Alqahtani, KMA; (2012) Comparison of the multi-lineage differentiation ability of craniofacial and limb skeletal muscle derived progenitor cells. Doctoral thesis , UCL (University College London).

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Abstract

Comparison of multi-lineage differentiation ability of craniofacial skeletal muscle and limb muscle progenitor cells Alqahtani, K, Wall, I, Lewis, M.P. Craniofacial Skeletal Muscles (CSkM), including masticatory muscles, have a number of properties that distinguish them from the majority of skeletal muscles. They have different embryological origin from other muscles; they have better regenerative abilities compared to other body muscles including limb muscles. In addition, they may have easier access for biopsies. These findings suggest that CSkM may contain highly active multipotent progenitor cells that may have enhanced differentiation abilities and survival rates. The aims of this study were to isolate craniofacial muscle progenitor cells based on their adhesion properties and to investigate their differentiation ability compared to cells derived from limb muscles. Material and methods: Cells derived from mouse masseter and hind-limb muscles were isolated using enzymatic digestion method, then serially plated into three different plates, pre-plate 1, 2 and 3 (PP1, PP2 and PP3), based on their adhesion properties. Cells in PP1, PP2 and PP3 adhered within the 1st hr, 24 hrs and 4 days respectively, of initial plating. Cells were investigated for their stem cell, neural crest cells (NCCs) gene expression profile, and also their differentiation along the osteogenic and myogenic lineages using osteogenic medium, or myogenic medium. Osteogenic differentiation was assessed by Alkaline Phosphatase (ALP) staining, Alizarin Red staining, Calcium deposition assay, and gene expression. The myogenic differentiation was assessed by the presence of multinucleate myotubes using bright field microscope and immune-fluorescent stating, and gene expression. Different growth behaviours and gene expression profile were seen in all isolated muscle cells. Cells in PP1 and PP2 had a fibroblastic appearance, whereas cells in PP3 were spindle-like and round in shape. Moreover, cells in PP3 had slower growth rate initially compared to cells in PP1 and PP2. The early adhered cells in both muscle groups showed higher stem cell gene expression compared to late adhered cells. Moreover, early adhered cells in CSkM showed higher expression of NCC genes compared to late adhered cells of the same group and all cells from LM. The differentiation abilities were also different among different pre-plates. In osteogenic differentiation, early adhered cells from both muscle groups had higher differentiation ability compared to late adhered cells while myotubes were less evident in early adhered cells compared to late adhered cells. Different population of cells that may have different osteogenic and myogenic differentiation abilities were isolated from craniofacial skeletal muscle (CSkM) based on their adhesion properties. There were no siginificant differences in differentiation abilities between cells isiolated from CSkM and their equivalent ceels isolated from LM. early adhered cells isolated from CSkM have higher expression of NCCs genes than cells isolated from LM.

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