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Bone marrow derived mesenchymal stromal cells differ from hepatic stellate cells and attenuate liver fibrosis
21st ISCT Annual Meeting
especially reactive oxygen species (ROS)-induced damage. The most frequently occurring oxidized DNA lesion is 8-oxoguanine (8-oxoG), the incorporation of which to the DNA can cause double strand breaks (DSB), characteristic features of cellular senescence. Recent studies, on the other hand, have demonstrated that telomerase has a telomere-length independent function that promotes cellular resistance against oxidative stress. Here, we studied the senescence as well as the response to the exposure to external acute oxidative stress of hMSCs from adipose tissue. We demonstrated that as cells senesce accumulate DNA damage (mainly 8oxoG and DSBs) and that senescent cells are unable to repair the damage induced by exogenous oxidative stress. We also generated mesenchymal cells from adipose tissue overexpressing the telomerase catalytic subunit (hTERT) gene. Cells were engineered using the Sleeping Beauty transposon 100X system, a non-viral dual plasmid system with minimum cytotoxicity. Preliminary data indicate that hMSCs overexpressing hTERT show no difference as compared with the control cells in the amount of damage they accumulate due to the acute external oxidative exposure. Nevertheless, senescence reaching hTERT overexpressing cells show significantly lower percentages of oxidative DNA lesions and DSB as compared to control cells of the same passage. This means that hTERT offers a significant protection against the DNA damage that accumulates in the nuclei of the cells during normal cellular ageing. These results offer novel insights regarding the limitations of ex vivo amplified cell preparations for therapeutic applications. 110 WILL NOT BE PRESENTED 111 WILL NOT BE PRESENTED 112 BONE MARROW DERIVED MESENCHYMAL STROMAL CELLS DIFFER FROM HEPATIC STELLATE CELLS AND ATTENUATE LIVER FIBROSIS R Chinnadurai, J Galipeau Emory University, Atlanta, Georgia, United States Rationale: Liver fibrosis is the end result of most chronic hepatic ailments and treatment options are limited. Mesenchymal stromal cells (MSCs) are under investigation for the treatment of liver fibrosis/cirrhosis. Hepatic stellate cells (HSCs), which are quiescent non-parenchymal cells in the healthy liver transform in to activated myofibroblasts, secrete collagen and promote fibrogenesis. Current unknown is the shared and unique immunomodulatory and pro/anti fibrotic properties of MSCs and HSCs. Methods: Murine primary MSCs were isolated from marrow of C57BL6 mice and HSCs were isolated by in vivo liver perfusion followed by density gradient separation. Bone marrow derived human MSCs were compared with established human hepatic stellate cell line (LX2). Immunophenotyping and T cell suppressive potential of MSCs were performed by flow cytometry. Responses to profibrotic (TGFb) and anti-fibrotic cytokine (IFNg) were analyzed by realtime PCR. Carbon Tetrachloride (CCL4) induced liver fibrosis was used in the animals to investigate the anti-fibrotic effect of MSCs. Results: HSCs express International Society for Cellular Therapy defined cell surface markers (CD105+CD73+CD90+CD45-) to define MSCs. Stimulation with IFNg upregulates MHC-I, Indoleamine 2,3 dioxygenase(IDO) and B7-H1, but not the costimulatory molecules B7-1 and B7-2 on both MSCs and HSCs. Although MSCs and HSCs inhibit T cell proliferation, MHC-II is upregulated only on MSCs not HSCs. In addition, TGFb stimulation upregulate collagen type I only on HSCs (10 fold) but not on MSCs (>2 fold). Infusion of MSCs attenuated collagen upregulation (Fold upegulation: CCL4¼41+28; CCL4+MSC¼16+9) in the fibrotic livers of CCL4 treated animals. Conclusion: Although phenotypes are similar, cell physiology, immune plasticity and anti-fibrotic properties of MSCs are distinct from HSCs and are an attractive cellular pharmaceutical for the treatment of liver fibrosis. 113 REDUCING UNCERTAINTY IN THE EVALUATION OF STEM CELL COLONIES A Plant1, M Halter1, S Lund2, A Peskin3, Y-S Li-Baboud3, D Hoeppner4, P Bajcsy3 1 Biosystems and Biomaterials, NIST, Gaithersburg, Maryland, United States, 2Statistical Engineering, NIST, Gaithersburg, Maryland, United
