iPSC INDUCTION
20230313148 · 2023-10-05
Inventors
Cpc classification
C12N5/0696
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
International classification
Abstract
A method of inducing pluripotency in somatic cells derived from a non-human domestic animal or farm animal comprises culturing neural stem cells (NSCs) in the presence of vectors that express one or more reprogramming factors. Canine, porcine and bovine iPSCs are obtained with distinct genetic marker profiles.
Claims
1.-27. (canceled)
28. A porcine, ovine or canine induced pluripotent stem cell (iPSC), wherein the iPSC expresses one or more or all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
29. The porcine, ovine or canine iPSC according to claim 28, wherein the iPSC expresses all of LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
30. The porcine, ovine or canine iPSC according to claim 28, derived from a neural stem cell (NSC).
31. A porcine iPSC according to claim 28.
32. An ovine iPSC according to claim 28.
33. A porcine iPSC according to claim 30.
34. An ovine iPSC according to claim 30.
35. A porcine or ovine iPSC according to claim 28, derived from an NSC, and wherein the iPSC expresses all of LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
36. The porcine, ovine or canine iPSC according to claim 28, wherein the iPSC additionally expresses all of NANOG, REX1, SSEA-3 and SSEA-4.
37. A population of porcine, ovine or canine iPSCs, wherein at least 50% of the iPSCs express all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
38. The population according to claim 37, wherein at least 90% of the iPSCs express one or more or all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
39. The population according to claim 37, wherein at least 95% of the iPSCs express all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
40. The population according to claim 37, derived from neural stem cells (NSCs).
41. The population according to claim 39, derived from neural stem cells (NSCs).
42. A method of inducing pluripotency, comprising culturing porcine, ovine or canine neural stem cells (NSCs) in the presence of non-integrating vectors that express one or more reprogramming factors.
43. The method according to claim 42, wherein the reprogramming factors are selected from two or more or all of Oct4, Sox2, cMyc and Klf4.
44. The method according to claim 42, wherein the reprogramming factors comprise at least Oct4 and cMyc.
45. The method according to claim 42, wherein the vectors are Sendai viral vectors.
46. The method according to claim 42, for producing porcine, ovine or canine iPSCs that express all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
47. The method according to claim 42, for producing porcine or ovine iPSCs that express all of the genes selected from LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
Description
EXAMPLES
[0052] The present invention is now described in more and specific details in relation to the production of specific induced pluripotent stem cells (iPSCs) and with reference to the accompanying drawings in which:
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[0055]
[0056]
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[0059]
[0060]
[0061]
[0062] DNA, RNA and amino acid sequences are referred to below, in which: [0063] SEQ ID NO: 1 is the porcine LMNA forward primer DNA sequence; [0064] SEQ ID NO: 2 is the porcine LMNA reverse primer DNA sequence; [0065] SEQ ID NO: 3 is the canine LMNA forward primer DNA sequence; [0066] SEQ ID NO: 4 is the canine LMNA reverse primer DNA sequence; [0067] SEQ ID NO: 5 is the porcine HTRA1 forward primer DNA sequence; [0068] SEQ ID NO: 6 is the porcine HTRA1 reverse primer DNA sequence; [0069] SEQ ID NO: 7 is the canine HTRA1 forward primer DNA sequence; [0070] SEQ ID NO: 8 is the canine HTRA1 reverse primer DNA sequence; [0071] SEQ ID NO: 9 is the porcine FGF1 forward primer DNA sequence; [0072] SEQ ID NO: 10 is the porcine FGF1 reverse primer DNA sequence; [0073] SEQ ID NO: 11 is the canine FGF1 forward primer DNA sequence; [0074] SEQ ID NO: 12 is the canine FGF1 reverse primer DNA sequence; [0075] SEQ ID NO: 13 is the porcine GASK1B forward primer DNA sequence; [0076] SEQ ID NO: 14 is the porcine GASK1B reverse primer DNA sequence; [0077] SEQ ID NO: 15 is the canine GASK1B forward primer DNA sequence; [0078] SEQ ID NO: 16 is the canine GASK1B reverse primer DNA sequence; [0079] SEQ ID NO: 17 is the porcine PHLDA1 forward primer DNA sequence; [0080] SEQ ID NO: 18 is the porcine PHLDA1 reverse primer DNA sequence; [0081] SEQ ID NO: 19 is the canine PHLDA1 forward primer DNA sequence; and [0082] SEQ ID NO: 20 is the canine PHLDA1 reverse primer DNA sequence.
