Embryonic Stem (ES) cells are derived from pre-implantation embryos and share two unique properties: the ability to be grown indefinitely in culture (self-renewal) and the ability to differentiate into any cell type of an organism (pluripotency). The pluripotency of ES cells is determined by the concerted action of external signalling molecules, intrinsic transcription factors and epigenetic regulators. In the last decade, the introduction in somatic cells of transcription factors (Oct4, Sox2, Klf4, c-myc), microRNAs and small molecules allowed them to reverse their development and become pluripotent (induced Pluripotent Stem cells, iPS). Due to their ability to give rise to any type of differentiated cells and tissues, embryonic and induced pluripotent stem cells hold great promise for modelling human diseases and development of regenerative medicine.
1. The role of Promyelocytic Leukemia (PML) in ES cells (C.Hadjimichaeland K.Chanoumidou)
PML is a tumor suppressor protein involved in leukemias. It is the core component of PML bodies, nuclear structures that regulate important cell processes such as proliferation, apoptosis, differentiation and senescence. In ES cells PML is expressed in a differentiation-specific manner and contributes to ESC self-renewal maintenance by controlling cell-cycle progression and sustaining the expression of crucial pluripotency factors. Transcriptomic analysis and gain- or loss-of-function approaches showed that PML-deficient ESC exhibit morphological, metabolic, and growth properties distinct to naive and closer to the primed pluripotent state. During differentiation of embryoid bodies, PML influences cell-fate decisions between mesoderm and endoderm by controlling the expression of Tbx3. PML loss compromises the reprogramming ability of embryonic fibroblasts to induced pluripotent stem cells by inhibiting the transforming growth factor β pathway at the very early stages.
2. The role of Aminoterminal Enhancer of Split (AES) in ESC (K.Chanoumidou)
AES/GRG5 is an interesting member of the Groucho family, which has the ability to act as both co-activator and co-repressor of transcription. It interferes with many signaling pathways and its role has been mainly studied in organogenesis and in cancer cells, where it is implicated in metastasis inhibition. Previous published microarray data in the lab have denoted AES/GRG5 as a factor with altered expression during mESCs differentiation. We investigate the role of AES in transcriptional regulation using knocking down and over-expression approaches. In addition we study the network of AES interacting protein factors by proteomics analysis.
3. Identification and characterization of novel microRNAs that regulate ES cell fate decisions (C.Hadjimichael)
Using the Ion Torrent platform (IMBB) we performed deep sequencing of miRNA expressed in mouse ESCs (mESCs) and differentiated embryoid bodies (EBs). Among differentially expressed miRNAs we characterized four novel miRNAs that are involved in ES cell differentiation choices acting as regulators of the TGF/Activin /BMP4 signaling pathway.
FIGURE Proposed mechanism for the regulation of mESC differentiation by miR-16-1, 191, 23a and 421.
4. Dynamic nuclear positioning of pluripotency gene loci in murine Embryonic Stem cells (K.Chanoumidou)
Embryonic Stem Cells (ESCs) are characterized by less structured nuclear organization and unique chromatin state, defined by a higher level of mobility. The above mentioned features make them an attractive system for studying the functional relevance of gene intra-nuclear positioning. We investigate the intra-nuclear localization of key pluripotency genes relative to landmarks of the nuclear architecture and try to correlate this with the ESC differentiation state. Techniques that are used include 3D-DNA FISH, Immuno-fish and Chromatin Immuno-precipitation (ChIP).
5. Derivation and characterization of induced Pluripotent Stem (iPS) cells from Umbilical Cord blood (A.Vogiatzoglou).
Umbilical cord (UC) blood is an established source of hematopoietic stem cells (HSCs) for transplantation in hematologic disorders and, together with the Wharton’s jelly, a promising source of non-HSCs such as endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs) and unrestricted somatic stem cells (USSCs) for potential use in regenerative medicine. UC blood banks have been established worldwide for collection and cryopreservation of UC blood for related/autologous (family/private commercial banks) or unrelated (public non-profit banks) transplantation
Participating in the UMBISTEM consortium we are trying to produce UC-derived induced Pluripotent Stem Cells (iPSCs) and define their properties for biobanking qualification. In addition we are characterizing transcription factors and small molecules that are important for efficient differentiation towards the osteogenic, adipogenic and chondrogenic lineages.