Method for scalable skeletal muscle lineage specification and cultivation

Abstract

The present disclosure relates to methods for enhancing cultured meat production, such as livestock-autonomous meat production. In certain aspects, the 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.

1 . A method for producing cultured muscle tissue, the method comprising:
 modifying a self-renewing cell line of an animal species with a myogenic transcription factor to produce a myogenic-transcription-factor-modified cell line, and inducing said modified cell line by exogenous regulation to maintain the cell line in self-renewal process or advance the cell line to myogenic differentiation process.   
     
     
         2 . The method of  claim 1 , wherein the muscle tissue is of a livestock or poultry species. 
     
     
         3 . The method of  claim 2 , wherein the muscle tissue is of a livestock species. 
     
     
         4 . The method of  claim 3 , wherein the livestock species is porcine or bovine. 
     
     
         5 . The method of  claim 1 , wherein the produced muscle tissue is fit for human and non-human dietary consumption. 
     
     
         6 . The method of  claim 1 , wherein said self-renewing cell line is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, extraembryonic cell lines, and somatic cell lines. 
     
     
         7 . The method of  claim 6 , wherein the self-renewing stem cell line is from a livestock or poultry species. 
     
     
         8 . The method of  claim 7 , wherein the self-renewing stem cell line is from a livestock species. 
     
     
         9 . The method of  claim 8 , wherein the livestock species is porcine or bovine. 
     
     
         10 . The method of  claim 6 , wherein the self-renewing stem cell line is from any animal species intended for human or non-human dietary consumption, is any companion animal, and/or is from any animal species used for research or development of therapeutics. 
     
     
         11 . The method of  claim 1 , wherein said myogenic transcription factor is selected from the group consisting of MYOD1, MYOG, MYF5, MYF6, PAX3, PAX7, paralogs, orthologs, and genetic variants thereof. 
     
     
         12 . The method of  claim 1 , wherein said myogenic transcription factor is MYOD1. 
     
     
         13 . The method of  claim 1  further comprising: maintaining the modified cell line in a first culture medium for undifferentiated cell stock expansion. 
     
     
         14 . The method of  claim 13  further comprising: maintaining the modified cell line in a first culture medium comprising doxycycline for cell stock expansion. 
     
     
         15 . The method of  claim 1  further comprising: transferring the modified cell line into a second culture medium for lineage-specific differentiation. 
     
     
         16 . The method of  claim 15  further comprising: treating the modified cell line in a second culture medium comprising E2 for lineage-specific differentiation. 
     
     
         17 . The method of  claim 15 , wherein said second culture medium further comprises a reagent for activating the canonical WNT signaling pathway, a reagent for epigenetic modulation, or a combination thereof. 
     
     
         18 . The method of  claim 17 , wherein said second culture medium further comprises a reagent for activating the canonical WNT signaling pathway. 
     
     
         19 . The method of  claim 18 , wherein said reagent for activating the canonical WNT signaling pathway comprises a GSK3P inhibitor. 
     
     
         20 . The method of  claim 18 , wherein said GSK3P inhibitor is selected from one or more members of the group consisting of CHIR99021, lithium chloride, BIO, CHIR-99021, SB216763, CHIR-98014, TWS119, Tideglusib, IM-12, 1-Azakenpullone, AR-A014418, and SB415286. 
     
     
         21 . The method of  claim 20 , wherein said GSK3P inhibitor is CHIR99021. 
     
     
         22 . The method of  claim 17 , wherein said second culture medium further comprises a reagent for epigenetic modulation. 
     
     
         23 . The method of  claim 22 , wherein said epigenetic modulator is selected from one or more members of the group consisting of 5AC, 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, histone deacetylase paralogs, histone acetyltransferase paralogs, tet-methylcytosine dioxygenase paralogs, histone demethylase paralogs, histone methyltransferase paralogs, and DNA methyltransferase paralogs, histones, and subunits of chromatin remodeling complexes including Mi-2/NuRD and SWI/SNF. 
     
     
         24 . The method of  claim 22 , wherein the epigenetic modulator is an inhibitor of DNA methylation. 
     
     
         25 . The method of  claim 1 , wherein the method comprises forming multinucleated myotubes. 
     
     
         26 . The method of  claim 1 , wherein the muscle tissue comprises skeletal muscle fibers. 
     
     
         27 . The method of  claim 1 , wherein the canonical WNT signaling pathway is activated by gene-editing inhibition of GSK3P or its substrate CTNNB1. 
     
     
         28 . An in vitro-produced muscle tissue made by the method of  claim 1 .