- Email: [email protected]
Phenotypic expressions of reprogrammed osteosarcoma cell lines
S64
Poster Abstracts
218 ENDOVASCULAR RETINAL INFUSION OF BONE MARROW HEMATOPOIETIC STEM CELLS FOR PIGMENTOUS RETINITIS J Arturo1, C Perez1, O Segura2, OS Guerrero2, Y Bastidas1, L Larios1 1 Molecular and Regenerative Medicine, Inmugen Corporation, Bogota, Colombia, 2Haemodinamia, Diagnosticos e Imagenes, Bogota, Colombia There is more than 75 eye and constitutional disorders that may be associated with Pigmentous Retinitis (PR), a disease characterized by progressive retinal degeneration and photoreceptors apoptosis , which affects 1 in 3,500 people. Symptoms include glare and photopsia, altered color perception, night blindness, tunnel vision , and progressive reduction of central vision. With an hereditary origin, in most cases may be involved a large number of genes and patterns ( Autosomal dominant , Autosomal recessive and sexlinked ). Today the current treatments are palliative with oral vitamins and macular vasodilators whereas gene therapies and surgical procedures are under evaluation without significant results. Considering the clinical history , retinography and visual field diagnostic of PR , previous ethical comitee aprobation, we performed a treatment in three patients , with implantation of autologous bone marrow autologous hematopoietic stem cells by catheterism through guide catheter platform No .6.0 F and guide endovascular navigation system under maping road No. 1.5 F , with supra selective ophthalmic artery infusion to to ensure the arrival of the cells to the retina During the first month of monitoring improvement in symptoms of phosphenes, photopsia, night blindness and color vision were persistently improved. Within 3 months after increase bilateral visual acuity, allowing patients to maintain a capacity for self-care and perform activities of daily living so far (48 months later). The amount of retinal lesions in comparative analysis was reduced in the 3 patients. The procedure does not produce any complications. We conclude after performing evaluation and follow treatment with autologous bone marrow hematopoietic stem cells is a safe therapeutic option in patients with pigmentous retinitis, with improve in life quality , which must be evaluated in future clinical trials.
219 ANALYSES OF CD90 ROLE IN THE GROWTH, OSTEOGENIC DIFFERENTIATION AND MORPHOLOGY, IMMUNOGENIC PROPERTY OF HUMAN MESENCHYMAL STEM CELLS (MSC) D Moraes1,2, O Toledo2, L Gamarra4, F Araújo3, T Sibov4, L Marti4, R Azevedo1, D Oliveira1 1 Genetics and Morphology, University of Brasilia, Brasília, Distrito Federal, Brazil, 2Ciencias da saúde, University of Brasília, Brasilia, Distrito Federal, Brazil, 3Farmacia, University of Brasília, Brasilia, Distrito Federal, Brazil, 4Instituto do Cerebro, Instituto Israelita Albert Eisntein, São Paulo, São Paulo, Brazil MSCs are isolated from several human tissues and expanded in vitro. These cells promptly differentiate into multiple connective tissue lineages, such as osteoblasts, chondrocytes and adipocytes. Studies showed that human MSC have unique immunological properties: they are not immunogenic, they do not simulate alloreactivity, they scape from T citotoxic and NK cells lysis activity. The imunophenotypic characterization of MSC is positive for expression of cell markers CD90, CD105, CD73, CD117, CD44, CD166, CD29 and STRO-1. This work has as goal the analysis of CD90 glycoprotein role in the morphology, growth, differentiation of dental pulp MSCs, and immunological response of CD90 through the reduction of CD90 expression using RNA interference mechanism. Lentivirus particles were used to create stable MSCs clones expressing shRNA against CD90 and a scramble shRNA control. Clones were isolated via puromycin selection. The reduction of CD90 expression, confirmed in flow cytometry assays, was analysed using FLOJO software. Morphology was analysed using phase contrast optical microscopy and flow citometry. For proliferative rate analyses the number of adherent cells was determined by hemocytometer. For differentiation analyses, cells were induced for osteogenic differentiation and their calcium deposits were stained with Alisarin red. We investigated calcium concentration through Espectometry. We found that a significant decrease in CD90 levels does not affect MSCs immunogenic properties, proliferation, differentiation and morphology. The partial reduction of CD90 expression leads: to a decrease in CD166 and CD44 expression and to an increase in calcium concentration and mineralization when compared to our control cells. This work supported by CNPQ.
