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US20160251625A1 - Method for scalable skeletal muscle lineage specification and cultivation - Google Patents

Method for scalable skeletal muscle lineage specification and cultivation Download PDF

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US20160251625A1
US20160251625A1 US15/033,207 US201415033207A US2016251625A1 US 20160251625 A1 US20160251625 A1 US 20160251625A1 US 201415033207 A US201415033207 A US 201415033207A US 2016251625 A1 US2016251625 A1 US 2016251625A1
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cell line
differentiation
myogenic
self
cell
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Nicholas J. Genovese
R. Michael Roberts
Bhanu Prakash V.L. TELUGU
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Upside Foods Inc
University of Missouri St Louis
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Memphis Meats Inc
University of Missouri St Louis
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Assigned to THE CURATORS OF THE UNIVERSITY OF MISSOURI reassignment THE CURATORS OF THE UNIVERSITY OF MISSOURI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: TELUGU, Bhanu Prakash V.L.
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    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present disclosure relates to methods for enhancing cultured meat production, such as livestock-autonomous meat production.
  • cultured meat e.g., animal-autonomous meat production by in vitro cell culture, tissue engineering, and food technology methods
  • the conceptual promises of “cultured meat” include increased production efficiency, reduced environmental impacts, expanded culinary application utility, enhanced nutritional value, poison-free production and improved food safety relative to conventionally produced meats.
  • Technologies, to date however, have not advanced sufficiently to support scalable, economically sustainable production.
  • the current laboratory-scale cultivation of prototype tissues has utilized primary animal components such as animal tissues and serum, thereby largely negating the advantages of animal-autonomous meat production.
  • current methods fail to resolve the animal dependence from cultured meat production sufficiently to realize the conceptual promises of “cultured meat” and provide a commercially advantageous product. Therefore, there is a need to provide new and improved methods for scalable meat cultivation from a self-renewing source in vitro for dietary nutrition and other applications.
  • the example embodiments provide a scalable platform for skeletal muscle cultivation that utilizes cell lines with the potential to differentiate as skeletal muscle.
  • the cell lines are from livestock such as domestic cattle, pigs, sheep, goats, camels, water buffalo, rabbits and the like.
  • the cells lines are from poultry such as domestic chicken, turkeys, ducks, geese, pigeons and the like.
  • the cell lines are from common game species such as wild deer, gallinaceous fowl, waterfowl, hare and the like.
  • the cell lines are from aquatic species or semi-aquatic species harvested commercially from wild fisheries or aquaculture operations, or for sport, including certain fish, crustaceans, mollusks, cephalopods, cetaceans, crocodilians, turtles, frogs and the like.
  • the cell lines are from exotic, conserved or extinct animal species.
  • the cell lines are from any metazoan species demonstrating the capacity for skeletal muscle tissue specification.
  • the cell lines are for research or for therapeutic purposes, such as humans, primates, rodents including rats and mice, and companion animals such as dogs, cats, horses, and the like.
  • the cell lines from any organisms are self-renewing stem cell lines.
  • the selected cell line is modified by a ‘genetic switch’ to induce rapid and efficient conversion of cells to skeletal muscle for cultured meat production.
  • a ‘genetic switch’ to induce rapid and efficient conversion of cells to skeletal muscle for cultured meat production.
  • the above or other aspects may be accomplished by a method comprising modifying a selected self-renewing cell line by a myogenic transcription factor to produce a myogenic-transcription-factor-modified cell line, and inducing such modified cell line by exogenous regulation to direct alternate self-renewal or differentiation processes.
  • the self-renewing cell line is selected from a group consisting of embryonic stem cells, induced pluripotent stem cells, somatic cell lines, or extra-embryotic cell lines with myogenic potential.
  • the cell line is derived from species intended for dietary consumption.
  • myogenic transcription factors include, alone or in combination, MYOD1, MYOG, MYF5, MYF6, PAX3, PAX7, paralogs, orthologs, genetic variants thereof, or transcriptional activation agonists of the respective promoter recognition DNA sequences of the myogenic transcription factors as further described herein.
  • an inducible MyoD transcription factor may be used as the differentiation lineage specifier.
  • the porcine induced pluripotent cell line O2K may be employed as the self-renewing cell line.
  • the method comprises modifying a O2K stem cell line with an inducible MyoD transcription factor to produce a myogenic-transcription-factor-modified O2KM cell line, and inducing such O2KM cell line by exogenous regulation to direct self-renewal or differentiation processes.
  • the aforementioned modifying step can further comprise modifying the cell line with a chromosomally integrated vector constitutively expressing an inducible fusion of the MYOD1 transcription factor and an ESR1 ligand binding domain from a constitutively active promoter region.
  • the inducible activity of the translated fusion transcript e.g., MyoDER
  • the ESR1 agonist e.g., 17-(3 Estradiol (E2)
  • the inducing step can further comprise the self-renewal sub-step and the differentiation sub-step regulated by a double-switch mechanism.
  • the self-renewal sub-step the modified cell line undifferentiated ground-state is preserved, such as in the presence of doxycycline (DOX), whereby the cell line is maintained in a stem cell self-renewal state by the induced expression of the pluripotency transgenes POU5F1 and KLF4.
  • DOX doxycycline
  • the modified cell line is treated, such as with E2 in the absence of DOX, whereas the cell line is efficiently specified to skeletal myocytes, i.e., the myogenic lineage, by the inducible MyoD transcription factor, resulting in characteristic elongated cells with spindle-like morphology.
