Patent Application
20220145264
A culture medium is provided for establishing expanded potential stem cell (EPSC) lines for mammals. Methods are provided using the medium for the in vitro conversion and maintenance of cells, including pluripotent cells into EPSCs.
Mammalian embryonic development begins when a sperm and an egg fuse to form a zygote, which undergoes a fixed number of divisions. Up to the 8 cells (8C) stage, an embryo has the capacity to differentiate to all lineages in the embryo proper and extraembryonic tissues and are considered totipotent (Ishiuchi et al 2013). Subsequent cell divisions produce two of the earliest lineages: the trophectoderm epithelium (TE) cells which are restricted to the trophoblast lineage and are essential for the formation of the placenta, and the inner cell mass (ICM) which are pluripotent and give rise to all cell types of the embryo proper, as well as to extra-embryonic endoderm and mesoderm, and embryonic stem (ES) cells (Gardner 1985, Rossant et al 2009, Yamanaka et al 2006).
Although ES cells are capable of differentiating into all germ cell layers of the embryo when returned to the blastocyst environment, they are generally unable to contribute to the trophoblast lineage. Conversely, trophoblast stem cells, which are derived from the trophectoderm can efficiently differentiate into trophoblasts in vitro and in vivo. However, they are unable to differentiate into all germ cell layers of the embryo.
Human embryonic stem cells have been reported to differentiate to trophoblasts in vitro under certain conditions, but there is debate as to whether these in vitro differentiated trophoblasts are bona fide trophoblasts (see, Roberts R M et al 2014) When cultured in vitro, human embryonic stem cells show distinct molecular and biological characteristics that are different from the paradigmatic embryonic stem cells. The terminology ‘naïve’ (or ‘ground state’) and ‘primed’ was introduced to describe the observed differences.
Recently, several researchers have reported alternative conditions for inducing a more ‘naïve’ pluripotent state in conventional human embryonic stem cells, for example, by culturing in a mix of inhibitors (summarised in Theunissen et al 2014). However, although cells produced by these methods display some characteristics which are comparable to naive cells, there are also significant differences.
Despite these findings, it remains unclear whether it is possible to experimentally generate and maintain bona fide pluripotent stem cells from important mammalian animal species, in particular large farm animals. The need remains for improved human pluripotent stem cells for studying human development, biology, and regenerative medicine remains.
Provided herein is a culture medium for establishing expanded potential stem cell (EPSC) lines which resemble naïve or ground state embryonic stem cells, but are also able to differentiate into placenta trophoblasts and the embryo proper.
In one embodiment of the present disclosure is a porcine stem cell culture medium, comprising a basal medium comprising SRC inhibitor, Vitamin C supplement, LIF protein, and ACTIVIN protein. In certain embodiments, the basal medium is DMEM/F-12. In certain embodiments, the basal medium is DMEM. In certain embodiments, the SRC inhibitor is WH-4-023 and XAV939. In certain embodiments, the medium further comprises N2 supplement, B27 supplement, Glutamine Penicillin-Streptomycin, NEAA, 2-mercaptoethanol, CHIR99021, and FBS.
In one embodiment of the present disclosure is a porcine stem cell culture medium, comprising a basal medium comprising SRC inhibitor, Vitamin C supplement, and LIF protein. In certain embodiments, the basal medium is DMEM/F-12. In certain embodiments, the basal medium is DMEM. In certain embodiments, the SRC inhibitor is A-419259 and XAV939. In certain embodiments, the medium further comprises N2 supplement, B27 supplement, Glutamine Penicillin-Streptomycin, NEAA, 2-mercaptoethanol, and CHIR99021.
In one embodiment of the present disclosure is a porcine stem cell culture medium, comprising a basal medium comprising ITS-X 200, Vitamin C supplement, Bovine Albumin Fraction V, Trace elements B, Trace elements C, Reduced glutathione, Defined lipids, SRC inhibitor, endo-IWR-1, SRK inhibitor, and Chiron 99021. In certain embodiments, the basal medium is DMEM/F-12. In certain embodiments, the basal medium is DMEM. In certain embodiments, the SRC inhibitor is XAV939. In certain embodiments, the SRK inhibitor is A-419259. In certain embodiments, the medium further comprises Neurobasal medium, Penicillin-Streptomycin-Glutamine, NEAA, Sodium Pyruvate, 2-Mercaptoethanol, N2, B27, Human Lif protein.
In one embodiment of the present disclosure is a porcine stem cell culture medium, comprising a basal medium comprising ITS-X 200, Vitamin C supplement, Bovine Albumin Fraction V, Trace elements B, Trace elements C, Reduced glutathione, SRC inhibitor, endo-IWR-1, Chiron 99021, Human Lif protein, and Activin A. In certain embodiments, the basal medium is DMEM/F-12. In certain embodiments, the basal medium is DMEM. In certain embodiments, the SRC inhibitor is WH-4-023 and XAV939. In certain embodiments, the medium further comprises Neurobasal medium, Penicillin-Streptomycin-Glutamine, NEAA, Sodium Pyruvate, 2-Mercaptoethanol, N2, and B27.
One embodiment of the present disclosure is a method for producing a population of porcine expanded potential stem cells (EPSCs) which comprises: (i) Providing a population of pluripotent cells, and (ii) Culturing the population in the stem cell disclosed herein.
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Importantly, the targeted pEPSCs retained a normal karyotype. h. Bright field and fluorescence images of the pEPSCEmb colonies with the H2B-mCherry correctly targeted to the ROSA26 locus. i. in vitro differentiation of pEPSCEmb to cells of the three somatic germ layers and the trophectoderm lineage (KRT7+). j. Confocal images of immunostaining SDC1-expressing cells in pEPSCEmb teratoma sections. DAPI stains the nucleus.
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Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. For purposes of the present disclosure, the following terms are defined below.
“iPSCs” are pluripotent cells which are derived from non-pluripotent, differentiated ancestor cells. Suitable ancestor cells include somatic cells, such as adult fibroblasts and peripheral blood cells. These ancestor cells are typically reprogrammed by the introduction of pluripotency genes (or RNA encoding them) or their corresponding proteins into the cell, or by re-activating the endogenous pluripotency genes. The introduction techniques include plasmid or viral transfection or direct protein delivery in certain embodiments.
“Feeder cells” or “feeders” are terms used to describe cells of one type that are co-cultured with cells of another type, to provide an environment in which the cells of the second type can grow. A feeder free culture will contain less than about 5% feeder cells. Compositions containing less than 1%, 0.2%, 0.05%, or 0.01% feeder cells (expressed as % of total cells in the culture) are increasingly more preferred.
A “growth environment” is an environment in which cells of interest will proliferate in vitro. Features of the environment include the medium in which the cells are cultured, and a supporting structure (such as a substrate on a solid surface) if present.
A “nutrient medium” is a medium for culturing cells containing nutrients that promote proliferation, including: isotonic saline, buffer, amino acids, serum or serum replacement, and other exogenously added factors.
A “conditioned medium” is prepared by culturing a first population of cells in a medium, and then harvesting the medium. The conditioned medium, along with anything secreted into the medium by the cells, may then be used to support the growth of a second population of cells. Where a particular ingredient or factor is described as having been added to the medium, the factor has been mixed into the medium by deliberate manipulation.
The term “antibody” as used in this disclosure refers to both polyclonal and monoclonal antibody of any species. The ambit of the term encompasses not only intact immunoglobulin molecules, but also fragments and genetically engineered derivatives of immunoglobulin molecules and equivalent antigen binding molecules that retain the desired binding specificity.
The terms “isolated” or “purified” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
The term “serum” as used herein means the liquid portion of the blood that remains after blood cells and fibrinogen/fibrin are removed. The term “serum-free culture medium” means a culture medium containing no serum or product extracted from sera of animals and especially those originating from mammals, birds, fish or crustaceans.
The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
Unless otherwise indicated by the terms “exactly”, “precisely”, or another equivalent term, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used herein, are to be understood as being modified in all instances by the term “about”, and thus to inherently include variations of up to 10% greater or less than the actual number stated. Accordingly, the numerical parameters herein are approximations depend upon the desired properties sought to be obtained by the present disclosure. At the very least, each numerical parameter should at least be construed given the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters describing the broad scope of the disclosure are approximations, the numerical values in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains standard deviations that necessarily result from the errors found in the numerical value's testing measurements.
Described herein is the production of expanded potential stem cells (EPSCs) from populations of pluripotent cells. EPSCs have ‘naïve’ or ground state properties and have an expanded potential to differentiate into extraembryonic cell lines (trophoblasts and extraembryonic endoderm in the yolk sac) as well as cells of the embryo proper. EPSCs may be produced from different pluripotent cell lines which are cultured in expanded potential stem cell media (EPSCM). EPSCs have been successfully differentiated into a range of cell types including somatic cells and trophoblast cells. EPSCs may be useful for studying the mechanisms of development and EPSCs or cells differentiated therefrom. This helps particularly with research and R&D in regenerative medicine, for example in disease modelling, screening for therapeutics, testing toxicity, studying genetic diseases and studying reproductive biology.
A population of expanded potential stem cells (EPSCs) may be produced by culturing a population of pluripotent cells (PSCs) in an expanded potential stem cell medium (EPSCM) to produce a population of EPSCs. Described herein is the derivation of porcine EPSC (pEPSC) lines either directly from preimplantation embryos or by reprogramming porcine fetal fibroblasts. Pluripotent cells may include embryonic stem cells (ESCs) and non-embryonic stem cells, for example fetal and adult stem cells, and induced pluripotent stem cells (iPSCs).
While porcine iPSCs are available, the use of these cells for the screen is confounded by the leaky expression of the transgenic reprogramming factors after reprogramming or by low levels of expression of the endogenous pluripotency genes [11-19]. To overcome this challenge, new porcine iPSCs are generated to express pluripotency genes such as Doxycycline (Dox)-inducible LIN28, NANOG, LRH1 and RARG, in concert with the four Yamanaka factors.
The pluripotency genes or proteins may comprise one, two, three, four, five or six of a LIN family member, NANOG family member, LRH family member, RAR family member.
The Lrh family member may be LRH1.
The Rar family member may be Rar-g.
In one embodiment, pluripotency genes or proteins may comprise Oct4, Sox2, Klf4 and c-Myc (Yamanaka factors).
Techniques for the production of iPSCs are well-known in the art (Yamanaka et al Nature 2007; 448:313-7; Yamanaka 6 2007 Jun. 7; 1(1):39-49; Kim et al Nature. 2008 Jul. 31; 454(7204):646-50; Takahashi Cell. 2007 Nov. 30; 131(5):861-72. Park et al Nature. 2008 Jan. 10; 451(7175):141-6; Kimet et al Cell Stem Cell. 2009 Jun. 5; 4(6):472-6; Dallier, L., et al. Stem Cells, 2009. 999(999A), Wang W, et al. PNAS. (2011) 108; 45; 18283-8. However, the strategy provided herein substantially improves the efficiency of reprogramming wild-type German Landrace porcine fetal fibroblasts (PFFs) and transgenic PFFs, in which a tdTomato cassette had been inserted into the 3′ UTR of the porcine OCT4 (POU5F1) locus (POT PFFs) [20], to putative iPSC colonies (Extended Data
Upon Dox removal, the iPSCs differentiated within 4-5 days, concomitant with rapid down-regulation of the exogenous reprogramming factors and endogenous pluripotency genes and with increased expression of both embryonic and extraembryonic cell lineage genes (Extended Data
Thus, a population of pluripotent stem cells may be obtained by reprogramming non-pluripotent cells, such as somatic cells into induced pluripotent stem cells (iPSCs) by introducing pluripotency genes or their corresponding proteins, or by reactivating the endogenous pluripotency genes, using techniques which are known in the art and discussed herein.
The iPSCs may be obtained from a mammalian individual. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors. In some embodiments, the iPSCs may be derived from somatic cells or other antecedent cells obtained from an individual. The iPSCs may be used to produce a population of EPSCs which share the genotype of that individual. In some embodiments the EPSCs or cells differentiated therefrom in vitro produced from an individual, may be useful in studying the mechanisms of a disease condition associated with that individual.
Suitable culture media for pluripotent cells are well-known in the art and include; Knockout Dulbecco's Modified Eagle's Medium (KO-DMEM) supplemented with 20% Serum Replacement, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 0.1 mM 0-mercaptoethanol and 4 ng/ml to 10 ng/ml FGF2; or Knockout (KS) medium supplemented with 4 ng/ml FGF2; or KO-DMEM supplemented with 20% Serum Replacement, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 0.1 mM (3-mercaptoethanol and 4 ng/ml to 10 ng/ml human FGF2; or DMEM/F12 supplemented with 20% knockout serum replacement (KSR), 6 ng/ml FGF2 (PeproTech), 1 mM L-Gln, 100 μm non-essential amino acids, 100 μM 2-mercaptoethanol, 50 U/ml penicillin and 50 mg/ml streptomycin.
In certain embodiments, a population of pluripotent cells for use in the present methods may be cultured in a chemically defined medium (CDM) which comprise a chemically defined basal medium comprising inhibitors for GSK3 (CHER99021), SRC (WH-4-023) and Tankyrases (XAV939) (the last two were inhibitors important for mouse EPSCs[1]) (#517, porcine EPSC medium: pEPSCM) (Extended Data
To maintain Dox-independent porcine iPSCs in the undifferentiated state (Extended Data
Suitable techniques for cell culture are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct. 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec. 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug. 2005) ISBN: 0471453293, Ho W Y et al J Immunol Methods. (2006) 310:40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430) Basic Cell Culture Protocols' by J. Pollard and J. M. Walker (1997), ‘Mammalian Cell Culture: Essential Techniques’ by A. Doyle and J. B. Griffiths (1997), ‘Human Embryonic Stem Cells’ by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside’ by A. Bongso (2005), Peterson & Loring (2012) Human Stem Cell Manual: A Laboratory Guide Academic Press and ‘Human Embryonic Stem Cell Protocols’ by K. Turksen (2006). Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be employed for the above culture steps, for example 37° C., 5% Carbon Dioxide.
A population of pluripotent cells for use may be cultured in the present expanded potential stem cell medium (EPSCM) described herein to produce a population of EPCSs. Once converted, the EPSCs may be cultured in an EPSC maintenance medium (EPSCMM). The maintenance medium may have a composition as described herein, for example, fewer inhibitors/modulators compared to the EPSCM which was used for converting the cells. Once converted, EPSCs may not require as many inhibitors/modulators to maintain them in culture as EPSCs.