220 A NOVEL ANIMAL COMPONENT-FREE CULTURE MEDIUM FOR EFFICIENT DERIVATION AND EXPANSION OF HUMAN MESENCHYMAL CELLS R Wagey1, J Yau1, E Hadley1, M Wong1, C Duronio1, A Sampaio1, C Miller1, T Thomas1, A Eaves1,2, SA Louis1 1 STEMCELL Technologies Inc., Vancouver, British Columbia, Canada,, 2Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada MesenCultÔ-ACF, a novel animal component-free (ACF) culture medium, was used to derive and expand mesenchymal progenitor cells (MPCs) from primary human bone marrow mononuclear cells (BMMC) and adipose tissues (AD). MPCs were isolated from primary BMMC by plating 10,000 - 50,000 cells/cm2 in MesenCultÔ-ACF or in a serum-containing control medium. Clonogenic growth was evaluated using the Colony-Forming Unit-Fibroblast (CFU-F) assay. To evaluate MPC expansion from primary BMMC, cells were plated at 3 e 5 x 104 cells/cm2 in MesenCultÔ-ACF or 5 x 104 e 1.0 x 105 cells/cm2 in control medium. For subsequent subcultures, cells were plated at 1500 e 3000 cells/cm2. Expansion cultures of MPCs from AD were initiated with 500 - 1250 cells/cm2 in either MesenCultÔACF or control medium. The proliferative potential of MPCs from either cell source in each medium was determined by counting cell number at each passage (P) up to P8 and paired t-test was used for statistical analysis. Total CFU-F derived per 1 x 106 BMMC was significantly higher in MesenCultÔ-ACF than in control medium (51 8 versus 29 5; mean SEM; n¼6; p<0.05). Average fold-expansion at each subculture of MPCs from BMMC from P1 to P8 (31-46 days) in MesenCultÔ-ACF was significantly higher than in control medium (6.6 1.0 versus 4.0 0.8; mean SEM; n¼6; p<0.05). Similarly, the average fold-expansion of ADderived MPCs from P1 to P8 (25 to 39 days) was also significantly higher in MesenCultÔ-ACF than in control medium (10.6 1.2 versus 2.1 0.2; mean SEM; n¼3; p<0.01). Cells cultured in MesenCultÔ-ACF differentiated robustly under the appropriate differentiation cultures into adipocytes, osteogenic cells and chondrocytes, as visualized by Oil Red O, Von Kossa, and Alcian Blue staining, respectively. In summary, MesenCultÔ-ACF efficiently derives and expands MPCs directly from primary tissues under animal component-free culture conditions. 221 PHENOTYPIC EXPRESSIONS OF REPROGRAMMED OSTEOSARCOMA CELL LINES P Choong1,2, H Teh2, H Teoh1, H Ong2, K Choo2, S Cheong1,2, T Kamarul3 1 Stem Cell Transplantation, PPUKM-MAKNA Cancer Centre, Universiti Kebangsaan Malaysia Medical Centre, Malaysia, Kuala Lumpur, Kuala Lumpur, Malaysia, 2Faculty of Medicine and Health Sciences, University Tunku Abdul Rahman (UTAR), Selangor, Selangor, Malaysia, 3Tissue Engineering Group, National Orthopaedic Centre of Excellence for Research and Learning, Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Kuala Lumpur, Malaysia Reprogramming of cancer cells have brought us closer to generate patient-specific induced pluripotent stem cells (iPSC) with cancer properties for understanding of cancer biology and drug screening for patient-specific cancer therapy. We used
20th Annual ISCT Meeting
Yamanaka factors, OCT4, SOX2, KLF4 and c-MYC, to transduce all osteosarcoma cells. Transduced cells were transferred to inactivated mouse embryonic fibroblast (iMEF) on Day 3 post transduction. Colonies were manually picked on Day 15 - Day 20 and transferred to new iMEF. Reprogrammed sarcomas were characterised by observation on morphology, alkaline phosphatase and pluripotency markers expression, embryoid body formation and directed differentiation into adipocytes and osteocytes. All four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotency. