Method for producing porcine embryonic stem cells

Abstract

The present invention relates to a method of producing porcine embryonic stem cells comprising the steps of (a) chemically activating mature porcine oocytes, (b) culturing the chemically activated oocytes until an embryo sac is formed, (c) removing trophoblast from the embryo sac to isolate an inner cell mass (ICM), and (d) transferring the ICM onto a culture plate for adherence and cell growth. The invention also relates to porcine embryonic stem cells obtained by these methods.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Priority Application No. 63 / 433,454. TECHNICAL FIELD

[0003] The present invention relates to the production of porcine embryonic stem cells by chemical activation. BACKGROUND ART

[0004] The stable and cost-effective production of porcine embryonic stem cells (“pES cells”) is of concern in multiple fields. Porcine embryonic stem cells are suitable for research purposes in animal and human studies, for the production of research or therapeutic antibodies, and for the production of laboratory-cultured tissues.

[0005] For example, the study of early developmental processes in livestock is difficult or impractical, although the comparative biology and physiology shared between pigs and humans are well recognized. Thus, in addition to testing applications in livestock, pES cells can also be used in the study of human disorders. Due to the physiological similarities between humans and pigs, porcine biomedical models are a viable method for studying human diseases and disorders.

[0006] As an alternative, pES cells can be used in complex research and development areas such as the study and production of antibodies for research or therapeutic use.

[0007] pES also provides benefits for livestock research, such as animal therapy, or even animal cloning in the context of endangered pig breeds. Additionally, the production of porcine embryonic stem cells is of concern in the food production field.

[0008] With the increasing global demand for food, the consequences of livestock farming and meat production are significant. The environmental impact of agriculture is substantial, both in terms of the space required to raise animals and the environmental impact of growing sufficient feed for animals.

[0009] In addition, ethical concerns regarding livestock farming have led to a significant number of individuals to forgo consuming animal products, either completely or in a restricted form.

[0010] In recent years, meat alternatives have come into focus. This includes both vegetarian alternatives specifically selected as meat substitutes and vegetarian “meat alternatives” that attempt to mimic animal products.

[0011] Lab-grown meat serves as an alternative to livestock farming but does not seek to be a “replacement” for meat. This allows for the production of meat with all the flavor and texture advantages, but is a more ethical and environmentally conscious choice compared to producing meat through livestock farming.

[0012] However, due to the difficulty in obtaining stable pES cells and the difficulty in obtaining in vitro cultured meat cells in a cost-effective manner, the large-scale production of in vitro cultured meat is an area that requires further research.

[0013] Regardless of the desired use of pES cells, there is a need to improve the production of stable and cost-effective pES cells. Summary of the Invention

[0014] The present invention relates to a method for producing porcine embryonic stem cells, comprising the steps of: (a) chemically activating mature porcine oocytes, (b) culturing the chemically activated oocytes until blastocysts are formed, (c) removing the trophoblast from the blastocysts to isolate the inner cell mass (ICM), and (d) transferring the ICM onto a culture plate for attachment and outgrowth of cells.

[0015] In one embodiment, the mature porcine oocytes are chemically activated by incubating them in the presence of a calcium ionophore.

[0016] In other embodiments, the mature porcine oocytes are chemically activated by incubating them in the presence of a calcium ionophore at a concentration of about 0.5 - 20 μM.

[0017] In other embodiments, the mature porcine oocytes are chemically activated in the presence of cytochalasin B and / or 6-dimethylaminopurine.

[0018] In other embodiments, the trophoblast is removed from the blastocysts by immunosurgery.

[0019] In other embodiments, the trophoblast is removed from the blastocysts by immunosurgery using rabbit anti-pig serum.

[0020] In other embodiments, the trophoblast is removed from the blastocysts by immunosurgery using guinea pig complement.

[0021] In other embodiments, the immunosurgery is performed at a temperature of about 35 - 40 °C for about 20 - 40 minutes.

