- Email: [email protected]
Isolation of wharton’s jelly mesenchymal stem cells and their differentiation to insulin producing cells
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
S66
Poster Abstracts
1 Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt, 2Obstetrics and Gynecology Department , Faculty of Medicine, Ain Shams University, Cairo, Egypt
Introduction: Now, cell therapy for Diabetes Mellitus (DM) is under extensive study. Recently, there has been much attention towards generation of insulin producing cells (IPCs) from stem cells. Mesenchymal stem cells isolated from umbilical cord wharton’s jelly (WJ-MSCs) offer several advantages over other stem cells. Objectives: We sought to investigate the outcome of differentiation of WJMSCs to IPCs using several extrinsic factors, since there is no standard method for induction till now. Materials and methods: WJ-MSCs were isolated and expanded for several passages. Expression of surface markers and differentiation towards MSCs lineages were used to verify MSCs identity. Afterwards, WJ-MSCs were induced to differentiate into IPCs using several protocols, involving various extrinsic factors and induction periods; namely nicotinamide, bemercaptoethanol and exendin-4. Differentiated IPCs were assessed by determining the expression of key markers of b-cells such as Pdx-1, Isl-1, Nkx2.2, together with both MafA and MafB using qRT-PCR, and functionally by measuring insulin secretion after glucose challenge; a hall mark of functional b-cells. Results and conclusions: WJ-MSCs differentiated successfully to functioning IPCs. Interestingly, nicotinamide together with exendin-4 had a synergistic effect during the induction process, resulting in higher expression levels of b-cells markers, as compared to the use of each of them alone. Furthermore, the levels of both MafA and MafB elevated during differentiation stages to IPCs, which highlights the possible role MafB could be playing during development and function of human b-cells. Unexpectedly, the levels of Oct-4 were found to be elevated in obtained IPCs, despite the significant decrease of Nestin. In conclusion, WJ-MSCs represent a potential source for cell therapy of DM. Yet, further research is warranted to understand differentiation mechanisms in order to improve maturation and therapeutic outcome of these cells.
225 COMPARING UMBILICAL CORD BLOOD STEM CELLS AND WHARTON’S JELLY MESENCHYMAL STEM CELLS REGARDING THEIR DIFFERENTIATION POTENTIAL TO INSULIN PRODUCING CELLS MM Kamal1, HO El-Mesallamy1, LN Hammad2, RF El-Demerdash2 1 Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt, 2Pharmacology and Toxicology Department, Misr International University, Cairo, Egypt Introduction: The number of patients suffering from Diabetes Mellitus (DM) is growing in an alarming rate which makes DM the most prevalent and serious metabolic disease. Now, cell therapy treatment options for diabetic patients are under extensive study. Interestingly, umbilical cord (UC) has been proved to be a good source of mesenchymal stem cells (MSCs), namely from umbilical cord blood (UCB-MSCs) and Wharton’s jelly (WJ-MSCs). Objectives: We thought to investigate the difference between these 2 important banking sources of stem cells and to compare their differentiation potentials towards insulin producing cells (IPCs) in vitro and their potential use for treatment of streptozotocin (STZ) induced diabetic rats invivo. Materials and methods: Both UCB-MSCs and WJ-MSCs were isolated from UC and expanded for several passages. Expression of typical MSCs surface antigens and adipogenic differentiation potential as an example of mesenchymal lineage was used to verify MSCs identity. Afterwards, both UCB-MSCs and WJ-MSCs were induced to differentiate into IPCs, then the differentiated cells were assessed both genetically by determining the expression of Nestin, as stem cell marker and key markers of mature b-cells such as Pdx-1, Mafa and Nkx2.2 using qRT-PCR, and functionally by measuring insulin secretion after glucose challenge (Glucose stimulated insulin secretion; GSIS); a hall mark of functional b-cells. Results and conclusions: WJ appeared to be a much more homogenous and potential source for MSCs as compared to UCB. Interestingly, both UCBstem cells and WJ-MSCs were successfully differentiated to IPCs. Yet, the resulting IPCs from WJ-MSCs were to a limited extent functioning better than those obtained from UCB-MSCs. Both cell types were able to decrease fasting blood glucose level transiently in STZ induced diabetic mice. Taken together, we can conclude that WJ could represent a potential source of cells in the field of DM cell therapy rather than UCB.
