Fibroblast growth factors, A potential game plan for regeneration of skeletal muscle

Gudagudi, Kirankumar (2019-12)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Introduction: Adult mammalian tissue regeneration recruits progenitor stem cells. In skeletal muscle, these are primary satellite cells. Primary satellite cells can be harvested from muscle tissue to investigate or even use as potential therapeutic application. Satellite cells exist in quiescence in the muscle tissue and only become activated following an insult. Most studies investigating satellite cells in vitro use already activated satellite cells, called myoblasts. Fibroblast Growth Factors (FGFs) are fundamental in embryonic development but also in adult skeletal muscle regeneration from injury or pathology. Understanding the role of specific members of this growth factor family could assist in improving the understanding of their influence on the regeneration sequence in skeletal muscle. Methods: Isolated satellite cells from human muscle biopsies were expanded in vitro creating primary human myoblast (PHM) clones. In order to distinguish the rate of proliferation between different PHM clones, a comparative index (CI) was established using the cell cycle and total RNA data of the two PHM clones. Two distinct index calculation models were also presented to determine if these may distinguish between the two clones with greater sensitivity. Secondly, the quiescent state is an integral part of stem cell regulation, therefore choosing the right protocol for inducing quiescence is important. In this study, two developed protocols were assessed, and a new blended protocol addressing the limitations of both protocols was established. This method involved the use of suspension culture (SuCu) with knock out serum replacement (KOSR). Finally, FGF6 and FGF2, both individually and sequentially, were used to treat quiescent myoblasts to determine their involvement in activation and proliferation with the use of cell cycle analysis and mRNA assessment of ki67, p21, myf5, and MyoD. Results and conclusion: The development of the CI was successful in determining the difference in proliferation rate for the different clones. Suspension culture with KOSR, the blended protocol method, resulted in reduced ki67 expression and improved quiescence compared to both the SuCu or KOSR alone. Unlike FGF2, individual treatment with FGF6 was adequate to activate the quiescent PHMs and aid their re-entry into cell cycle with consistency in all three PHM clones by upregulating ki67 expression. However, FGF2 did impede the cell cycle inhibition factor p21, indirectly influencing proliferation. Sequential treatment of FGF6 and FGF2 allowed to determine whether the sequence of treatment would be important. The potential for significantly improving proliferation was found for the sequence: FGF6 followed by FGF2. The inverse sequential treatment order did not demonstrate any significant effect on both activation and proliferation of the quiescent cells. In conclusion, using clones that were distinctly different as assessed by the comparative index, this thesis illuminates that the two FGF family members investigated, act on cell cycle in different ways, thus would influence their utilization in experimental or therapeutic applications.

AFRIKAANSE OPSOMMING: Inleiding: Regenerasie van volwasse weefsel behels die werwing van voorloper stamselle. In skeletspier is hierdie primêre satellietselle. Dit kan vanuit spierweefsel versamel word vir ondersoeke of selfs as potensiële terapeutiese aanwending. Satellietselle bestaan in ʼn rustoestand in die spierweefsel en word slegs na ʼn aanslag geaktiveer. Meeste studies wat satellietselle in vitro ondersoek, maak van reeds- geaktiveerde satellietselle gebruik, naamlik mioblaste. Fibroblastgroeifaktore (FGFe) is noodsaaklik vir embrioniese ontwikkeling, maar ook vir skeletspierregenerasie na besering of patologie. Om die rol van spesifieke lede van hierdie groei faktor familie te verstaan, kan tot verbeterde begrip van hul invloed op die regenerasie proses in skeletspier lei. Metodes: Geïsoleerde satellietselle vanaf menslike spierbiopsies is in vitro gegroei om primêre menslike mioblast (PMM) klone te genereer. Om die tempo van proliferasie tussen verskillende PMM klone te onderskei, is ʼn vergelykende indeks (VI) opgestel met die selsiklus- en totale RNS data van twee PMM klone. Twee afsonderlike indeks berekeningsmodelle is ook voorgestel om te ondersoek watter van hierdie twee modelle met groter sensitiwiteit tussen die twee klone kan onderskei. Tweedens is die metode waarmee die rustoestand geïnduseer word ʼn integrale deel van die rustoestand/aktivering, dus is die keuse van protokol om die rustoestand te induseer, belangrik. In hierdie studie was twee ontwikkelde protokolle ondersoek, asook ʼn nuwe verbonde protokol wat die tekortkominge van die ander twee protokolle bespreek, was gevestig. In hierdie metode is ʼn suspensie kultuur met uitslaan serum vervanging (USV) gebruik. Laastens is FGF6 en FGF2 beide individueel en opeenvolgend, gebruik om rustende selle te behandel om hul betrokkenheid in aktivering en proliferasie te ondersoek met behulp van selsiklus analise en assessering van mRNS-vlakke van ki67, p21, myf5 en MyoD. Resultate en gevolgtrekking: Die ontwikkeling van die VI om die verskille in proliferasie tempo tussen die verskillende klone vas te stel, was suksesvol. Suspensie kultuur met USV, die gemengde protokol metode, het gelei tot verlaagde ki67 uitdrukking en verbeterde rustende toestand in vergelyking met beide die (SuCu) of USV alleen (p<0.05). Anders as FGF2 was individuele behandeling met FGF6 genoeg om rustende PMMe te aktiveer en om hul hertoetrede tot die selsiklus te ondersteun met konsekwentheid tussen al drie PMM klone deur opgereguleerde ki67 uitdrukking. FGF2 het egter die selsiklus inhibisie faktor p21 belemmer en so indirek proliferasie beïnvloed. Opeenvolgende behandeling met FGF6 en FGF2 het toegelaat dat daar bepaal kan word of die volgorde van behandeling belangrik is. Die potensiaal vir beduidende verbetering van proliferasie is gevind vir die behandelingsvolgorde: FGF6 gevolg deur FGF2. Die omgekeerde opeenvolgende behandelingsorde het nie enige beduidende effek op beide aktivering of proliferasie van rustende mioblaste getoon nie. In opsomming, deur die gebruik van klone wat uitdruklik verskillend was, soos gemeet deur die vergelykende indeks, illumineer hierdie tesis dat die twee FGF familie lede wat ondersoek was, op verskillende maniere die selsiklus beïnvloed. Dit sal hul gebruik in eksperimentele of terapeutiese aanwending beïnvloed.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/106920
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