral particles are cleared from the cytoplasm after several cell divisions, allowing for screening for full clearance using a qPCR assay.

[0080] The day before the introduction, 3 x 10 5Actively growing NSCs were seeded in RHB-A medium on a laminin 511 matrix in one well of a 6-well plate (as described in Examples 1-3), allowing the cells to adhere and spread and reach 50-80% confluence before transfection.

[0081] CytoTune 2.0 reprogramming vector titers are lot-dependent and lot-specific Certificates of Analysis (CoA) are available for download at: https: / / www.thermofisher.com / order / catalog / product / A16517

[0082] The lot-specific CoA gave the volume of viral vector per well to achieve an MOI of 5:5:3 (KOS:hc-Myc:hKlf4).

[0083] One ml of warmed RHB-A medium was added per well of transfected cells. Cytotune 2.0 vials (containing vectors) were removed from -80°C storage and warmed by hand. The vials were centrifuged to collect the contents and placed on ice. The calculated amount of each vector was added to the RHB-A medium in each well and mixed with a pipette. The cells were then incubated at 37°C for 24 hours, at which point the transfection medium was aspirated and replaced with fresh RHB-A (1 ml per well). RHB-A medium was replaced every 24 hours until day 6 of culture.

[0084] The transduced cells were recovered using 0.3 ml / well of Accutase at 37°C for 5 minutes. This incubation time was observed due to the sensitivity of the cells to the enzyme. During dissociation (cell rounding), 2 ml of RHB-A was added to protect the cells from the enzyme. The cells were collected in a 15 ml tube and centrifuged at 200 g for 4 minutes. The cells were then resuspended in canine iPSC medium. The preparation method is as follows:

[0085] To a 500ml bottle of DMEM / F12 (Thermo Fisher cat. 11520396), add 100ml of KOSR (Thermo Fisher 10828028), 5ml of Non-Essential Amino Acids 100X (Thermo Fisher 11140035), 5ml of 100mM Sodium Pyruvate (Thermo Fisher 11360039), 1ml of 2-mercaptoethanol (Thermo Fisher 31350010), and 5ml of Antibiotic (Sigma A5955). Immediately prior to use, add 62µl of huFGF (Peprotech 100-18B), 62µl of huLIF (Peprotech 300-05), and 500µl of 3mM Chiron stock (Tolcris - final concentration 3µM). Shake before use.

[0086] Cells were counted before seeding and incubating in new culture vessels. To optimize reprogramming efficiency, cells were grown at a relatively high cell density, typically 1 × 10 per 100 mm culture dish. 5 ~5×10 5 The cells were seeded at 1000 x g.

[0087] The culture medium for canine iPSCs was changed every 24 hours until colony formation was observed, which typically occurred within 12 days to 4 weeks.

[0088] Colonies were selected based on morphological characteristics. The day before colony selection, 24-well plates of irradiated mouse embryonic fibroblast (MEF) feeder cells (pre-coated with 0.2% gelatin / PBS) were prepared in MEF medium (1 ml per well) (4 × 10 cells per 24 wells). 6 The preparation method is as follows:

[0089] To a 500ml bottle of DMEM / F12 (Thermo Fisher cat. 11520396), add 50ml of FCS (Sigma F2442), 5ml of Non-Essential Amino Acids 100X (Thermo Fisher 11140035), 5ml of 100mM Sodium Pyruvate (Thermo Fisher 11360039), 1ml of 2-mercaptoethanol (Thermo Fisher 31350010), and 5ml of Antibiotic antimycotic (Sigma A5955). Shake before use.

[0090] The extracted colonies were transferred to separate wells of a prepared 24-well plate containing canine iPSC medium. After colonies grew, they were disaggregated using Accutase and replated into a single well of a prepared 6-well plate of irradiated MEFs. After reaching confluence, the cells were split into six wells of a prepared 6-well plate of irradiated MEFs using Accutase. After reaching confluence, the cells were cryopreserved in banks of 12 vials (half a well per vial). In this way, 12 vials of cells were banked per colony.

[0091] When passaging canine iPSCs embedded in MEFs, cell types may be dissociated by gentle pipetting. Then, place the cell mixture into a tube and centrifuge at 1500 rpm (0.4 rcf) for 3 minutes. Aspirate the medium and resuspend the canine iPSCs in canine iPSC medium. Before adding the cells to freshly pre-seeded MEFs, aspirate the MEF medium and replace it with canine iPSC medium.