  • the derivative myocytes can fuse into multinucleated myotubes, precursors to skeletal muscle fibers.
  • the differentiation sub-step can further comprise adding certain reagents in the culture medium for activating the canonical WNT signaling pathway to prevent cell death and facilitate myogenic differentiation, and adding epigenetic modulators to the culture medium to alter the chromatin structure for enhanced myogenic gene expression.
  • Certain aspects of the disclosure employ genetically enhanced cells for unlimited renewal capacity and efficient conversation to skeletal muscle, the predominant tissue lineage constituting non-offal meat products, in serum-free culture medium.
  • tissue engineering approach When coupled with a scalable tissue engineering approach, such methods can revolutionize the way meat is produced and marketed for consumers by enabling cultivation of animal tissue in unlimited quantities for animal-autonomous cultured meat production. Additional applications contemplated include in vivo xeno-transplantation use and in vitro models for drug screening, developmental physiology, and developmental biology.
  • FIG. 1 shows a MyoDER DNA sequence
  • FIG. 2 is a schematic illustration of a method comprising a double-switch regulation mechanism for expansion of the undifferentiated cell line, or skeletal muscle lineage specification.
  • FIG. 3 is a schematic illustration showing a double-switch mechanism applied to the myogenic modified O2KM cell line regulated by DOX or E2.
  • FIG. 4A is a schematic illustration of the selectable MyoDER transgene expression cassette. Arrows and boxes respectively indicate promoter and gene sequences.
  • FIG. 4B shows Western blot image detection of MyoDER transgene expression in blastidicin-selected O2K by an anti-MYOD1 antibody.
  • Transgene expression cassette modified O2K is designated as O2KM.
  • TUBA alpha-tubulin
  • FIG. 5 is an image showing O2KM cells exhibiting stable, compact-colony morphology in self-renewal conditions as the parental O2K cell line.
  • FIG. 6 is a panel of images showing O2KM cultured on Poly-D-Lysine+Laminin+MATRIGEL coated dishes+/ ⁇ 0.25 ⁇ M 5-Aza-Cytidine (5AC) for phenol-free self-renewal medium (SRM) under 5% O 2 for three days followed with or without 17- ⁇ Estradiol (E2) induction of the MyoDER fusion protein under 20% O 2 for two days in phenol red-free myogenic induction medium (MIM) supplemented with 3 ⁇ M CHIR99021.
  • C Phase-contrast images of O2KM on E2-induction day 2.
  • FIG. 7 shows Western blot analysis of MYOD1 (MyoD), MYF5 (Myf5), and MYOG (myogenin) in differentiated O2KM cell lysates harvested following indicated 2-day E2 induction regimens.
  • MyoDER migration ⁇ 75 kD.
  • Expected endogenous MYOD1 migration 45-50 kD.
  • FIG. 8 shows images of immunofluorescent detection of myocyte cell surface marker NCAM (Alexa568) and nuclei (DAPI) in 5AC-exposed O2KM cultures prior to, and following, a 2-day 10 ⁇ M E2 induction time-course.
  • NCAM myocyte cell surface marker NCAM
  • DAPI nuclei
  • FIG. 9 shows a panel of phase-contrast images of i. ground state undifferentiated O2K colonies cultured on Poly-D Lysine+Gelatin+Laminin under 5% O 2 in SRM, ii.-vi. adherent colonies differentiating from the ground state, as shown in panel i. for two days in differentiation medium (DM) under 20% O 2 supplemented with ii. 0 ⁇ M, iii. 1 ⁇ M, iv. 3 ⁇ M, v. 6 ⁇ M or vi. 9 ⁇ M CHIR99021.
  • Non-adherant colonies were prevalent as embryoid bodies in cultures exposed to 6 ⁇ M (vii.) or 9 ⁇ M (viii.) CHIR99021.
  • FIG. 11 shows a bar graph of flow cytometric analysis of Annexin V labeled cells. Undifferentiated O2K colonies were cultured under 20% O 2 in differentiation medium the presence of 0, 1, 3 or 6 ⁇ M CHIR99021 for one day prior to analysis.
  • FIG. 12 shows Western Blot analysis of relative CTNNB1 ( ⁇ -catenin) levels and phosphorylation (p-CTNNB1) at GSK3 ⁇ substrates serine 33, 37 and threonine 41 in cultures differentiated from the ground-state (as shown in FIG. 9 ) in the presence of CHIR99021 at the concentrations indicated. Cultures were exposed to 50 nM Calyculin A and 30 ⁇ M MG-132 for 3 hours prior to harvest to stabilize detectable levels of p-CTNNB1 for comparative analysis.
  • FIG. 13A shows a graph illustrating the densitomentric ratios of p-CTNNB1/CTNNB1 bands as shown in FIG. 12 .
  • FIG. 13B shows a graph illustrating the densitometric ratios of CTNNB1/TUBA bands as shown in FIG. 12 .
  • FIG. 14 shows an image illustrating the differentiation marker time-course Western blot analysis. Expression levels of pluripotency markers, POU5F1 and KLF4, and the pre-myogenic paraxial mesoderm marker PAX3 in O2K cultures differentiated from the ground state in the presence of CHIR99021.
  • FIG. 15 shows images of terminal differentiation of O2KM myocytes differentiated in the absence (left panel) or presence (right panel) of 5-Aza-Cytidine (5AC). Note the myocyte derivatives with flattened morphology in the left panel ( ⁇ 5AC) in contrast to the elongated, multinucleated myotubes in the left panel (+5AC).