[0022] In other embodiments, the culture plate contains mouse embryonic fibroblasts.

[0023] In an alternative embodiment, the present invention relates to porcine embryonic stem cells obtained by a production method comprising (a) chemically activating mature porcine oocytes, (b) culturing the chemically activated oocytes until blastocysts are formed, (c) removing the trophoblast from the blastocysts to isolate the inner cell mass (ICM), and (d) transferring the ICM onto a culture plate for attachment and outgrowth of cells.

[0024] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes chemically activating mature porcine oocytes by incubating them in the presence of a calcium ionophore.

[0025] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes chemically activating mature porcine oocytes by incubating them in the presence of a calcium ionophore at a concentration of about 0.5 - 20 μM.

[0026] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes incubating mature porcine oocytes in the presence of cytochalasin B and / or 6-dimethylaminopurine.

[0027] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes removing the trophoblast from the blastocyst by immunosurgery.

[0028] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes removing the trophoblast from the blastocyst by immunosurgery using an antiserum against pigs derived from rabbits.

[0029] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes removing the trophoblast from the blastocyst by immunosurgery using a complement derived from guinea pigs.

[0030] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes performing an immunosurgery step at a temperature of about 35 - 40 °C for about 20 - 40 minutes.

[0031] In other embodiments, porcine embryonic stem cells are obtained by a production method that includes a culture plate that contains mouse embryonic fibroblasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following figures illustrate the steps and elements of the present invention described below.

[0033] Figure 1 An image of a blastocyst (upper left) that has hatched after leaving the zona pellucida (lower right) is shown, where the inner cell mass (ICM) is visible inside the blastocyst.

[0034] Figure 2 A porcine embryo after removal of the zona pellucida with acidic Tyrode's buffer is shown.

[0035] Figure 3Shows the immunosurgery procedure for a single embryo (identified by the arrow in Plate i). This includes: ii) zona pellucida removal; iii) antibody binding; iv) complement lysis of the trophoblast; and v) isolated ICM.

[0036] Figure 4 Shows pre-grinding (left) and post-grinding (right) blastocysts with dead cells, debris, and remaining zona pellucida removed from the isolated ICM (arrow) using a narrow-bore glass pipette.

[0037] Figure 5 Shows initial ICM attachment. Putative stem cells are indicated by asterisks.

[0038] Figure 6 Shows initial outgrowth of the inoculated ICM. Putative pES cells are recognizable by their large nuclei and prominent nucleoli.

[0039] Figure 7 Shows the proliferation of the initial outgrowths (such as those in Figure 6 ) after an additional few days of culture.

[0040] Figure 8 Shows the manual removal of feeder cells approximately 1 week later. Preferably, ES colonies are separated with a glass needle and the clumps are removed from the plate.

[0041] Figure 9 Shows a clump of non-attached pES cells on a fresh feeder cell plate.

[0042] Figure 10 Shows the rapid growth of pES approximately three days after introduction to the feeder cell plate.

[0043] Figure 11 Shows ES cell colonies expressing active alkaline phosphatase, as demonstrated by the red color (visible to the naked eye within the well plate) formed when incubated with a fast violet substrate.

[0044] Figure 12 Shows ES cell colonies (left) stained for the pluripotency marker OCT4 (right).

[0045] Figure 13 Shows ES cell colonies stained for the pluripotency markers NANOG (left) and SOX2 (right).

[0046] Figure 14 Shows ES cell clusters differentiated into motor neurons, which display a morphology consistent with dorsal root ganglia.

