The role of adipose stem cells in myogenic differentiation in vitro
Jokinen, Vilma (2023)
Jokinen, Vilma
2023
Master's Programme in Biomedical Technology
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
Hyväksymispäivämäärä
2023-05-22
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202305055312
https://urn.fi/URN:NBN:fi:tuni-202305055312
Tiivistelmä
Skeletal muscle is a hierarchically arranged and interactive tissue that contributes to important physiological processes and is responsible for movement and posture of the body. Its normal function and structure, as well as the regenerative capacity is impaired in various conditions and disorders and in vitro models of skeletal muscle are needed for studying these conditions and possible treatment for them. Typically, skeletal muscle modelling in vitro is conducted with rodent-origin cells that do not truly recapitulate human physiology or pathophysiology. As the use of human primary muscle cells in vitro is limited in some respects, other alternatives, such as mesenchymal stem cells, should be investigated as an alternative human cell source for skeletal muscle modeling. Additionally, the structure and function of skeletal muscle cannot be mimicked in two-dimension (2D) and thus three-dimensional (3D) models are needed. In this thesis, the aim was to contribute to the development of human 3D skeletal muscle model by studying the potential and role of human adipose stem cells (hASCs) and engineered myobundles in myogenic differentiation and skeletal muscle modeling.
In this study, the myogenic potential of hASCs was investigated with different variables in growth and differentiation medium. These included low and high glucose basal medium, low and high serum differentiation medium and growth factors, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2). hASCs were grown in co-culture with mouse myoblasts to promote myogenic differentiation of hASCs. The effects of those factors on proliferation, myotube formation and differentiation were assessed with CyQuant proliferation assay, immunofluorescence stainings and gene expression analysis with quantitative polymerase chain reaction (q-PCR). In addition, 3D engineered fibrin hydrogel -based co-culture myobundle was constructed and its structural organization and differentiation ability was investigated with immunofluorescence staining and gene expression analysis.
High serum concentration seemed to affect proliferation of all cells positively but did not promote myotube formation. Addition of IGF-2 in differentiation medium increased the cell number in most cases and seemed to support myotube maturation. No clear effects on proliferation or myotube formation between low and high glucose concentration or with IGF-1 were observed. hASCs did not express any myogenic differentiation indicating genes in monoculture or co-culture with mouse myoblasts in 2D or in engineered myobundles. Myoblast culture in engineered myobundles were shown to express mature muscle cell indicating proteins.
In this study hASCs did not undergo myogenic differentiation, even in direct co-culture with myoblasts. However, hASCs seemed to support proliferation of myoblasts. As ASCs do not undergo myogenic differentiation, other potential roles of them in myogenic co-cultures need to be studied further. Engineered myobundles showed promise as a 3D model for skeletal muscle modeling but needs to be developed further.
In this study, the myogenic potential of hASCs was investigated with different variables in growth and differentiation medium. These included low and high glucose basal medium, low and high serum differentiation medium and growth factors, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2). hASCs were grown in co-culture with mouse myoblasts to promote myogenic differentiation of hASCs. The effects of those factors on proliferation, myotube formation and differentiation were assessed with CyQuant proliferation assay, immunofluorescence stainings and gene expression analysis with quantitative polymerase chain reaction (q-PCR). In addition, 3D engineered fibrin hydrogel -based co-culture myobundle was constructed and its structural organization and differentiation ability was investigated with immunofluorescence staining and gene expression analysis.
High serum concentration seemed to affect proliferation of all cells positively but did not promote myotube formation. Addition of IGF-2 in differentiation medium increased the cell number in most cases and seemed to support myotube maturation. No clear effects on proliferation or myotube formation between low and high glucose concentration or with IGF-1 were observed. hASCs did not express any myogenic differentiation indicating genes in monoculture or co-culture with mouse myoblasts in 2D or in engineered myobundles. Myoblast culture in engineered myobundles were shown to express mature muscle cell indicating proteins.
In this study hASCs did not undergo myogenic differentiation, even in direct co-culture with myoblasts. However, hASCs seemed to support proliferation of myoblasts. As ASCs do not undergo myogenic differentiation, other potential roles of them in myogenic co-cultures need to be studied further. Engineered myobundles showed promise as a 3D model for skeletal muscle modeling but needs to be developed further.