Mesenchymal Stem Cell Group

The major research interest of the group is focused on the systematic characterization of the specific features of amniotic fluid MSCs at the cellular, molecular, proteomic and pre-clinical level. (hMSCs) derived from amniotic fluid. Human MSCs constitute a population of multipotent adherent cells able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. The group has systematically characterized the phenotypic, molecular and cellular aspects of these stem cells. Our studies were extended to post-transcriptional  analysis of AF-MSCs  and also to the use of AF-MSCs in therapeutic applications of acute hepatic failure and bladder cancer. Considering that the amniotic fluid can be obtained through routine prenatal diagnosis without ethical concerns, it may represent a valuable source of MSCs for autologous transplantation and future clinical applications.

AF-MSC characterization and biological properties

Post transcriptional regulation mechanisms of AF-MSC at miRNA level

Therapeutic role of AF-MSCs in acute hepatic failure (AHF)

Therapeutic role of AF-MSCs as delivery vehicle for anti-tumor agents in bladder cancer

Publications

AF-MSC characterization and biological properties
(Roubelakis, Bitsika, Zagoura)

Human mesenchymal progenitor cells (MSCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MSCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MSCs. We found that early colonies of AF-MSCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MSCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MSCs and RS-AF-MSCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MSCs. Furthermore, SS-AF-MSCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MSCs and thus we further evaluated SS-AF-MSCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MSCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MSCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. These results demonstrated that AF-MSCs consisted of at least two different MSC populations. Additionally, SS-AF-MSCs, isolated based on their colony morphology and CD90 expression, represented the only MSC population that can be expanded easily in culture.

(Roubelakis et al Stem cells and Dev 2007, Roubelakis et al J Cell Mol Med 2010)

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Post transcriptional regulation mechanisms of AF-MSC at miRNA level
(Trohatou, Roubelakis)

To further decipher the molecular mechanisms as they relate to the MSCs from bone marrow (BM) and umbilical cord blood (UCB), we are currently investigating the comparative post-transcriptional regulation mechanisms of MSCs from the three sources at the miRNA level. More specifically, the objectives of the study are i) the detection of miRNA populations in AF, BM and UCB-MSCs, ii) the validation of their expression levels using Real Time PCR, iii) the use of GOmir, an algorithm generated by our group in collaboration with Dr Kossida (Bioinformatics Unit) for the in silico detection of miRNA target-genes, iv) the validation of miRNA binding on specific targets predicted by the algorithm application and v) the determination of specific miRNA functional role.

(Roubelakis et al BMC Bioinformatics 2009, Zotos et al Curr Bioinformatics 2012)

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Therapeutic role of AF-MSCs in acute hepatic failure (AHF)
(Zagoura, Roubelakis)

Recent interest is focused on the use of AF-MSCs as an efficient tool for future in vivo therapeutic applications, especially in diseases such as acute hepatic failure (AHF). We transplanted SS-AF-MSCs, hepatic progenitor-like (HPL) and hepatocyte-like (HL) cells, derived from SS-AF-MSCs, into CCl₄-injured NOD/SCID mice that depict the AHF phenotype. Subsequently, we delivered intra-hepatically conditioned medium (CM) derived from SS-AF-MSCs or HPL cells in order to determine whether the engraftment of the cells or their secreted molecules are the most important agents for liver repair. Both HPL cells and SS-AF-MSCs were incorporated into CCl₄-injured livers, whereas HPL cell transplantation had a more efficient therapeutic effect. In contrast, HL cells failed to engraft and contribute to recovery. Additionally, HPL-CM, compared to CM derived from SS-AF-MSCs, was found to be more efficient in liver therapy. Proteome profile analysis of HPL-CM indicated the presence of anti-inflammatory factors such as IL-10, IL-1ra, IL-13 and IL-27, that may induce liver recovery. Indeed, blocking studies of IL-10 secretion from HPL cells confirmed the therapeutic significance of this cytokine in AHF mouse model. Human SS-AF-MSCs or HPL cells might be valuable tools to induce liver repair and support liver function by cell transplantation. More importantly the factors they release can also play an important role in cell treatment in liver diseases.