EXAMPLE 1—DERIVATION OF PRIMARY CANINE NEURAL STEM CELLS
[0083] Neural stem cells (NSCs) were derived from the brain of a 6-year-old dog.
[0084] A large sandwich box was washed, cleaned and transferred to a class II cabinet before being sprayed with 70% industrial methylated spirit and left to air dry. The UV light was turned on and the box left for 20 minutes. Separately, two 10 cm.sup.2 tissue culture dishes were re-coated with iMatrix Laminin 511 and stored at 4° C. overnight.
[0085] Upon receipt, the canine brain was placed in the sterile sandwich box in phosphate buffered saline (PBS) without calcium and magnesium. The brain was cut in half into its two lobes using a scalpel. The area of the brain comprising the subventricular zone (lining the lateral ventricles of the forebrain) was isolated.
[0086] The excised subventricular zone was cut into smaller pieces that were then placed into a 50 ml tube with 10 ml accutase. Shaking intermittently, the tube was incubated for 10 minutes at 37° C. A pipette was then used to help dissociate the cells from the tissue. 20 ml PBS was added to the tube and the larger pieces of tissue were allowed to settle at the bottom of the tube, before the supernatant was removed and placed into a fresh tube. The accutase process was then repeated in the tube with the larger pieces of tissue.
[0087] The fresh tubes comprising supernatant were centrifuged at 1800 rpm for 4 minutes. The resulting supernatant in these tubes was removed and resuspended in 10 ml PBS, before being passed through a 70 μm cell strainer. The cells were then plated out into two 10 cm.sup.2 laminin coated dishes (each with 20 ml RHB-A medium+10 ng/ml huEGF+10 ng/ml HuFGF+penicillin, dihydrostreptomycin and primocin).
[0088] The growth media was replaced every 1-2 days until the cultures were around 70% confluent (around 9-14 days). Each dish was then split into two 75 cm.sup.2 laminin coated flasks.
[0089] NSC morphology was assessed via microscopy; the cells appeared to grow as single cells but, as they became more confluent, looked like a network with thin dendritic processes.
[0090] Before Culture Day 20, the NSCs were frozen down in vials according to standard laboratory practice.
EXAMPLE 2—DERIVATION OF PRIMARY PORCINE NEURAL STEM CELLS
[0091] Neural stem cells (NSCs) were derived from the brain of a 1-day-old piglet.
[0092] A large sandwich box was washed, cleaned and transferred to a class II cabinet before being sprayed with 70% industrial methylated spirit and left to air dry. The UV light was turned on and the box left for 20 minutes. Separately, two 10 cm.sup.2 tissue culture dishes were re-coated with iMatrix Laminin 511 and stored at 4° C. overnight.
[0093] Upon receipt, the porcine brain was placed in the sterile sandwich box in phosphate buffered saline (PBS) without calcium and magnesium. The brain was cut in half into its two lobes using a scalpel. The area of the brain comprising the subventricular zone (lining the lateral ventricles of the forebrain) was isolated.
[0094] The excised subventricular zone was cut into smaller pieces that were then placed into a 50 ml tube with 10 ml accutase. Shaking intermittently, the tube was incubated for 10 minutes at 37° C. A pipette was then used to help dissociate the cells from the tissue. 20 ml PBS was added to the tube and the larger pieces of tissue were allowed to settle at the bottom of the tube, before the supernatant was removed and placed into a fresh tube. The accutase process was then repeated in the tube with the larger pieces of tissue.