  • FIG. 16 shows Annexin V labeling of apoptotic cells prior to, and following 24 h transition of cultures, as in FIG. 9 , panels i-ii.
  • FIG. 17A shows Western blot detection of full-length CPP32 ( ⁇ 32 kD procaspase 3a) and the large cleaved fragment ( ⁇ 17 kD cleaved-caspase 3a) in ground-state colonies prior to (0 h) and following (12-48 h) differentiation milieu transition (12-48 h).
  • FIG. 17B shows Western blot detection of full-length CPP32 and the cleaved fragment in colonies following 42 h transition to the differentiation milieu in the presence of CHIR99021 levels indicated.
  • FIG. 18 shows outgrowth morphology of embryoid bodies formed in a differentiation milieu containing 6 ⁇ M CHIR99021 for two days and following transfer to a Poly-D Lysine+Laminin+MATRIGEL coated substrate for one (d3, left panel) or three (d5, right panel) additional days.
  • FIG. 19A shows Western Blot of lysates from unmodified piPSC cultured in the absence of 3i, DOX, hLIF and E2 in differentiation milieu containing KOSR.
  • FIG. 19B shows Western Blot of lysates from MyoDER-modified piPSC cultured in the presence of DOX, LIF and 3i. MYODER-modified piPSC were cultured 3 days in self-renewal or expansion milieu.
  • FIG. 20A is a schematic of the O2KM expansion and induction regimens, followed by the terminal differentiation regimen.
  • FIG. 20B shows myotube morphology and conformation.
  • Post-induction (d2), piPSC developed as elongated, anisotropic, refractive myotubes when exposed to 5AC during the expansion and induction regimens.
  • FIG. 20C shows uniform expression of myosin heavy chain by d6.
  • FIG. 20D shows myotube multinucleation.
  • Left panel enlarged image of d4 terminal differentiation cultures. Bracketed arrows indicate multiple nuclei within a single myotube.
  • FIG. 20E Western blots show increasing expression of desmin (DES) and myogenin (MYOG) over the 8d course.
  • FIG. 20F shows cell-cycle withdrawal concomitant with terminal differentiation.
  • FIG. 20G shows Transmission Electron Microscopy of d6 myotubes. Sarcomeric structural units were aligned in single (left panels) and staggered, parallel rows (right panel).
  • FIG. 21A shows asynchronous, single-cell transient cycles (left, middle and right panels) were observed in spontaneously contracting d6 myotube subpopulations.
  • FIG. 21B shows FOV activation and synchronization of calcium transient cycles by 1.0 Hz field stimulation in d6 myotubes.
  • FIG. 21C shows FOV calcium transient activation of d6 myotubes by 10 mM caffeine.
  • FIG. 21D single-cell analysis of calcium transient activation in a d7 myotubes by 100 nM acetylcholine.
  • a or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • meat is any metazoan tissue or cell-derived comestible product intended for use as a comestible food or nutritional component by humans, companion animals, domesticated or captive animals whose carcasses are intended for comestible use, service animals, conserved animal species, animals used for experimental purposes, or cell cultures.
  • the cell source is a stem cell source, for example, a self-renewable stem cell line.
  • Certain aspect of the methods employ a myogenic, inducible transgene-modified self-renewable cell line derived from an intended species.
  • the intended species can be any edible species including livestock and poultry species.
  • the intended species are livestock species such as domestic cattle, pigs, sheep, goats, camels, water buffalo, rabbits and the like.
  • the intended species are poultry species such as domestic chicken, turkeys, ducks, geese, pigeons and the like.
  • the intended species are common game species such as wild deer, gallinaceous fowl, waterfowl, hare and the like.
  • the intended species are aquatic species or semi-aquatic species harvested commercially from wild fisheries or aquaculture operations, or for sport, including certain fish, crustaceans, mollusks, cephalopods, cetaceans, crocodilians, turtles, frogs and the like.
  • the intended species are exotic, conserved or extinct animal species.
  • the intended species are any metazoan species demonstrating the capacity for skeletal muscle tissue specification.
  • the intended species are for research or for therapeutic purposes, such as humans, primates, rodents including rats and mice, and companion animals such as dogs, cats, horses, and the like.
  • the cell line is regulated by a double-switch mechanism to either maintain the cell line in self-renewal process or direct myogenic differentiation.
  • a parent/host cell line of aspects disclosed herein has the properties of being immortal (self-renewing) and having the potential to differentiate, reprogram, specify or otherwise convert to skeletal muscle lineage, such as following regimens comprising one or more components that direct myogenic conversion.
  • Three classes of stem cell may be employed as cell sources for scalable cultivation: (1) lineage-restricted primary adult progenitor stem cell isolations, (2) lineage-restricted immortalized cell lines, and (3) pluripotent stem cells lines. It has been determined that each of these approaches has advantages and disadvantages in serving as a cell source for cultured meat production.
  • Skeletal muscle progenitor cells include, but are not limited, to satellite cells, myoblasts and myocytes.
  • Their advantages include: i) primary adult progenitor cells are restricted to specific lineages and require little or no in vitro specification to desired lineages; and ii) primary adult progenitor cells do not require genetic modification for lineage specification.
  • Their disadvantages include: i) they must either be harvested from a freshly slaughtered animal carcass or procured from an invasive biopsy. Either method conveys dependence on livestock and compromises the benefit of livestock-autonomous production to the extent that livestock are used in the process; ii) primary cell isolation is a highly inefficient process.