A suitable porcine EPSCM of 500 ml comprise one or more:
0.3 μM WH-4-023 (SRC inhibitor, TOCRIS, Cat. No. 5413),
2.5 μM XAV939 (Sigma, Cat. No. X3004) or 2.0 μM IWR-1 (TOCRIS, Cat. No. 3532),
50 μg/ml Vitamin C (Sigma, Cat. No. 49752-100G),
10 ng/ml LIF (Stem Cell Institute, University of Cambridge. SCI),
20 ng/ml ACTIVIN (SCI).
Optionally the EPSCM may also contain LIE The EPSCM may contain a nutrient medium.
A suitable EPSCM or EPSCMM comprise nutrient medium and a GSK3 inhibitor.
A suitable EPSCM or EPSCMM may contain one or more of the following ingredients: 482.5 ml DMEM/F-12 (Gibco, Cat. No. 21331-020), 2.5 ml N2 supplement (Thermo Fisher Scientific, Cat. No. 17502048), 5 ml B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044), 5 ml 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 5 ml 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016), 110 μM 2-mercaptoethanol (Sigma, Cat. No. M6250), and 0.2 μM CHIR99021(GSK3i, TOCRIS, Cat. No. 4423), 0.3% FBS (Gibco, Cat. No. 10270).
A suitable porcine EPSCM of 500 ml comprise one or more of the following ingredients:
ITS-X 200× (thermos, 51500056), add 2.5 ml;
Vitamin C(Sigma, 49752-100G), working concentration 64 μg/ml;
Bovine Albumin Fraction V (7.5% solution) (Thermo, 15260037), 3 ml;
Trace elements B(Corning, MT99175CI) 1000×
Trace elements C(Corning, MT99176CI) 1000×
reduced glutathione(sigma, G6013-5G) 10 mg/ml, add 165 ul
XAV939 (Sigma X3004), working concentration 2.5 μM;
endo-IWR-1(Tocris, Cat. No. 3532), working concentration 1 μM
WH-4-023 (Tocris, Cat. No. 5413), working concentration 0.16 μM;
Chiron 99021 (Tocris Bioscience, 4423), working concentration 0.2 μM;
Human Lif, working concentration 10 ng/ml; and
Activin A(S TEM CELL TECHNOLOGY, Catalog #78001.1) 20 ng/ml.
A suitable EPSCM or EPSCMM may contain one or more of the following ingredients: F12 DMEM (Gibco, 21331-020), add 240 ml; Neurobasal medium (Life Technologies, 21103-049) 240 ml; Penicillin-Streptomycin-Glutamine (100×) (Gibco, 10378016), add 5 ml; NEAA 100× (Gibco, 11140050), add 5 ml; Sodium Pyruvate100× (gibco, 11360070), add 5 ml; 14.3M 2-Mercaptoethanol (M6250 Aldrich, Sigma), add 3.8 μl (working concentration 110 μM); 200×N2 (Thermo 17502048), add 2.5 ml; and 100×B27 (Thermo 17504044), add 5 ml.
A suitable human EPSCM of 500 ml comprise one or more of the following ingredients:
0.1 μM A-419259 (SRC inhibitor, TOCRIS, Cat. No. 3914),
2.5 μM XAV939 (Sigma, Cat. No. X3004) or 2.5 μM IWR-1 (TOCRIS, Cat. No.
3532),
50 μg/ml Vitamin C (Sigma, Cat. No. 49752-100G),
10 ng/ml LIF (SCI).
Optionally the EPSCM may also contain LIF. The EPSCM may contain a nutrient medium.
A suitable EPSCM or EPSCMM comprise a nutrient medium together with a GSK3 inhibitor.
A suitable EPSCM or EPSCMM may contain one or more of the following ingredients: 482.5 ml DMEM/F-12 (Gibco, Cat. No. 21331-020), 2.5 ml N2 supplement (Thermo Fisher Scientific, Cat. No. 17502048), 5 ml B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044), 5 ml 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 5 ml 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016), 110 μM 2-mercaptoethanol (Sigma, Cat. No. M6250), and 1.0 μM CHIR99021(GSK3 inhibitor, TOCRIS, Cat. No. 4423).
A suitable human EPSCM of 500 ml may comprise one or more of the following ingredients:
ITS-X 200× (thermos, 51500056), add 2.5 ml
Vitamin C (Sigma, 49752-100G), working concentration 64 μg/ml;
Bovine Albumin Fraction V (7.5% solution) (Thermo, 15260037), 3 ml;
Trace elements B (Corning, MT99175CI) 1000×
Trace elements C (Corning, MT99176CI) 1000×
reduced glutathione (sigma, G6013-5G) 10 mg/ml, add 165 μl
defined lipids (Invitrogen, 11905031) 500×
XAV939 (Sigma X3004), working concentration 2.5 μM;
endo-IWR-1(Tocris, Cat. No. 3532), working concentration 2.5 μM
A419259 (Tocris Bioscience, 3748), working concentration 0.1 μM;
Chiron 99021 (Tocris Bioscience, 4423), working concentration 1.0 μM.
A suitable EPSCM or EPSCMM may contain one or more of the following ingredients: F12 DMEM (Gibco, 21331-020), add 240 ml; Neurobasal medium (Life Technologies, 21103-049) 240 ml; Penicillin-Streptomycin-Glutamine (100×) (Gibco, 10378016), add 5 ml; NEAA 100× (Gibco, 11140050), add 5 ml; Sodium Pyruvate100×(gibco, 11360070), add 5 ml; 14.3M 2-Mercaptoethanol (M6250 Aldrich, Sigma), add 3.8 μl (working concentration 110 μM); 200×N2 (Thermo 17502048), add 2.5 ml; 100×B27 (Thermo 17504044), add 5 ml; and Human Lif, working concentration 10 ng/ml.
In one embodiment, porcine EPSC media comprises:
DMEM/F-12 (Gibco, Cat. No. 21331-020), or knockout DMEM (Gibco, Cat. No. 10829-018), basal media, 98%;
N2 supplement (Thermo Fisher Scientific, Cat. No. 17502048), range from 0.1 to 1%, between 0.25 to 0.75%, between 0.4-0.6%;
B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044), range from 0.1 to 2%, between 0.5 to 1.5%, between 0.8-1.0%;
Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), basal supplement, 1%;
NEAA (Thermo Fisher Scientific, Cat. No. 10378-016), basal supplement, 1%;
2-mercaptoethanol (Sigma, Cat. No. M6250), basal supplement, 110 μM;
CHIR99021(GSK3i, TOCRIS, Cat. No. 4423), range from 0.05 to 0.5 μM, between 0.1 to 0.5 μM, between 0.2 to 0.3 μM;
WH-4-023 (SRC inhibitor, TOCRIS, Cat. No. 5413), range from 0.1 to 1.0 μM, between 0.2 to 0.8 μM, between 0.3 to 0.5 μM;
XAV939 (Sigma, Cat. No. X3004), range from 1 to 10 μM, between 2 to 5 μM, even between 2.5 to 4.5 μM; or IWR-1 (TOCRIS, Cat. No. 3532), range from 1 to 10 μM, between 2 to 5 μM, between 2.5 to 4.5 μM;
Vitamin C (Sigma, Cat. No. 49752-100G), range from 10 to 100 μg/ml, between 20 to 80 μg/ml, between 50 to 70 μg/ml;
LIF (Stem Cell Institute, University of Cambridge. SCI), range from 1 to 20 ng/ml, between 5 to 15 ng/ml, between 8 to 12 ng/ml;
ACTIVIN (SCI), range from 10 to 50 ng/ml, between 15 to 30 ng/ml, even between 20 to 25 ng/ml;
FBS (Gibco, Cat. No. 10270) range from 0.1 to 0.5%, preferably between 0.2 to 0.4%, between 0.25-0.35% and
ITS-X (thermos, 51500056), range from 0.1 to 2%, preferably between 0.2 to 0.8%, between 0.4-0.6%.
In another embodiment, human EPSC media comprises:
DMEM/F-12 (Gibco, Cat. No. 21331-020), or knockout DMEM (Gibco, Cat. No. 10829-018), basal media, 98%;
N2 supplement (Thermo Fisher Scientific, Cat. No. 17502048), range from 0.1 to 1%, between 0.25 to 0.75%, between 0.4-0.6%;
B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044), range from 0.1 to 2%, between 0.5 to 1.5%, between 0.8-1.0%;
Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), basal supplement, 1%;
NEAA (Thermo Fisher Scientific, Cat. No. 10378-016), basal supplement, 1%;
2-mercaptoethanol (Sigma, Cat. No. M6250), basal supplement, 110 μM;
CHIR99021(GSK3 inhibitor, TOCRIS, Cat. No. 4423), range from 0.2 to 2 μM, between 0.5 to 1.5 μM, between 0.8 to 1.2 μM;
A-419259 (SRC inhibitor, TOCRIS, Cat. No. 3914), range from 0.05 to 0.5 μM, between 0.1 to 0.5 μM, between 0.15 to 0.3 μM XAV939 (Sigma, Cat. No. X3004) range from 1 to 10 μM, between 2 to 5 μM, between 2.5 to 4.5 μM or IWR-1 (TOCRIS, Cat. No. 3532), range from 1 to 10 μM, between 2 to 5 μM, between 2.5 to 4.5 μM;
Vitamin C (Sigma, Cat. No. 49752-100G), range from 10 to 100 μg/ml, between 20 to 80 μg/ml, between 50 to 70 μg/ml;
LIF (SCI), range from 1 to 20 ng/ml, between 5 to 15 ng/ml, between 8 to 12 ng/ml;
In another embodiment, human EPSC media comprises:
F12 DMEM (Gibco, 21331-020), basal media, 48%
Neurobasal medium (Life Technologies, 21103-049), basal media, 48%
Penicillin-Streptomycin-Glutamine (Gibco, 10378016), basal supplement, 1%
NEAA (Gibco, 11140050), basal supplement, 1%
Sodium Pyruvate (gibco, 11360070), basal supplement, 1%
2-Mercaptoethanol (Aldrich, Sigma), basal supplement, 110 μM
N2 (Thermo 17502048), range from 0.1 to 1%, between 0.25 to 0.75%, between 0.4-0.6%
B27 (Thermo 17504044), range from 0.1 to 2%, between 0.5 to 1.5%, between 0.8-1.0%
ITS-X (thermos, 51500056), range from 0.1 to 1%, between 0.25 to 0.75%, between 0.4-0.6%
Vitamin C (Sigma, 49752-100G), range from 10 to 100 μg/ml, between 20 to 100 μg/ml, between 50 to 70 μg/ml
Bovine Albumin Fraction V (7.5% solution) (Thermo, 15260037), range from 0.1% to 1%, between 0.2 to 0.8%, between 0.4-0.6%
trace elements B (Corning, MT99175CI) basal supplement, 0.1%
trace elements C (Corning, MT99176CI) basal supplement, 0.1%
reduced glutathione (sigma, G6013-5G) range from 1 to 20 μg/ml, between 1 to 10 μg/ml, between 2 to 5 μg/ml
defined lipids (Invitrogen, 11905031) basal supplement, 0.2%
XAV939 (Sigma X3004), range from 1 to 10 μM, between 2 to 5 μM, between 2.5 to 4.5 μM
endo-IWR-1(Tocris, Cat. No. 3532), range from 1 to 10 μM, between 2 to 5 μM, between 2.5 to 4.5 μM
A419259 (Tocris Bioscience, 3748), range from 0.05 to 0.5 μM, between 0.1 to 0.5 μM, between 0.15 to 0.3 μM
Chiron 99021 (Tocris Bioscience, 4423), range from 0.2 to 2 μM, between 0.5 to 1.5 μM, between 0.8 to 1.2 μM
Human Lif(Stem Cell Institute, University of Cambridge. SCI), range from 1 to 20 ng/ml, between 5 to 15 ng/ml, between 8 to 12 ng/ml
In one embodiment, Porcine EPSC media comprises:
F12 DMEM (Gibco, 21331-020), basal media, 48%
Neurobasal medium (Life Technologies, 21103-049), basal media, 48%
Penicillin-Streptomycin-Glutamine (Gibco, 10378016), basal supplement, 1%
NEAA (Gibco, 11140050), basal supplement, 1%
Sodium Pyruvate (gibco, 11360070), basal supplement, 1%
2-Mercaptoethanol (Aldrich, Sigma), basal supplement, 110 μM
N2 (Thermo 17502048), range from 0.1 to 1%, between 0.25 to 0.75%, between 0.4-0.6%
B27 (Thermo 17504044), range from 0.1 to 2%, between 0.5 to 1.5%, between 0.8-1.0%
ITS-X (thermos, 51500056), range from 0.1 to 1%, between 0.25 to 0.75%, between 0.4-0.6%
Vitamin C (Sigma, 49752-100 G), range from 10 to 100 μg/ml, between 20 to 100 μg/ml, between 50 to 70 μg/ml
Bovine Albumin Fraction V (7.5% solution) (Thermo, 15260037), range from 0.1% to 1%, between 0.2 to 0.8%, between 0.4-0.6%
trace elements B (Corning, MT99175CI) basal supplement, 0.1%
trace elements C (Corning, MT99176CI) basal supplement, 0.1%
reduced glutathione (sigma, G6013-5G) range from 1 to 20 μg/ml, between 1 to 10 μg/ml, between 2 to 5 μg/ml
XAV939 (Sigma X3004), range from 1 to 10 μM, between 2 to 5 μM, between 2.5 to 4.5 μM
endo-IWR-1 (Tocris, Cat. No. 3532), range from 1 to 10 μM, between 1 to 5 μM, between 1 to 2 μM
WH-4-023 (Tocris, Cat. No. 5413), range from 0.1 to 1.0 μM, between 0.1 to 0.5 μM, between 0.1 to 0.2 μM
Chiron 99021 (Tocris Bioscience, 4423), range from 0.05 to 0.5 μM, between 0.1 to 0.5 μM, between 0.2 to 0.3 μM
Human Lif(Stem Cell Institute, University of Cambridge. SCI), range from 1 to 20 ng/ml, between 5 to 15 ng/ml, between 8 to 12 ng/ml
Activin A (STEM CELL TECHNOLOGY, Catalog #78001.1). range from 10 to 50 ng/ml, between 15 to 30 ng/ml, between 20 to 25 ng/ml.