[0047] Figure 15ES cells differentiated into myocytes are shown to exhibit multinucleated myotubes (left panel), which are positive for staining of the muscle-specific transcription factor myogenin (middle panel) and myosin heavy chain protein (right panel). Detailed Description

[0048] Given the unlimited self-renewal capacity of porcine embryonic stem cells (pES), compared to adult stem cells isolated from muscle biopsies, porcine embryonic stem cells (pES) are highly suitable as an input cell source for the production of cultured meat. pES cell lines can be established from ovaries and boar semen sourced from abattoirs. The in vitro production of blastocyst-stage embryos and chemical modifiers of intracellular signal transduction pathways capable of maintaining the porcine pluripotency network have been previously described (see, e.g., "Chemically Defined Media Can Maintain Pig Pluripotency Network In Vitro" Choi KH, Lee DK, Kim SW et al. Stem Cell Reports, 2019, incorporated herein by reference). However, the information provided by these protocols is insufficient to generate pES cells and does not provide a suitable source of materials.

[0049] It has been found that pES cells can be prepared according to the present invention. By using the present invention, pES cells can be generated and differentiated into desired cells that can be used in downstream research, development, or production methods.

[0050] In Vitro Production of Porcine Embryos

[0051] Porcine embryos for the preparation of pES cells according to the present invention can be generated by in vitro production methods, such as those described in "Culture of pig embryos" (Petters RM and Wells KD. J. Reprod. Fertil. Suppl., 1993); "Birth of Piglets Derived from Porcine Zygotes Cultured in a Chemically Defined Medium" (Yoshioka K, Suzuki C, Tanaka A et al. Biol. Reprod., 2002); and "Removal of hypotaurine from porcine embryo culture medium does not impair development of in vitro-fertilized or somatic cell nuclear transfer-derived embryos at low oxygen tension" (Chen PR, Spate LD, Leffeler EC et al. Mol. Reprod. Dev., 2020), each of which is incorporated by reference.

[0052] In vitro production of embryos is beneficial because embryos from in vivo sources are not required.

[0053] Porcine ovaries are generally available from commercial sources, such as by-products of pork processing in slaughterhouses. In one embodiment, porcine ovaries are obtained and embryos with antral follicles having a diameter greater than 4 mm are punctured with an 18G needle and a 10 ml syringe to remove the oocyte-cumulus complex (COC) and cell debris.

[0054] The cell debris is exposed to two saline wash cycles, the COC is isolated therefrom under a dissecting microscope, and it is placed in a maturation medium supplemented with hormones and growth factors (see, for example, Table 2). After culturing in humidified air in 5% CO2 for about 40 - 50 hours, preferably 42 - 48 hours, and more preferably 44 - 48 hours, mature oocytes are obtained, as exemplified by the presence of polar bodies. In one embodiment, oocyte maturation is carried out at about 30 - 40 °C, preferably about 35 - 38 °C, and more preferably at about 38 °C.

[0055] In some embodiments, once mature, the cumulus cells are stripped from the oocytes and ground using 0.1% hyaluronidase. In other embodiments, other enzymes (such as trypsin) and / or other mechanical methods (such as vortexing) are used to strip the cumulus cells from the oocytes.

[0056] The mature oocytes can be further processed by using in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), electrical activation, or chemical activation. In a preferred embodiment, the mature oocytes are further processed by chemical activation.

[0057] Chemical activation of mature oocytes

[0058] It has been found that compared with other in vitro production methods (such as IVF or ICSI), parthenogenesis by chemical activation increases the number of embryos obtainable per oocyte.

[0059] Chemical activation is carried out by incubating the mature oocytes in an embryo culture (EC) medium. A suitable EC medium for the present invention is shown in Table 3.

[0060] It has been found that when formulating the EC medium, chemical activation can be best achieved by using a modified NCSU basal medium. In one embodiment, chemical activation is carried out by a calcium-binding compound, preferably a cell-permeable calcium-binding compound, such as a calcium ionophore. In a particularly preferred embodiment, the calcium ionophore used is "calcium ionophore A23187".

[0061] The calcium ionophore is present at a concentration of 0.5 - 20 μM, preferably 1 - 10 μM, more preferably 5 μM. Preferably, the oocytes are incubated in the presence of the calcium ionophore for 1 - 30 minutes, more preferably 5 - 20 minutes, more preferably 5 - 10 minutes, and most preferably 7 minutes.