[0092] Example 5 - Reprogramming of porcine neural stem cells Porcine neural stem cells (NSCs) were reprogrammed using the CytoTune 2.0 reprogramming kit. This kit uses a modified, non-integrating Sendai virus delivery system to deliver reprogramming vectors to primary cells, enabling the generation of iPSCs. The Sendai virus used in this kit is non-integrating and remains in the cytoplasm. Viral particles are cleared from the cytoplasm after several cell divisions, allowing for screening with full clearance using a qPCR assay.

[0093] The day before the introduction, 3 x 10 5 Actively growing NSCs were seeded in RHB-A medium on a laminin 511 matrix in one well of a 6-well plate (as described in Examples 1-3), allowing the cells to adhere and spread and reach 50-80% confluence before transfection.

[0094] CytoTune 2.0 reprogramming vector titers are lot-dependent and lot-specific Certificates of Analysis (CoA) are available for download at: https: / / www.thermofisher.com / order / catalog / product / A16517

[0095] The lot-specific CoA gave the volume of viral vector per well to achieve an MOI of 5:5:3 (KOS:hc-Myc:hKlf4).

[0096] One ml of warmed RHB-A medium was added per well of transfected cells. Cytotune 2.0 vials (containing vectors) were removed from -80°C storage and warmed by hand. The vials were centrifuged to collect the contents and placed on ice. The calculated amount of each vector was added to the RHB-A medium in each well and mixed with a pipette. The cells were then incubated at 37°C for 24 hours, at which point the transfection medium was aspirated and replaced with fresh RHB-A (1 ml per well). The RHB-A medium was replaced every 24 hours until day 6 of culture.

[0097] The transduced cells were recovered using 0.3 ml / well of Accutase at 37°C for 5 minutes. This incubation time was observed due to the sensitivity of the cells to the enzyme. During dissociation (cell rounding), 2 ml of RHB-A was added to protect the cells from the enzyme. The cells were collected in a 15 ml tube and centrifuged at 200 g for 4 minutes. The cells were then resuspended in porcine iPSC medium. The preparation method is as follows:

[0098] To a 500 ml bottle of DMEM / F12 (Thermo Fisher cat. 11520396), add 100 ml of KOSR (Thermo Fisher cat. 10828028), 5 ml of Non-Essential Amino Acids 100X (Thermo Fisher cat. 11140035), 5 ml of 100 mM Sodium Pyruvate (Thermo Fisher cat. 11360039), 1 ml of 2-mercaptoethanol (Thermo Fisher cat. 31350010), and 5 ml of Antibiotic (Sigma A5955). Immediately prior to use, add 62 μl of huFGF (Peprotech cat. 100-18B) and 62 μl of huLIF (Peprotech cat. 300-05). Shake well before use.

[0099] Cells were counted before seeding and incubating in new culture vessels. To optimize reprogramming efficiency, cells were grown at a relatively high cell density, typically 1 × 10 per 100 mm culture dish. 5 ~5×10 5 The cells were seeded at 1000 x g.

[0100] The culture medium for porcine iPSCs was changed every 24 hours until colony formation was observed, which typically occurred within 12 days to 4 weeks.

[0101] Colonies were selected based on morphological characteristics. The day before colony selection, 24-well plates of irradiated mouse embryonic fibroblast (MEF) feeder cells (pre-coated with 0.2% gelatin / PBS) were prepared in MEF medium (1 ml per well) (4 × 10 cells per 24 wells). 6 The preparation method is as follows:

[0102] To a 500ml bottle of DMEM / F12 (Thermo Fisher cat. 11520396), add 50ml of FCS (Sigma F2442), 5ml of Non-Essential Amino Acids 100X (Thermo Fisher 11140035), 5ml of 100mM Sodium Pyruvate (Thermo Fisher 11360039), 1ml of 2-mercaptoethanol (Thermo Fisher 31350010), and 5ml of Antibiotic (Sigma A5955). Shake before use.

[0103] Selected colonies were transferred to separate wells of a prepared 24-well plate containing porcine iPSC medium. After colonies grew, they were disaggregated using Accutase and replated into a single well of a prepared 6-well plate of irradiated MEFs. After reaching confluence, the cells were split into six wells of a prepared 6-well plate of irradiated MEFs using Accutase. After reaching confluence, the cells were cryopreserved in banks of 12 vials (half a well per vial). In this way, 12 vials of cells were banked per colony.

[0104] When passaging porcine iPSCs embedded in MEFs, cell types may be dissociated by gentle pipetting. The cell mixture is then placed in a tube and centrifuged at 1500 rpm (0.4 rcf) for 3 minutes. The medium is then aspirated and the porcine iPSCs are resuspended in porcine iPSC medium. Before adding the cells to newly seeded MEFs, the MEF medium is aspirated and replaced with porcine iPSC medium.