In one embodiment, 500 ml porcine EPSC media comprises:
482.5 ml DMEM/F-12 (Gibco, Cat. No. 21331-020),
2.5 ml N2 supplement (Thermo Fisher Scientific, Cat. No. 17502048),
5 ml B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044),
5 ml 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050),
5 ml 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016),
110 μM 2-mercaptoethanol (Sigma, Cat. No. M6250),
0.2 μM CHIR99021(GSK3i, TOCRIS, Cat. No. 4423),
0.3 μM WH-4-023 (SRC inhibitor, TOCRIS, Cat. No. 5413),
2.5 μM XAV939 (Sigma, Cat. No. X3004) or 2.0 μM IWR-1 (TOCRIS, Cat. No. 3532),
50 μg/ml Vitamin C (Sigma, Cat. No. 49752-100G),
10 ng/ml LIF (Stem Cell Institute, University of Cambridge. SCI),
20 ng/ml ACTIVIN (SCI),
1 ml ITS-X 200× (thermos, 51500056), and
0.3% FBS (Gibco, Cat. No. 10270).
In another embodiment, 500 ml human EPSC media comprises:
482.5 ml DMEM/F-12 (Gibco, Cat. No. 21331-020),
2.5 ml N2 supplement (Thermo Fisher Scientific, Cat. No. 17502048),
5 ml B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044),
5 ml 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050),
5 ml 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016),
110 μM 2-mercaptoethanol (Sigma, Cat. No. M6250),
1.0 μM CHIR99021(GSK3 inhibitor, TOCRIS, Cat. No. 4423),
0.1 μM A-419259 (SRC inhibitor, TOCRIS, Cat. No. 3914),
2.5 μM XAV939 (Sigma, Cat. No. X3004) or 2.5 μM IWR-1 (TOCRIS, Cat. No. 3532),
50 μg/ml Vitamin C (Sigma, Cat. No. 49752-100 G), and 10 ng/ml LIF (SCI).
In another embodiment, 500 ml human EPSC media comprises:
F12 DMEM (Gibco, 21331-020), add 240 ml,
Neurobasal medium (Life Technologies, 21103-049) 240 ml,
Penicillin-Streptomycin-Glutamine (100×) (Gibco, 10378016), add 5 ml,
NEAA 100× (Gibco, 11140050), add 5 ml,
Sodium Pyruvate100× (gibco, 11360070), add 5 ml,
14.3M 2-Mercaptoethanol (M6250 Aldrich, Sigma), add 3.8 μl (working concentration 110 μM),
200×N2 (Thermo 17502048), add 2.5 ml,
100×B27 (Thermo 17504044), add 5 ml,
ITS-X 200×(thermos, 51500056), add 2.5 ml,
Vitamin C (Sigma, 49752-100G), working concentration 64 ug/ml,
Bovine Albumin Fraction V (7.5% solution) (Thermo, 15260037), 3 ml,
trace elements B, (Corning, MT99175CI) 1000×
trace elements C, (Corning, MT99176CI) 1000×
reduced glutathione (sigma, G6013-5G) 10 mg/ml, add 165 ul,
defined lipids, (Invitrogen, 11905031) 500×
XAV939 (Sigma X3004), working concentration 2.5 μM,
endo-IWR-1(Tocris, Cat. No. 3532), working concentration 2.5 μM,
A419259 (Tocris Bioscience, 3748), working concentration 0.1 μM,
Chiron 99021 (Tocris Bioscience, 4423), working concentration 1.0 μM, and
Human Lif, working concentration 10 ng/ml.
In one embodiment, 500 ml Porcine EPSC media comprises:
F12 DMEM (Gibco, 21331-020), add 240 ml,
Neurobasal medium (Life Technologies, 21103-049) 240 ml,
Penicillin-Streptomycin-Glutamine (100×) (Gibco, 10378016), add 5 ml,
NEAA 100× (Gibco, 11140050), add 5 ml,
Sodium Pyruvate100× (gibco, 11360070), add 5 ml,
14.3M 2-Mercaptoethanol (M6250 Aldrich, Sigma), add 3.8 μl (working concentration 110 μM),
200×N2 (Thermo 17502048), add 2.5 ml,
100×B27 (Thermo 17504044), add 5 ml,
ITS-X 200×(thermos, 51500056), add 2.5 ml,
Vitamin C (Sigma, 49752-100 G), working concentration 64 ug/ml,
Bovine Albumin Fraction V (7.5% solution) (Thermo, 15260037), 3 ml,
trace elements B, (Corning, MT99175CI) 1000×
trace elements C, (Corning, MT99176CI) 1000×
reduced glutathione (sigma, G6013-5G) 10 mg/ml, add 165 ul,
XAV939 (Sigma X3004), working concentration 2.5 μM,
endo-IWR-1(Tocris, Cat. No. 3532), working concentration 1 μM,
WH-4-023 (Tocris, Cat. No. 5413), working concentration 0.16 μM,
Chiron 99021 (Tocris Bioscience, 4423), working concentration 0.2 μM,
Human Lif, working concentration 10 ng/ml, and
Activin A (STEM CELL TECHNOLOGY, Catalog #78001.1) 20 ng/ml. Suitable chemically defined basal media are described above and include Iscove's Modified Dulbecco's Medium (IMDM), Ham's F12, Advanced Dulbecco's modified eagle medium (DMEM/F12) (Price et al Focus (2003), 25 3-6), RPMI-1640 (Moore, G. E. and Woods L. K., (1976) Tissue Culture Association Manual. 3, 503-508). A preferred chemically defined basal medium is DMEM/F12.
The basal medium may be supplemented by serum-containing or serum-free culture medium supplements and/or additional components. Suitable supplements and additional components are described above and may include L-glutamine or substitutes, such as GlutaMAX-1™, chemically defined lipids, albumin, 1-thiolglycerol, polyvinyl alcohol, insulin, vitamins, such as vitamin C, antibiotics such as penicillin and/or streptomycin and transferrin.
Each of the inhibitors or modulators may be added to the EPSCM to an amount ranging from 0.1 μM to 150 μM; in certain embodiments, in an amount of 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, or 150 μM.
Each of the inhibitors or modulators may be added to the EPSCM to an amount ranging from 0.05 μM to 0.1 μM, 0.1 μM to 1 μM, 1 μM to 2 μM, 2 μM to 3 μM, 3 μM to 4 μM, 4 μM to 5 μM, 5 μM to 6 μM, 6 μM to 7 μM, 7 μM to 8 μM, 8 μM to 9 μM, 9 μM to 10 μM, 10 μM to 15 μM, 15 μM to 20 μM, 20 μM to 30 μM, 30 μM to 40 μM, 40 μM to 50 μM, 50 μM to 60 μM, 60 μM to 70 μM, 70 μM to 80 μM, 80 μM to 90 μM, 90 μM to 100 μM, 100 μM to 110 μM, 110 μM to 120 μM, 120 μM to 130 μM, 130 μM to 140 μM, 140 μM to 150 μM, or 150 μM to 160 μM.
Suitable inhibitors or modulators include natural and synthetic small molecule inhibitors or antibodies. Suitable Mek-ERK, JNK, p38, Src, GSK3 and Wnt pathway inhibitors are known in the art and are commercially available. The Mek-ERK pathway is chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. The major proteins in this pathway are MEK and ERK. Inhibiting these proteins will disrupt signaling in this pathway. Thus, the inhibitor may directly or indirectly inhibit MEK or ERK such that signaling in this pathway is disrupted. For example, the inhibitor may be a MEK inhibitor or ERK inhibitor.
Suitable Jun N-Terminal Kinase (JNK) inhibitors include JNK Inhibitor VIII (catalogue number sc-202673), RWJ 67657 (catalogue number sc-204251), Antibiotic LL Z1640-2 (catalogue number sc-202055), SX 011 (sc-358841), Bentamapimod (sc-394312), AEG 3482 (sc-202911), from www.scbt.com or SP600125 JNK inhibitor from www.invivogen.com. In one embodiment, the JNK Inhibitor is SP600125.
Suitable p38 inhibitors include sB203580 which inhibits both the a and R isoforms of p38 MAPK available from www.invivogen.com, p38 MAP Kinase Inhibitor IV (catalogue number sc-204159), LY2228820 (catalogue number sc-364525), PH-797804 (catalogue number sc-364579), p38 MAP Kinase Inhibitor (catalogue number sc-204157), SX 011 (sc-358841) and 2-(4-Chlorophenyl)-4-(fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one (sc-220665) available from www.scbt.com. In one embodiment, the p38 Inhibitor is sB203580.
The Src family kinases (SFK) are a family of non-receptor tyrosine kinases that included nine highly related members. Broad spectrum Src Kinase family inhibitors which inhibit multiple src family members are available and known in the art. Suitable Src Kinase family inhibitors include A-419259 which is a broad spectrum Src family kinase inhibitor (available from Sigma-Aldrich). Other suitable SRK inhibitors include PP1, PP2 and CGP77675 also available from Sigma-Aldrich (www.sigmaaldrich.com), and A419259 trihydrochloride or KB SRC 4 available from Tochris Bioscience (www.tochris.com). In one embodiment, the Src Kinase family inhibitor is WH-4-023 or A-419259.
Suitable GSK3 inhibitors include CHIR99021, a selective and potent GSK3 inhibitor available from Tocris Bioscience(cat 4423), or BIO (cat 3194), A 1070722 (cat 4431), 3F8 (cat 4083), AR-A 014418 (cat 3966), L803-mts (cat 2256) and SB 216763 (cat 1616) also available from Tocris Bioscience(www.tochris.com). Other suitable GSK inhibitors include GSK-3 Inhibitor IX (available from Santa Cruz Biotechnology sc-202634). In one embodiment, the GSK-3 Inhibitor is CHIR99021.
In addition, Wnt inhibitor may be added to the presently disclosed composition. Wnt inhibitor is an antagonist of the Wnt/13-catenin signalling pathway.
The Wnt/13-catenin signaling pathway is the Wnt pathway that causes an accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus. In the absence of wnt signaling β-catenin is degraded by a destruction complex which includes the proteins Axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase In (CK1α).
The wnt inhibitor may be a tankyrase inhibitor. Tankyrase inhibition inhibits axin ubiquitinization and stabilises axin protein (Huang et al 2009), therefore inhibiting wnt signalling.
A suitable tankyrase inhibitor is XAV939 (www.sigmaaldrich.com). Additional published tankyrase inhibitors include WIKI4, TC-E 5001 and JW 55, all commercially available from Tocris (www.tocris.com).
An effective amount of an inhibitor may be added to the presently disclosed composition. An effective amount is an amount which is sufficient to inhibit signaling in the pathway or by the protein which is targeted.
The expanded potential stem cell medium (EPSOM) may be a chemically defined medium (CDM).
A chemically defined medium (CDM) is a nutritive solution for culturing cells which contains only specified components, components of known chemical structure in certain embodiments. Therefore, a CDM is devoid of undefined components or constituents which include undefined components, such as feeder cells, stromal cells, serum, matrigel, serum albumin and complex extracellular matrices. Suitable chemically defined basal medium, such as Advanced Dulbecco's modified eagle medium (DMEM) or DMEM/F12 (Price et al Focus (2003) 25 3-6), Iscove's Modified Dulbecco's medium (IMDM) and RPMI-1640 (Moore, G. E. and Woods L. K., (1976) Tissue Culture Association Manual. 3, 503-508; see Table 1), knockout serum replacement (KSR) are known in the art and available from commercial sources (e.g. Sigma-Aldrich MI USA; Life Technologies USA).
In one embodiment, the basal medium is DMEM/F12. The basal medium may comprise or may be supplemented with, AlbuMAX II, which is a commercially available BSA or knockout serum replacement (KSR). The basal medium may also be supplemented with any or all of N2, B27, L-Glutamine, antibiotics (in certain embodiments, Penicillin and Streptomycin); Non-Essential Amino Acids; vitamins (in certain embodiments, vitamin C) and basal medium eagle (bME), all of which are commercially available (for example from Sigma-Aldrich). Other suitable supplements are known in the art and described herein.
In certain embodiments, the following additives may be present in the composition described below
Glutamine, Penicillin and Streptomycin are commercially available as a Penicillin-Glutamine-Streptomycin mix (Cat. No. 11140-050) for example from Thermo Fisher Scientific.
An example of an EPSCM comprises DMEM/F12 basal medium; supplemented with AlbuMAX II or Knockout Serum Replacement and the inhibitors and modulators described herein. The EPSCM may also comprise any of human insulin; N2, B27; Glutamine-Penicillin-Streptomycin; Non-Essential Amino Acids; vitamin C and basal medium eagle (bME), and LIF.
In some embodiments, the population of EPSCs is produced by culturing a population of pluripotent stem cells in the EPSCM for one or more (for example two or more, three or more, four or more, five or more) repeated “passages” to produce a descendent population of EPSCs. Passaging is also referred to as sub-culturing, and is the transfer of cells from a previous culture into fresh growth medium. Cells in a culture follow a characteristic growth pattern of lag phase, log phase and stationary phase. The timings of these phases may vary depending on the cell used (e.g. mammalian cells vs non-mammalian cells). Methods to determine the stage of cell growth are well known in the art. Generally cells are passaged in log phase. In some embodiments the pluripotent stem cells may be passaged (i.e. sub-cultured) one to ten times, three to ten times, three to five times in the EPSCM, to produce the population of EPSCs. In one embodiment, the population is passaged at least three times to produce the population of EPSCs.
EPSCM as described herein may be formulated into a kit for sale.
The one or more culture media in the kit may be formulated in deionized, distilled water. The one or more media will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. The one or more media may be frozen (e.g. at 20° C. or 80° C.) for storage or transport. The one or more media may contain one or more antibiotics to prevent contamination.
The one or more media may be a 1× formulation or a more concentrated formulation, e.g. a 2× to 250× concentrated medium formulation. In a 1× formulation each ingredient in the medium is at the concentration intended for cell culture, for example a concentration set out above. In a concentrated formulation one or more of the ingredients is present at a higher concentration than intended for cell culture. Concentrated culture media are well known in the art, such as salt precipitation or selective filtration. A concentrated medium may be diluted for use with water (in certain embodiments, deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.
The one or more media in the kit may be contained in hermetically-sealed vessels which prevent contamination. Hermetically-sealed vessels may be preferred for transport or storage of the culture media. The vessel may be any suitable vessel, such as a flask, a plate, a bottle, a jar, a vial or a bag.
The kit may also include instructions for use, e.g. for using the EPSCM to obtain EPSCs.
