Human pluripotent stem cell biology and applications
We investigate the fundamental biology and translational applications of stem cells, with particular emphasis on induced pluripotent stem cells (iPSCs) derived from patients carrying different mutations related to diseases. Our research focuses on differentiation protocols towards the three lineages, especially towards vascular progenitor cells through mesodermal intermediates.
Generation of human induced pluripotent stem cells in defined, feeder-free conditions
We have established a modified protocol for the generation of human induced pluripotent stem cells (hiPS) and expansion under defined, serum free and feeder free conditions. These cells exhibit a high level of plasticity towards various differentiation pathways both in vitro and in vivo. Ultimately, hiPS-derived lines achieved high standards of three dimensional differentiations on biomaterial scaffolds and promoted in vivo regeneration of complex organs, such as Anterior Cruciate Ligament (in swine ACL-rupture models) and other tissues as well.
A Kyrkou et al., Stem cell research 17 (2), 458 (2016)
Reprogramming strategy
Heat map analysis of the differentially regulated genes following reprogramming among hiPSC lines, original human fibroblasts (hFs) and embryonic HUES1 stem cell line.
Culturing Human Pluripotent Stem Cells on Micropatterned Silicon Surfaces
Human pluripotent stem cell culture conditions are continually being refined, offering valuable insight into the environmental cues that influence cell fate decisions. A broad range of culture media, coating materials, and substrates is now available to address diverse experimental requirements. Increasing evidence indicates that physical parameters such as substrate stiffness, surface roughness, and topography can contribute to—or even dictate—the acquisition of specific cellular phenotypes. In this context, we describe the use of patterned silicon substrates coated with Matrigel for the propagation and differentiation of human pluripotent stem cells.
H1 cells cultured on silicon substrates.
(A) H1 colonies on low (a), medium (b), and high (c) roughness substrates. Scale bar is set at 100 μm. (B) H1 colonies on low (a), medium (b), and high (c) roughness substrates, lateral views were also obtained, scale bar is set at 100 μm.
V Chalmantzi et al., Methods in molecular biology 2454, 49 (2022).
Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells
Model of transition of hESCs into differentiated cells
hESCs differentiate into mesoderm intermediates and then into CD34+ VPCs and CD34− cells, under feeder-free conditions, using defined media. CD34+ synthesize known vascular cell markers, as well as proteins related to vasculogenesis and smooth muscle differentiation, suggesting a bipotent phenotype. Indeed, CD34+ can differentiate further to both directions depending on the growth conditions. CD34− cells consist of a mixed population oversynthesizing secreted factors that can promote angiogenesis, suggesting a paracrine effect on CD34+ cells and also synthesizing proteins that can lead to differentiation to other lineages (muscle, bone, cartilage).
Bagli et al., J Proteome Res 15 (6), 1995 (2016).
Endocytosis and trafficking pathways of Activin A in Human Embryonic Stem Cells
Ligand–receptor complexes formed at the plasma membrane are internalised via various endocytic pathways that influence the ultimate signalling output by regulating the selection of interaction partners by the complex along the trafficking route. We report that, in differentiated cells, activin A–receptor complexes are internalised via clathrin-mediated endocytosis (CME) and macropinocytosis (MP), whereas in human embryonic stem cells (hESCs) internalisation occurs via CME. We further show that hESCs are devoid of MP, which becomes functional upon differentiation towards endothelial cells through mesoderm mediators. Our results reveal, for the first time, that MP is an internalisation route for activin A in differentiated cells, and that MP is not active in hESCs and is induced as cells differentiate.
Rabankyrin-5 localisation during hESCs differentiation.
N Kostopoulou et al., Journal of cell science 134 (13) (2021).