  • the desired cells comprise a fraction of the source tissue. A subfraction of the desired cells survive the isolation process. Desired cell lineages must be isolated from mixed populations of surviving cells, requiring additional purification and expansion steps; iii) primary adult progenitor cells are subject to the ‘Hayflick Limit’, wherein cells can divide only limited number of times before they lose their capacity to proliferate. Moreover, primary adult progenitor cells lose their ability to terminally differentiate in a manner concordant with extended passage.
  • additional cells must be procured from primary cell isolations, thereby limiting cultivation scalability from a single isolation; and iv) primary cell culture of lineages of tissues applicable to cultured meat production, such as skeletal muscle, are anchorage dependent-limiting methods for volumetric scalability of cultures. In suspension culture, these cells may be susceptible to cell death by anoikis.
  • lineage-committed primary cells that are genetically altered to self-renew indefinitely while retaining their capacity to terminally differentiate or lineage-restricted.
  • Their advantages include: i) “perpetually self-renewing” (i.e. not subject to the ‘Hayflick Limit’) and can expand indefinitely for scalable and livestock-autonomous cultivation; ii) restricted to specific lineages and require little or no further in vitro specification.
  • Their disadvantages include: i) immortalized, lineage-restricted cell lines from certain species with the capacity to differentiate along lineages applicable to cultured meat production (e.g. skeletal muscle) may require development; ii) cultures of lineage-committed cell lines are anchorage dependent, limiting scalability.
  • lineage-committed cell lines may be susceptible to cell death by anoikis; and iii) cellular transformation(s) enabling ‘immortalization’ necessitates genetic modification.
  • the necessary genetic modifications that immortalize applicable primary cell populations without interfering with their capacity to terminally differentiate are not well characterized.
  • Pluripotent stem cell lines include embryonic stem cells or induced pluripotent stem cells (iPSC) that maintain the capacity to self-renew in the undifferentiated state, or alternately differentiate to any tissue lineage.
  • iPSC induced pluripotent stem cells
  • Their advantages include: i) in general, pluripotent stem cell lines proliferate at a higher rate than primary or immortalized lineage-restricted cell lines, reducing the time required for biomass expansion in production processes; ii) pluripotent stem cells may be cultivated as embryoid bodies in suspension culture, thereby enhancing culture scalability per unit of culture volume.
  • embryoid bodies may be cultured as ‘bio-ink’ compatible with micromold and bioprinting tissue assembly methods; and iii) like immortalized lineage-restricted cell lines, pluripotent stem cells are not subject to the ‘Hayflick Limit’ and can expand indefinitely for scalable, livestock-autonomous cultivation.
  • Their disadvantages include: i) authentic embryonic stem cell lines derived from certain species may require development; ii) methods for reprogramming and self-renewal of iPSC may be transgene-dependent. Hence, iPSC pluripotency may require genetic modification for induction and self-renewal of the undifferentiated state.
  • Efficient iPSC differentiation requires mechanisms for silencing the transgenes used for reprogramming and maintenance of the undifferentiated state mutually exclusive to the differentiated state to avoid conflicting transcription network activation disadvantageous to desired lineage specification; and iii) relative to lineage-restricted primary adult progenitor stem cells and immortalized cell lines, pluripotent stem cells, in general, require additional lineage specification steps to develop and enrich the desired lineage specification.
  • induced trophoblast cell lines (representing non-pluripotent, non-somatic immortalized cells of extra-embryonic type), whose myogenic potential was established previously by the teratoma assay, may be suitable for myogenic conversion as well.
  • somatic cell lines partially reprogrammed to pluripotency may possess myogenic potential but fail to form teratomas representing three embryonic germ layers.
  • STAP cell lines (stimulus-triggered acquisition of pluripotency) may be myo-potent and self-renewing.
  • the O2K cell line is an induced pluripotent stem cell line established from the inner cell mass of a pre-implantation porcine embryo.
  • the O2K cell line has been studied and it was discovered that the self-renewal state of O2K can be maintained by transcriptional activation of POU5F1 and KLF4 transgenes by doxycycline (i.e. DOX) using a ‘Tet-On’ induction system.
  • DOX doxycycline
  • MYOD1 (i.e. MyoD) is a dominant regulator of skeletal muscle lineage commitment.
  • the MyoDER construct has been described previously, consisting of a genetic fusion of the murine MYOD1 gene and the sequence encoding the ligand binding domain of the human estrogen receptor ⁇ , shown in FIG. 1 (SEQ ID NO: 1).
  • the MyoDER consists of a genetic fusion between the murine MYOD1 gene at the Nar I restriction endonuclease digest site with the ligand binding domain coding sequence of the ESR1 (i.e. human estrogen receptor ⁇ ) nucleotides 844-1781.
  • ESR1 i.e. human estrogen receptor ⁇
  • MyoDER fusion construct The myogenic specification activity of the MyoDER fusion construct is post-translationally induced by addition of the estrogen receptor ⁇ ligand, 17 ⁇ -Estradiol (i.e., E2). In the absence of the 17 ⁇ -estradiol, MyoDER remains in an inactive state.
  • the MyoDER construct is herein referred to as “inducible MyoD.”
  • one aspect is cell-stock-expansion, i.e., expansion of the cell line in self-renewal conditions necessary for the maintenance of cell stocks for continued scalable cultivation.
  • Another aspect is the lineage-specification/differentiation, i.e., inducing myogenic lineage differentiation for further tissue cultivation process.