[0095] The fresh tubes comprising supernatant were centrifuged at 1800 rpm for 4 minutes. The resulting supernatant in these tubes was removed and resuspended in 10 ml
[0096] PBS, before being passed through a 70 μm cell strainer. The cells were then plated out into two 10 cm.sup.2 laminin coated dishes (each with 20 ml RHB-A medium+10 ng/ml huEGF+10 ng/ml HuFGF+penicillin, dihydrostreptomycin and primocin).
[0097] The growth media was replaced every 1-2 days until the cultures were around 70% confluent (around 9-14 days). Each dish was then split into two 75 cm.sup.2 laminin coated flasks.
[0098] NSC morphology was assessed via microscopy throughout the culture period; the cells appeared to grow as single cells but, as they became more confluent, looked like a network with thin dendritic processes.
[0099] Before Culture Day 20, the NSCs were frozen down in vials according to standard laboratory practice.
EXAMPLE 3—DERIVATION OF PRIMARY BOVINE NEURAL STEM CELLS
[0100] Neural stem cells (NSCs) were derived from the brains of a 1-year-old cow and a 2-year-old cow (both chemically euthanized).
[0101] Two large sandwich boxes were washed, cleaned and transferred to a class II cabinet before being sprayed with 70% industrial methylated spirit and left to air dry. The UV light was turned on and the boxes left for 20 minutes. Separately, four 10 cm.sup.2 tissue culture dishes were re-coated with iMatrix Laminin 511 and stored at 4° C. overnight.
[0102] Upon receipt, the bovine brains were placed in sterile sandwich boxes in phosphate buffered saline (PBS) without calcium and magnesium. The brains were cut in half into two lobes using a scalpel. The area of the brains comprising the subventricular zone (lining the lateral ventricles of the forebrain) was isolated.
[0103] The excised subventricular zone was cut into smaller pieces that were then placed into a 50 ml tube with 10 ml accutase. Shaking intermittently, the tube was incubated for 10 minutes at 37° C. A pipette was then used to help dissociate the cells from the tissue. 20 ml PBS was added to the tube and the larger pieces of tissue were allowed to settle at the bottom of the tube, before the supernatant was removed and placed into a fresh tube. The accutase process was then repeated in the tube with the larger pieces of tissue.
[0104] The fresh tubes comprising supernatant were centrifuged at 1800 rpm for 4 minutes. The resulting supernatant in these tubes was removed and resuspended in 10 ml PBS, before being passed through a 70 μm cell strainer. The cells were then plated out into two 10 cm.sup.2 laminin coated dishes (each with 20 ml RHB-A medium+10 ng/ml bovine EGF+10 ng/ml bovine FGF+penicillin, dihydrostreptomycin and primocin).
[0105] The growth media was replaced every 1-2 days until the cultures were around 70% confluent (around 9-14 days). Each dish was then split into two 75 cm.sup.2 laminin coated flasks.
[0106] NSC morphology was assessed via microscopy throughout the culture period; the cells appeared to form (1) densely packed colonies without processes (like epithelial cells), (2) long stretched out cells in a looser network with dendritic processes, and (3) smaller single cells that developed into a network with thin dendritic processes as they became more confluent.
[0107] Before Culture Day 20, the NSCs were frozen down in vials according to standard laboratory practice.
EXAMPLE 4—REPROGRAMMING OF CANINE NEURAL STEM CELLS
[0108] Canine neural stem cells (NSCs) were reprogrammed using the CytoTune 2.0 Reprogramming kit. This kit uses a modified, non-transmissible form of the Sendai virus delivery system to introduce reprogramming vectors into primary cells, in order to enable the generation of iPSCs. The Sendai virus used in the kit is non-integrating and remains in the cell cytoplasm. The viral particles are cleared from the cell cytoplasm over generations of cell division and can be screened for full clearance using qPCR assays.
[0109] One day before transduction, 3×10.sup.5 actively growing NSCs were plated in 1 well of a 6-well plate on a laminin 511 matrix in RHB-A medium (as described in Examples 1-3). This allowed the cells to adhere and extend, as well as reach a 50-80% confluence before transduction.
