Development and validation of an in vitro porcine skeletal muscle model
Dan-Jumbo, Susan Omuboba
The projected rise in world population to 9.7 billion by 2050 will require an increased production of meat globally to meet human requirements for protein without compromising environmental and financial sustainability. Efficient increase in meat production will require an in-depth knowledge of muscle growth and development. The ability of sows to produce large numbers of piglets per litter coupled with a farrowing rate of an average of two litters per year makes the pig a highly efficient source of meat. In addition, the anatomical and physiological similarities between pig and humans makes the pig an excellent experimental model to study human physiology and disease. The availability of robust in vitro models of skeletal muscle from livestock species is critical for understanding muscle development and disease, and to make progress towards improving animal meat production. A current technical limitation in this regard is that stem/precursor cells routinely obtained from muscle are a mixed population with low proliferative potential and low differentiation efficiency in vitro thus limiting their use in large-scale studies. This thesis aimed to isolate, purify, and characterise myogenic cells from porcine skeletal muscle. It also aimed to develop and characterise an explant-based culture model for the isolation and maintenance in vitro of muscle stem cells from pigs. Finally, with the aim to provide proof-of-concept of the usefulness of the novel in vitro model, studies were carried out to investigate the effects of β-Klotho (KLB) and its ligand, fibroblast growth factor 21 (FGF21) on adipogenic differentiation of pig muscle-derived progenitor cells using both a siRNA mediated knockdown and CRISPR/Cas9-mediated knockout of KLB. Using fluorescent activated cell sorting (FACS), I isolated porcine myogenic cells based on the expression of CD146 and obtained CD45-/CD31-/CD146+ and CD45-/CD31-/CD146- as myogenic and non-myogenic cell fractions, respectively. Analysis by RT-qPCR revealed that CD45-/CD31-/CD146+ fraction was indeed enriched for myogenic cells showing high expression of muscle stem cell markers, PAX7 and CD56, and low expression of preadipocyte marker, PDGFRα which was highly expressed in the CD45-/CD31-/CD146- fraction. In addition, when placed in myogenic differentiation media the CD45-/CD31-/CD146+ cells were able to fuse to form mulinucleaded myotubes with increased expression of muscle markers MYH3 and MYOG. Interestingly, no myotubes were observed in the CD45-/CD31-/CD146- fraction. On the contrary, both CD45-/CD31-/CD146+ and CD45-/CD31-/CD146- fractions were able to accumulate lipids and differentiate into adipocytes when placed in adipiogenic media. However, CD45-/CD31-/CD146- displayed a higher adipogenic capacity with significantly increased levels of expression of fatty acid binding protein, FABP4 when compared to CD45-/CD31-/CD146+ fraction. To establish an explant-based in vitro model, muscle tissue fragments were seeded on matrigel coated cell culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Expression of lineage markers in MDPCs was determined with the use of RT-qPCR and flow cytometry. In addition, their ability to differentiate into myogenic and adipogenic lineages during long term culture was also tested followed by RT-qPCR analysis of relevant lineage markers. The results showed that MDPCs displayed long-term expansion in vitro, showing an average doubling time of 48 hours. The MDPCs also expressed key muscle stem cell markers, PAX 7, MYOD, MYF5 and CD56 in the early passages while the adipogenic cell markers CD105 and PDGFRα were highly expressed at the later passages. In addition, the MDPCS were able to efficiently form myotubes over several passages. Eventually, these cells lost their myogenic potential and acquired adipogenic potential following prolonged culturing. Finally, to demonstrate the usefulness of this in vitro model for functional molecular studies in porcine muscle, I investigated the role of fibroblast growth factor 21-βKlotho (FGF21-KLB) signalling in regulating adipogenic differentiation of porcine MDPCs using both gain of function and loss of function approaches. RT-qPCR and Immunocytochemistry analysis revealed that KLB expression was upregulated in differentiating porcine MDPCs synchronous to the formation of adipocytes and upregulation of the adipocyte markers, PPARy and FABP4. Stimulation of porcine MDPCs with FGF21 increased adipogenic differentiation while siRNA mediated knockdown and CRISPR/Cas9 mediated knockout of KLB inhibited adipogenesis by porcine MDPCs indicating that FGF21-KLB signalling is a regulator of adipogenesis in pig muscle. In conclusion, these results show that CD146 is a suitable marker for isolation of myogenic cells from pig skeletal muscle using FACS. Moreover, I describe a simple and efficient method to purify muscle stem cells from pig skeletal muscle tissue and characterise their growth and differentiation potentials. This, together with novel know-how on gene targeting using CRISPRs provides valuable information towards future research applications to understand pig skeletal muscle development and improve meat production. Moreover, the novel in vitro model I developed could have applications in the expanding field of cultured meat. Lastly, the methods developed in this thesis may be transferable to other species provided culture conditions are appropriately optimised.