226 QUEST FOR CIRCULATING MESENCHYMAL STEM CELLS IN MAN MJ Hoogduijn1, AU Engela1, MM Verstegen2, SS Korevaar1, M Roemeling-van Rhijn1, M Franquesa1, J de Jonge2, JN IJzermans2, W Weimar1, M Betjes1, LJ van der Laan2, CC Baan1 1 Internal Medicine, Erasmus Medical Center, Rotterdam, Netherlands, 2Surgery, Erasmus Medical Center, Rotterdam, Netherlands Mesenchymal stem cells (MSC) are present in the bone marrow, from where they are thought to migrate via the bloodstream to sites of injury. On the other hand, virtually all tissues contain resident MSC that may contribute to local regenerative and immunomodulatory processes, thereby hypothetically pre-empting the need for recruitment of MSC via the bloodstream. Although there is some indication from animal models, the question remains whether there is solid evidence for the mobilization and migration of MSC in human. In the present study we investigated whether circulating MSC were present in the peripheral blood of healthy individuals and patients with organ injury. We were unable to detect MSC in the blood of healthy individuals by flow cytometry and cell culture techniques. We then analyzed the presence of MSC in the blood of patients with end-stage renal disease (n¼10), end-stage liver disease (n¼10) and in heart transplant patients with biopsy proven rejection (n¼8), by culturing of mononuclear cells under MSC-supporting culture conditions. In none of these patients MSC were identified in the blood. In the stromal vascular fraction of adipose tissue and in liver transplant perfusion fluid we were able to detect MSC, indicating that the methods used enabled the detection of MSC. The conclusion of this study is that MSC are not detectable in the circulation in patients with injured solid organs and during aggressive immune responses. 227 HUMAN MESENCHYMAL STEM CELLS SELECTIVELLY RECOGNIZE AND ADHERE TO APOPTOTIC ENDOTHELIAL CELLS S Doronin1, IA Potapova2, IS Cohen3 1 Physiology and Biophysics, Stony Brook University, Stony Brook, New York, United States, 2Physiology and Biophysics, Stony Brook University, Stony Brook, New York, United States, 3Physiology and Biophysics, Stony Brook University, Stony Brook, New York, United States The majority of clinical applications of mesenchymal stem cells rely on their ability to home to sites of injury. However, little is known about the mechanism of mesenchymal stem cell homing to injured tissues. We investigated the adhesion of human mesenchymal stem cells to apoptotic endothelial cells in vitro. Our results show that the development of apoptosis in endothelial cells stimulates endothelial cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells to apoptotic endothelial cells depends on the activation of caspases and p38 MAPK in endothelial cells. Inhibition of p38 MAPK in endothelial cells completely abolishes the stimulation of the mesenchymal stem cell adhesion. The inhibition of caspases in endothelial cells partially inhibits the stimulation of the mesenchymal stem cell adhesion. We hypothesize that the activation of caspases potentiates p38 MAPK-dependent adhesion of human mesenchymal stem cells to apoptotic endothelial cells. Overall, our study demonstrates that human mesenchymal stem cells selectively recognize and adhere to distressed/apoptotic endothelial cells. 228 ENHANCED IDO ACTIVITY AND REDUCED IL8 PRODUCTION OF HBM-MSC IN INFLAMMATORY CONDITION BY PRETREATMENT WITH CYTOKINES INCLUDING INTERFERON-GAMMA J Jang1, S Suh2, J Kim2 1 School of Medicine, The Catholic University of Korea, Seocho-Gu, Seoul, Korea, Republic of, 2Catholic Institute of Cell Therapy, Catholic Medical Center, Seocho-Gu, Seoul, Korea, Republic of Mesenchymal stem cells (MSCs) have recently been studied and used in many fields of regenerative medicine including regeneration of body tissues such as bone, cartilage and heart, enhancement of engrafting efficiency of hematopoietic stem cells, amelioration of allograft rejection, and alleviation of inflammatory responses in autoimmune diseases including rheumatoid arthritis. Although MSCs have been reported to produce anti-inflammatory paracrine factors in vitro, results from in vivo researches were not always successful. Some research reported that MSCs, in some situations, may produce pro-inflammatory cytokines and sometimes deteriorate the inflammatory symptoms.