[0110] The titre of each CytoTune 2.0 reprogramming vector is lot-dependent, with the lot number specific certificate of analysis (CoA) downloadable from:— [0111] https://www.thermofisher.com/order/catalog/product/A16517
[0112] The Lot specific CoA gave the volumes of viral vector per well to achieve an MOI of 5:5:3 (KOS:hc-Myc:hKlf4).
[0113] 1 ml of warm RHB-A medium was provided per well of cells to be transduced. The Cytotune 2.0 vials (containing the vector) were removed from −80° C. storage and thawed by hand. The vials were centrifuged to collect the contents and then placed on ice. The calculated volume of each vector was added to the RHB-A medium in each well and then mixed with a pipette. The cells were then incubated at 37° C. for 24 hours before the transduction medium was aspirated and replaced with fresh RHB-A (1 ml per well). The RHB-A medium was then changed every 24 hours until Day 6 of the culture.
[0114] The transduced cells were harvested using 0.3 ml/well accutase for 5 minutes at 37° C. The incubation time was adhered to due to the sensitivity of the cells to the enzyme. During dissociation (rounding-up of the cells), 2 ml of RHB-A were added to protect the cells against the enzyme. The cells were collected into 15 ml tubes and centrifuged at 200 g for 4 minutes. The cells were then resuspended in canine iPSC medium, the recipe for which is as follows:—
[0115] To a 500 ml bottle of DMEM/F12 (Thermo Fisher cat 11520396), add 100 ml KOSR (Thermo Fisher 10828028), 5 ml Non Essential Amino Acids 100× (Thermo Fisher 11140035), 5 ml Sodium Pyruvate 100 mM (Thermo Fisher 11360039), 1 ml 2-Mercaptoethanol (Thermo Fisher 31350010), and 5 ml Antibiotic antimycotic (Sigma A5955). Just prior to use, add 62 μl huFGF (Peprotech 100-18B), 62 μl huLIF (Peprotech 300-05), and 500 ul of 3 mM Chiron stock (Tolcris—final conc. 3 μM). Swirl to mix before use.
[0116] The cells were counted before being seeded into the new culture vessels and incubated. In order to optimize reprogramming efficiency, the cells were plated at a relatively high density, typically 1×10.sup.5-5×10.sup.5 cells per 100 mm culture dish.
[0117] The canine iPSC culture medium was changed every 24 hours until colony formation was observed. This colony formation was typically observed within 12 days to 4 weeks.
[0118] Colonies were picked based on morphological properties. The day before picking colonies, a 24 well plate (pre-coated with 0.2% Gelatin/PBS) of irradiated mouse embryonic fibroblasts (MEF Feeder Cells) was prepared (4×10.sup.6 MEF for 24 wells) in MEF media (1 ml per well), the recipe for which is as follows:—
[0119] To a 500 ml bottle of DMEM/F12 (Thermo Fisher cat 11520396), add 50 ml FCS (Sigma F2442), 5 ml Non Essential Amino Acids 100× (Thermo Fisher 11140035), 5 ml Sodium Pyruvate 100 mM (Thermo Fisher 11360039), 1 ml 2-Mercaptoethanol (Thermo Fisher 31350010), and 5 ml Antibiotic antimycotic (Sigma A5955). Swirl to mix before use.
[0120] The picked colonies were each transferred into separate wells of the prepared 24 well plate with canine iPSC media. After colony growth, the colonies were disaggregated using accutase and re-plated in single wells of a prepared 6-well plate of irradiated MEFs. Following confluence, accutase was used and the cells were split into six wells of a prepared 6-well plate of irradiated MEFs. Following confluence, the cells were frozen down in a bank of 12 vials (half a well per vial). As such, each colony resulted in 12 vials of cells being banked.
[0121] When passaging canine iPSCs embedded in MEFs, gentle pipetting of the cells often helps to dissociate the cell types. The cell mixture can then be placed in tubes and centrifuged at 1500 rpm (0.4 rcf) for 3 minutes, before aspirating the media and resuspending the canine iPSCs in canine iPSC media. Before the cells are added to the new pre-plated MEFs, the MEF media is aspirated and replaced with canine iPSC media.