  • certain aspects may be summarized as comprising two main steps: i) modifying a selected self-renewing cell line with a myogenic transcription factor to produce an myogenic-transcription-factor-modified cell line, and ii) inducing such modified cell line by exogenous regulation to maintain in self-renewal process or advance to differentiation process.
  • modifying refers to inserting a nucleic acid vector or construct operably encoding a myogenic transcription factor (such as by transfection, transduction, transformation, and the like) into the cell line, wherein the modified cell line expresses the myogenic transcription factor.
  • the inserted myogenic transcription factor is inducibly-expressed to produce an inducible-myogenic transcription factor cell modified cell line.
  • inducibly refers to any genetically engineered approaches that may be used to exogenously regulate the activities of a gene product such as a myogenic transcription factor.
  • Inducible approaches include, but are not limited to, regulation of myogenic transcription factor activity by ligand inducible transcription factor technology (e.g., tet-on, tet-off, RheoSwitch), site-directed recombination technology (e.g., Cre-LoxP, flp-FRT), transposon technology (e.g. Sleeping Beauty, PiggyBac), ligand binding receptor fusion technology (e.g., estrogen, progesterone, androgen, thyroid hormone, glucocorticoid hormone, tamoxifen ligand agonists), and transient transfection of extrachromosomal expression vectors bearing a myogenic transcription factor gene.
  • ligand inducible transcription factor technology e.g., tet-on, tet-off, RheoSwitch
  • site-directed recombination technology e.g., Cre-LoxP, flp-FRT
  • transposon technology
  • the nucleic acid construct or vector is chromosomally integrated into the modified cell line.
  • Representative examples of self-renewing cell lines include those selected from a group consisting of embryonic stem cells, induced pluripotent stem cells, and immortal lineage-restricted cell lines. In certain aspects, such self-renewing cell lines are derived from species intended for dietary consumption or for research or for therapeutic purposes.
  • Representative examples of myogenic transcription factors include, used alone or in combination, MYOD1, MYOG, MYF5, MYF6, PAX3, PAX7, paralogs, orthologs, genetic variants thereof, or transcriptional activation agonists of the respective promoter recognition DNA sequences of the myogenic transcription factors disclosed herein.
  • FIG. 3 includes exemplary schematic illustrations of the double-switch mechanism of the myogenic modified O2KM cell line regulated by DOX or E2.
  • SRM self-renewal medium
  • myogenic differentiation is activated by the inducible MyoD transgene in the presence of E2 in the myogenic induction medium (MIM), while such directed differentiation is inactive when E2 is absent.
  • Certain aspects provide differentiation/specification methods comprising additional reagents other than E2 in the MIM to prevent cell death and to modulate the epigenetic state of chromatin.
  • additional reagents other than E2 in the MIM for example, a Glyogen Synthase Kinase-3 ⁇ (GSK3 ⁇ ) inhibitor can be added to the SRM and MIM to activate the canonical WNT signaling pathway, in turn, enhance myogenic differentiation and reduce cell death at the time of DOX withdrawal.
  • GSK3 ⁇ Glyogen Synthase Kinase-3 ⁇
  • the epigenetic modular alters the chromatin activation by myogenic transcription factors and/or enhances expression of myogenic transcription factors, such as MYF5.
  • GSK3 ⁇ inhibition includes targeting with small-molecules.
  • Gene editing is also a promising approach to enhance skeletal muscle specification by WNT signaling activation.
  • GSK3 ⁇ may be inhibited in the host/parental cell line by mutating the GSK3 ⁇ alleles either by sequence-specific insertion or deletion technology (i.e. Zinc-Finger Nuclease, TALEN, CRISPR). Mutating the endogenous promotor region can be used to repress expression of GSK3 ⁇ .
  • the GSK3 ⁇ open reading frame may be deleted or mutated using the same methods to abolish GSK3 ⁇ activity.
  • downstream phosphorylation target of GSK3 ⁇ beta-catenin (CTNNB1) may be mutated at the codons coding for residues phosphorylated by GSK3 ⁇ , thereby preventing phosphorylation of CTNNB1 by GSK3 ⁇ , resulting in a constitutively active, stable CTNNB1.
  • CTNNB1 beta-catenin
  • Such “gene-editing” methods would reduce the cost of GSK3 ⁇ inhibition by small-molecule targeting and potentially improve the safety profile of the meat product, as additional chemicals would not be required to inhibit GSK3 ⁇ during the process.
  • a third approach to Wnt-signaling inhibition includes the use of anti-sense nucleic acid inhibitors to GSK30 or other factors antagonistic to the WNT pathway. These may include RNA interference methods using sequence-targeting shRNA or miRNA.
  • GSK3 ⁇ inhibitor to promote cell survival, for example at the time of DOX withdrawal.
  • a GSK30 inhibitor is CHIR99021.
  • Additional representative GSK30 inhibitors may include, without limitation: lithium chloride, BIO, SB216763, CHIR-98014, TWS119, Tideglusib, IM-12, 1-Azakenpullone, AR-A014418, and SB415286. Without being bound by theory, it is believed that concomitant with DOX, both self-renewal of undifferentiated cells is maintained by a WNT signaling pathway involving the inhibition of GSK3 ⁇ .
  • the GSK3 ⁇ inhibitor enhanced expression of the myogenic transcription factor MYF5 in differentiating O2K.
  • a GSK3 ⁇ inhibitor such as CHIR99021, enhanced the terminal differentiation of the myogenic murine C2C12 cell line into multi-nucleated myotubes, as shown in Table 1.