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction for all four cell lines. Morphology of the colonies resembles ESC colonies with defined border and tightly packed cells. We then characterised our reprogrammed sarcomas and found all the reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, similar to ESC. In our observation, all reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension condition in a low attachment dish for up to 10 days. We further test the differentiation capacity of our reprogrammed sarcomas by performing directed differentiation into adipocytes and osteocytes. Our directed differentiation results showed that all four reprogrammed sarcoma could differentiate into adipocytes as shown with Oil Red O staining. While, only reprogrammed Saos-2-REP, MG-63-REP and G-292REP could differentiate into osteocytes as shown by Alizarin Red staining. These results support the ability of cancer cells to be reprogrammed. However, further works need to be done to fully characterise the reprogrammed sarcomas. 222 DUX4 EXPRESSION DURING OSTEOGENIC DIFFERENTIATION IN MESENCHYMAL STROMAL CELLS (MSCS) L de la Kethulle de Ryhove1, E Ansseau1, M Geens3, F Coppee1, KD Sermon3, L Lagneaux2, A Belayew1 1 Laboratory of Molecular Biology, University of Mons, Mons, Belgium, 2Laboratory of Clinical Cell Therapy, ULB, Brussels, Belgium, 3Department of Embryology and Genetics, VUB, Brussels, Belgium Our group has identified the Double Homeobox 4 (DUX4) gene within repeated DNA elements in the 4q35 chromosome region linked to the FSHD
S65
muscular dystrophy. In healthy individuals Dr. S. Tapscott’s group has detected a full length DUX4 mRNA (fl-DUX4) in induced pluripotent stem (iPS) cells and human testis, and a longer mRNA where the gene contains 4 additional exons and a more distal polyadenylation signal than in FSHD muscles. Our preliminary data suggested DUX4 was expressed at a very low level in MSC isolated from Bone Marrow (BM-MSC) and more abundant in Wharton jelly (Wj-MSC). We wanted to evaluate whether DUX4 expression changed during BM-MSC differentiation. We added an osteogenic differentiation medium to BM-MSCs cultures, collected cells after 0, 7, 14 and 21 days and performed an immunodetection on western blot. To confirm the differentiation process we stained calcium deposits in the cell culture dishes with alizarin red. We observed an increase of DUX4 expression after 14 and 21 days. We then investigated whether DUX4 was involved in the differentiation process. We transfected MSCs with antisense oligonucleotides (2’O Methyl phosphorothiate, DUX4-AO) targeting the DUX4 mRNA and previously shown to interfere with the protein expression3. The cells transfected with a DUX4-AO presented weaker alizarin red staining after switch to differentiation medium (Fig.1). In conclusion we show that DUX4 expression is increased upon MSC differentiation to osteoblasts. This observation is in contrast with the data published about iPS cells differentiation to embryoid bodies in which DUX4-fl expression disappeared. However Dr. M. Kyba’s group has recently shown DUX4 implication in neurogenesis. They transfected murine Embryonic stem cells with a DUX4 inducible vector and observed after DUX4 induction that differentiated cells expressed neuronal expression markers. We hypothesize that DUX4 could generally be implicated in the mechanism of early differentiation. 223 MYELOID DERIVED SUPPRESSOR CELLS ARE EXPANDED IN PATIENTS WITH MULTIPLE MYELOMA, INDUCE TREG CELLS AND DELAY T-CELL RECOVERY POST TRANSPLANTATION K Zarkos, J Favaloro, T Liyadipitiya, R Brown, S Yang, H Suen, C Weatherburn, J Gibson, P Ho, D Joshua rpa hospital, institute of haematology, Sydney, New South Wales, Australia Background: Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells expressing immature myeloid markers and have been implicated as inhibitors of lymphopoiesis. We determined the number of MDSC in patients with multiple myeloma (MM), the impact of G-CSF on MDSC prior to stem cell collection, the ability of MDSC to induce Treg cells and the impact of MDSC on lymphocyte regeneration posttransplant. Methods: MDSC were detected