Methods for generating skeletal muscle progenitor cells
Inventors
Pyle, April D. • Hicks, Michael R.
Assignees
University of California San Diego UCSD
Publication Number
US-12365875-B2
Publication Date
2025-07-22
Expiration Date
2037-12-15
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Abstract
The present disclosure provides methods of generating skeletal muscle progenitor cells (SMPCs). The present disclosure provides methods of generating immature SMPCs and multinucleated muscle cells, and matured SMPCs and multinucleated muscle cells. The present disclosure provides engraftment methods and treatment methods, involving generating SMPCs and introducing the SMPCs into an individual.
Core Innovation
The present disclosure provides methods of generating skeletal muscle progenitor cells (SMPCs) from pluripotent stem cells (PSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The disclosed methods comprise culturing PSCs in chemically defined liquid culture media through sequential steps involving specific inhibitors and growth factors to yield a population of cells expressing the paired box protein-7 (PAX7), a key SMPC marker. The culture periods range from about 20 to 70 days, with particular sequences and media compositions detailed for different culture intervals.
The methods further include enriching the generated populations for cells positive for nerve growth factor receptor (NGFR) and Erb-B2 receptor tyrosine kinase 3 (ERBB3), which are identified as markers that enrich for more myogenic, PAX7-expressing SMPCs. This enrichment elevates the proportion of PAX7+ cells in the population to at least 50%, and up to about 95%. Negative selection against markers such as HNK1 further enhances the purity of SMPCs. The disclosure also describes expansion protocols for these enriched SMPC populations using culture media containing cell cycle regulators, inhibitors of DNA damage or stress, STAT3 activators, ERBB3 and NGFR pathway activators, and migration factors to achieve substantial cell population increases while maintaining myogenic potential.
Additionally, methods are provided to generate multinucleated muscle cells that express myosin heavy chain proteins by culturing SMPCs with inhibitors of TGF-β receptor activity, which promotes differentiation and fusion of myotubes. The methods also encompass engraftment procedures whereby generated SMPCs, optionally enriched and matured via TGF-β inhibition, are introduced into a subject for potential therapeutic benefit, including treatment of musculoskeletal diseases or disorders.
Claims Coverage
The patent includes one independent claim that covers a method for generating skeletal muscle progenitor cells with specific culture steps and subsequent enrichment procedures.
Method for generating skeletal muscle progenitor cells (SMPCs) from pluripotent stem cells
A stepwise culture protocol involving: i) culturing PSCs with a Rho-associated kinase inhibitor from day 1 to day 2 only; ii) culturing with a glycogen synthase kinase-3 (GSK3) inhibitor from day 2 to day 3 only; iii) culturing in chemically defined medium from day 3 to day 12 only; iv) culturing with fibroblast growth factor from day 12 to day 20 only; v) culturing in chemically defined medium from day 20 to day 35 only; and vi) culturing with insulin-like growth factor-1 from day 35 to day 50 only, thereby generating a cell population with at least 50% PAX7+ SMPCs which also express NGFR, ERBB3, or both.
Enrichment of SMPCs based on surface markers
Sorting the generated cell population to select for nerve growth factor receptor (NGFR)-positive and Erb-B2 receptor tyrosine kinase 3 (ERBB3)-positive cells to increase the proportion of PAX7+ SMPCs.
Chemically defined culture conditions excluding BMP inhibitor and including specific supplements
Providing that the liquid culture medium used in the step of culturing from day 3 to day 12 does not include a bone morphogenetic protein (BMP) inhibitor and optionally includes transferrin, a transferrin substitute, L-glutamine, an L-glutamine substitute, and monothioglycerol.
Expansion of SMPC populations with specified growth factors and regulators
Expanding the enriched SMPC population in liquid culture media comprising one or more of a cell cycle or developmental regulator, inhibitor of DNA damage or cell stress, STAT3 pathway activator, ERBB3 pathway activator, NGFR signaling activator, and a migration factor to achieve at least a 5-fold increase in SMPCs.
Generation of multinucleated muscle cells expressing myosin heavy chain using TGF-β receptor inhibitors
Culturing the expanded SMPC population in liquid culture medium comprising an inhibitor of TGFβ receptor activity to induce formation of multinucleated muscle cells expressing myosin heavy chain.
Use of mutations in pluripotent stem cells
Including pluripotent stem cells comprising mutations associated with deleterious muscle phenotypes, such as dystrophin gene mutations, in the generation of SMPCs by the method.
Introduction of generated SMPCs into subjects
Introducing the SMPC populations into subjects, potentially with co-administration of TGF-β inhibitors, survival factors, vascular permeabilization factors, extracellular matrix components, or hydrogels for treatment of muscle diseases or disorders.
The independent claim delineates a multi-step culture method to generate PAX7-expressing SMPCs enriched for NGFR and ERBB3 markers from PSCs, with optional selection steps and culture compositions. Additional features include expansion protocols with specific growth factors and generation of multinucleated muscle cells by TGF-β receptor inhibition. The claim also encompasses using mutated PSCs and methods for therapeutic introduction into subjects.
Stated Advantages
Generation of SMPCs with increased proportion of PAX7+ cells enriched for NGFR and ERBB3 markers enhances myogenic potential.
Expansion methods allow substantial increase in SMPC numbers while maintaining stemness and differentiation capacity.
TGF-β inhibition promotes maturation of SMPC-derived myotubes, improving fusion and expression of adult myosin heavy chain isoforms.
Enrichment combined with maturation via TGF-β inhibition improves in vivo engraftment efficiency of SMPCs, restoring dystrophin protein near levels seen with uncultured fetal muscle.
Documented Applications
Generating skeletal muscle progenitor cells from pluripotent stem cells for research or therapeutic cell engraftment.
Treating musculoskeletal diseases or disorders in subjects by introducing SMPCs to restore or improve muscle function.
Using enriched and matured SMPCs for engraftment into skeletal muscle tissue in animals or humans.
Potential correction of genetic muscle disorders by using PSCs carrying or corrected for mutations related to the muscle phenotype.
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