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 - 28 . (canceled) 
     
     
         29 . An in vitro method for producing a cultured meat product for dietary consumption,
 the method comprising:
 providing a cell line demonstrating the capacity for skeletal muscle tissue specification; 
 modifying said cell line with an inducible myogenic transcription factor to produce a myogenic transcription-factor-modified cell line; 
 inducing myogenic differentiation of said modified cell line; and 
 culturing the differentiated modified cell line, thereby producing a cultured meat product for dietary consumption. 
   
     
     
         30 . The method of  claim 29 , wherein the cell line is from a livestock or poultry species. 
     
     
         31 . The method of  claim 30 , wherein the livestock species is porcine or bovine. 
     
     
         32 . The method of  claim 29 , wherein the myogenic transcription factor is MYOD1, MYOG, MYF5, MYF6, PAX3, PAX7, or a paralog, ortholog, or genetic variant thereof, 
     
     
         33 . The method of  claim 29 , wherein the myogenic transcription factor is a transcriptional activation agonist of the respective promoter recognition DNA sequences of a myogenic transcription factor. 
     
     
         34 . The method of  claim 29 , wherein the cell line is a self-renewing cell line. 
     
     
         35 . The method of  claim 34 , wherein the self-renewing cell line comprises embryonic stem cells, induced pluripotent stem cells, somatic cells, or extra-embryotic cells with myogenic potential. 
     
     
         36 . The method of  claim 34 , wherein the inducing step further comprise a self-renewal sub-step and a differentiation sub-step regulated by a double-switch mechanism. 
     
     
         37 . The method of  claim 36 , wherein in the self-renewal sub-step the undifferentiated state of the modified cell line is maintained. 
     
     
         38 . The method of  claim 36 , wherein in the differentiation sub-step, the modified cell line is treated, and the cell line is specified to skeletal myocytes. 
     
     
         39 . The method of  claim 38 , wherein the differentiation sub-step further comprises contacting the cell line with a reagent for activating the canonical WNT signaling pathway to prevent cell death and facilitate myogenic differentiation. 
     
     
         40 . The method of  claim 38 , wherein the differentiation sub-step further comprises contacting the cell line with an epigenetic modulator to alter the chromatin structure for enhanced myogenic gene expression. 
     
     
         41 . The method of  claim 29 , wherein said inducing is carried out by exogenous regulation to direct a differentiation processes in the cell line. 
     
     
         42 . The method of  claim 29 , wherein culturing of the differentiated modified cell line forms myocytes and multinucleated myotubes. 
     
     
         43 . The method of  claim 42 , wherein the cell line is cultured in a low-mitogen culture medium. 
     
     
         44 . The method of  claim 42 , wherein the myocytes and multinucleated myotubes both comprise myonuclei, and wherein greater than 50% of the total myonuclei are within the multinucleated myotubes. 
     
     
         45 . The method of  claim 42 , further comprising culturing the myocytes and multinucleated myotubes to generate skeletal muscle fibers.