by flow cytometry as CD11b+CD33+HLADR lo/-. Treg cells were identified as CD4+CD25+CD127 low/neg. pSTAT3 was determined by phosphoflow. Results: Granulocytic MDSC (G-MDSC: CD14-CD15+, CD33+CD11b+HLA-DRlo/-) were significantly higher in the blood of patients with MM (n¼25; mean: 9.2%) compared to age matched controls (n¼11; mean: 2.1%) (U¼78.5; p¼ 0.04) and greater still in patients with active disease (n¼9; mean: 50.1%) (U¼0.0; p¼0.0002). Flow-sorted MM MDSC (n¼7) cocultured 1:1 with autologous mononuclear cells induced a greater proportion of Treg cells (p¼ 0.05) than MDSC from age matched controls (n¼4). G-MDSC were significantly increased in the blood of patients undergoing autologous transplant following G-CSF administration with a pre G-CSF mean¼3.8% and post G-CSF mean¼16.4%). The proportion of G-MDSC in the PBSC (mean ¼ 18%) was significantly higher than in matched blood samples (t¼3.24; p¼ 0.018). Although the number of G-MDSC infused had no apparent influence on lymphocyte recovery, there was a correlation (R2¼0.59; p<0.01) between pSTAT3 expression in G-MDSC in the PBSC collection reinfused and the lymphocyte count rising above 0.0x109/L post-transplant suggesting it was the activity of G-MDSC that delayed lymphocyte recovery. Conclusion: MDSC are increased in the blood of patients with MM and increase after G-CSF stem cell mobilisation. Their activation status as demonstrated by pSTAT3 expression suggests that they contribute to the inhibition of lymphocyte regeneration post-transplant. 224 ISOLATION OF WHARTON’S JELLY MESENCHYMAL STEM CELLS AND THEIR DIFFERENTIATION TO INSULIN PRODUCING CELLS DH Kassem1, MM Kamal1, HO El-Mesallamy1, A El-Kholy2
Poster Abstracts
218 ENDOVASCULAR RETINAL INFUSION OF BONE MARROW HEMATOPOIETIC STEM CELLS FOR PIGMENTOUS RETINITIS J Arturo1, C Perez1, O Segura2, OS Guerrero2, Y Bastidas1, L Larios1 1 Molecular and Regenerative Medicine, Inmugen Corporation, Bogota, Colombia, 2Haemodinamia, Diagnosticos e Imagenes, Bogota, Colombia There is more than 75 eye and constitutional disorders that may be associated with Pigmentous Retinitis (PR), a disease characterized by progressive retinal degeneration and photoreceptors apoptosis , which affects 1 in 3,500 people. Symptoms include glare and photopsia, altered color perception, night blindness, tunnel vision , and progressive reduction of central vision. With an hereditary origin, in most cases may be involved a large number of genes and patterns ( Autosomal dominant , Autosomal recessive and sexlinked ). Today the current treatments are palliative with oral vitamins and macular vasodilators whereas gene therapies and surgical procedures are under evaluation without significant results. Considering the clinical history , retinography and visual field diagnostic of PR , previous ethical comitee aprobation, we performed a treatment in three patients , with implantation of autologous bone marrow autologous hematopoietic stem cells by catheterism through guide catheter platform No .6.0 F and guide endovascular navigation system under maping road No. 1.5 F , with supra selective ophthalmic artery infusion to to ensure the arrival of the cells to the retina During the first month of monitoring improvement in symptoms of phosphenes, photopsia, night blindness and color vision were persistently improved. Within 3 months after increase bilateral visual acuity, allowing patients to maintain a capacity for self-care and perform activities of daily living so far (48 months later). The amount of retinal lesions in comparative analysis was reduced in the 3 patients. The procedure does not produce any complications. We conclude after performing evaluation and follow treatment with autologous bone marrow hematopoietic stem cells is a safe therapeutic option in patients with pigmentous retinitis, with improve in life quality , which must be evaluated in future clinical trials.