[0105] Example 6 - Reprogramming of bovine neural stem cells Bovine neural stem cells (NSCs) were reprogrammed using the CytoTune 2.0 Reprogramming Kit. This kit uses a modified, non-recombinant Sendai virus delivery system to deliver reprogramming vectors to primary cells, enabling the generation of iPSCs. The Sendai virus used in this kit is non-integrating and remains in the cytoplasm. Viral particles are cleared from the cytoplasm after several cell divisions, allowing for screening for full clearance using a qPCR assay.

[0106] The day before the introduction, 3 x 10 5 Actively growing NSCs were seeded in RHB-A medium on a laminin 511 matrix in one well of a 6-well plate (as described in Examples 1-3), allowing the cells to adhere and spread and reach 50-80% confluence before transfection.

[0107] CytoTune 2.0 reprogramming vector titers are lot-dependent and lot-specific Certificates of Analysis (CoA) are available for download at: https: / / www.thermofisher.com / order / catalog / product / A16517

[0108] The lot-specific CoA gave the volume of viral vector per well to achieve an MOI of 5:5:3 (KOS:hc-Myc:hKlf4).

[0109] One ml of warmed RHB-A medium was added per well of transfected cells. Cytotune 2.0 vials (containing vectors) were removed from -80°C storage and warmed by hand. The vials were centrifuged to collect the contents and placed on ice. The calculated amount of each vector was added to the RHB-A medium in each well and mixed with a pipette. The cells were then incubated at 37°C for 24 hours, at which point the transfection medium was aspirated and replaced with fresh RHB-A (1 ml per well). The RHB-A medium was replaced every 24 hours until day 6 of culture.

[0110] The transduced cells were recovered using 0.3 ml / well of Accutase at 37°C for 5 minutes. This incubation time was observed due to the sensitivity of the cells to the enzyme. During dissociation (cell rounding), 2 ml of RHB-A was added to protect the cells from the enzyme. The cells were collected in a 15 ml tube and centrifuged at 200 g for 4 minutes. The cells were then resuspended in bovine iPSC medium. The preparation method is as follows:

[0111] To a 500 ml bottle of DMEM / F12 (Thermo Fisher cat. 11520396), add 100 ml of KOSR (Thermo Fisher cat. 10828028), 5 ml of Non-Essential Amino Acids 100X (Thermo Fisher cat. 11140035), 5 ml of 100 mM Sodium Pyruvate (Thermo Fisher cat. 11360039), 1 ml of 2-mercaptoethanol (Thermo Fisher cat. 31350010), and 5 ml of Antibiotic (Sigma A5955). Immediately prior to use, add 62 μl of huFGF (Peprotech cat. 100-18B) and 62 μl of huLIF (Peprotech cat. 300-05). Shake well before use.

[0112] Cells were counted before seeding and incubating in new culture vessels. To optimize reprogramming efficiency, cells were grown at a relatively high cell density, typically 1 × 10 per 100 mm culture dish. 5~5×10 5 The cells were seeded at 1000 x g.

[0113] The culture medium for bovine iPSCs was changed every 24 hours until colony formation was observed, which typically occurred within 12 days to 4 weeks.

[0114] Colonies were selected based on morphological characteristics. The day before colony selection, 24-well plates of irradiated mouse embryonic fibroblast (MEF) feeder cells (pre-coated with 0.2% gelatin / PBS) were prepared in MEF medium (1 ml per well) (4 × 10 cells per 24 wells). 6 The preparation method is as follows:

[0115] To a 500ml bottle of DMEM / F12 (Thermo Fisher cat. 11520396), add 50ml of FCS (Sigma F2442), 5ml of Non-Essential Amino Acids 100X (Thermo Fisher 11140035), 5ml of 100mM Sodium Pyruvate (Thermo Fisher 11360039), 1ml of 2-mercaptoethanol (Thermo Fisher 31350010), and 5ml of Antibiotic (Sigma A5955). Shake before use.

[0116] Selected colonies were transferred to separate wells of a prepared 24-well plate containing bovine iPSC medium. After colonies grew, they were disaggregated using Accutase and replated into a single well of a prepared 6-well plate of irradiated MEFs. After reaching confluence, the cells were split into six wells of a prepared 6-well plate of irradiated MEFs using Accutase. After reaching confluence, the cells were cryopreserved in banks of 12 vials (half a well per vial). In this way, 12 vials of cells were banked per colony.