[0062] In one embodiment, the EC medium further contains one or more antibiotics or antimicrobial components, preferably penicillin and / or streptomycin.

[0063] In another embodiment, the chemical activation further includes the presence of one or more of cytochalasin B and 6-dimethylaminopurine (DMAP) to prevent the extrusion of the second polar body and allow the development of diploid parthenogenetic blastocysts.

[0064] The concentration of cytochalasin B can be in the range of 1 - 10 μg / ml, preferably 5 - 9 μg / ml, and more preferably 7.5 μg / ml. The concentration of DMAP can be in the range of 0.5 - 5 mM, preferably 1 - 3 mM, and more preferably 2 mM.

[0065] Preferably, the activated oocytes are incubated with CB and / or DMAP for about 2 - 10 hours, preferably about 4 - 8 hours, and more preferably about 4 - 5 hours.

[0066] In a preferred embodiment, EC medium is used for the chemical activation step. In a particularly preferred embodiment, the EC medium used is as described in Table 3.

[0067] In other embodiments, 24 h after chemical activation, the fertilized eggs are dissected and transferred onto an embryo culture plate, and cultured in humidified air at 38.5 °C, 5% CO2, 5% O2 in groups of 30 - 100 for an additional 5 days. Within 8 days of incubation, the embryos reach the blastocyst stage.

[0068] It has been found that the above method provides a suitable supply of blastocysts for further use according to the present invention.

[0069] Derivation of porcine embryonic stem cells

[0070] It has further been found that pES cells can be obtained from porcine blastocysts, such as those produced according to the above embodiments.

[0071] After 5 - 10 days, preferably 5 - 8 days, and more preferably 6 - 8 days of development after activation, blastocysts are selected.

[0072] As Figure 1 shown, the blastocysts at this stage after activation should have hatched out of the zona pellucida. In one embodiment, the zona pellucida can be removed or sufficiently diluted by washing with acidic Tyrode's lactate (ATL) buffer. By incubating the blastocysts in ATL for about 1 to 5 minutes, the zona pellucida can be diluted or removed. In one embodiment, the blastocysts are incubated until the zona pellucida thickness is less than 5 μm. In a preferred embodiment, as Figure 2 shown, the blastocysts are incubated until there is no zona pellucida.

[0073] In another embodiment, the zona pellucida can also be removed mechanically or enzymatically, such as by using a glass needle, laser microdissection, or other known techniques. In a preferred embodiment, mechanical removal is performed after the zona pellucida has been first diluted as described above.

[0074] It has been found that preferably the blastocysts are treated to remove the trophoblast by antibody - mediated complement cell lysis (also known as "immunological surgery"). Immunological surgery enables the isolation of the inner cell mass (ICM) to obtain pES cells, as Figure 3 shown. Similar to the above zona pellucida removal, in addition to or as an alternative to the said method, the ICM can be isolated by mechanically dissecting or enzymatically digesting the blastocysts. In any method, care should be taken to ensure that the ICM is not damaged by the method.

[0075] Immunosurgery according to the present invention can be carried out using any anti - porcine serum (or recombinant antibody) and any complement diluted in a suitable medium. Preferred sources of anti - porcine serum are obtained from rabbits, goats or sheep. Preferred sources of recombinant antibodies (monoclonal or polyclonal) are obtained from immortalized rodent or rabbit cells. Preferred sources of complement are obtained from guinea pigs, mice, rats, rabbits, monkeys and humans. In a particularly preferred embodiment, rabbit serum and guinea pig complement are used for immunosurgery.

[0076] In a preferred embodiment, immunosurgery is carried out by incubating blastocysts in anti - porcine rabbit serum (most preferably, serum diluted 1:10 with wash buffer). Preferably, the incubation is carried out for 20 - 40 minutes, preferably 30 minutes. Preferably, the incubation is carried out at about 35 - 40 °C, more preferably at 37 °C.