219 ANALYSES OF CD90 ROLE IN THE GROWTH, OSTEOGENIC DIFFERENTIATION AND MORPHOLOGY, IMMUNOGENIC PROPERTY OF HUMAN MESENCHYMAL STEM CELLS (MSC) D Moraes1,2, O Toledo2, L Gamarra4, F Araújo3, T Sibov4, L Marti4, R Azevedo1, D Oliveira1 1 Genetics and Morphology, University of Brasilia, Brasília, Distrito Federal, Brazil, 2Ciencias da saúde, University of Brasília, Brasilia, Distrito Federal, Brazil, 3Farmacia, University of Brasília, Brasilia, Distrito Federal, Brazil, 4Instituto do Cerebro, Instituto Israelita Albert Eisntein, São Paulo, São Paulo, Brazil MSCs are isolated from several human tissues and expanded in vitro. These cells promptly differentiate into multiple connective tissue lineages, such as osteoblasts, chondrocytes and adipocytes. Studies showed that human MSC have unique immunological properties: they are not immunogenic, they do not simulate alloreactivity, they scape from T citotoxic and NK cells lysis activity. The imunophenotypic characterization of MSC is positive for expression of cell markers CD90, CD105, CD73, CD117, CD44, CD166, CD29 and STRO-1. This work has as goal the analysis of CD90 glycoprotein role in the morphology, growth, differentiation of dental pulp MSCs, and immunological response of CD90 through the reduction of CD90 expression using RNA interference mechanism. Lentivirus particles were used to create stable MSCs clones expressing shRNA against CD90 and a scramble shRNA control. Clones were isolated via puromycin selection. The reduction of CD90 expression, confirmed in flow cytometry assays, was analysed using FLOJO software. Morphology was analysed using phase contrast optical microscopy and flow citometry. For proliferative rate analyses the number of adherent cells was determined by hemocytometer. For differentiation analyses, cells were induced for osteogenic differentiation and their calcium deposits were stained with Alisarin red. We investigated calcium concentration through Espectometry. We found that a significant decrease in CD90 levels does not affect MSCs immunogenic properties, proliferation, differentiation and morphology. The partial reduction of CD90 expression leads: to a decrease in CD166 and CD44 expression and to an increase in calcium concentration and mineralization when compared to our control cells. This work supported by CNPQ.
220 A NOVEL ANIMAL COMPONENT-FREE CULTURE MEDIUM FOR EFFICIENT DERIVATION AND EXPANSION OF HUMAN MESENCHYMAL CELLS R Wagey1, J Yau1, E Hadley1, M Wong1, C Duronio1, A Sampaio1, C Miller1, T Thomas1, A Eaves1,2, SA Louis1 1 STEMCELL Technologies Inc., Vancouver, British Columbia, Canada,, 2Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada MesenCultÔ-ACF, a novel animal component-free (ACF) culture medium, was used to derive and expand mesenchymal progenitor cells (MPCs) from primary human bone marrow mononuclear cells (BMMC) and adipose tissues (AD). MPCs were isolated from primary BMMC by plating 10,000 - 50,000 cells/cm2 in MesenCultÔ-ACF or in a serum-containing control medium. Clonogenic growth was evaluated using the Colony-Forming Unit-Fibroblast (CFU-F) assay. To evaluate MPC expansion from primary BMMC, cells were plated at 3 e 5 x 104 cells/cm2 in MesenCultÔ-ACF or 5 x 104 e 1.0 x 105 cells/cm2 in control medium. For subsequent subcultures, cells were plated at 1500 e 3000 cells/cm2. Expansion cultures of MPCs from AD were initiated with 500 - 1250 cells/cm2 in either MesenCultÔACF or control medium. The proliferative potential of MPCs from either cell source in each medium was determined by counting cell number at each passage (P) up to P8 and paired t-test was used for statistical analysis. Total CFU-F derived per 1 x 106 BMMC was significantly higher in MesenCultÔ-ACF than in control medium (51 8 versus 29 5; mean SEM; n¼6; p<0.05). Average fold-expansion at each subculture of MPCs from BMMC from P1 to P8 (31-46 days) in MesenCultÔ-ACF was significantly higher than in