Enhanced Differentiation of Human Embryonic Stem Cells into Multipotential progenitors by Inhibition of TGF-b/Activin/Nodal signalling using SB-431542

Amer Mahmoud

Post Doctoral, Msc, Phd
Research Scientist
Molecular Endocrinology (KMEB)
University of Southern Denmark

Abstract

Human embryonic stem cells (hESCs) are established from the inner cell mass of pre-implantation embryos. HESCs hold the ability to differentiate into every known tissue in the body, and self-renewable capacity makes them an unlimited source of cells for therapy in many degenerative diseases, such as Parkinson’s disease, diabetes, myocardial infarction, muscle dystrophy, osteoporosis and traumatic spinal cord injuries. In this context, we are interested in studying the differentiation of hESC into mesenchymal/osteogenic lineage in order to understand the early human bone development and to provide an unlimited supply of osteogenic cells for cell therapy in many bone diseases. In general, differentiation of hESC in currently available protocols is always random and uncontrolled due to less knowledge of their differentiation regulatory mechanisms and due to cellular heterogeneity. Different research groups have derived osteogenic population from both human and mouse ESCs, however these studies are based on co-culture with mouse cells, or animal serum has been used. The current work aimed to design a protocol for directing the differentiation of hESC into a multipotent mesenchymal progenitor population and further to osteoblastic cells that can be used for bone tissue engineering. Firstly, blocking TGF-β signaling through embryoid body formation and outgrowth culture in serum free media enhanced mesoderm formation. The formed mesoderm was mainly of presomitic origin and had muscle progenitor phenotype. These cells were analyzed and characterized thoroughly by basic molecular biology methods in combination with Affymatrix microarray. In addition, different derivatives of hESCs were tested for their ability to survive, differentiate and integrate into host tissue of immune deficient mice. Secondly, muscle progenitors were stimulated to differentiate into mesenchymal stem cells (MSC). The hESC-derived MSCs could in addition be differentiated in vitro into osteoblasts and adipocytes and in vivo into bone and cartilage. The results of these experiments recapitulate many known observations and events in differentiation of hESCs into mesoderm, furthermore these studies demonstrated a novel protocol for directing the differentiation of hESCs into muscle and mesenchymal progenitors.