[0077] In a preferred embodiment, the blastocysts are placed in a 1:10 dilution of complement in embryo wash buffer (as shown in Table 5) and further incubated for 20 - 40 minutes, more preferably 30 minutes, at an incubation temperature of about 35 - 40 °C, more preferably about 37 °C. The type of complement used can be the same as or different from the type of serum. In a preferred embodiment, different species are used. In a particularly preferred embodiment, guinea pig complement is used.

[0078] In other embodiments, the immunosurgery step includes a washing step between the step of adding serum (or recombinant antibody) and the step of adding complement to remove any residual and unbound antibody. In a preferred embodiment, the washing step is carried out more than once, up to about 5 minutes each time. In a preferred embodiment, the washing step is carried out three times, about 5 minutes each time.

[0079] After immunosurgery and optional washing steps, the ICM must be cleaned to better adhere to the culture plate and grow out from the culture plate. As Figure 4 shown, preferably, the ICM is cleaned by grinding it up to 50 times with a narrow - bore glass pipette to remove residual zona pellucida fragments, dead / dying trophoblast cells. As an alternative or in addition, other removal methods can be used, such as using a glass needle or laser dissection.

[0080] When ICM cleaning is completed, individual ICMs are placed on a culture plate to allow attachment and cell outgrowth, which represents pES cells, as Figure 5 shown. In one embodiment, the culture plate contains extracellular proteins such as fibronectin or laminin. In a preferred embodiment, the culture plate contains a feeder culture of fibroblasts, such as mouse, rat, rabbit or porcine fibroblasts. In a particularly preferred embodiment, the culture plate contains a feeder culture of mouse embryonic fibroblasts.

[0081] After the ICM adheres, outgrowths (i.e., small cell clusters) must be observed, such as those shown in Figures 5 - 7 . Then, the outgrowths of the cells are lifted from the culture plate and re-inoculated onto a fresh culture plate, either chemically, enzymatically, or mechanically. In a preferred embodiment, as shown in Figure 8 and 9 , the outgrowths are lifted mechanically and inoculated onto mouse fibroblast feeder cells.

[0082] In a preferred embodiment, the medium for ICM adhesion consists of embryo harvesting medium (such as the medium in Table 6).

[0083] In another preferred embodiment, as shown in Figure 10 , the medium is not changed during the first seven days of adhesion to provide maximum adhesion and outgrowth.

[0084] Confirming pES cell differentiation

[0085] When processed according to the present invention as described above, it was unexpectedly found that stable and differentiated pES could be obtained. pES differentiation can be confirmed by a variety of techniques.

[0086] In one embodiment, as shown in Figure 11 , when the cells are incubated with a fast violet substrate, pES differentiation is observed visually by the presence of red color. The red color is obtained due to the presence of expressed active alkaline phosphatase.

[0087] In another embodiment, as shown in Figure 12 and 13 , the pES cells can be stained for one or more of the pluripotency markers OCT4, NANOG, or SOX2.

[0088] In other embodiments, as shown in Figure 14 , when observed under a microscope, the pES cells will show differentiated motor neurons that display a morphology consistent with dorsal root ganglia.

[0089] In yet another embodiment, the pES cells can be stained for the presence of myosin heavy chain protein and / or the muscle-specific transcription factor myogenin, see Figure 15 .

[0090] List of materials and reagents for the disclosed invention

[0091] The following materials, solutions, and media are suitable for preparing the materials according to the present invention. Suitable alternatives to the listed reagents are also known, and these changes will not detract from the present invention.