control medium (6.6 1.0 versus 4.0 0.8; mean SEM; n¼6; p<0.05). Similarly, the average fold-expansion of ADderived MPCs from P1 to P8 (25 to 39 days) was also significantly higher in MesenCultÔ-ACF than in control medium (10.6 1.2 versus 2.1 0.2; mean SEM; n¼3; p<0.01). Cells cultured in MesenCultÔ-ACF differentiated robustly under the appropriate differentiation cultures into adipocytes, osteogenic cells and chondrocytes, as visualized by Oil Red O, Von Kossa, and Alcian Blue staining, respectively. In summary, MesenCultÔ-ACF efficiently derives and expands MPCs directly from primary tissues under animal component-free culture conditions. 221 PHENOTYPIC EXPRESSIONS OF REPROGRAMMED OSTEOSARCOMA CELL LINES P Choong1,2, H Teh2, H Teoh1, H Ong2, K Choo2, S Cheong1,2, T Kamarul3 1 Stem Cell Transplantation, PPUKM-MAKNA Cancer Centre, Universiti Kebangsaan Malaysia Medical Centre, Malaysia, Kuala Lumpur, Kuala Lumpur, Malaysia, 2Faculty of Medicine and Health Sciences, University Tunku Abdul Rahman (UTAR), Selangor, Selangor, Malaysia, 3Tissue Engineering Group, National Orthopaedic Centre of Excellence for Research and Learning, Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Kuala Lumpur, Malaysia Reprogramming of cancer cells have brought us closer to generate patient-specific induced pluripotent stem cells (iPSC) with cancer properties for understanding of cancer biology and drug screening for patient-specific cancer therapy. We used
20th Annual ISCT Meeting
Yamanaka factors, OCT4, SOX2, KLF4 and c-MYC, to transduce all osteosarcoma cells. Transduced cells were transferred to inactivated mouse embryonic fibroblast (iMEF) on Day 3 post transduction. Colonies were manually picked on Day 15 - Day 20 and transferred to new iMEF. Reprogrammed sarcomas were characterised by observation on morphology, alkaline phosphatase and pluripotency markers expression, embryoid body formation and directed differentiation into adipocytes and osteocytes. All four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotency. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction for all four cell lines. Morphology of the colonies resembles ESC colonies with defined border and tightly packed cells. We then characterised our reprogrammed sarcomas and found all the reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, similar to ESC. In our observation, all reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension condition in a low attachment dish for up to 10 days. We further test the differentiation capacity of our reprogrammed sarcomas by performing directed differentiation into adipocytes and osteocytes. Our directed differentiation results showed that all four reprogrammed sarcoma could differentiate into adipocytes as shown with Oil Red O staining. While, only reprogrammed Saos-2-REP, MG-63-REP and G-292REP could differentiate into osteocytes as shown by Alizarin Red staining. These results support the ability of cancer cells to be reprogrammed. However, further works need to be done to fully characterise the reprogrammed sarcomas. 222 DUX4 EXPRESSION DURING OSTEOGENIC DIFFERENTIATION IN MESENCHYMAL STROMAL CELLS (MSCS) L de la Kethulle de Ryhove1, E Ansseau1, M Geens3, F Coppee1, KD Sermon3, L Lagneaux2, A Belayew1 1 Laboratory of Molecular Biology, University of Mons, Mons, Belgium, 2Laboratory of Clinical Cell Therapy, ULB, Brussels, Belgium, 3Department of Embryology and Genetics, VUB, Brussels, Belgium Our group has identified the Double Homeobox 4 (DUX4) gene within repeated DNA elements in the 4q35 chromosome region linked to the FSHD
S65