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
S66
Poster Abstracts
1 Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt, 2Obstetrics and Gynecology Department , Faculty of Medicine, Ain Shams University, Cairo, Egypt
Introduction: Now, cell therapy for Diabetes Mellitus (DM) is under extensive study. Recently, there has been much attention towards generation of insulin producing cells (IPCs) from stem cells. Mesenchymal stem cells isolated from umbilical cord wharton’s jelly (WJ-MSCs) offer several advantages over other stem cells. Objectives: We sought to investigate the outcome of differentiation of WJMSCs to IPCs using several extrinsic factors, since there is no standard method for induction till now. Materials and methods: WJ-MSCs were isolated and expanded for several passages. Expression of surface markers and differentiation towards MSCs lineages were used to verify MSCs identity. Afterwards, WJ-MSCs were induced to differentiate into IPCs using several protocols, involving various extrinsic factors and induction periods; namely nicotinamide, bemercaptoethanol and exendin-4. Differentiated IPCs were assessed by determining the expression of key markers of b-cells such as Pdx-1, Isl-1, Nkx2.2, together with both MafA and MafB using qRT-PCR, and functionally by measuring insulin secretion after glucose challenge; a hall mark of functional b-cells. Results and conclusions: WJ-MSCs differentiated successfully to functioning IPCs. Interestingly, nicotinamide together with exendin-4 had a synergistic effect during the induction process, resulting in higher expression levels of b-cells markers, as compared to the use of each of them alone. Furthermore, the levels of both MafA and MafB elevated during differentiation stages to IPCs, which highlights the possible role MafB could be playing during development and function of human b-cells. Unexpectedly, the levels of Oct-4 were found to be elevated in obtained IPCs, despite the significant decrease of Nestin. In conclusion, WJ-MSCs represent a potential source for cell therapy of DM. Yet, further research is warranted to understand differentiation mechanisms in order to improve maturation and therapeutic outcome of these cells.
225 COMPARING UMBILICAL CORD BLOOD STEM CELLS AND WHARTON’S JELLY MESENCHYMAL STEM CELLS REGARDING THEIR DIFFERENTIATION POTENTIAL TO INSULIN PRODUCING CELLS MM Kamal1, HO El-Mesallamy1, LN Hammad2, RF El-Demerdash2 1 Biochemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt, 2Pharmacology and Toxicology Department, Misr International University, Cairo, Egypt Introduction: The number of patients suffering from Diabetes Mellitus (DM) is growing in an alarming rate which makes DM the most prevalent and serious metabolic disease. Now, cell therapy treatment options for diabetic patients are under extensive study. Interestingly, umbilical cord (UC) has been proved to be a good source of mesenchymal stem cells (MSCs), namely from umbilical cord blood (UCB-MSCs) and Wharton’s jelly (WJ-MSCs). Objectives: We thought to investigate the difference between these 2 important banking sources of stem cells and to compare their differentiation potentials towards insulin producing cells (IPCs) in vitro and their potential use for treatment of streptozotocin (STZ) induced diabetic rats invivo. Materials and methods: Both UCB-MSCs and WJ-MSCs were isolated from UC and expanded for several passages. Expression of typical MSCs surface antigens and adipogenic differentiation potential as an example of mesenchymal lineage was used to verify MSCs identity. Afterwards, both UCB-MSCs and WJ-MSCs were induced to differentiate into IPCs, then the differentiated cells were assessed both genetically by determining the expression of Nestin, as stem cell marker and key markers of mature b-cells such as Pdx-1, Mafa and Nkx2.2 using qRT-PCR, and functionally by measuring insulin secretion after glucose challenge (Glucose stimulated insulin secretion; GSIS); a hall mark of functional b-cells. Results and conclusions: WJ appeared to be a much more homogenous and potential source for MSCs as compared to UCB. Interestingly, both UCBstem cells and WJ-MSCs were successfully differentiated to IPCs. Yet, the resulting IPCs from WJ-MSCs were to a limited extent functioning better than those obtained from UCB-MSCs. Both cell types were able to decrease fasting blood glucose level transiently in STZ induced diabetic mice. Taken together, we can conclude that WJ could represent a potential source of cells in the field of DM cell therapy rather than UCB.