EXAMPLE 5—REPROGRAMMING OF PORCINE NEURAL STEM CELLS
[0122] Porcine neural stem cells (NSCs) were reprogrammed using the CytoTune 2.0 Reprogramming kit. This kit uses a modified, non-transmissible form of the Sendai virus delivery system to introduce reprogramming vectors into primary cells, in order to enable the generation of iPSCs. The Sendai virus used in the kit is non-integrating and remains in the cell cytoplasm. The viral particles are cleared from the cell cytoplasm over generations of cell division and can be screened for full clearance using qPCR assays.
[0123] One day before transduction, 3×10.sup.5 actively growing NSCs were plated in 1 well of a 6-well plate on a laminin 511 matrix in RHB-A medium (as described in Examples 1-3). This allowed the cells to adhere and extend, as well as reach a 50-80% confluence before transduction.
[0124] The titre of each CytoTune 2.0 reprogramming vector is lot-dependent, with the lot number specific certificate of analysis (CoA) downloadable from:— [0125] https://www.thermofisher.com/order/catalog/product/A16517
[0126] The Lot specific CoA gave the volumes of viral vector per well to achieve an MOI of 5:5:3 (KOS:hc-Myc:hKlf4).
[0127] 1 ml of warm RHB-A medium was provided per well of cells to be transduced. The Cytotune 2.0 vials (containing the vector) were removed from −80° C. storage and thawed by hand. The vials were centrifuged to collect the contents and then placed on ice. The calculated volume of each vector was added to the RHB-A medium in each well and then mixed with a pipette. The cells were then incubated at 37° C. for 24 hours before the transduction medium was aspirated and replaced with fresh RHB-A (1 ml per well). The RHB-A medium was then changed every 24 hours until Day 6 of the culture.
[0128] The transduced cells were harvested using 0.3 ml/well accutase for 5 minutes at 37° C. The incubation time was adhered to due to the sensitivity of the cells to the enzyme. During dissociation (rounding-up of the cells), 2 ml of RHB-A was added to protect the cells against the enzyme. The cells were collected into 15 ml tubes and centrifuged at 200 g for 4 minutes. The cells were then resuspended in porcine iPSC medium, the recipe for which is as follows:—
[0129] To a 500 ml bottle of DMEM/F12 (Thermo Fisher cat 11520396), add 100 ml KOSR (Thermo Fisher 10828028), 5 ml Non Essential Amino Acids 100× (Thermo Fisher 11140035), 5 ml Sodium Pyruvate 100 mM (Thermo Fisher 11360039), 1 ml 2-Mercaptoethanol (Thermo Fisher 31350010), and 5 ml Antibiotic antimycotic (Sigma A5955). Just prior to use, add 62 μl huFGF (Peprotech 100-18B), and 62 μl huLIF (Peprotech 300-05). Swirl to mix before use.
[0130] The cells were counted before being seeded into the new culture vessels and incubated. In order to optimize reprogramming efficiency, the cells were plated at a relatively high density, typically 1×10.sup.5-5×10.sup.5 cells per 100 mm culture dish.
[0131] The porcine iPSC culture medium was changed every 24 hours until colony formation was observed. This colony formation was typically observed within 12 days to 4 weeks.
[0132] Colonies were picked based on morphological properties. The day before picking colonies, a 24 well plate (pre-coated with 0.2% Gelatin/PBS) of irradiated mouse embryonic fibroblasts (MEF Feeder Cells) was prepared (4×10.sup.6 MEF for 24 wells) in MEF media (1 ml per well), the recipe for which is as follows:—
[0133] To a 500 ml bottle of DMEM/F12 (Thermo Fisher cat 11520396), add 50 ml FCS (Sigma F2442), 5 ml Non Essential Amino Acids 100× (Thermo Fisher 11140035), 5 ml Sodium Pyruvate 100 mM (Thermo Fisher 11360039), 1 ml 2-Mercaptoethanol (Thermo Fisher 31350010), and 5 ml Antibiotic antimycotic (Sigma A5955). Swirl to mix before use.