muscular dystrophy. In healthy individuals Dr. S. Tapscott’s group has detected a full length DUX4 mRNA (fl-DUX4) in induced pluripotent stem (iPS) cells and human testis, and a longer mRNA where the gene contains 4 additional exons and a more distal polyadenylation signal than in FSHD muscles. Our preliminary data suggested DUX4 was expressed at a very low level in MSC isolated from Bone Marrow (BM-MSC) and more abundant in Wharton jelly (Wj-MSC). We wanted to evaluate whether DUX4 expression changed during BM-MSC differentiation. We added an osteogenic differentiation medium to BM-MSCs cultures, collected cells after 0, 7, 14 and 21 days and performed an immunodetection on western blot. To confirm the differentiation process we stained calcium deposits in the cell culture dishes with alizarin red. We observed an increase of DUX4 expression after 14 and 21 days. We then investigated whether DUX4 was involved in the differentiation process. We transfected MSCs with antisense oligonucleotides (2’O Methyl phosphorothiate, DUX4-AO) targeting the DUX4 mRNA and previously shown to interfere with the protein expression3. The cells transfected with a DUX4-AO presented weaker alizarin red staining after switch to differentiation medium (Fig.1). In conclusion we show that DUX4 expression is increased upon MSC differentiation to osteoblasts. This observation is in contrast with the data published about iPS cells differentiation to embryoid bodies in which DUX4-fl expression disappeared. However Dr. M. Kyba’s group has recently shown DUX4 implication in neurogenesis. They transfected murine Embryonic stem cells with a DUX4 inducible vector and observed after DUX4 induction that differentiated cells expressed neuronal expression markers. We hypothesize that DUX4 could generally be implicated in the mechanism of early differentiation. 223 MYELOID DERIVED SUPPRESSOR CELLS ARE EXPANDED IN PATIENTS WITH MULTIPLE MYELOMA, INDUCE TREG CELLS AND DELAY T-CELL RECOVERY POST TRANSPLANTATION K Zarkos, J Favaloro, T Liyadipitiya, R Brown, S Yang, H Suen, C Weatherburn, J Gibson, P Ho, D Joshua rpa hospital, institute of haematology, Sydney, New South Wales, Australia Background: Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells expressing immature myeloid markers and have been implicated as inhibitors of lymphopoiesis. We determined the number of MDSC in patients with multiple myeloma (MM), the impact of G-CSF on MDSC prior to stem cell collection, the ability of MDSC to induce Treg cells and the impact of MDSC on lymphocyte regeneration posttransplant. Methods: MDSC were detected by flow cytometry as CD11b+CD33+HLADR lo/-. Treg cells were identified as CD4+CD25+CD127 low/neg. pSTAT3 was determined by phosphoflow. Results: Granulocytic MDSC (G-MDSC: CD14-CD15+, CD33+CD11b+HLA-DRlo/-) were significantly higher in the blood of patients with MM (n¼25; mean: 9.2%) compared to age matched controls (n¼11; mean: 2.1%) (U¼78.5; p¼ 0.04) and greater still in patients with active disease (n¼9; mean: 50.1%) (U¼0.0; p¼0.0002). Flow-sorted MM MDSC (n¼7) cocultured 1:1 with autologous mononuclear cells induced a greater proportion of Treg cells (p¼ 0.05) than MDSC from age matched controls (n¼4). G-MDSC were significantly increased in the blood of patients undergoing autologous transplant following G-CSF administration with a pre G-CSF mean¼3.8% and post G-CSF mean¼16.4%). The proportion of G-MDSC in the PBSC (mean ¼ 18%) was significantly higher than in matched blood samples (t¼3.24; p¼ 0.018). Although the number of G-MDSC infused had no apparent influence on lymphocyte recovery, there was a correlation (R2¼0.59; p<0.01) between pSTAT3 expression in G-MDSC in the PBSC collection reinfused and the lymphocyte count rising above 0.0x109/L post-transplant suggesting it was the activity of G-MDSC that delayed lymphocyte recovery. Conclusion: MDSC are increased in the blood of patients with MM and increase after G-CSF stem cell mobilisation. Their activation status as demonstrated by pSTAT3 expression suggests that they contribute to the inhibition of lymphocyte regeneration post-transplant. 224 ISOLATION OF WHARTON’S JELLY MESENCHYMAL STEM CELLS AND THEIR DIFFERENTIATION TO INSULIN PRODUCING CELLS DH Kassem1, MM Kamal1, HO El-Mesallamy1, A El-Kholy2