226 QUEST FOR CIRCULATING MESENCHYMAL STEM CELLS IN MAN MJ Hoogduijn1, AU Engela1, MM Verstegen2, SS Korevaar1, M Roemeling-van Rhijn1, M Franquesa1, J de Jonge2, JN IJzermans2, W Weimar1, M Betjes1, LJ van der Laan2, CC Baan1 1 Internal Medicine, Erasmus Medical Center, Rotterdam, Netherlands, 2Surgery, Erasmus Medical Center, Rotterdam, Netherlands Mesenchymal stem cells (MSC) are present in the bone marrow, from where they are thought to migrate via the bloodstream to sites of injury. On the other hand, virtually all tissues contain resident MSC that may contribute to local regenerative and immunomodulatory processes, thereby hypothetically pre-empting the need for recruitment of MSC via the bloodstream. Although there is some indication from animal models, the question remains whether there is solid evidence for the mobilization and migration of MSC in human. In the present study we investigated whether circulating MSC were present in the peripheral blood of healthy individuals and patients with organ injury. We were unable to detect MSC in the blood of healthy individuals by flow cytometry and cell culture techniques. We then analyzed the presence of MSC in the blood of patients with end-stage renal disease (n¼10), end-stage liver disease (n¼10) and in heart transplant patients with biopsy proven rejection (n¼8), by culturing of mononuclear cells under MSC-supporting culture conditions. In none of these patients MSC were identified in the blood. In the stromal vascular fraction of adipose tissue and in liver transplant perfusion fluid we were able to detect MSC, indicating that the methods used enabled the detection of MSC. The conclusion of this study is that MSC are not detectable in the circulation in patients with injured solid organs and during aggressive immune responses. 227 HUMAN MESENCHYMAL STEM CELLS SELECTIVELLY RECOGNIZE AND ADHERE TO APOPTOTIC ENDOTHELIAL CELLS S Doronin1, IA Potapova2, IS Cohen3 1 Physiology and Biophysics, Stony Brook University, Stony Brook, New York, United States, 2Physiology and Biophysics, Stony Brook University, Stony Brook, New York, United States, 3Physiology and Biophysics, Stony Brook University, Stony Brook, New York, United States The majority of clinical applications of mesenchymal stem cells rely on their ability to home to sites of injury. However, little is known about the mechanism of mesenchymal stem cell homing to injured tissues. We investigated the adhesion of human mesenchymal stem cells to apoptotic endothelial cells in vitro. Our results show that the development of apoptosis in endothelial cells stimulates endothelial cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells to apoptotic endothelial cells depends on the activation of caspases and p38 MAPK in endothelial cells. Inhibition of p38 MAPK in endothelial cells completely abolishes the stimulation of the mesenchymal stem cell adhesion. The inhibition of caspases in endothelial cells partially inhibits the stimulation of the mesenchymal stem cell adhesion. We hypothesize that the activation of caspases potentiates p38 MAPK-dependent adhesion of human mesenchymal stem cells to apoptotic endothelial cells. Overall, our study demonstrates that human mesenchymal stem cells selectively recognize and adhere to distressed/apoptotic endothelial cells. 228 ENHANCED IDO ACTIVITY AND REDUCED IL8 PRODUCTION OF HBM-MSC IN INFLAMMATORY CONDITION BY PRETREATMENT WITH CYTOKINES INCLUDING INTERFERON-GAMMA J Jang1, S Suh2, J Kim2 1 School of Medicine, The Catholic University of Korea, Seocho-Gu, Seoul, Korea, Republic of, 2Catholic Institute of Cell Therapy, Catholic Medical Center, Seocho-Gu, Seoul, Korea, Republic of Mesenchymal stem cells (MSCs) have recently been studied and used in many fields of regenerative medicine including regeneration of body tissues such as bone, cartilage and heart, enhancement of engrafting efficiency of hematopoietic stem cells, amelioration of allograft rejection, and alleviation of inflammatory responses in autoimmune diseases including rheumatoid arthritis. Although MSCs have been reported to produce anti-inflammatory paracrine factors in vitro, results from in vivo researches were not always successful. Some research reported that MSCs, in some situations, may produce pro-inflammatory cytokines and sometimes deteriorate the inflammatory symptoms.