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Presenter: Denis, Dufrane, Brussels, Belgium
Authors: Denis Dufrane1, Hélène Antoine-Poirel3, Pierre-Louis Docquier2, Wivine Andre1, Najima Aouassar1, Daela Xhema1, Marie-Sophie Hanet1, Rose-Marie Goebbels1, Genevieve Ameye3, Line Verhaeghe3, Sandrine Nonckreman3, Christian Delloye2
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Genetic characterization of human autologous adipose mesenchymal stem cells differentiated in 3-dimensional osteogenic graft
Denis Dufrane1, Hélène Antoine-Poirel3, Pierre-Louis Docquier2, Wivine Andre1, Najima Aouassar1, Daela Xhema1, Marie-Sophie Hanet1, Rose-Marie Goebbels1, Genevieve Ameye3, Line Verhaeghe3, Sandrine Nonckreman3, Christian Delloye2
1Endocrine Cell Therapy; 2Orthopaedic Surgery Service; 3Human Molecular Genetics, University Clinical Hospital Saint-Luc, Brussels, Belgium
Introduction: Osteogenic differentiated autologous adipose mesenchymal stem cells (AMSCs) could be proposed for bone reconstruction. This work studied the safety of the procedure in term of human AMSCs isolation to differentiation and genetic stability.
Methods: AMSCs isolation (by collagenase digestion of subcutaneous adipose tissue) /differentiation into an original 3-dimensional « bone-like » structure were performed from 8 independent patients. Group 1: Five patients with bone tumour history (3 osteosarcomas, 1 chondroblastoma, 1 Ewing’s sarcoma) characterized by several clonal cytogenetic alterations (Karyotyping and FISH study of tumor suppressor gene loci such as TP53/17p13, CDKN2/9p21, RB1/13q14) of the original tumour. Group 2: Three patients with pseudarthrosis (Neurofibromatosis and Diamond Blackfan syndrome). Graft characterization (Osteocalcin,Von Kossa staining) and genetic analysis were performed on AMSCs proliferation and differentiation phases. In addition, microarrays analysis studied the gene expression for osteogenic (RUNX2, BMP2, OPN3, FGF2, ALPL, SP7, FGF23, SMAD9, MEN1) and senescence/tumorogenic (c-Myc,TP53, NFKB,Cyclin D) development between un-/and osteogenic-differentiated states of AMSCs.
Results: A mean of 74 ± 30 days was required to achieve the autologous bone-like 3-D structure (osteocalcin expression, collagen deposition and mineralization). Microarrays demonstrated the up-regulation of osteogenic genes for differentiated cells (p<0.05) and no sign of upregulation for TP53,NFKB, Cyclin D, c-Myc for both un-/differentiated AMSCs. In Group 1, no native tumour anomalies was found prior and after osteogenic differentiation of AMSCs. However, AMSCs culture (up to Passage 4th) can induced, in both Group 1 and 2, tri-/tetraploidies (0,5-14% of cells), recurrent clonal alterations as trisomy 7 (in 6-20% of cells for 3 patients) and chromosomal breakage cht(3)(q13.3) (for 4 patients) for undifferentiated AMSCs in proliferation phase. Interestingly, the osteogenic differentiation reduced significantly anomalies found in proliferation state (trisomy 7: <2-5.5% of cells).
Conclusion: These preliminary results demonstrated, for the first time, the impact of proliferation and osteogenic differentiation on the genetic stability of human AMSCs.
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