  • Table 1 lists the influences of extracellular matrix effectors (i.e. Gelatin, Poly-D-Lysine, Laminin, MATRIGEL) and soluble factors (E2, CHIR99021) on the terminal differentiation of the murine C2C12 myoblast by assessment of the size and extent of myotube formation proceeding a 5-day differentiation time course. (Cultures were scored from [*****], indicating robust myotube formation to [-] indicating non-detectable myotube formation.)
  • a GSK3 ⁇ inhibitor when retained in the culture medium during differentiation (in the absence of DOX), is deemed compatible with the inducible myogenic transcription factor directed lineage specification and subsequent terminal differentiation conditions by derivative myocytes.
  • CHIR99021 was retained in the O2KM cultures following DOX withdrawal during subsequent E2-induced lineage-specification and terminal differentiation processes.
  • MYF5 expression was enhanced and cells exhibited a refractive myofibril morphology, whereas myocytes derived in the absence of 5AC expressed reduced MYF5 and exhibited an flattened morphology atypical to mature myofibrils. This distinction may explained by the enhanced expression of the myogenic transcription factor MYF5 observed only in the 5AC-exposed O2KM prior to and following 48 hours of DOX withdrawal
  • non-phosphorylated myogenin isoform known to be the active transactivator
  • the morphological distinction among these cultures may be explained by MYF5 expression enhanced by 5AC exposure.
  • epigenetic modulation entails alteration of chromatin structure influencing transcription factor binding and targeted transcriptional activation by altering the DNA methylation patterns and post-translational modification of nucleosome-associated histones.
  • epigenetic modulation may entail small-molecule agonists or antagonists targeting epigenetic pathways or expressed proteins comprising epigenetic machinery.
  • a small-molecule epigenetic modulator is 5-Aza-Cytidine (5AC).
  • small molecule epigenetic modulators include 5-Aza-2′-deoxycytidine, RG108, Scriptaid, sodium butyrate, trichostatin A, Suberoylanilide Hydroxamic Acid, MS-275, CI-994, BML-210, M344, MGCD0103, PXD101, LBH-589, Tubastatin A, NSC3825, NCH-51, NSC-3852, HNHA, BML-281, CBHA, Salermide, Pimelic Diphenylamide, ITF-2357, PCI-24781, APHA Compound 8, Droxinostat, and SB-939.
  • proteins involved in epigenetic modulation include histone deacetylase paralogs, histone acetyltransferase paralogs, tet-methycytosine dioxygenase paralogs, histone demethylase paralogs, histone methyltransferase paralogs, and DNA methyltransferase paralogs, histones, and subunits of chromatin remodeling complexes including Mi-2/NuRD (and its components such as methyl-CpG-binding domain protein 3 (MBD2)) and SWI/SNF (and its components such as BAF60 and BAF60C).
  • Mi-2/NuRD and its components such as methyl-CpG-binding domain protein 3 (MBD2)
  • SWI/SNF and its components such as BAF60 and BAF60C
  • respective activities of protein epigenetic modulators may be influenced by representative modalities such as targeting by small-molecule factors, over-expression of a respective exogenous transcript, anti-sense RNA-targeted respective transcript degradation, RNAi, and targeted mutation at the genetic locus.
  • the O2KM cell line was derived from the parental O2K cell line by lentiviral insertion of a blastidicin-selectable transgene cassette containing the MyoDER open reading frame sequence (ORF).
  • ORF MyoDER open reading frame sequence
  • O2K cells/cell line and piPSC cells are used interchangeably.
  • O2K was transduced with pseudovirus concentrated from the 293FT supernatant.
  • Transduced O2K were cultured in phenol-red free culture medium and selected four days with 10 ⁇ g/mL blasticidin followed by two additional days with 15 ⁇ g/mL blasticidin. Selected cells were designated as O2KM. Expression of MyoDER in the O2KM stock was verified by Western blot, FIG. 4B
  • O2KM stem cell renewal milieu was conducted as for the parental O2K line, with the following exception: phenol-red free formulations of DMEM/F-12 and neurobasal medium were substituted for the phenol-red containing formulations to avert pleotropic agonistic effects on MyoDER (i.e. activation).
  • the O2KM cell stock self-renewal medium consisted of the following components: phenol-red-free neurobasal medium (Life Technologies #12348-017), phenol-red free DMEM-F12 (Life Technologies #11039-021), 1 ⁇ non-essential amino acids (Sigma-Aldrich #M7145), 0.5 ⁇ Glutamax (Life Technologies #35050061), 0.000007% ⁇ -Mercaptoethanol, 0.5 ⁇ N2 Supplement (Life Technologies #17502048), 0.5 ⁇ B27 Supplement Minus Vitamin A (Life Technologies #12587010), 0.1 mg/mL Bovine Serum Albumin, 2 ⁇ g/mL doxacycline hyclate (i.e.
  • DOX 10 ng/mL human leukemia inhibitory factor (hLIF, Millipore #LIF1050), 3 ⁇ M CHIR99021, 0.8 ⁇ M PD032591 and 0.1 ⁇ M PD173074.
  • the three inhibitors CHIR99021, PD032591 and PD173074 are collectively regarded as ‘3i’.
  • N-2 and B-27 serum replacements were substituted using 15% KnockOut Serum Replecement (KOSR; Life Technologies #A15870).