[0092] Table 1. In Vitro Maturation (IVM) Medium:

[0093]

[0094]

[0095] Table 2. Modified North Carolina State University (mNCSU) Medium:

[0096]

[0097] Table 3. Embryo Culture (EC) Medium:

[0098]

[0099] Table 4. Acidic Tyrode's Lactate (ATL) Buffer:

[0100]

[0101] Table 5. Embryo Wash Buffer:

[0102]

[0103]

[0104] Table 6. Embryo Derived Medium:

[0105]

[0106] References

[0107] The following references may be helpful to the reader, and these references are incorporated herein by reference in their entirety:

[0108] a. J.A. Piedrahita, G.B. Anderson, and R.H. Bondurant, Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines. Theriogenology (1990).

[0109] b. M. S. Serrano Albal, G. Silvestri, L. G. Kiazim, et al., Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality. Zygote (2022).

[0110] c. S. Liu, K. Cui, H. L. Li, et al., Comparison of chemical, electrical, and combined activation methods for in vitro matured porcine oocytes. In Vitro Cell Dev Biol Anim (2015).

[0111] d. M. A. Silvestre, J. Alfonso, E. García-Mengual, et al., Effect of recombinant human follicle-stimulating hormone and luteinizing hormone on in vitro maturation of porcine oocytes evaluated by the subsequent in vitro development of embryos obtained by in vitro fertilization, intracytoplasmic sperm injection, or parthenogenetic activation. J Anim Sci (2007).

[0112] e. Y. Yuan, L. D. Spate, B. K. Redel, et al., Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation. Proc Natl Acad Sci U S A (2017).

[0113] f.K.-H.Choi,D.-K.Lee,S.W.Kim,et al.,Chemically Defined Media CanMaintain Pig Pluripotency Network In Vitro.Stem Cell Reports(2019).

[0114] g.Petters RM and Wells KD.J.,Culture of pig embryos,Reprod.Fertil.Suppl.,1993.

[0115] h.Yoshioka K,Suzuki C,Tanaka A,et al.,Birth of Piglets Derived fromPorcine Zygotes Cultured in a Chemically Defined Medium,Biol.Reprod.,2002

[0116] i.Chen PR,Spate LD,Leffeler EC,et al.,Removal of hypotaurine fromporcine embryo culture medium does not impair development of in vitro-fertilized or somatic cell nuclear transfer-derived embryos at low oxygentension,Mol.Reprod.Dev.,2020

[0117] It should be understood that the present disclosure is not limited to the specific compositions, methods, and experimental conditions described, as these may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the present disclosure will be defined only by the appended claims. It will be apparent to those skilled in the art that various embodiments and modifications can be made to the disclosure of this application without departing from the invention.

Claims

1. A method for producing porcine embryonic stem cells, the method comprising: a. Chemically activating mature porcine oocytes; b. Culturing the chemically activated oocytes until blastocysts are formed; c. Removing the trophoblast from the blastocysts to isolate the inner cell mass (ICM); and d. Transferring the ICM to a culture plate for attachment and cell outgrowth.

2. The method according to claim 1, wherein The mature porcine oocytes are chemically activated by incubating the mature porcine oocytes in the presence of a calcium ionophore.

3. The method according to claim 1 or 2, wherein, The calcium ionophore is present at a concentration of about 0.5 - 20 μM.

4. The method according to any one of claims 1 to 3, wherein The mature porcine oocytes are chemically activated in the presence of cytochalasin B and / or 6-dimethylaminopurine.

5. The method according to any one of claims 1 to 4, wherein The trophoblast is removed from the blastocysts by immunological surgery.

6. The method according to claim 5, wherein The immunological surgery includes using rabbit anti-pig serum.

7. The method according to claim 5 or 6, wherein The immunological surgery includes using complement derived from guinea pigs.

8. The method according to any one of claims 4 to 7, wherein The immunological surgery is performed at a temperature of about 35 - 40 °C for about 20 - 40 minutes.

9. The method according to any one of claims 1 to 8, wherein The culture plate contains mouse embryonic fibroblasts.

10. A porcine embryonic stem cell obtained by the method according to any one of claims 1 to 9.

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