[0134] The picked colonies were each transferred into separate wells of the prepared 24 well plate with porcine iPSC media. After colony growth, the colonies were disaggregated using accutase and re-plated in single wells of a prepared 6-well plate of irradiated MEFs. Following confluence, accutase was used and the cells were split into six wells of a prepared 6-well plate of irradiated MEFs. Following confluence, the cells were frozen down in a bank of 12 vials (half a well per vial). As such, each colony resulted in 12 vials of cells being banked.
[0135] When passaging porcine iPSCs embedded in MEFs, gentle pipetting of the cells often helps to dissociate the cell types. The cell mixture can then be placed in tubes and centrifuged at 1500 rpm (0.4 rcf) for 3 minutes, before the aspirating the media and resuspending the porcine iPSCs in porcine iPSC media. Before the cells are added to the new pre-plated MEFs, the MEF media is aspirated and replaced with porcine iPSC media.
EXAMPLE 6—REPROGRAMMING OF BOVINE NEURAL STEM CELLS
[0136] Bovine neural stem cells (NSCs) were reprogrammed using the CytoTune 2.0 Reprogramming kit. This kit uses a modified, non-transmissible form of the Sendai virus delivery system to introduce reprogramming vectors into primary cells, in order to enable the generation of iPSCs. The Sendai virus used in the kit is non-integrating and remains in the cell cytoplasm. The viral particles are cleared from the cell cytoplasm over generations of cell division and can be screened for full clearance using qPCR assays.
[0137] One day before transduction, 3×10.sup.5 actively growing NSCs were plated in 1 well of a 6-well plate on a laminin 511 matrix in RHB-A medium (as described in Examples 1-3). This allowed the cells to adhere and extend, as well as reach a 50-80% confluence before transduction.
[0138] The titre of each CytoTune 2.0 reprogramming vector is lot-dependent, with the lot [0139] number specific certificate of analysis (CoA) downloadable from:—https://www.thermofisher.com/order/catalog/product/A16517
[0140] The Lot specific CoA gave the volumes of viral vector per well to achieve an MOI of 5:5:3 (KOS:hc-Myc:hKlf4).
[0141] 1 ml of warm RHB-A medium was provided per well of cells to be transduced. The Cytotune 2.0 vials (containing the vector) were removed from −80° C. storage and thawed by hand. The vials were centrifuged to collect the contents and then placed on ice. The calculated volume of each vector was added to the RHB-A medium in each well and then mixed with a pipette. The cells were then incubated at 37° C. for 24 hours before the transduction medium was aspirated and replaced with fresh RHB-A (1 ml per well). The RHB-A medium was then changed every 24 hours until Day 6 of the culture.
[0142] The transduced cells were harvested using 0.3 ml/well accutase for 5 minutes at 37° C. The incubation time was adhered to due to the sensitivity of the cells to the enzyme. During dissociation (rounding-up of the cells), 2 ml of RHB-A was added to protect the cells against the enzyme. The cells were collected into 15 ml tubes and centrifuged at 200 g for 4 minutes. The cells were then resuspended in bovine iPSC medium, the recipe for which is as follows:—
[0143] To a 500 ml bottle of DMEM/F12 (Thermo Fisher cat 11520396), add 100 ml KOSR (Thermo Fisher 10828028), 5 ml Non Essential Amino Acids 100× (Thermo Fisher 11140035), 5 ml Sodium Pyruvate 100 mM (Thermo Fisher 11360039), 1 ml 2-Mercaptoethanol (Thermo Fisher 31350010), and 5 ml Antibiotic antimycotic (Sigma A5955). Just prior to use, add 62 μl huFGF (Peprotech 100-18B), and 62 μl huLIF (Peprotech 300-05). Swirl to mix before use.
[0144] The cells were counted before being seeded into the new culture vessels and incubated. In order to optimize reprogramming efficiency, the cells were plated at a relatively high density, typically 1×10.sup.5-5×10.sup.5 cells per 100 mm culture dish.
[0145] The bovine iPSC culture medium was changed every 24 hours until colony formation was observed. This colony formation was typically observed within 12 days to 4 weeks.