  • O2KM maintained by enzymatic dissociation of colonies and passages of cells onto culture dishes coated with poly-D lysine and murine laminin in phenol-free SRM under 5% O 2 every 3d. In these self-renewal conditions, O2KM maintained compact, stem cell-like morphology as the parental O2K line, as shown in FIG. 5 .
  • Differentiation of the parental O2K line in the absence of SRM culture medium components that support self-renewal hLIF, DOX, CHIR99021, PD032591 and PD173074, and KOSR resulted in massive cell death as determined by (1) phase contrast microscopy as shown in FIG. 9 , panels i.-ii., (2) CPP32 cleavage, as shown in FIG. 17A , and (3) Annexin V labeling shown in FIG. 16 .
  • a culture medium formulation including CHIR99021 when retained in the SRM basal medium in the absence of hLIF, DOX, PD032591 and PD173074, supported both cell survival during differentiation as determined by (1) phase-contrast microscopy, as shown in FIG.
  • FIG. 9 panels iii.-viii., (2) cell adhesion assay, as shown in FIG. 10 , (3) CPP32 cleavage inhibition as shown in FIG. 17B , and (4) Annexin V labeling, as shown in FIG. 11 .
  • CHIR99021 exposure during primordial differentiation stabilizes and modulates the phosphorylation status of the GSK3 ⁇ substrate, CTNNB1, as shown in FIGS. 12, 13A and 13B , the phospho-regulated downstream effector of the canonical WNT signaling pathway known to direct mesodermal differentiation during embryonic lineage specification, myogenic enrichment of mesodermal progenitors, and terminal differentiation of skeletal myocytes.
  • CHIR99021 supplemented basal medium supported pre-myogenic paraxial mesoderm lineage specification of differentiating O2K, as shown in FIG. 14 and when included in low-mitogen differentiation cultures (2% horse serum/DMEM) of the myogenic murine C2C12 cell line, enhanced terminal differentiation into skeletal myotubes, listed in Table. 1.
  • CHIR99021 repressed cell death supported differentiation toward paraxial mesoderm by the differentiating O2K cell line and enhanced terminal differentiation by the C2C12 cell line, precedent was established to retain the compound in all culture stages.
  • 3 ⁇ M CHIR99021 was retained in the culture medium during expansion, induction and terminal differentiation steps ( FIG. 20A ) unless stated otherwise.
  • O2KM cells were seeded onto culture dishes coated with poly-D lysine, murine laminin and MATRIGEL at a density of 4.1 ⁇ 10 3 cells/cm 2 and cultured under 5% O 2 in self-renewal medium for 3d.
  • cultures were transferred to a 20% O 2 basal differentiation milieu, designated by withdrawal of PD032591, PD173074, DOX, hLIF and ⁇ -mercaptoethanol.
  • 10 ⁇ M E2 was added to the medium.
  • E2-directed myogenic lineage specification following 2d induction culture was confirmed by (1) adoption of spindle-like morphology characteristic of skeletal myocytes in treated cultures, as shown in FIG. 6 , (2) expression of endogenous the MYOG skeletal muscle transcription factor, as shown in FIG. 7 and (3) uniform expression of the skeletal myocyte cell surface marker, NCAM, as shown in FIG. 8 .
  • the gene expression program in the O2KM-derived myocytes was not sufficient to enable terminal differentiation as per the established conditions.
  • 5AC a small-molecule epigenetic modulator
  • 5AC exposure was further determined to enhance terminal differentiation by the C2C12 cell line.
  • 250 nM 5AC the highest dose tolerated by undifferentiated O2KM, was included during in the proliferative O2KM expansion and induction regimens, as shown in FIG. 20A .
  • TDM terminal differentiation medium
  • FIG. 20A Cultures exposed to 5AC during expansion and induction regimens formed refractive, anisotropic myotubes during the terminal differentiation regimen, shown in FIG. 20B .
  • FIG. 20B Cultures exposed to 5AC during expansion and induction regimens formed refractive, anisotropic myotubes during the terminal differentiation regimen, shown in FIG. 20B .
  • FIG. 20A Cultures exposed to 5AC during expansion and induction regimens formed refractive, anisotropic myotubes during the terminal differentiation regimen, shown in FIG. 20B .
  • FIG. 20C shows uniform expression of myosin heavy chain by d6, and FIG. 20E shows increasing expression of desmin (DES) and myogenin (MYOG) over the 8d course.
  • DES desmin
  • MYOG myogenin
  • Myotube polyploidy was observed during terminal differentiation, as shown in FIG. 20D , left panel.
  • the relative distribution of myonuclei in d8 myotubes according to ploidy is shown in FIG. 20D , right panel.
  • Relative prevalence of S-phase nuclei in the renewal milieu (d3 colonies), expansion milieu (d0 cultures, FIG. 20A ), and terminal differentiation milieu (d8 cultures, FIG. 20A ), as shown in FIG. 20F indicated cell cycle withdrawal following terminal differentiation.
  • Contractile potential of terminally differentiating skeletal muscle myotubes was validated by (1) structural development of well-organized sarcomeres, as shown in FIG. 20G ; (2) asynchronous spontaneous contraction, as shown in FIG. 21A ; (3) contractile stimulation and synchronization by field stimulation, as shown in FIG. 21B ; (4) caffeine-stimulated contraction, as shown in FIG. 21C ; and (5) acetylcholine-stimulated contraction, as shown in FIG. 21D .
  • FIG. 9 shows phase-contrast images of ground-state piPSC colonies cultured under 5% O 2 in the self-renewal milieu (i.) and following 48 h under 20% O 2 in the absence of DOX, LIF and 3i, (ii.)