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States, 3Software and Systems, NIST, Boulder, Colorado, United States, 4Lieber Institute for Brain Development, Baltimore, Maryland, United States Visual inspection of stem cell colonies by microscopy is often the method used to assess the pluripotency quality of colonies. Decisions are then made about selecting the colonies for further expansion. Because the method is qualitative and subject to operator bias and fatigue, we examined the possibility of finding an automated algorithmic approach to the ranking of stem cell colony quality. Two experts examined a set of 480 images of hESC colonies and scored them on a scale of 1 (poor) to 5 (maximum pluripotency character). The experts’ rankings agreed only 60% of the time, and the absence of ‘ground truth’ complicated the development of a classification scheme. We compared two classes of models, linear and Random Forest, and a number of parameters including identity of expert, colony segmentation method, and image features. We treated the scores as a continuous scale of pluripotency, and used all experts’ scores to define the trend with a normally distributed uncertainty around each score. A linear model based on both experts predicted the scores from expert 1 with a mean squared error (MSE) of 0.42, which is significantly better than the MSE between experts scores (0.62). The same model predicts the scores from expert 2 with a MSE of 0.65. Analysis indicates that our feature set accounts for only about 70% of the experts’ decisions, meaning that we have not identified all of the features that the experts used. However, it also indicates that scoring by expert 2 exhibits variability, and probably inconsistency, that is not shared with expert 1. This method allows the use of an imperfect training set to develop and evaluate a classification algorithm that can allow automation of the process of stem cell colony expansion. Similar classification algorithms may be useful for characterizing cell products for release assays. 114 CONVERSION OF HEPATOCYTES INTO MULTI-POTENT MESENCHYMAL STROMAL CELLS BY TRANSFORMING GROWTH FACTOR BETA-1 C-W Lee1, OK Lee1,2,3,4,5 1 Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan, 2Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, 3Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan, 4Taipei City Hospital, Taipei, Taiwan, 5Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan It is well known that lineage conversion can be induced by exogenous delivery of ectopic genes or small molecules. However, it is less clear whether such phenomenon can be achieved by defined growth factors. In the present study, we design a stepwise protocol with progressively decreasing amounts of transforming growth factor beta 1 (TGF-b1) to induce lineage reprogramming in mouse primary hepatocytes. Stepwise TGF-b1 treatment not only down-regulated the expression level of hepatogenic specific genes, but also altered morphology, cell surface phenotype and transcriptome of hepatocytes into MSC-like state. After exogenous TGF-b1 treatment, expression of endogenous TGF-b1 in MSC-like was induced and remained even after removal of exogenous TGF-b1. Upon induction, MSC-like cells derived from hepatocytes could further differentiate into hepatocytes, osteoblasts, and adipocytes with characteristic tissue-specific gene signatures. The results indicate that the converted MSC-like cells acquire multilineage differentiation potentials in vitro. In summary, plasticity of terminally differentiated cells, hepatocytes in this case, may be way more than what have been previously appreciated. Besides, lineage conversion may occur without forced ectopic gene expression. Such findings have opened an new avenue for future studies of reprogramming and lineage conversion. 115 ESTABLISHMENT AND CHARACTERIZATION OF A TUMOR CELL LINE, BML01, DERIVED FROM BONE MARROW OF PATIENT WITH MYELODYSPLASTIC SYNDROME B-Y Tsai1, J-L Tang2, C-C Li2, C-T Lin2, B-L Chiang3 1 NAVI BIO-THERAPEUTICS INC., Taipei, Taiwan, 2Tai Cheng Stem Cell Therapy Center, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, 3Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Cell line BML01 was established from the bone marrow of a patient with myelodysplastic syndrome (MDS) that ineffectively produces myeloid class of