(Zagoura et al, Gut 2011)

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Therapeutic role of AF-MSCs as delivery vehicle for anti-tumor agents in bladder cancer
(Bitsika, Roubelakis)

Concurrent studies from our group in collaboration with Dr Vlahou (Biotechnology Lab) supported that SS-AF-MSCs can be used as delivery vehicles for anti-tumor agents, by migrating to the region of neoplasia without causing damage to the neighbouring tissues. We investigated, for the first time, the AF-MSCs tropism and capability to transport interferon-β (IFNβ) to the region of neoplasia in a bladder cancer model. It has been shown that high concentrations of IFNβ repressed the malignant cell growth in vitro, whereas in vivo there are limitations due to its cytotoxicity. To this end, we used the T24M bladder cancer cell line, previously generated from our studies, and developed a disease progression model in immunosupressed mice, that can recapitulate the molecular events of bladder carcinogenesis. Our results documented that SS-AF-MSCs exhibited a high motility when migrated to T24M cells. Furthermore, lentivirus transduced SS-AF-MSCs expressing IFNβ or GFP were administered intravenously to T24M tumor bearing animals at multiple doses in order to examine any therapeutic effect. GFP- and IFNβ-AF-MSCs successfully migrated and colonized at the tumor site. Notably, a significant inhibition of tumor growth and a prolonged survival were observed in the presence of IFNβ-AF-MSCs.

(Bitsika et al, Stem Cells and Dev 2011, Makridakis et al, J Proteome Res 2010, Makridakis et al, Proteomics 2008, Zoidakis et al, Mol Cell Ptoteomics 2011)

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Publications

1. Roubelakis MG, Pappa KI, Bitsika V, Zagoura D, Vlahou A, Papadaki HA, Antsaklis A, Anagnou NP. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells.Stem Cells and Dev 2007 Dec;16(6):931-52.
2. Makridakis M, Gagos S, Petrolekas A, Roubelakis MG, Bitsika V, Stravodimos K, Pavlakis K, Anagnou NP, Coleman J, Vlahou A. Chromosomal and proteome analysis of a new T24-based cell line model for aggressive bladder cancer. Proteomics. 2009 Jan;9(2):287-98.
3. Roubelakis MG, Zotos P, Papachristoudis G, Michalopoulos I, Pappa KI, Anagnou NP, Kossida S. Human microRNA target analysis and gene ontology clustering by GOmir, a novel stand-alone application. BMC Bioinformatics. 2009 Jun 16;10 Suppl 6:S20.
4. Makridakis M, Roubelakis MG, Bitsika V, Dimuccio V, Samiotaki M, Kossida S, Panayotou G, Coleman J, Candiano G, Anagnou NP, Vlahou A. Analysis of secreted proteins for the study of bladder cancer cell aggressiveness. J Proteome Res. 2010 Jun 4;9(6):3243-59.
5. Roubelakis MG, Bitsika V, Zagoura D, Trohatou O, Pappa KI, Makridakis M, Antsaklis A, Vlahou A, Anagnou NP. In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells. J Cell Mol Med. 2011 Sep;15(9):1896-913. doi: 10.1111/j.1582-4934.2010.01180.x.
6. Gazouli M, Roubelakis MG, Theodoropoulos GE, Papailiou J, Vaiopoulou A, Pappa KI, Nikiteas N, Anagnou NP. OCT4 spliced variant OCT4B1 is expressed in human colorectal cancer. Mol Carcinog. 2012 Feb;51(2):165-73. doi: 10.1002/mc.20773. Epub 2011 Apr 7.
7. Bitsika V, Roubelakis MG, Zagoura D, Trohatou O, Makridakis M, Pappa KI, Marini FC, Vlahou A, Anagnou NP. Human amniotic fluid-derived mesenchymal stem cells as therapeutic vehicles: a novel approach for the treatment of bladder cancer. Stem Cells Dev. 2012 May 1;21(7):1097-111. Epub 2011 Oct 11.
8. Zagoura DS, Roubelakis MG, Bitsika V, Trohatou O, Pappa KI, Kapelouzou A, Antsaklis A, Anagnou NP.Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure. Gut. 2012 Jun;61(6):894-906. Epub 2011 Oct 13.
9. Zoidakis J, Makridakis M, Zerefos PG, Bitsika V, Esteban S, Frantzi M, Stravodimos K, Anagnou NP, Roubelakis MG, Sanchez-Carbayo M, Vlahou A. Profilin 1 is a potential biomarker for bladder cancer aggressiveness. Mol Cell Proteomics. 2012 Apr;11(4):M111.009449. Epub 2011 Dec 8.

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