Provided herein are repeated PFF reprogramming experiments by directly culturing the primary colonies in pEPSCM (Extended Data
The pEPSCsEmb and pEPSCsiPS expressed pluripotency genes at levels comparable to the blastocysts (Extended Data
pEPSCs are tested to see if they had the potential to produce PGC-like cells (PGCLCs) in vitro, similar to mouse and human pluripotent stem cells [25-27]. In early-primitive streak (PS)-stage porcine embryos (E11.5E12), the first cluster of porcine PGCs can be detected as SOX17+ cells in the posterior end of the nascent primitive streak, and these cells later co-express OCT4, NANOG, BLIMP1 and TFAP2C [26]. NANOS3 is an evolutionarily conserved PGC-specific factor [28, 29] and human NANOS3 reporter cells have been used for studying the derivation of PGCLCs from pluripotent stem cells [26, 27]. To facilitate identification of putative porcine PGCLCs, the H2BmCherry reporter cassette are targeted to the 3′ UTR of the NANOS3 locus in pEPSCsEmb (Line K3, male) (Extended Data
The derivation of putative porcine PGCLCs was BMP2/4 dependent, as removal of BMP2 from the EB culture or inhibition of the BMP2/4 signaling by inhibitor LDN-193189 abrogated the formation of mCherry+/TNAP+ cell clusters (
The mCherry+ (NANOS3+) putative PGCLCs within the EBs expressed PGC-specific genes NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT and OCT4 [33], which were detected in RT-qPCR and was confirmed by immunofluorescence at single cell resolution (
Human ESCs have been widely used in studying human embryo development in vitro and hold great potential for regenerative medicine. [36-37] The finding that inhibition of SRC and Tankyrases is sufficient to convert mouse ESCs to mEPSCs [1] and that these two inhibitors are required for the generation of pEPSCs raises the possibility that similar in vitro culture conditions may also work for other mammalian species. To explore this possibility, four established human ES cell (hESC) lines (H1, H9, Man1 or M1, and Man10 or M10 cells) [30-32] are cultured in pEPSCM and passaged them up to three times. The cells displayed diverse morphologies and heterogeneous expression of OCT4 (Extended Data
When human primary iPSC colonies reprogrammed from dermal fibroblasts were directly cultured in hEPSCM, around 70% of the picked colonies could be established as stable iPSC lines (iPSC-EPSCs) (Extended Data
These results demonstrate that human and porcine EPSCs could be derived and maintained using the similar set of small molecule inhibitors. Global gene expression profiling revealed that pEPSCs and hEPSCs were clustered together, and were distinct from PFFs or other human pluripotent stem cells [1, 42, 43] (
The biological significance of the high histone gene expression in hEPSCs and in human 8-cell and morula stage embryos remains to be further investigated. Single cell RNA-seq (scRNAseq) of porcine and human EPSCs revealed uniform expression of the core pluripotency factors: OCT4, SOX2, NANOG and SALL4 (
hEPSCs and pEPSCs shared similar signalling requirements as revealed by the impacts after removal of individual components from the culture medium. Removal of the SRC inhibitor WH-4-023 or A419259 reduced expression of pluripotency factors in both EPSCs (Extended Data
The differentiation of hEPSCs to trophoblast cells was tracked by expression of CDX2-Venus reporter (T2A-Venus inserted into the 3′ UTR of the CDX2 locus) (Extended Data
To further infer the identity of the differentiated hEPSCs by TGFβ inhibition, we performed Pearson correlation coefficient analysis of the transcriptome of cells differentiated from H1-EPSCs, iPSC-EPSCs or H1-ESCs with external reference data including primary human trophoblasts (PHTs) and human placenta tissues, [50] which again revealed the similarity between cells differentiated from hEPSCs and PHTs and the placenta (Extended Data
When hEPSCs (ESC-converted-EPSCs and iPSC-EPSCs) were cultured in human trophoblast stem cell (hTSC) conditions [53] with low cell density (2,000 cells/3.5 cm dish), colonies with TSC morphology formed after 7-9 days (
One of the key mechanisms for derivation and maintenance of EPSCs of mouse, porcine and human is blocking poly(ADP-ribosyl)ation activities of PARP family members TNKS1/2 using small molecule inhibitors such as XAV939. [57, 58] In human cells, poly(ADP-ribose) in proteins is removed by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). [59] Genetic inactivation of Parp1/2 and TIVKS1/2 in the mouse caused trophoblast phenotypes, [60] whereas inactivating Parg led to loss of functional trophectoderm and TSCs. [61] PARG is tested whether it was of any relevance to hEPSCs developmental potential to derive trophoblasts. In hEPSCs, PARG-deficiency did not appear to cause noticeable changes in EPSC culture but adversely affected trophoblast differentiation (Extended Data
The present subject matter described herein will be illustrated more specifically by the following non-limiting examples, it being understood that changes and the variations can be made therein without deviating from the scope and the spirit of the disclosure as hereinafter claimed. It is also understood that various theories as to why the disclosure works are not intended to be limiting.
The experiments of using human ESCs and human cells were approved by HMDMC of the Wellcome Trust Sanger Institute, Cambridge UK. The experiments using porcine embryos were approved by the Niedersaechsisches Landesamt fuer Verbraucherschutz and Lebensmittelsicherheit, LAVES, Oldenburg Germany. The mouse teratoma Experiments were performed in accordance with UK Home Office regulations and the Animals (Scientific Procedures) Act 1986 (license number 80/2552), and were approved by the Animal Welfare and Ethical Review Body of the Wellcome Genome Campus, and the Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong (CULATR, HKU). At the end of the study, mice were euthanized by cervical dislocation, in accordance with stated UK Home Office regulations
Porcine and human EPSC cultures were routinely maintained on STO feeders. STO feeder plates were prepared 3-4 days before passaging by thawing and plating the mitomycin C inactivated STO cells on 0.1% gelatinised plates at the density of ˜1.1×104 cells/cm2. Porcine/human EPSC cells were maintained on STO feeder layers and enzymatically passaged every 3-5 days by a brief PBS wash followed by treatment for 3-5 minutes with 0.25% trypsin/EDTA (Gibco, Cat. No. 25500-054). The cells were dissociated and centrifuged (300 g×5 minutes) in M10 medium. M10: knockout DMEM (Gibco, Cat. No. 10829-018), 10% FBS (Gibco, Cat. No. 10270), 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140050) and 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016). After removing supernatant, the porcine/human EPSCs were re-suspended and seeded in pEPSCM/hEPSCM supplemented with 5 μM ROCK inhibitor Y-27632 (Tocris, Cat. No. 1254). 5% FBS (Gibco, Cat. No. 10270) and 10% KnockOut Serum Replacement (KSR) (Gibco, Cat. No. 10828028) were added in pEPSCM and hEPSCM respectively to improve cells survive. 12-24 hours later, medium was switched to pEPSCM/hEPSCM only. Both pEPSCM and hEPSCM are N2B27 based media. N2B27 basal media (500 ml) was prepared by inclusion of the following components: 482.5 ml DMEM/F-12 (Gibco, Cat. No. 21331-020), 2.5 ml N2 supplement (Thermo Fisher Scientific, Cat. No. 17502048), 5 ml B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044), 5 ml 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), and 5 ml 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016), 0.1 mM 2-mercaptoethanol (Sigma, Cat. No. M6250). pEPSCM (500 ml) was generated by adding the following small molecules and cytokines into 500 ml N2B27 basal media: 0.2 μM CHIR99021(GSK3i, TOCRIS, Cat. No. 4423), 1 μM WH-4-023 (SRC inhibitor, TOCRIS, Cat. No. 5413), 2.5 μM XAV939 (Sigma, Cat. No. X3004) or 2.5 μM IWR-1 (TOCRIS, Cat. No. 3532), 50 ng/ml Vitamin C (Sigma, Cat. No. 49752-100G), 10 ng/ml LIF (Stem Cell Institute, University of Cambridge. SCI) and 20 ng/ml ACTIVIN (SCI). hEPSCM (500 ml) was generated by adding the following components into 500 ml N2B27 basal media: 1.0 μM CHIR99021(GSK3 inhibitor, TOCRIS, Cat. No. 4423), 0.5 μM A-419259 (SRC inhibitor, TOCRIS, Cat. No. 3914), 2.5 μM XAV939 (Sigma, Cat. No. X3004), 50 ng/ml Vitamin C (Sigma, Cat. No. 49752-100G), 10 ng/ml LIF (SCI). Although both targeting SRC family kinases (SFKs), WH-4-023 and A419259 were preferred for porcine and human EPSCs, respectively. Both porcine and human EPSCs need CHIR99021 for improved proliferation. The high concentration of CHIR99021 (e.g. 3.0 μM) used for mouse ES cells culture induces porcine and human EPSC differentiation. The concentrations of CHIR99021 for porcine and human EPSC cultures are 0.2 μM and 1.0 μM, respectively. The human EPSC culture condition does not contain 0.3% FBS. 0.25 μM SB 590885 (BRAF inhibitor, R&D, Cat. No. 2650) and 2.0 μM SP600125 (INK inhibitor, TOCRIS, Cat. No. 1496) were included to improve porcine and human EPSC cultures, but they were not essential for the routine maintenance of porcine and human EPSCs. All cell cultures in this paper were performed under conditions of 37° C. and 5% CO2 unless stated otherwise.
Germany Landrace [1] and China TAIHU OCT4-TD-tomato [2] Porcine fetal fibroblasts (PFFs) were plated on gelatinized 15-cm tissue culture plates and cultured in M20 media. They were trypsinized with 0.25% trypsin/EDTA solution (Gibco, Cat. No. 25500-054) and harvested for electroporation at 80% confluence. M20: knockout DMEM (Gibco, Cat. No. 10829-018), 20% FBS (Gibco, Cat. No. 10270), 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050) and 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016). The transfections were performed using an Amaxa Nucleofector machine (Lonza) according to the manufacturer's protocol (NHDF Nucleofector® Kit, Cat. No. VPD-1001, program U-20). piggyBac transposition was used to achieve stable integration of reprogramming factors. The expression of the reprogramming factors was under the transcriptional control of the tetO2 tetracycline/doxycycline inducible promoter. 1.5 million PFFs and 6.0 μg DNA (2.0 μg PB-TRE-pOSCK, Porcine OCT4, SOX2, cMYC and KLF4; 1.0 μg PB-TRE-pNhL, 1.0 μg PB-TRE-hRL: human RARG and TRH1, 1.0 μg PB-EF1a-transposase and 1.0 μg PB-EF1a-rTTA) were used in each electroporation reaction. PB-TRE-pOSCK: cDNAs of porcine OCT4, SOX2, cMYC and KLF4 linked by 2A sequence were expressed as a single transcript [3] from the tetO2 promoter. PB-TRE-pNhL contains cDNAs of porcine NANOG and human LIN28, also linked with 2A sequence [3]. PB-TRE-RL has 2A linked human RARG and TRH1 cDNAs [4]. EF1a promoter was employed to drive the PB transposase expression. Reverse tetracycline controlled transactivator (rtTA) was expressed to induce the expression of the reprogramming factors upon Dox addition. After transfection, 0.2 million PFFs were seeded on mitomycininactivated STO feeders in M15 supplemented with LIF (10 ng/ml, SCI) and Vitamin C (Sigma, Cat. No. 49752-100G) in 10-cm dishes. M15: knockout DMEM (Gibco, Cat. No. 10829-018), 15% FBS (Gibco, Cat. No. 10270), 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016) and 0.1 mM 2-mercaptoethanol (Sigma, Cat. No. M6250). Doxycycline (Dox) (1.0 μg/mL, Sigma, Cat. No. D9891) was added for induction of reprogramming factor expression. The culture media was changed each other day. For transgene dependent iPSC generation, the colonies were picked in M15 at day 12 supplemented with Dox, 50 μg/ml Vitamin C and 10 ng/ml bFGF (SCI) and maintained in the same media. For directly establishing transgene independent iPSCs lines in pEPSCM, Dox was removed at day 9 and the media was switch to pEPSCM immediately. The Dox independent iPSCs colonies were picked in pEPSCM supplemented with 5μM ROCK inhibitor Y27632 (Tocris, Cat. No. 1254) on day 14-15. Y26537 was removed from the culture media 24 hours later and pEPSCM was refreshed every day subsequently.
Dox dependent porcine iPSCs were dissociated in 0.25% trypsin/EDTA solution (Gibco, Cat. No. 25500-054) and seeded in 24-well STO feeder plates at a density of 1×104 cells per well. The cells were cultured in M15 supplemented with Dox (Sigma, Cat. No. D9891), Vitamin C (Sigma, Cat. No. 49752-100G) and 10 ng/ml bFGF (SCI) for two days before the culture media was switched to medium supplemented with indicated small molecules and cytokines (Supplementary Table 1). M15 and N2B27 media: see above. AlbumMax media: DMEM/F12 (Gibco, Cat. No. 21331-020), 20% AlbumMax II (Gibco, Cat. No. 11021-037), 25 mg/mL Human Insulin (Sigma, Cat. No. 91077C), 2×B27 Supplement, 100 ug/mL IGFII (R&D, Cat. No. 292-G2-250), 1×Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016) and 0.1 mM 2mercaptoethanol (Sigma, Cat. No. M6250). 20% KSR media: DMEM/F-12 (Gibco, Cat. No. 21331-020), 20% KnockOut Serum Replacement (KSR) (Gibco, Cat. No. 10828-028), 1× glutamine penicillin-streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016) and 0.1 mM 2-mercaptoethanol (Sigma, Cat. No. M6250). Small molecules and cytokines were supplemented as indicated at the following final concentrations: CHIR99021 (0.2 or 3 μM, TOCRIS, Cat. No. 4423), PD0325901 (0.1 μM and 1 μM, TOCRIS, Cat. No. 22854192); WH-4-023 (4 μM, TOCRIS, Cat. No. 5413), PKC inhibitor Go6983 (5 μM. TOCRIS, Cat. No. 2285); SB203580 (p38 inhibitor, 10 μM. TOCRIS, Cat. No. 1202); SP600125 (JNK inhibitor, 4 μM. TOCRIS, Cat. No. 1496); Vitamin C (50 μg/ml. Sigma, Cat. No. 49752-100G), SB590885 (BRAF inhibitor, 0.25 μM, R&D, Cat. No. 2650), XAV939 (2.5 μM, Cat. No. X3004), R04929097 (Notch signaling inhibitor, 10 μM, Selleckchem, Cat. No. S1575), LDN193189 (BMP inhibitor, 0.1 μM, Sigma, Cat. No. SML0559), Y27632 (ROCKi, 5 μM, Tocris, Cat. No. 1254), Verteporfin (YAP inhibitor, 10 μM, Tocris, Cat. No. 5305). LIF (10 ng/ml, SCI), BMP4 (10 ng/ml, R&D, Cat. No. 5020-BP), SCF (50 ng/ml, R&D, Cat. No. 255-SC-010), EGF (50 ng/ml, R&D, Cat. No. 236-EG-200), TGFβ (10 ng/ml, Cat. No. 7754-BH-005), bFGF (10 ng/ml, SCI), ACTIVIN (20 ng/ml, SCI). The medium was refreshed every day and the surviving cells were passaged at day 6. In the first 24 hours after passaging, 5 uM of ROCKi Y27632 (Tocris, Cat. No. 1254) was supplemented in the media and removed 24 hours later. After 4 days of growing, the colonies survived were collected for RT-qPCR analysis to check the endogenous porcine OCT4 and NANOG expression.