especially reactive oxygen species (ROS)-induced damage. The most frequently occurring oxidized DNA lesion is 8-oxoguanine (8-oxoG), the incorporation of which to the DNA can cause double strand breaks (DSB), characteristic features of cellular senescence. Recent studies, on the other hand, have demonstrated that telomerase has a telomere-length independent function that promotes cellular resistance against oxidative stress. Here, we studied the senescence as well as the response to the exposure to external acute oxidative stress of hMSCs from adipose tissue. We demonstrated that as cells senesce accumulate DNA damage (mainly 8oxoG and DSBs) and that senescent cells are unable to repair the damage induced by exogenous oxidative stress. We also generated mesenchymal cells from adipose tissue overexpressing the telomerase catalytic subunit (hTERT) gene. Cells were engineered using the Sleeping Beauty transposon 100X system, a non-viral dual plasmid system with minimum cytotoxicity. Preliminary data indicate that hMSCs overexpressing hTERT show no difference as compared with the control cells in the amount of damage they accumulate due to the acute external oxidative exposure. Nevertheless, senescence reaching hTERT overexpressing cells show significantly lower percentages of oxidative DNA lesions and DSB as compared to control cells of the same passage. This means that hTERT offers a significant protection against the DNA damage that accumulates in the nuclei of the cells during normal cellular ageing. These results offer novel insights regarding the limitations of ex vivo amplified cell preparations for therapeutic applications. 110 WILL NOT BE PRESENTED 111 WILL NOT BE PRESENTED 112 BONE MARROW DERIVED MESENCHYMAL STROMAL CELLS DIFFER FROM HEPATIC STELLATE CELLS AND ATTENUATE LIVER FIBROSIS R Chinnadurai, J Galipeau Emory University, Atlanta, Georgia, United States Rationale: Liver fibrosis is the end result of most chronic hepatic ailments and treatment options are limited. Mesenchymal stromal cells (MSCs) are under investigation for the treatment of liver fibrosis/cirrhosis. Hepatic stellate cells (HSCs), which are quiescent non-parenchymal cells in the healthy liver transform in to activated myofibroblasts, secrete collagen and promote fibrogenesis. Current unknown is the shared and unique immunomodulatory and pro/anti fibrotic properties of MSCs and HSCs. Methods: Murine primary MSCs were isolated from marrow of C57BL6 mice and HSCs were isolated by in vivo liver perfusion followed by density gradient separation. Bone marrow derived human MSCs were compared with established human hepatic stellate cell line (LX2). Immunophenotyping and T cell suppressive potential of MSCs were performed by flow cytometry. Responses to profibrotic (TGFb) and anti-fibrotic cytokine (IFNg) were analyzed by realtime PCR. Carbon Tetrachloride (CCL4) induced liver fibrosis was used in the animals to investigate the anti-fibrotic effect of MSCs. Results: HSCs express International Society for Cellular Therapy defined cell surface markers (CD105+CD73+CD90+CD45-) to define MSCs. Stimulation with IFNg upregulates MHC-I, Indoleamine 2,3 dioxygenase(IDO) and B7-H1, but not the costimulatory molecules B7-1 and B7-2 on both MSCs and HSCs. Although MSCs and HSCs inhibit T cell proliferation, MHC-II is upregulated only on MSCs not HSCs. In addition, TGFb stimulation upregulate collagen type I only on HSCs (10 fold) but not on MSCs (>2 fold). Infusion of MSCs attenuated collagen upregulation (Fold upegulation: CCL4¼41+28; CCL4+MSC¼16+9) in the fibrotic livers of CCL4 treated animals. Conclusion: Although phenotypes are similar, cell physiology, immune plasticity and anti-fibrotic properties of MSCs are distinct from HSCs and are an attractive cellular pharmaceutical for the treatment of liver fibrosis. 113 REDUCING UNCERTAINTY IN THE EVALUATION OF STEM CELL COLONIES A Plant1, M Halter1, S Lund2, A Peskin3, Y-S Li-Baboud3, D Hoeppner4, P Bajcsy3 1 Biosystems and Biomaterials, NIST, Gaithersburg, Maryland, United States, 2Statistical Engineering, NIST, Gaithersburg, Maryland, United