  • FIG. 16 shows Annexin V labeling of apoptotic cells prior to, and following 24 h transition of cultures.
  • FIG. 17A shows Western blot detection of full-length CPP32 ( ⁇ 32 kD procaspase 3a) and the large cleaved fragment ( ⁇ 17 kD cleaved-caspase 3a) in ground-state colonies prior to (0 h) and following (12-48 h) differentiation milieu transition (12-48 h).
  • FIG. 9 shows phase contrast images of adherent cultures (iii, iv, vi, and vi) and non-adherent embryoid body cultures (vii and viii) following 48 h culture in differentiation milieu supplemented with CHIR99021 as indicated.
  • FIG. 11 shows Annexin V labeling of apoptotic cells following 24 h transition of cultures to differentiation milieu supplemented with CHIR99021 as indicated.
  • FIG. 17B shows Western blot detection of full-length CPP32 and the cleaved fragment in colonies following 42 h transition to the differentiation milieu in the presence of CHIR99021 levels indicated.
  • TUBA is detected an internal protein loading control.
  • CHIR99021 Stabilizes CTNNB1 and Supports Differentiation from the Ground State.
  • FIG. 12 shows Western blot detection of CTNNB1 and p-CTNNB1 (total and phospho-S33,37,T41 ⁇ -catenin, respectively) following 24 h differentiation milieu transition in the presence of CHIR99021, as indicated.
  • TUBA detected as an internal protein loading control.
  • FIG. 13A indicates ratios of p-CTNNB1/CTNNB1 bands.
  • FIG. 13B indicates ratios of CTNNB1/TUBA bands.
  • FIG. 18 shows outgrowth morphology of embryoid bodies formed in a differentiation milieu containing 6 ⁇ M CHIR99021 for two days and following transfer to a Poly-D Lysine+Laminin+MATRIGEL coated substrate for one (d3, left panel) or three (d5, right panel) additional days.
  • FIG. 14 shows Western blot analysis of PAX3, POU5F1 and KLF4 expression in ground-state milieu (d0) and differentiation milieu cultures (d1-d5) according to one regimen aspect described elsewhere herein.
  • TUBA detection is shown as an internal protein loading control.
  • FIG. 4A shows a Blasticidin (BLAST)-selectable MyoDER expression cassette. Arrows and boxes indicate promoter and gene sequences, respectively.
  • FIG. 4B shows Western blot detection of MyoDER in the unmodified (O2K) and MyoDER expression cassette-modified (O2KM) piPSC line. MyoDER was detected with an antibody raised against a MyoD peptide.
  • FIG. 7 shows Western blot detection of MyoDER ( ⁇ 75 kD), MYF5 and MYOG following 2d piPSC induction. Endogenous MYOD1 ( ⁇ 45 kD, expected) was not detected.
  • FIGS. 19A and 19B Determinants of MYF5 activation: Western blot analyses.
  • FIG. 19B shows MYF5 activation in the presence of DOX, LIF and 3i.
  • MYODER-modified piPSC were cultured 3 days in self-renewal or expansion milieu.
  • 5AC exposure supported detectable MYF5 expression levels, as shown in FIG. 19B , after 3 days of expansion culture.
  • MYF5 was not detected following 3 days of renewal culture ( ⁇ 5AC), as shown in FIG. 19B .
  • TUBA is detected as an internal protein loading control.
  • FIG. 6 shows the morphology of selected piPSC cultures following lineage specification induction culture regimens ( FIG. 20A ) described elsewhere herein.
  • FIG. 8 shows immunocytoflourescent detection of nuclei (DAPI) and NCAM in piPSC-MyoDER cultures prior to (d0, left panel) and following (d2, right panel) 10 ⁇ M E2 exposure, in the presence of 5AC.
  • FIG. 20A Prior to terminal differentiation, cultures were expanded for 3 days in the presence of 5AC, induced for 2 days in the presence of E2 & 5AC, and terminally differentiated in the absence of 5AC & E2 as shown in FIG. 20A .
  • FIG. 20B shows myotube morphology and conformation.
  • Post-induction (d2), piPSC developed as elongated, anisotropic, refractive myotubes. Where 5AC was not included in the expansion and induction steps, cells exhibited a flattened, non-refractive morphology.
  • FIG. 20C and FIG. 20E show terminal myogenesis protein expression.
  • FIG. 20D shows myotube multinucleation.
  • Left panel enlarged image of d4 terminal differentiation cultures. Bracketed arrows indicate multiple nuclei within a single myotube.
  • FIG. 20E shows Western blot analyses. MyoD (MYOD1), myogenin (MYOG) and desmin (DES) expression, d0-d8.
  • TUBA is detected as an internal protein loading control.
  • FIG. 20F shows cell-cycle withdrawal coinciding with terminal differentiation.
  • FIG. 20G Transmission Electron Microscopy, d6 myotubes. Sarcomeric structural units were aligned in single (left panels) and staggered, parallel rows (right panel).
  • FIG. 21A shows representative asynchronous, single-cell transient cycles (left, middle and right panels) were observed in spontaneously contracting d6 myotube subpopulations.
  • FIG. 21B shows FOV activation and synchronization of calcium transient cycles by 1.0 Hz field stimulation in d6 myotubes.
  • FIG. 21C shows FOV calcium transient activation of d6 myotubes by 10 mM caffeine.
  • FIG. 21D single-cell analysis of representative calcium transient activation in a d7 myotubes by 100 nM acetylcholine.

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