Peripubertal German Landrace gilts (approx. 7-9 months of age, 90-120 kg bodyweight) served as embryo donors. Gilts were synchronized by feeding 5 ml/day/gilt altrenogest (Regumate®, 4 mg/ml, MSD Animal Health, Germany) for 13 days. Followed by an injection of 1500 IU PMSG (Intergonan® 240 I.E./ml, MSD Animal Health, Germany) on the last day of Altrenogest feeding [5]. Ovulation was induced by intramuscular injection of 500 IU of hCG (Ovogest® 300 I.E./ml, MSD Animal Health, Germany) 76 hours later.
Semen was collected from Germany Landrace boars [1] via the hand-gloved method using phantom and was immediately diluted in Androhep□Plus solution (Minitube, Tiefenbach, Germany). The sows were artificially inseminated twice at 40 hours and 48 hours, after hCG administration. Five days after the second insemination, sows were slaughtered and the uterus was excised and flushed with Dulbecco's PBS medium (AppliChem, Cat. No. A0964) supplemented with 1% Newborn Calf Serum (NBCS, Gibco™, Cat. No. 16010159). Collected morulae were either directly used for injection experiments or cultured overnight in PZM-3 medium to blastocyst stage and used for ICM isolation (PZM-3 medium: 108 mM Sodium chloride (NaCl, Sigma-Aldrich, Cat. No. S5886), 10 mM Potassium chloride (KCl, Sigma-Aldrich, P-5405), 0.35 mM Potassium phosphate monobasic (KH2PO4, SigmaAldrich, Cat. No. P5655), 0.40 mM Magnesium Sulfate heptahydrate (MgSO4×7 H2O, Sigma-Aldrich, Cat. No. M5921), 25.07 mM Sodium bicarbonate (NaHCO3, Sigma-Aldrich, S4019), 2 mM L(+) Lactic acid calcium salt pentahydrate (C6H10CaO6×5 H2O, Roth, Cat. No. 4071), 0.2 mM Sodium pyruvate (Sigma-Aldrich, Cat. No. P2256), 1 mM L-Glutamine (AppliChem, Cat. No. A3704), 0.05 mg/ml Gentamicin sulfate salt (Sigma-Aldrich, Cat. No. G3632), 0.55 mg/ml Hypotaurine (Sigma-Aldrich, Cat. No. H1384), 20 μl/ml BME amino acids solution (Sigma-Aldrich, Cat. No. B6766), 10 μl/ml MEM Non-essential Amino Acid Solution (Sigma-Aldrich, Cat. No. M7145) and 3 mg/ml Bovine Serum Albumin (BSA, Sigma-Aldrich, A7030)).
Porcine ovaries from prepubertal gilts were transported at 30° C. from a local abattoir and washed three times with 0.9% Sodium Chloride (NaCl, Sigma-Aldrich, Cat. No. S5886) containing 0.06 mg/ml Penicilin G potassium salt (AppliChem, Cat. No. A1837) and 0.131 mg/ml Streptomycin sulfate (AppliChem, Cat. No. A1852). Oocytes were aspirated from follicles with a diameter of 2-6 mm using an 18-gauge needle and washed in Dulbecco's PBS medium (AppliChem, Cat. No. A0964) supplemented with 0.33 mM Sodium Pyruvate (Sigma-Aldrich, Cat. No. P2256), 5.56 mM D(+)-Glucose Monohydrate (Roth, Cat. No. 6887), 0.9 mM Calcium chloride dihydrate (AppliChem, Cat. No. A3587), 50 mg/ml Streptomycin sulfate (AppliChem, Cat. No. A1852), 6 mg/ml Penicillin G potassium salt (AppliChem, Cat. No. A1837) and 1% Newborn Calf Serum (NBCS, Gibco™, Cat. No. 16010159). Cumulus-oocytes-complexes with multiple layers of compacted cumulus were matured in vitro in 1:1 DMEM High Glucose (Biowest, Cat. No. L0101-500) and Ham's F-12 Medium (Merck, Cat. No. F0815) supplemented with 60 μg/ml Penicilin G potassium salt (AppliChem, Cat. No. A1837), 50 ng/ml Streptomycin sulfate (AppliChem, Cat. No. A1852), 2.5 mM L-glutamine (AppliChem, Cat. No. A3704), 10% Fetal Bovine Serum (FCS, Gibco®, Lot 42Q0154K, Cat. No. 10270-106), 50 ng/ml murine Epidermal growth factor (EGF, SigmaAldrich, Cat. No. E4127), 10 I.E./ml Pregnant Mare's Serum Gonadotropin (PMSG, Intergonan® 240 I.E./ml, MSD Animal Health, Germany), 10 I.E./ml human Chorionic Gonadotropin (hCG, Ovogest® 300 I.E./ml, MSD Animal Health, Germany), 100 ng/ml human recombinant Insulin-like Growth Factor 1 (IGF1, R&D Systems, Cat. No. 291-G1), 5 ng/ml recombinant human FGF-basic (bFGF, Peprotech, Cat. No. 100-18B) for 40 h in humidified air with 5% CO2 at 38.5° C.
After maturation, the oocytes were freed from cumulus cells by 5 min incubation with 0.1% Hyaluronidase (Sigma-Aldrich, Cat. No. H3506) in TL-Hepes 321+Ca2+medium composed of 114 mM Sodium chloride (NaCl, Sigma-Aldrich, Cat. No. S5886), 3.2 mM Potassium chloride (KCl, Sigma-Aldrich, P-5405), 2 mM Calcium chloride dihydrate (CaCl2×2 H2O; AppliChem, Cat. No. A3587), 0.4 mM Sodium dihydrogen monohydrate (NaH2PO4×H2O, Merck, Cat. No. 106346), 0.5 mM Magnesium chloride hexahydrate (MgCl2×6 H2O, Roth, Cat. No. HN03.2), 2 mM Sodium hydrogen carbonate (NaHCO3, Roth, Cat. No. HN01.2), 10 mM HEPES (Roth, Cat. No. 9105.3), 10 mM Sodium DL-lactate solution (60%) (SigmaAldrich, Cat. No. L1375), 100 U/L Penicilin G potassium salt (AppliChem, Cat. No. A1837), 50 mg/L Streptomycin sulfate (AppliChem, Cat. No. A1852), 0.25 mM Sodium Pyruvate (Sigma-Aldrich, Cat. No. P2256), 57 mM Sucrose (Merck, Cat. No. 107653) and 0.4% Bovine Serum Albumin (Sigma-Aldrich, Cat. No. A9647). After washing with TL-Hepes 321+Ca2+ medium oocytes with visible first polar body were exposed to a single pulse of 24 V for 45 μs in SOR activation medium (182.2 g/mol Sorbitol (Sigma-Aldrich, Cat. No. S1876), 158.2 g/mol Calcium acetate hydrate (Sigma-Aldrich, Cat. No. C4705), 214.5 g/mol Magnesium Acetate Tetrahydrate (Sigma-Aldrich, Cat. No. M5661), 0.1% Bovine Serum Albumin (Sigma-Aldrich, Cat. No. A9647)). Thereafter oocytes were incubated for 3 hours in 2 mM 6Dimethylaminopurine (6-DMAP, Sigma-Aldrich, Cat. No. D2629) in PZM-3 medium.
After activation, oocytes were cultured in PZM-3 medium at 39° C. in 5% CO2 and 5% O2 for 6 days. For isolation of ICM, porcine blastocysts from day 6 were cultured for an additional 24 h in D15 medium containing DMEM High Glucose (Biowest, Cat. No. L0101-500), and 2 mM L-Glutamine (AppliChem, Cat. No. A3704), 15% Fetal Bovine Serum (FCS, Gibco®, Lot 42Q0154K, Cat. No. 10270-106), 1% Penicillin/Streptomycin Solution (Corning, Cat. No. PS-B), 1% MEM Nonessential Amino Acids Solution (Corning, Cat. No. NEAA-B), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, Cat. No. M7522) supplemented with 1000 U/ml ESGRO® Recombinant Mouse LIF Protein (Millipore, Cat. No. ESG1107).
Porcine parthenogenetic blastocysts from day 7 and in vivo derived blastocysts from day 5 were used for the establishment of porcine PSC lines. Blastocysts were washed twice in TLHepes 296+Ca2+ medium composed of 114 mM Sodium Chloride (NaCl, Sigma-Aldrich, Cat. No. S5886), 3.2 mM Potassium chloride (KCl, Sigma-Aldrich, Cat. No. P-5405), 2 mM Calcium chloride dihydrate (CaCl2×2 H2O, AppliChem, Cat. No. A3587), 0.4 mM Sodium dihydrogen phosphate monohydrate (NaH2PO4×H2O, Merck, Cat. No. 106346), 0.5 mM Magnesium chloride hexahydrate (MgCl2×6 H2O, Roth, Cat. No. HN03.2), 2 mM Sodium bicarbonate (NaHCO3, Sigma-Aldrich, Cat. No. S4019), 10 mM HEPES (Roth, Cat. No. 9105.3), 10 mM Sodium DL-lactate solution (60%) (Sigma-Aldrich, Cat. No. L1375), 100 U/L Penicilin G potassium salt BioChemica (AppliChem, Cat. No. A1837), 50 mg/L Streptomycin sulfate BioChemica (AppliChem, Cat. No. A1852), 0.25 mM Sodium Pyruvate (Sigma-Aldrich, Cat. No. P2256), 32 mM Sucrose (Merck, Cat. No. 107653) and 0.4% Bovine Serum Albumin (BSA, Sigma-Aldrich, Cat. No. A9647). ICMs were separated from the trophectoderm in 100 μl drops of TL-Hepes 296+Ca2 medium using ophthalmic scissors (Bausch & Lomb GmbH, Germany). Isolated ICMs were cultured on a monolayer of Mitomycin C-treated STO cells in pEPSCM medium, supplemented with 10 μM Y27632 (ROCKi, Tocris, Cat. No. 1254) for 7 days, until initial outgrowths could be observed. Subsequently, pEPSCM medium without ROCKi was used for further culture. Medium was changed every day. 12-14 days after plating, ICM colonies were mechanically removed from the STO feeder cells using fine-pulled glass capillary pipettes and reseeded onto fresh feeder cells. Growth of colonies was evaluated daily and approximately three days later cells began to form well-defined porcine EPSCEmb colonies. These cells were sub-cultured using 0.05% trypsin-EDTA (GE Healthcare, Cat. No. L11-003) every 3-4 days.
To investigate the developmental capacity of the derived cells lines, porcine EPSCsEmb and EPSCsiPS labelled with mCherry expression were injected into parthenogenetic blastocysts and the incidence of chimerism was assessed. Stem cells were detached from feeders with 0.05% trypsin-EDTA (GE Healthcare, Cat. No. L11-003) and re-suspended in Fetal Bovine Serum (FBS, Gibco®, Lot 42Q0154K, Cat. No. 10270-106). After centrifugation, stem cells were re-suspended and stored at room temperature in D15 medium supplemented with 1000 U/ml ESGRO® Recombinant Mouse LIF Protein (Millipore, Cat. No. ESG1107) and 10 μM Y27632 (ROCKi, Tocris, Cat. No. 1254). Small clumps containing 6-8 cells were injected into day 4 or day 6 old porcine parthenogenetic embryos with the aid of a piezo-driven micromanipulator (Zeiss, Eppendorf) in Opti-MEM® I (1×)+GlutamMAX™-I Reduced Serum Medium (Gibco®, Cat. No. 51985-026) supplemented with 10% FBS (Gibco®, Lot 42Q0154K, Cat. No. 10270-106). After injection, embryos were cultured in D15 medium supplemented with 1000 U/ml ESGRO® Recombinant Mouse LIF Protein (Millipore, Cat. No. ESG1107) and 10 μM Y27632 (ROCKi, Tocris, Cat. No. 1254) at 39° C. in 5% CO2 and 5% O2 for 24 hours (for blastocysts day 6) or 48 hours (for day 4 embryos). Non-injected porcine parthenogenetic embryos day 4 or day 6 cultured in the above medium were used as controls for embryo development.