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States, 3Software and Systems, NIST, Boulder, Colorado, United States, 4Lieber Institute for Brain Development, Baltimore, Maryland, United States Visual inspection of stem cell colonies by microscopy is often the method used to assess the pluripotency quality of colonies. Decisions are then made about selecting the colonies for further expansion. Because the method is qualitative and subject to operator bias and fatigue, we examined the possibility of finding an automated algorithmic approach to the ranking of stem cell colony quality. Two experts examined a set of 480 images of hESC colonies and scored them on a scale of 1 (poor) to 5 (maximum pluripotency character). The experts’ rankings agreed only 60% of the time, and the absence of ‘ground truth’ complicated the development of a classification scheme. We compared two classes of models, linear and Random Forest, and a number of parameters including identity of expert, colony segmentation method, and image features. We treated the scores as a continuous scale of pluripotency, and used all experts’ scores to define the trend with a normally distributed uncertainty around each score. A linear model based on both experts predicted the scores from expert 1 with a mean squared error (MSE) of 0.42, which is significantly better than the MSE between experts scores (0.62). The same model predicts the scores from expert 2 with a MSE of 0.65. Analysis indicates that our feature set accounts for only about 70% of the experts’ decisions, meaning that we have not identified all of the features that the experts used. However, it also indicates that scoring by expert 2 exhibits variability, and probably inconsistency, that is not shared with expert 1. This method allows the use of an imperfect training set to develop and evaluate a classification algorithm that can allow automation of the process of stem cell colony expansion. Similar classification algorithms may be useful for characterizing cell products for release assays. 114 CONVERSION OF HEPATOCYTES INTO MULTI-POTENT MESENCHYMAL STROMAL CELLS BY TRANSFORMING GROWTH FACTOR BETA-1 C-W Lee1, OK Lee1,2,3,4,5 1 Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan, 2Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, 3Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan, 4Taipei City Hospital, Taipei, Taiwan, 5Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan It is well known that lineage conversion can be induced by exogenous delivery of ectopic genes or small molecules. However, it is less clear whether such phenomenon can be achieved by defined growth factors. In the present study, we design a stepwise protocol with progressively decreasing amounts of transforming growth factor beta 1 (TGF-b1) to induce lineage reprogramming in mouse primary hepatocytes. Stepwise TGF-b1 treatment not only down-regulated the expression level of hepatogenic specific genes, but also altered morphology, cell surface phenotype and transcriptome of hepatocytes into MSC-like state. After exogenous TGF-b1 treatment, expression of endogenous TGF-b1 in MSC-like was induced and remained even after removal of exogenous TGF-b1. Upon induction, MSC-like cells derived from hepatocytes could further differentiate into hepatocytes, osteoblasts, and adipocytes with characteristic tissue-specific gene signatures. The results indicate that the converted MSC-like cells acquire multilineage differentiation potentials in vitro. In summary, plasticity of terminally differentiated cells, hepatocytes in this case, may be way more than what have been previously appreciated. Besides, lineage conversion may occur without forced ectopic gene expression. Such findings have opened an new avenue for future studies of reprogramming and lineage conversion. 115 ESTABLISHMENT AND CHARACTERIZATION OF A TUMOR CELL LINE, BML01, DERIVED FROM BONE MARROW OF PATIENT WITH MYELODYSPLASTIC SYNDROME B-Y Tsai1, J-L Tang2, C-C Li2, C-T Lin2, B-L Chiang3 1 NAVI BIO-THERAPEUTICS INC., Taipei, Taiwan, 2Tai Cheng Stem Cell Therapy Center, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, 3Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Cell line BML01 was established from the bone marrow of a patient with myelodysplastic syndrome (MDS) that ineffectively produces myeloid class of