[0117] When passaging bovine iPSCs embedded in MEFs, cell types may be dissociated by gentle pipetting. The cell mixture is then placed into a tube and centrifuged at 1500 rpm (0.4 rcf) for 3 minutes. The medium is then aspirated and the bovine iPSCs are resuspended in bovine iPSC medium. Before adding the cells to freshly pre-seeded MEFs, the MEF medium is aspirated and replaced with bovine iPSC medium.

[0118] FIG. 9 depicts both the derivation of bovine NSCs and their subsequent reprogramming into iPSC cells.

[0119] Example 7 - Confirmation of iPSC markers The iPSC induction method of the present invention (demonstrated in Examples 4, 5, and 6) was found to produce thousands of iPSC clones with high efficiency from canine NSCs (Example 4), porcine NSCs (Example 5), and bovine NSCs (Example 6) in a manner that cannot be achieved by Sendai virus infection under standard conditions.

[0120] Colonies generated using this method had distinct edges and morphology typical of pluripotent stem cells, were easily cloned by selection, and were positive for stem cell markers such as homologous alkaline phosphatase and Oct4, as well as increased expression of the pluripotency markers NANOG and REX1 (see Figure 1 for canine iPSCs and Figure 2 for porcine iPSCs).

[0121] Figure 3 is a heat map showing the expression of pluripotent stem cell markers in canine and porcine fibroblasts, NSCs, and iPSCs; NANOG, PRDM14, and REX1 are all clearly shown to be expressed at much higher levels in iPSCs than in any other cell type.

[0122] Figure 4 shows heat maps showing the expression of somatic markers for endoderm (GATA6, GATA4, and CDX2), ectoderm (GATA3), and mesoderm (BRACHYURY) in canine and porcine iPSCs and embryoid bodies (EBs). It is clear that, unlike EBs, somatic markers are expressed at very low levels in iPSCs.

[0123] Example 8 - Determination of SSEA-3 and SSEA-4 Marker Profiles Canine and porcine iPSCs prepared as described above were dissociated into single cells and stained with antibodies specific to two cell surface antigens (SSEA-3 and SSEA-4) associated with the pluripotency of human iPSCs. The flow cytometry results are shown in Figure 5: the top two panels show canine iPSCs, and the bottom two panels show porcine iPSCs.

[0124] More than 60% of canine iPSCs and more than 80% of porcine iPSCs were positive for SSEA-4 expression. + Within the iPSC population, over 55% of the canine iPSCs were also SSEA-3 + More than 50% of the porcine iPSCs were also SSEA-3 + Furthermore, the iPSC populations analyzed for SSEA-3 and -4 expression were impure because they also contained MEFs (negative for each marker) in the culture medium, and therefore the SSEA-3 and -4 marker expression in canine and porcine iPSCs in this experiment may be underestimated.

[0125] Furthermore, at the time of initial description of this example, the iPSCs have been maintained in culture for over a year. These iPSCs have been extensively passaged and successfully cloned and subcloned multiple times without difficulty. The iPSCs have also been shown to form EBs, express differentiation markers, and can be directly induced to differentiate into all three cell lineages (ectoderm, endoderm, and mesoderm). RNA-seq data demonstrate that both canine and porcine iPSCs produced by this invention share endogenous gene expression consistent with a shared self-renewal phenotype.

[0126] Example 9 - Induction of iPSCs from porcine cells (fibroblast vs. NSC) Biopsies were taken from the skin and brain of the same piglet, as seen in Figure 6. Fibroblast and neural stem cell cultures were derived separately and reprogrammed using the Sendai Cytotune 2.0 reprogramming kit (Thermo Fisher).

[0127] Visible colonies were counted on day 14; smooth-edged colonies were observed in neuronal reprogramming plates, whereas irregular cell patches were seen in fibroblast plates.

[0128] Alkaline phosphatase staining of the reprogramming plates revealed uniform staining of neural-derived iPS colonies (569 colonies counted), while irregularly shaped stained patches (38 patches counted) were observed on the fibroblast reprogramming plates.

[0129] All six colonies selected from neurally reprogrammed cells established iPS cell lineages after selection and passaging (stained with alkaline phosphatase), whereas none of the six patches of fibroblasts established iPS cell clones (none stained with alkaline phosphatase).

[0130] This indicates that iPS cells were successfully produced from pig neural stem cells, but not from skin fibroblasts.

[0131] Example 10 - Induction of iPSCs from porcine neural stem cells using Oct4 As seen in Figure 7, either Oct4 or eGFP episomal plasmids were transfected into porcine neural stem cells.

[0132] Expression from the vector was confirmed by luminescence from the GFP vector within 24 hours after transfection.

[0133] Sustained expression of the construct was confirmed by day 6 post-transfection through GFP expression. Cultures transfected with Oct4 episomes showed increased cell death by day 6 and morphological changes in cell appearance, including the formation of clusters.