[0146] Colonies were picked based on morphological properties. The day before picking colonies, a 24 well plate (pre-coated with 0.2% Gelatin/PBS) of irradiated mouse embryonic fibroblasts (MEF Feeder Cells) was prepared (4×10.sup.6 MEF for 24 wells) in MEF media (1 ml per well), the recipe for which is as follows:—
[0147] To a 500 ml bottle of DMEM/F12 (Thermo Fisher cat 11520396), add 50 ml FCS (Sigma F2442), 5 ml Non Essential Amino Acids 100× (Thermo Fisher 11140035), ml Sodium Pyruvate 100 mM (Thermo Fisher 11360039), 1 ml 2-Mercaptoethanol (Thermo Fisher 31350010), and 5 ml Antibiotic antimycotic (Sigma A5955). Swirl to mix before use.
[0148] The picked colonies were each transferred into separate wells of the prepared 24 well plate with bovine iPSC media. After colony growth, the colonies were disaggregated using accutase and re-plated in single wells of a prepared 6-well plate of irradiated MEFs. Following confluence, accutase was used and the cells were split into six wells of a prepared 6-well plate of irradiated MEFs. Following confluence, the cells were frozen down in a bank of 12 vials (half a well per vial). As such, each colony resulted in 12 vials of cells being banked.
[0149] When passaging bovine iPSCs embedded in MEFs, gentle pipetting of the cells often helps to dissociate the cell types. The cell mixture can then be placed in tubes and centrifuged at 1500 rpm (0.4 rcf) for 3 minutes, before the aspirating the media and resuspending the bovine iPSCs in bovine iPSC media. Before the cells are added to the new pre-plated MEFs, the MEF media is aspirated and replaced with bovine iPSC media.
[0150]
EXAMPLE 7—iPSC MARKER CONFIRMATION
[0151] The iPSC induction method of the invention (as demonstrated in Examples 4, 5 and 6) was found to be highly efficient and generate thousands of iPSC clones from dog NSCs (Example 4), pig NSCs (Example 5) and cow NSCs (Example 6) in a manner not achievable with Sendai infection under standard conditions.
[0152] The colonies generated using this method had discrete edges and morphology typical of pluripotent stem cells. They could be easily cloned by picking, were positive for stem cell markers such as homogenous alkaline phosphatase expression and Oct4, as well as having increased expression of the pluripotency markers NANOG and REX1 (see
[0153]
[0154]
EXAMPLE 8—DETERMINATION OF SSEA-3 AND SSEA-4 MARKER PROFILES
[0155] Canine and porcine iPSCs prepared as per above examples were disaggregated into single cells and stained with antibodies specific for two cell surface antigens associated with pluripotency in human iPSCs (SSEA-3 and SSEA-4). The flow cytometry results are shown in
[0156] In excess of 60% of canine iPSCs and in excess of 80% of porcine iPSCs were positive for SSEA-4 expression, while of that SSEA-4.sup.+ population of iPSCs in excess of 55% of the canine iPSCs were also SSEA-3.sup.+ and in excess of 50% of the porcine iPSCs were also SSEA-3.sup.+. Furthermore, the iPSC populations analyzed for SSEA-3 and -4 expression were impure, as they also included MEFs (negative for each of the markers) from the culture medium, and hence the SSEA-3 and -4 marker expression in the canine and porcine iPSCs is likely undervalued in this experiment.
[0157] Furthermore, at the time of first writing this Example, the iPSCs have been maintained for over a year in culture. These iPSCs have been passaged extensively and have been successfully cloned and subcloned multiple times without difficultly. It has also been found that the iPSCs can be differentiated to form EBs, express differentiation markers and undergo directed differentiation into all three cell lineages (ecto-, endo- and meso-derm). RNAseq data demonstrates that both canine and porcine iPSCs generated according to the invention have endogenous gene expression consistent with a common self-renewing phenotype.
EXAMPLE 9—DERIVATION OF IPSCS FROM PORCINE CELLS (FIBROBLAST VS NSC)
[0158] As can be seen in
[0159] Visible colonies were counted at day 14; smooth edged colonies were observed on neural cell reprogramming plates while irregular cell patches were seen on fibroblast plates.