Procedures for superovulation, insemination and embryo collection were described above. Porcine morulae day 5 collected from eight gilts were stored in Opti-MEM® I (1×)+GlutamMAX™-I Reduced Serum Medium (Gibco®, Cat. No. 51985-026) supplemented with 10% FBS (Gibco®, Lot 42Q0154K, Cat. No. 10270-106) in thermostatically controlled incubator at 37° C. before injection. Porcine EPSC lines at passage 2-8 after mCherry+ colonies picking were used for the embryo injection. Porcine EPSCs were cultured either on mitotically inactivated STO feeder or MEFs cells in pEPSCM medium. Two days before injection the medium was switch to pEPSCM medium without WH-4-023 (SRCi, TOCRIS, Cat. No. 5413). One day before injection medium was replaced with pEPSCM medium without WH-4-023 and additionally supplemented with Heparin (5 ng/ml, R&D, Cat. No. 9041-08-1) and 10 ng/ml bFGF (SCI). Four hours before injection medium was replaced with pEPSCM medium without WH-4-023, supplemented with 5 ng/ml Heparin, 10 ng/ml bFGF (SCI—Stem Cell Institute, the University of Cambridge), 10 ng/ml Lif (SCI), 5 μM Y27632 (ROCKi, Tocris, Cat. No. 1254), 20 ng/ml Human Recombinant ACTIVIN A (StemCell Technologies, Cat. No. 78001) and 10% Fetal Bovine Serum (FCS, Gibco®, Lot 42Q0154K, Cat. No. 10270-106). For the injection EPSCs were detached from culture dish with 0.05% trypsin-EDTA (GE Healthcare, Cat. No. L11-003), carefully re-suspended and plated in 500 p1 drop of M15 medium supplemented with 50 μg/ml Vitamin C (Sigma, Cat. No. 49752), 0.1 μM CHIR99021 (GSK3i, TOCRIS, Cat. No. 4423), 20 ng/ml Human Recombinant Activin A (StemCell Technologies, Cat. No. 78001), 10 ng/ml bFGF (SCI), 10 ng/ml Lif (SCI), 5 ng/ml Heparin and 5 μM Y27632 (ROCKi, Tocris, Cat. No. 1254). Porcine embryos were washed once and placed in a 5000 drop of Opti-MEMO I (1×)+GlutamMAX™-I Reduced Serum Medium (Gibco®, Cat. No. 51985026) supplemented with 20 ng/ml Human Recombinant Activin A (StemCell Technologies, Cat. No. 78001), 10 ng/ml bFGF (SCI), 5 μM Y27632 (ROCKi, Tocris, Cat. No. 1254) and 10% FBS (Gibco®, Lot 42Q0154K, Cat. No. 10270-106). Injection drops were plated onto injection plate under phase-contrast inverted microscope (Axiovert 35M, Carl Zeiss, Oberkochen, Germany) equipped with a microinjection system (Transferman and CellTram Vario micromanipulators, Eppendorf) and covered with mineral oil. Stem cell clumps containing approximately 6-8 cells were injected between blastomeres of porcine morulae. Thereafter, embryos were washed twice in M15 medium supplemented with 50 μg/ml Vitamin C (Sigma-Aldrich, Cat. No. 49752), 0.1 μM CHIR99021 (GSK3i, TOCRIS, Cat. No. 4423), 20 ng/ml Human Recombinant Activin A (StemCell Technologies, Cat. No. 78001), 10 ng/ml bFGF (SCI), 10 ng/ml Lif (SCI), 5 ng/ml Heparin and 5 μM Y27632 (ROCKi, Tocris, Cat. No. 1254) and either incubated 4 hours until the embryo transfer or cultured overnight and then fixed for confocal microscopy analysis.
Porcine chimeric blastocysts were fixed in 3.7% formaldehyde solution (Honeywell Riedel-de Haen™, Cat. No. 1635) for 15 min at room temperature. Thereafter embryos were incubated with 0.2 μM SiR-DNA (Spirochrome, Switzerland) for 30 min at 37° C. to visualize the nuclei. Localization and proliferation of porcine stem cells in blastocysts were analysed using confocal screening microscope (LSM 510, Zeiss). Remaining embryos were stored in DPBS supplemented with 0.5% FBS (Gibco®, Lot 42Q0154K, Cat. No. 10270-106) and 1% Penicillin/Streptomycin Solution (Corning, Cat. No. PS-B) in 4° C. for future analysis.
Day 25-27 porcine fetuses were dissected from pregnant sows and cut into two halves along head-tail axis. The first half fetuses were fixed in 4% paraformaldehyde (Sigma, Cat. No. P6148) at 4° C. overnight and subsequently transferred to 30% sucrose solution (Sigma, Cat. No. 0389) for cryopreservation. The second halves were subjected to FACS and genotyping analysis. The fixed half fetuses were embedded in OCT compound (CellPath, Cat. No. 15212776) and frozen on dry ice. Sections (10 μm thick) were cut on a Leica cryostat. The sections were permeabilized with 0.1% Triton-100 (Sigma, Cat. No. T8787) for 30 minutes and then blocked for 30 minutes with 5% donkey serum (Sigma, Cat. No. D9663) and 1% BSA (Sigma, Cat. No. A2153). Co-immunofluorescences of mCherry and other antibodies were performed to check the co-localisation of injected donor porcine EPSCs expressing mCherry and host lineage markers. For immunofluorescence staining of cryosections of PGCLC EBs, the EBs were fixed in 4% PFA for about 4 hours or overnight at 4° C. and embedded in OCT compound for frozen sections. The thickness of each section was 10 μm. Sections were first permeabilized with 0.1% Triton and blocked with 5% donkey serum plus 1% BSA followed by incubations with primary antibodies for 1-2 hours at room temperature or overnight in a cold room. Fluorescence-conjugated secondary antibodies were used to incubate the slides at room temperature for 1 hour. After antibody treatment, samples were counter-stained with 10 μg/ml DAPI (Thermo Fisher Scientific, Cat. No. 62248) for 10 minutes to mark nuclei and were observed under a fluorescence microscope. The antibodies are listed in Supplementary Table 9.
To analyse the contribution of donor mCherry+ porcine EPSCs in day 25-27 chimeras, the half fetuses were dissected into small pieces representing several body parts (head, trunk and tail). The dissected tissues and placenta were digested with 1.0 mg/ml collagenase IV (Thermo Fisher Scientific, Cat. No. 17104019) for 1-3 hours at 37° C. on a shaker. A pipette was used to blow the tissue blocks and dissociate them into single cells. The dissociated cells were filtered with a 35 μm nylon mesh (Corning, Cat. No. 352235) to remove tissues clumps. After centrifugation, the cells were fixed using Fixation Medium according to the manufacturers' manual (BD Cytofix, Cat. No. 554655) and the washed cells were stored at 4° C. in PBS supplemented with 0.1% NaN3 (Sigma, Cat. No. 199931) and 5% FBS (Gibco, Cat. No. 10270) before analysed with flow cytometry. All the samples were analysed using BD LSR Fortessa cytometer. 561 nm (610/20 bandpass filter) and 488 nm (525/50 bandpass filter) channels were used to detect mCherry and excluded autofluorescence. PGC EBs were trypsinized with 0.25% trypsin/EDTA Gibco, Cat. No. 25500-054) at 37° C. for 15 mins and stained with PerCP-Cy5.5 conjugated anti-TNAP antibody. 561 nm (610/20 bandpass filter) and 488 nm (710/50 bandpass filter) channels were used to detect NANOS3-H2BmCherry+/TNAP+ cells. FACS data were analysed by Flowjo software. The antibodies used in these experiments are listed in Supplementary Table 9.
Genomic DNA of porcine fetuses were extracted from the dissociated cells of dissected body parts as described above and of placentas that were prepared for FACS using DNA Releasy kit (Anachem, Cat. No. LS02). Genomic DNA PCR of H2BmCherry was employed to detect the presences of donor cells. Amplification of a region in the porcine PRDM1 locus served as the genomic DNA quality and PCR control. All PCR primers are listed in Supplementary Table 10.
For transcription factor mediated porcine PGCLC induction experiments, the piggyBac based PB-TRE-NANOG, PB-TRE BLIMP1, PB-TRE-TFAP2C and PB-CAG-SOX17-GR expression constructs were co-electroporated into the porcine NANOS3-2A-H2BmCherry reporter EPSCsemb (Line K3, male) with PB-CAGG-rtTA-IRES-Puromycin and transposase expressing plasmids. pEPSCsEmb harbouring the plasmids were selected by adding 0.3 μg/ml puromycin (Sigma, Cat. No. P8833) for two days. Thereafter the expressions of transgenic NANOG, BLIMP1 and TFAP2C were induced by 1.0 μg/ml Dox (Sigma, Cat. No.D9891) for indicated periods. As the SOX17 expressing plasmid has the hygromycin selection cassette, 150 μg/ml hygromycin (Gibco, Cat. No. 10687010) was used to select PB-CAG-SOX17-GR transfected cells. The SOX17 protein was fused with GR (human glucocorticoid receptor ligand-binding domain). This system allows inducing the nuclear translocation of SOX17 by addition of 2 μg/ml dexamethasone (Dex) (Sigma, Cat. No. D2915). For the pre-induction, pEPSCsEmb were detached from the STO feeder layer by 0.1% Type 2 collagenase (Thermo Fisher Scientific, Cat. No. 17101015) without dissociation and seeded on gelatinised plates in M15 media supplemented with 5 μM ROCKi Y-27632 (Tocris, Cat. No. 1254), 20 μg/ml ACTIVIN A (SCI) and 1.0 μg/ml Dox or 1.0 mg/ml Dex. After the 12 hours of induction and pre-differentiation, the cells were collected using 0.25% trypsin/EDTA (Gibco, Cat. No. 25500-054) and plated to ultra-low attachment U-bottom 96-well plates (Corning, Cat. No. 7007) at a density of 5,000-6,000 cells/well in 100 ml PGCLC medium. 3-4 days later, the EBs were collected for analysis. PGCLC medium is composed of Advanced RPMI 1640 (GIBCO, Cat. No. 12633-12), 1% B27 Supplement (Thermo Fisher Scientific, Cat. No. 17504044), 1× glutamine penicillin-streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016), 0.1 mM 2-mercaptoethanol (Sigma, Cat. No. M6250) and the following cytokines: 500 ng/ml BMP2 (SCI), 10 ng/ml human LIF (SCI), 100 ng/ml SCF (R&D, Cat. No. 255-SC-010), 50 ng/ml EGF (R&D, Cat. No. 236-EG-200) and 10 μM ROCK inhibitor (Y-27632, Tocris, Cat. No. 1254).
For human PGCLCs, the PGC differentiation potential of two hEPSC lines are tested with the sequential induction method [6]. Human pre-mesoderm (pre-ME) was first induced in pre-ME media (Advanced RPMI 1640 Medium, 1% B27 supplement, 1×NEAA and 1× glutamine penicillin-streptomycin supplemented with 100 ng/ml Activin A (SCI), 3 μM CHIR99021 and 10 μM of ROCKi Y-27632) for 12 hours. Pre-ME were trypsinized into single cells and seeded into Corning Costar Ultra-Low attachment multi well 96-well plates (Corning, Cat. No. 7007) 4,000-5,000 cells per well in the 100 μM PGCLC medium which was used for porcine PGCLC induction. To improve the cell aggregation, in all PGCLC induction experiments, 0.25% (v/v) poly-vinyl alcohol (Sigma, Cat. No. 341584) are added in the basal medium.
Porcine and human EPSCs were re-suspended in PBS supplemented with 30% matrigel (Corning, Cat. No. 354230) and 5 μM Rock inhibitor Y-27632 (Tocris, Cat. No. 1254). 5×106 porcine or human EPSCs were injected subcutaneously into both dorsal flanks of 8-weekold male NSG mice (NOD.Cg-Prkdcscid Il2rgtml Wjl/SzJ, The Jackson Laboratory) (100 ul per injection). Human and porcine EPSCs formed visible teratomas within 8 and 10 weeks. When the size of the teratomas reached 1.2 cm2, they were dissected, fixed overnight in 10% phosphate-buffered formalin and embedded in paraffin before sectioning.
Porcine and human EPSCs were trypsinised and seeded in gelatinised 6-well plates at a density of 4×106 cells/well for pre-differentiation. M15 media supplemented with 20 ng/ml ACTIVIN A (SCI) and 5 μM Rock inhibitor Y-27632 were used to culture the replated cells. The next day, the cells were detached using 0.25% trypsin/EDTA (Gibco, Cat. No. 25500-054) and plated to ultra-low cell attachment U-bottom 96-well plates (Corning, Cat. No. 7007) at a density of 5,000-6,000 cells/well in 200 μl M10 medium. After 7-8 days of growing, the EBs were collected for analysis. 0.25% (v/v) poly-vinyl alcohol (Sigma, Cat. No. 341584) was added in the medium to help cells aggregation.
pEPSCM without SRC inhibitor WH-4-023 (pEPSCM-SRCi) needs to be prepared in advance for pEPSCs transfection. Once pEPSCs reached 40-50% confluence, the media was switched to pEPSCM-SRCi and cells cultured for one more day (day −2). The next day (day −1), 5% FBS was added into pEPSCM-SRCi media and cells were cultured overnight. On the transfection day (day 0), porcine EPSCs were trypsinized with 0.25% trypsin/EDTA (Gibco, Cat. No. 25500-054) and dissociated into single cells with M10 media. After centrifugation, 1-1.5×106 cells were resuspended in 100 μl Opti-MEM (Gibco, Cat. No. 31985062) containing 5-6 μg plasmid DNA. Amaxa Nucleofector machine (Lonza) was used to perform the electroporation with program A-023. After transfection, half of transfected cells were seeded on drug resistant STO feeders in 10-cm dishes and the pEPSCM supplemented with 5 μM ROCKi Y-27632 (Tocris, Cat. No. 1254) and 5% FBS were used to culture the transfected cells. Y-27632 and FBS was removed from the media on day 1. The drugs were added into pEPSCM media from day 2 to select the transfected colonies. The drug concentrations used for selection are: Puromycin (0.3 n/ml, Sigma, Cat. No. P8833); G418 (150 ng/ml, Gibco, Cat. No. 10131027); Hygromycin (150 ng/ml, Gibco, Cat. No. 10687010). After 3 days of selection (day 5), the medium was changed to pEPSCM-SRCi supplemented with drugs for continuous selection. The survived colonies were picked at day 7-8. During transfection and selection, the culture media should be refreshed daily. For human EPSCs transfection, 10% KSR and 5% FBS were added into hEPSCM to culture hEPSCs (70%-80% confluence) overnight before collection using 0.05% trypsin-EDTA the next day. M10 media was also used to dissociate the cells and neutralize the trypsin. Once centrifuged, 300-400 μl PBS solution containing plasmid DNA was used to resuspend the cells at a density of 10 million cells per ml. 300-400 μl cells/DNA mixture was taken out and added into 0.4-cm electroporation cuvettes for electroporation (Gene Pulser Xcell System; Bio-Rad; 320 V, 500 μF, 0.4-cm cuvettes). 5×105 transfected cells were plated on drug resistant STO feeders in 10-cm dishes containing hEPSCM supplemented with 5 μM ROCKi Y-27632 (Tocris, Cat. No. 1254) and 10% KSR. Y-27632 and KSR were removed from the culture from day 1 and Puromycin was added for selection from day 2. Colonies were picked at around day 7-8. Follow the methods described above to expand the selected porcine and human EPSC colonies.