[0134] Seven days after transfection, transfected cells were replated onto feeders in stem cell medium. At day 14 after transfection, no iPS-like colonies were observed in either GFP or Oct4-transfected cultures. Alkaline phosphatase staining revealed some spindle-shaped, positively stained cells in both GFP and Oct4 cultures; however, no iPS cell colonies were present. This indicates that Oct4 alone is insufficient to generate iPS cells from porcine neural stem cells.

[0135] Example 11 - Gene Expression Profiling By performing RNA sequencing (RNA-seq) analysis, we identified a set of genes known to be involved in pluripotency; these genes are common to our iPSCs and other iPSCs for which RNA-seq data are publicly available. These genes include endogenous OCT4, NANOG, STAT3, REX1, and PDMR14.

[0136] This RNA-seq analysis confirmed that our iPSCs share all expression patterns of known ground-state iPSC populations. Gene expression was confirmed by qRT-PCR.

[0137] In addition to the gene expression patterns described above, numerous uniquely expressed genes were identified in the iPSCs of the present invention. Pairwise comparison of paired-end RNAseq datasets of the porcine iPSCs of the present invention versus other publicly available RNAseq datasets (NCBI Short Read Archive; Run Accession Numbers: DRR124546, DRR124547, DRR161385, DRR161386, ERR3153959, ERR3153960, SRR10677611, SRR10677612, SRR10677613, SRR10677614, SR R10677615, SRR10677616, SRR10677617, SRR10677618, SRR10677619, SRR10677620, SRR10677621, SRR10677622, ​​SRR4296448, SRR4296 Comparison of the 449, SRR4296450, SRR4296451, SRR5130116, SRR5130117, SRR5130118, SRR5130119, SRR5130120, SRR5130121, SRR8539521, SRR8539522, SRR8539523, SRR8539524, SRR8539525, SRR8539526, SRR8539527, and SRR8539528 provided a list of differentially expressed genes (adjusted p-value < 0.1).

[0138] A total of 21 differentially expressed genes were retained (adjusted p-value < 0.1), including GLDN, PTK2B, LOC110260197, ANGPT1, LY96, NYAP2, THBS2, ULK4, CRSP3, CHST8, SKOR1, KCNMB2, LMNA, HTRA1, PHLDA1, FGF1, and GASK1B.

[0139] Of the 21 differentially expressed genes, five were identified as being highly expressed in canine iPSCs. These differentially expressed genes include LMNA, HTRA1, PHLDA1, FGF1, and GASK1B, which are unique markers of the iPSCs of the present invention. As is known in the art, these genes have diverse functions, including DNA repair, tumor suppression, and cell proliferation, all of which are thought to contribute to sustained proliferation and subsequent differentiation potential.

[0140] Five additional genes (LMNA, HTRA1, PHLDA1, FGF1, and GASK1B) were found to be expressed in the canine and porcine iPSCs of the present invention, as confirmed by RT-PCR and qRT-PCR. Figure 8 shows standard RT-PCR showing the expression of LMNA, HTRA1, FGF1, GASK1B, and PHLDA1 in both porcine and canine iPSCs, as well as confirmation via qPCR with calculated C values. The primers used are listed below each graph. Appropriate gene expression controls were used to verify and normalize the expression of these genes. [Industrial Applicability]

[0141] The present invention therefore provides a method for inducing pluripotency in relatively low potency cells derived from domestic or livestock animals.

Claims

1. Porcine induced pluripotent stem cells (iPSCs) expressing all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1, and GASK1B.

2. The porcine iPSC of claim 1 , which is derived from a neural stem cell (NSC).

3. The porcine iPSCs according to claim 1 or 2, wherein the iPSCs further express all of NANOG, REX1, SSEA-3 and SSEA-4.

4. A population of porcine iPSCs, wherein at least 50% of the iPSCs express all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.

5. The population of porcine iPSCs of claim 4, wherein at least 90% of the iPSCs express one or more or all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.

6. The population of porcine iPSCs of claim 4 or 5, wherein at least 95% of the iPSCs express all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.

7. The population of porcine iPSCs according to any one of claims 4 to 6, which are derived from neural stem cells (NSCs).

8. A method for inducing pluripotency, comprising culturing porcine neural stem cells (NSCs) in the presence of a non-integrating vector expressing the reprogramming factors Oct4, Sox2, cMyc and Klf4.

9. The method of claim 8 , wherein the vector is a Sendai virus vector.

10. The method according to claim 8 or 9 for producing porcine iPSCs that express all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.

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