[0160] Alkaline phosphatase staining of reprogramming plates showed uniform staining of neural derived iPS colonies (569 colonies counted), and irregular-shaped stained patches on fibroblast reprogramming plates (38 patches counted).
[0161] All six colonies picked from neural reprogrammed cells established iPS cell lines after picking and passaging (stained with Alkaline Phosphatase), while none of six picked fibroblast patches established iPS cell clones (none stained with Alkaline Phosphatase).
[0162] This showed success in generating iPS cells from neural stem cells of the pig but not from skin fibroblast cells.
EXAMPLE 10—DERIVATION OF iPSCs FROM PORCINE NEURAL STEM CELLS USING OCT4
[0163] As can be seen in
[0164] Expression from the vectors was confirmed by fluorescence from GFP vector within 24 hrs of transfection.
[0165] Sustained expression of the constructs was confirmed through GFP expression by day 6 after transfection. By day 6 cultures transfected with Oct4 episome showed increased cell death as well as morphological changes in the appearance of cells including the formation of clusters.
[0166] Transfected cells were replated onto feeders in stem cell media on day 7 after transfections. On day 14 after transfection no iPS like colonies were visible on either
[0167] GFP or Oct4 transfected conditions. Staining with alkaline phosphatase showed some spindle like positive stained cells within both GFP and Oct4 cultures; however, no iPS cell colonies were present. This showed Oct 4 alone was insufficient to generate iPS cells from neural stem cells of the pig.
EXAMPLE 11—GENE EXPRESSION PROFILING
[0168] By performing an RNA-sequencing (RNAseq) analysis, a series of genes known to be implicated in pluripotency were identified; these genes being common to the iPSCs of the invention and other iPSCs (for which RNAseq data is publicly available). These genes include endogenous OCT4, NANOG, STAT3, REX1 and PDMR14.
[0169] This RNAseq analysis confirmed that the iPSCs of the invention share all the expression patterns of known ground-state iPSC populations. Gene expression was confirmed by qRT-PCR.
[0170] In addition to the above gene expression pattern, a number of uniquely expressed genes were identified in the iPSCs of the invention. RNAseq datasets were compared to provide a list of differentially expressed genes (adjusted p value<0.1) by pairwise comparison of pig iPSCs according to the invention vs other publicly available pig iPSC paired-end RNAseq datasets (see NCBI Short Read Archive; Run Accession Numbers: DRR124546, DRR124547, DRR161385, DRR161386, ERR3153959, ERR3153960, SRR10677611, SRR10677612, SRR10677613, SRR10677614, SRR10677615, SRR10677616, SRR10677617, SRR10677618, SRR10677619, SRR10677620, SRR10677621, SRR10677622, SRR4296448, SRR4296449, SRR4296450, SRR4296451, SRR5130116, SRR5130117, SRR5130118, SRR5130119, SRR5130120, SRR5130121, SRR8539521, SRR8539522, SRR8539523, SRR8539524, SRR8539525, SRR8539526, SRR8539527 and SRR8539528).
[0171] In total, 21 differentially expressed genes were retained (adjusted p value<0.1). These genes included GLDN, PTK2B, LOC110260197, ANGPT1, LY96, NYAP2, THBS2, ULK4, CRSP3, CHST8, SKOR1, KCNMB2, LMNA, HTRA1, PHLDA1, FGF1 and GASK1B.
[0172] From the 21 differentially expressed genes, 5 genes were identified as also been highly expressed in the canine iPS cells. These differentially expressed genes include high levels of expression of LMNA, HTRA1, PHLDA1, FGF1 and GASK1B as unique markers of the iPSCs of the invention. As is known in the art, these genes have diverse functions from DNA repair, tumour suppression and cell growth, all of which may contribute to sustained growth and subsequent differentiation potential.
[0173] The 5 genes (LMNA, HTRA1, PHLDA1, FGF1 and GASK1B) were additionally found to be expressed in the canine and porcine iPSCs of the invention, as confirmed by RT-PCR and qRT-PCR.
[0174] The invention thus provides a method of inducing pluripotency in a cell of lower relative potency that is derived from a domestic animal or a farm animal.