To target an EF1a-H2BmCherry-iRES-Puro cassette to the porcine ROSA26 locus, the targeting vector with the cassette flanked by Rosa 5′ and 3′ homology arms was constructed. 5′ and 3′ homology arms were synthesised from IDT Company (650-bp 5′arm, Chr13: 65756272-65756923; 648-bp 3′arm, Chr13: 65755620-65756267). The sequence 5′CAATGCTAGTGCAGCCCTCATGG-3′ was designed as the target of gRNA/CAS9. After electroporation, Puromycin (0.3 μ/ml, Sigma, Cat. No. P8833) was used to select the targeted cells. Genotyping analysis of picked colonies revealed that the targeting efficiency was about 25%-30%. To investigate pPGCLC differentiation from pEPSCs, the T2A-H2BmCherry expression cassette was knocked-in immediately downstream and in frame with the coding sequence of porcine NANOS3. Homology arms were also synthesised from IDT company (699-bp 5′arm, chr2: 65275456-65276148; 699 bp-3′ arm chr2: 65274749-65275447). 20-bp (5′-TCCACTTCTGCCTAAGAGGCTGG-3′) sequence preceding the stop codon was targeted by gRNA/CAS9 to introduce the cut and mediate homologous recombination. After selection with G418 (150 μg/ml, Gibco, Cat. No. 10131027), genomic DNA was extracted from picked colonies and subjected to genotyping PCR revealing a comparable targeting efficiency of about 25%-30%. Karyotyping analysis of correctly targeted clones was performed to confirm normal karyotype in the clones used. The same strategy was employed to make human OCT4-T2A-H2B-Venus and CDX2-T2A-H2B-Venus reporter EPSC lines. For human OCT4 locus, homology arms are 619-bp 5′arm (chr6: 31164604-31165222) and 636-bp 3′arm (chr6:31163965-31164600). The gRNA/CAS9 targeting sequence is 5′ TCTCCCATGCATTCAAACTGAGG-3′. CDX2 homology arms are 478-bp 5′arm (chr13: 27963118-27963595) and 557-bp 3′arm (chr13: 27962558-27963114). The gRNA/CAS9 targeting sequence is 5′-CCGTCACCCAGTGACCCACCGGG-3′. For each electroporation, 5 μg plasmid DNA was used: 1.5 μg of CAS9, 1.5 μg of gRNA and 2 μg of donor vector.
For the TOPflash assay, 2.0×106 cells were transfected with 10 μg TOPflash plasmid. 5 μg pRL-TK (Renilla) vectors were also transfected for normalization. Cells were split 1:9 into a 24-well plate in pEPSCM and hEPSCM with or without XAV939 (WNTi, 2.5 μM, Cat. No. X3004) for 48 h. Cell lysates were collected for luciferase assays. For determining the regulation pattern of Oct4 expression in porcine EPSCs, 10 μg reporter constructs were electroporated into 1.5×106 pEPSCs with 5 μg pRL-TK. Assays were performed 48 h later. All luciferase assays were performed using the Dual-Glo Luciferase Assay System (Promega, Cat. No. E2920).
Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Cat. No. 74106) for cultured cells or RNeasy Micro Kit (Qiagen, Cat. No. 74034) for sorted NANOS3-mCherry+ cells. RNA was subsequently quantified and treated with gDNA WipeOut to remove genomic DNA. Complementary DNA (cDNA) was prepared using a QuantiTect Reverse Transcription Kit (Qiagen, Cat. No. 205311). RT-qPCR primers or TaqMan Gene Expression Assays (Life Technologies) are listed in Supplementary Table 10 and 11. ABsolute Blue qPCR ROX Mix (ABgene, Cat. No. AB4138B) were used for probe based qPCR assays and SYBR Green ROX qPCR Mastermix (Qiagen, Cat. No. 330523) were used for primer based qPCR assays. All qPCR reactions were performed on ABI 7900 HT Sequence Detection System (Life Technologies). Information on all primers and probes used for qPCR analysis are provided in Supplementary Table 10 and 11. Gene expression was determined relative to GAPDH using the A Ct method. Data are shown as the mean and s.d.
Bisulfite treatment was performed using the EpiTect Bisulfite Kit (Qiagen, Cat. No. 59124) according to the manufacturer's recommendations. Genomic DNA PCR for human ELF5 and porcine OCT4 and NANOG promoter regions was performed using primer pairs described before [7-9]. PCR products were cloned into pGEM-T Easy Vector (Promega, Cat. No. A1360) and sequenced from both ends. Randomly selected clones were sequenced with the M13 forward and M13 reverse primers for each promoter. The primers used in this analysis are provided in Supplementary Table 10.
For dual staining of KRT7 with TFAP2C and GATA3, the differentiated hEPSCs were fixed in 4% paraformaldehyde (Sigma, Cat. No. P6148) solution, blocked with 3% goat serum and 1% BSA and incubated with mouse anti-KRT7 antibody at 4° C. overnight. Cells were then rinsed with PBS solution, incubated with Alexa 488-conjugated goat anti-mouse IgG secondary antibody (Abcam, Cat. No. AB150109) for 1 h at room temperature. After permeabilization with PBST (PBS solution with 0.3% Triton), cells were incubated with rabbit anti-TFAP2C and GATA3 antibodies at 4° C. overnight. The third day, cells were rinsed with PBST, incubated with Alexa 594-conjugated goat anti-rabbit IgG (Invitrogen, Cat. No. A21207) for 1 hour at room temperature, and counterstained with DAPI. For Tuj1, α-SMA, AFP and KRT7 immunostaining in differentiated porcine and human EPSCs, the cells were fixed and incubated with mouse-anti TUJ1, α-SMA, AFP and KRT7 antibodies, respectively, at 4° C. overnight. Cells were rinsed with PBS solution and incubated with Alexa 488-conjugated goat anti-mouse IgG (Abcam, Cat. No. AB150109) and 594-goat anti-mouse IgG (Invitrogen, Cat. No. A21207). After antibody treatment, samples were stained with 10 μg/ml DAPI (Thermo Fisher Scientific, Cat. No. 62248) to mark nuclei. For porcine and human pluripotency marker immunostaining, porcine and human EPSCs were fixed in 4% PFA/PBS solution, blocked in PBS solution with 3% goat serum (Sigma, Cat. No. G9023-10ML) and 1% BSA (Sigma, Cat. No. A2153) (for cell surface markers) or PBS solution with 3% goat serum, 1% BSA and 0.1% Triton (Sigam, Cat. No. T8787) (for intracellular markers, incubated with cell surface antibodies, SSEA-1, SSEA-4, Tra-1-60, Tra-1-81 or intracellular antibodies, OCT4, NANOG and SOX2 at 4° C. overnight. Cells were rinsed and incubated with Alexa 488 or 594-conjugated goat anti-mouse IgG, mouse IgM, rabbit IgG, and counterstained with DAPI. The antibodies used in these experiments is provided in Supplementary Table 9.
Whole-cell extracts were prepared from cells with indicated treatments in lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 0.15M NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate and complete mini EDTA free protease inhibitor cocktail (Roche Applied Science, Cat. No. 11836170001). The cells for the experiment were collected from the same batch of culture when the culture had reached 70-80% confluence. The biological replicates were included to allow the meaningful conclusions. 10 μg protein were used for electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk and treated with antibodies.
Primary antibodies of mouse or rabbit anti AXIN1, SMAD2/3, p-SMAD2/3 and ALPHA-TUBULIN were used. Horseradish peroxidase-conjugated secondary antibodies against rabbit or mouse IgG were added. After antibody treatment, blots were developed using ECL Western Blotting Detection System (Thermo Fisher Scientific, Cat. No. 32106). The antibodies used in these experiments is provided in Supplementary Table 9.
For conversion of primed human ESC lines, 5×104 trypsinized single cells were seeded on a 10-cm STO feeder plate in bFGF-containing standard media supplemented with 5 μM ROCK inhibitor Y-27632 (Tocris, Cat. No. 1254). Standard human ESC media: DMEM/F-12 (Gibco, Cat. No. 21331-020), 20% KnockOut Serum Replacement (KSR) (Gibco, Cat. No. 10828028), 1× Glutamine Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016), and 0.1 mM 2-Mercaptoethanol (Sigma, Cat. No.M6250) and 10 ng/ml bFGF (SCI). One day later, medium was switched to hEPSCM and then refreshed every day. Following the initial differentiation of the majority cells, dome-shaped hEPSC colonies emerged in about 5-6 days, which could be expanded in bulk using 3-5 minutes treatment with 0.25% trypsin/EDTA (Gibco, Cat. No. 25500-054) on STO feeder layer at a density of 5×104 cells/10-cm dish. 5-6 days later, stable dome-shaped single colonies could be picked and expanded following the method described above.
M20 media was used to culture human adult fibroblasts GM00013. The cells were collected by 0.25% trypsin/EDTA from ˜80% confluent T75 flask and washed once with PBS solution. The transfection was performed using an Amaxa Nucleofector machine (Lonza) according to the manufacturer's protocol (NHDF Nucleofector® Kit, Cat. No. VPD-1001). 5.0 μg of DNA were premixed in 100 μl transfection buffer. The DNA mixture consists of 2.0 μg of PB-TRE-hOCKS, 1.0 μg PB-TRE-RL, 1.0 μg PB-EF1a-transposase and 1.0 μg PB-EF1a-rtTA Among them, hOCKS were made with human cDNAs of OCT4, cMYC, KLF4 and SOX2 linked by 2A peptide. 1×106 washed human adult fibroblasts were resuspended in 100 μl solution/DNA mixture and electroporated using program U-20. 0.2×106 transfected cells were seeded on a STO feeder layer (10 cm-dish) in M15 media supplemented with 50 μg/ml Vitamin C (Sigma, Cat. No. 49752-100G). Dox (Sigma, Cat. No. D9891) was added in the media to 1.0 μg/ml final concentration to induce the reprogramming factors expression. After 12-14 days of induction, Dox was removed and the media was switched to hEPSCM for selecting the Dox independent human iPSC colonies. The survived colonies were picked to hEPSCM at ˜day 21 and expanded to stable iEPSC lines.
hEPSCs were dissociated with 0.25% trypsin/EDTA and seeded in gelatinised 6-well plates at a density of 0.1×106 cells/well. The cells were cultured in 20% KSR media supplemented with 5 μM ROCK inhibitor Y-27632 for 1 day. 20% KSR media: DMEM/F-12 (Gibco, Cat. No. 21331-020), 20% KnockOut Serum Replacement (KSR) (Gibco, Cat. No. 10828-028), 1× glutamine penicillin-streptomycin (Thermo Fisher Scientific, Cat. No. 11140-050), 1×NEAA (Thermo Fisher Scientific, Cat. No. 10378-016) and 0.1 mM 2-mercaptoethanol (Sigma, Cat. No. M6250). From the second day, different combinations of the TGFβ inhibitor SB431542 (10 μM, Tocris, Cat. No. 1514), BMP4 (50 ng/ml, R&D, Cat. No. 5020-BP) and the FGF receptor inhibitor PD173074 (0.1 μM, Tocris, Cat. No. 3044) were added into 20% KSR media to start the trophoblast differentiation. The cells were collected at indicated time points for analysis.
Single-dissociated hEPSCs and pEPSCEmb were plated on 6-well plates pre-coated with 1 mg/ml Col W (Corning, Cat. No. 354233) at a density of 2,000 cells per well and cultured in hTSC media as described [10] with a minor modification. hTSC media: DMEM/F12 (Gibco, Cat. No. 21331-020) supplemented with 0.1m M2-mercaptoethanol, 0.2% FBS (Gibco, Cat. No. 10270), 0.5% Penicillin-Streptomycin, 0.3% BSA (Gibco, Cat. No. 15260037), 1% ITSX supplement (Gibco, Cat. No. 51500056), 50 μg/ml Vc (Sigma, Cat. No. 49752-100G), 50 ng/ml EGF (R&D, Cat. No. 236-EG-200), 2 μM CHIR99021 (GSK3i, TOCRIS, Cat. No. 4423), 0.5 μM A83-01 (TOCRIS, Cat. No. 2939), 1 μM SB431542 (Tocris, Cat. No. 1514), 0.8 μM VPA (STEMCELL, Cat. No. 72292) and 5 μM Y27632 (Tocris, Cat. No. 1254). After 7-9 days of culture, the colonies with TSC-like morphologies were picked, dissociated in TrypLE (Gibco, Cat. No. 12605036) and replated on the plate pre-coated with 1 mg/ml Col IV After 4-5 passage, the cells were collected for syncytiotrophoblast (ST) and extravillous trophoblast (EVT) differentiation tests with the methods described [10].
Two porcine TSCs lines (pK3-TSC-#1 and pK3-TSC-#3) transfected with H2BmCherry (EF1a-H2BmCherry and CAGG-H2BmCherry) were used for embryo injection experiments. Cells at passage 20 were briefly treated with TrypLE (Gibco, Cat. No. 12605036), gently tapped out from culture dish, and re-suspended in human TSCs medium. After centrifugation, TSCs were re-suspended in TL-Hepes 296 Ca-free medium composed of 114 mM Sodium Chloride (NaCl, Sigma-Aldrich, Cat. No. S5886), 3.2 mM Potassium chloride (KCl, Sigma-Aldrich, Cat. No. P-5405), 0.4 mM Sodium dihydrogen phosphate monohydrate (NaH2PO4×H2O, Merck, Cat. No. 106346), 0.5 mM Magnesium chloride hexahydrate (MgCl2×6 H2O, Roth, Cat. No. HN03.2), 2 mM Sodium bicarbonate (NaHCO3, Sigma-Aldrich, Cat. No. S4019), 10 mM HEPES (Roth, Cat. No. 9105.3), 10 mM Sodium DL-lactate solution (60%) (Sigma-Aldrich, Cat. No. L1375), 100 U/L Penicilin G potassium salt BioChemica (AppliChem, Cat. No. A1837), 50 mg/L Streptomycin sulfate BioChemica (AppliChem, Cat. No. A1852), 0.25 mM Sodium Pyruvate (Sigma-Aldrich, Cat. No. P2256), 32 mM Sucrose (Merck, Cat. No. 107653), 0.4% Bovine Serum Albumin (BSA, Sigma-Aldrich, Cat. No. A9647) and 10 μM Y27632 (ROCKi, Tocris, Cat. No. 1254). For the injection, TSCs were incubated in 400 μl drops of TL-Hepes 296 Ca-free medium supplemented with 10 μM Y27632 (ROCKi, Tocris, Cat. No. 1254). Thereafter 8-10 single TSCs were injected into 6-day porcine parthenogenetic or IVF embryos with the aid of a piezo-driven micromanipulator (Zeiss, Eppendorf) in Opti-MEM I (1×)+GlutamMAX™-I Reduced Serum Medium (Gibco®, Cat. No. 51985-026) supplemented with 10% FBS (Gibco®, Lot42Q0154K, Cat. No. 10270-106)) and 10 μM Y27632 (ROCKi, Tocris, Cat. No. 1254). After injection, embryos were washed twice and cultured in D15 medium supplemented with 1000 U/ml ESGRO® recombinant mouse LIF protein (Millipore, Cat. No. ESG1107) and 10 μM Y27632 (ROCKi, Tocris, Cat. No. 1254) for 1-2 days at 39° C. in 5% CO2 and 5% 02. Thereafter embryos were fixed with 3.8% paraformaldehyde for 15 min at room temperature and stored in DPBS supplemented with 0.5% FBS (Gibco®, Lot 42Q0154K, Cat. No. 10270-106) and 1% Penicillin/Streptomycin Solution (Corning, Cat. No. PS-B) in 4° C.
Fixed parthenogenetic blastocysts were washed three times in DPBS (Sigma, Cat. No. D5652-10X1L) supplemented with 0.5% FCS (Gibco®, Lot 42Q0154K, Cat. No. 10270-106) and permeabilized in DPBS supplemented with 0.5% Triton® X-100 (Merck, Cat. No. 108603) and 0.5% FCS for 1 h. Thereafter, embryos were washed three times in DPBS and blocked for 1 h at room temperature in blocking solution (co-staining GATA3/CDX2/mCherry: 5% horse serum (Sigma, Lot 14M175, Cat. No. H1270) and 0.2% Triton® X-100 in PBS. After blocking, embryos were incubated with primary antibodies diluted in DPBS and 0.5% FCS for overnight at 4° C. On the following day, embryos were transferred through several washes in DPBS supplemented with either 0.5% horse serum for GATA3/CDX2/mCherry. Secondary antibodies (mCherry: donkey anti-rabbit IgG (H+L) Alexa Fluor Plus 555, A32794, Invitrogen. GATA3/CDX2: donkey-anti-goat IgG (H+L) Alexa Fluor Plus 488, A32814, Invitrogen) were diluted in PBS supplemented with 0.5% horse serum at 1:1000 and the incubation occurred at room temperature for 1 h followed by washing as described above. To visualize nuclei, embryos were incubated in SiR-Hoechst (Spirochrome, SiR-DNA kit, Cat. No. SC007,) at 1:500 dilution in DPBS for 1 h at 37° C. and examined immediately using a confocal imaging system LSM510 (Carl Zeiss MicroImaging GmbH, Germany).
Porcine and human TS cells were dissociated with TrypLE (Gibco, Cat. No. 12605036) and re-suspended in PBS supplemented with 30% matrigel (Corning, Cat. No. 354230) and 10 μM Rock inhibitor Y-27632 (Tocris, Cat. No. 1254). 5×106 porcine or human TSCs were injected subcutaneously into both dorsal flanks of 8-week-old male SCID mice (100 ul per injection). Human and porcine TSCs formed visible lesion within 7-10 days. The lesions were dissected, fixed overnight in 4% phosphate-buffered formalin and embedded in OCT compound (CellPath, Cat. No. 15212776) and paraffin for sectioning
Enzyme-linked immunosorbent assay kits for human VEGF, P1GF, sFlt-1, CGA and sEng were obtained from R&D Systems and Human Chorionic Gonadotropin ELISA assay kits were sourced from ALPCO Diagnostics and performed according to the manufacturer's specifications.
The cells for RNA preparation were collected from the same batch of culture when the culture had reached 70-80% confluence. The biological replicates were included to allow the meaningful conclusions. For human data, protein coding transcripts from GENCODE v27 were used, and transcripts from PAR Y regions were removed from the reference; for mouse data, protein coding transcripts from GENCODE vM16 were used; for porcine data, Ensembl build Sscrofa11.1 was used. Transcript fasta files were downloaded from GENCODE or Ensembl, and ERCC sequences were added into each build. Then the transcripts plus ERCC fasta files were indexed using salmon (version 0.9.1) [11], using the default parameter. When using GENCODE transcript reference, ‘---gencode’ flag was included during indexing to make sure salmon correctly handled the transcript id. For human naïve and primed ESC RNA-seq [12], fastq files were downloaded from ENA (Study accession PRJNA326944); for human embryo single cell data fastq files were downloaded from ENA (Study accession PRJEB8994) [13, 14]. For mouse EPSC data, fastq files from the previous study [15] were used. All the reads were directly quantified against the transcriptomes of the corresponding species using salmon (version 0.9.1) with the flags ‘--useVBOpt --numBootstraps 100 --posBias --seqBias --gcBias -1 ISR -g gene_map.tsv’ where gene_map.tsv was a tab delimited file mapping transcript ids to gene ids to get gene level expression values. The expression levels of each selected histone gene in different types of human cells and early embryos were extracted from expression matrix and visualized as a heatmap generated by GraphPad Prism 7.04 (https://www.graphpad.com/scientific-software/prism/). Gene expression values are linearly transformed into colours (as indicated by the colour legend below each matrix) in which blue colour represents low gene expression, red represents higher gene expression and no colour is equivalent to the highest level of the gene that was expressed. For single cell RNA-seq, an extra quality control step is added, where cells with less than 10,000 total reads, or less than 4,000 detected genes (at least 1 read), or more than 80% of reads mapped to ERCC or more than 60% of non-mappable reads were removed before downstream analyses.
Gene count from each sample was collected together, and log 10 transformed. Then batch effect (batches here mean different studies) and sequencing depth (total number of reads per sample) were regressed out using the “regress out” function from the NaiveDE package (https://github.com/Teichlab/NaiveDE/tree/master/NaiveDE). Principal component analyses were done on the regressed matrix using scikit-learn (Scikit-learn: Machine Learning in Python, Pedregosa et al., JMLR 12, pp. 2825-2830, 2011.). For cross-species comparison, only the one-to-one orthologous genes were used.
Gene expression matrix: reference index was created based on hg38 from GENCODE database [16]. Gene expression matrices for H1-ESC, H1-EPSC, hiPSC-EPSC, PHTu and PHTd were generated using Salmon [11] with following parameters: salmon quant --noversion-check -q -p 6 --useVBOpt --numBootstraps 100 --posBias --seqBias --gcBias. t-SNE (t-distributed stochastic neighbor embedding) analysis: R package ‘Rtsne’ was used for the dimension reduction of gene expression matrices (genes with maximum TPM<=1 were filtered out) and the corresponding result was visualized using a custom R script. Pearson correlation: the RNA-seq data for reference tissues was downloaded from Chang et al. paper [17], the data for reference cells (uESCs, uPHTs, dESCs, dPHTs) was downloaded from Yabe et al. paper [18]. A list of tissue specific genes (n=2293) defined by Chang et al. were selected for Pearson correlation coefficients analysis. Pairwise calculation was performed between the provided data (H1-ESC, H1-EPSC and hiPSC-EPSC) and external references. The result was visualized as a heatmap with high similarity in red colors while low similarity in blue colors. Expression dynamics of 37 trophoblast marker genes were analysed. The expression levels of each marker gene were extracted from expression matrix and normalized using the following method. The TPM of a given gene was divided by the highest gene expression level of that gene in a row (12 data points for each cell line, in total 36 values for H1-ESC, H1-EPSC and hiPSC-EPSC). Through this method, each TPM was transformed into a value between 0 and 1. The overall gene signatures were plotted as a heatmap using color keys ranging from blue (lowly expressed genes) to red (highly expressed genes). The cells for RNA preparation were collected from the same batch of culture when the culture had reached 70-80% confluence. The biological replicates were included to allow the meaningful conclusions.
“Factoextra” R package is applied for PCA analysis and “limma” R package for batch effect removal. Genes whose TPM values were lower than 1 in all samples were removed from the TPM expression matrix.
The single-cell mRNA-seq library was generated following the SMART-seq2 protocol described [19]. In short, single porcine and human EPSCs were sorted into 96-well plates prefilled with lysis buffer and external RNA spike-ins (Ambion) (1:500,000). First-strand synthesis and template-switching were then performed, followed by 25-cycle of pre-amplification. Complementary DNAs were purified by AMPure XP magnetic beads (Agencourt) using an automated robotic workstation (Zephyr). Quality of cDNAs was checked with the Bioanalyzer (Agilent) using high sensitivity DNA chip. Multiplex (96-plex) libraries were constructed and amplified using Nextera XT library preparation kit (Illumina). The libraries were then pooled and purified with AMPure XP magnetic beads. The quality of the library was then assessed by the Bioanalyzer (Agilent) before submission to the DNA sequencing pipeline at the Wellcome Trust Sanger Institute. Pair-ended 75-bp reads were generated by HiSeq2000 sequencers. Porcine and human scRNA seq data can be downloaded from: ftp://ngs.sanger.ac.uk/production/teichmann/xi/xuefei_epsc/single_cell_expr_matrix
Expression violin plot for all genes from scRNAseq: Porcine EPSCs: ftp://ngs.sanger.ac.uk/production/teichmann/xi/xuefei_epsc/porcine_sc_vplot/index.htm
Human EPSCs: ftp://ngs.sanger.ac.uk/production/teichmann/xi/xuefei_epsc/human_sc_vplot/index.html
The H3K4me3, H3K27me3, H3K27ac and input ChIP libraries of porcine and human EPSCs were prepared based on a modified ChIP protocol from Lee et al [20]. In short, about 20 million cells were cross-linked in 1% formaldehyde for 10 mins at room temperature. Cross-linking was then quenched with 0.125 M glycine for 5 minutes at room temperature. Cell pellets were washed with PBS, snap frozen by liquid nitrogen and stored in −80° C. until further processing. Chromatin was sheared by Bioruptor Pico (Diagenode) for 5-7 cycles: 30 sec on and off cycles. Immunoprecipitation were performed with 1 μg antibody pre-washed and pre-attached to protein A Dynaebeads (Invitrogen, Cat. No. 10002D) overnight at 4° C. Antibodies: H3K4me3, H3K27me3, H3K27ac are listed in Supplemental Table 9. The beads were then washed and cross-linking was reversed with the elution buffer at 65° C. for 4 hours. Immunoprecipitated DNAs were purified with proteinase K digestion and the Qiagen minElute PCR Purification kit (Qiagen, Cat. No. 28004). The multiplex sequencing libraries were prepared with the microplex library construction kit (Diagenode, Cat. No. 005010014) following manufacturer's instruction. DNA was amplified for 11 cycles and the quality of the library was checked on a bioanalyzer (Ailgent) using a high sensitivity DNA kit. Library concentration was check by qPCR using KAPA Library Quantification Kit (KK4824), and equal molar of different libraries were pooled and sequenced on 2 lanes of HiSeq2500. 50 base pair single end reads were mapped to the UCSC reference genomes (build susScr11 for porcine and hg38 for human) using bowtie2 (version 2.3.4) [21] with default setting. For the human reference hg38, all the alternative loci were removed (chr*_alt) before mapping. Reads mapped to the mitochondrial genome were removed, and reads mapped to the nuclear genome were filtered by samtools [22] with flags ‘-q 30’ to filter reads with relatively low mapping quality (MAPQ less than 30). For the ChIP-seq data from human naïve and primed ESCs [12], raw reads were downloaded from ENA (Study accession PRINA255308) and processed in the same manner. Peak calling was performed using MACS2 (2.1.1.20160309) [23]. For identification of enriched regions of punctate marks (H3K4me3 and H3K27ac) from porcine samples, peak calling was performed with flags ‘-t chip.bam -c input.bam -g 2.7e9 -q 0.01 -f BAM --nomodel -extsize 200 -B --SPMR’. For identification of enriched regions of broad marks (H3K27me3), peak calling was performed with flags ‘-t chip.bam -c input.bam -g 2.7e9 -q 0.01 -f BAM -nomodel --extsize 200 -B --SPMR --broad’. For human data, peak calling was done in the same way, with a change of genome size ‘-g hs’ during the peak calling. The resulting bedGraph files were converted to bigWig files using the script bdg2bw (https://gist.github.com/j132587/34370c995460f9d5ad65). The bigWig files were visualised using UCSC genome browser[24]. To compare the H3K4me3 signal around naive and primed genes, the differentially expressed gene list between human naive and primed ESCs was downloaded from the Supplementary Table of Theunissen et al. [12]. Genes were sorted by log 2 fold change, and then the top 1000 naïve or primed genes were selected. The H3K4me3 signals of human EPSCs were directly quantified around the transcriptional start sites of those 2000 genes using HOMER (v4.9) [25]. For porcine data, the one-to-one orthologues of those 2000 genes were first extracted from ensembl genome browser [26], and then porcine H3K4me3 signals were quantified in the same way as in human. The cells for histone modification profiles were collected from the same batch of culture when the culture had reached 70-80% confluence. The biological replicates were included to allow the meaningful conclusions.
DNA methylation levels were measured by whole genome bisulfate sequencing [27]. DNA was purified (Qiagen Blood DNA Extraction kit), sonicated using a covaris sonicator. Approximately 500 ng DNA per sample was processed using the NEBNext Ultra DNA library prep kit (NEB E7370) using methylated adapters (NEB or Illumina). Bisulfite conversion was performed using EZ DNA methylation Gold kit (Zymo) prior to final PCR amplification. Libraries were sequenced using Illumina MiSeq platform to generate 100 bp paired end reads. Raw sequence reads were trimmed to remove both poor quality calls and adapters using Trim Galore (v0.4.1, www.bioinformatics.babraham.ac.uk/projects/trim_galore/, Cutadapt version 1.8.1, parameters: --paired) and aligned to the human or porcine genome using Bismark v0.18.2 (Krueger and Andrews, 2011). Data were quantitated using SeqMonk (www.bioinformatics.babraham.ac.uk/projects/seqmonk/) using 500 CpG running windows and a minimum coverage of 100 CpG per window. The cells in this analysis were collected from the same batch of culture when the culture had reached 70-80% confluence.
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. The statistical analysis was conducted with Microsoft Excel or Prism 7.04 (GraphPad). P values were calculated using a two-tailed Student's t-test.
Sequencing data are deposited into ArrayExpress, and the accession numbers are E-MTAB-7252 (CMP-seq), E-MTAB-7253 (bulk RNA-seq) and E-MTAB-7254 (single cell RNA-seq). Human cell sequencing raw data (including ChIP-seq and bulk/single cell RNA-seq) files can be accessed via ftp://ngs.sangerac.uk/production/teichmann/xi/xuefei_epsc/human_fastqi; Porcine cell sequencing raw data (including ChIP-seq and bulk/single cell RNA-seq) files can be accessed via ftp://ngs.sangerac.uk/production/teichmann/xi/xuefei_epsc/pig_fastq/. All other relevant data are available from the corresponding author on request.
The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the relevant art(s) (including the contents of the documents cited and incorporated by reference herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one skilled in the relevant art(s).
While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of examples, and not limitation. It would be apparent to one skilled in the relevant art(s) that various changes in form and detail could be made therein without departing from the spirit and scope of the disclosure. Thus, the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/081594 | 3/27/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62829904 | Apr 2019 | US |
