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743. Proliferation and Multilineage Differentiation Potential of Adipose-Derived Stem Cells Isolated from Dystrophin and Utrophin Knockout Mice
MESENCHYMAL STEM CELLS 742. Tissue Factor-Triggered Procoagulant Activity of Murine/Human Mesenchymal Stem Cells Kohei Tatsumi,1 Kazuo Ohashi,1 Yoshinori Matsubara,2 Ayako Kohori,2 Hiroshi Kakidachi,2 Akihiro Horii,2 Takanori Iwata,1 Teruo Okano.1 1 Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Shinjuku-ku, Tokyo, Japan; 2 Corporate R&D Center, Olympus Corporation, Hachioji, Tokyo, Japan.
(Objectives) Mesenchymal stem cells (MSCs) have been highlighted in the field of regenerative medicine for their availabilities and multipotencies, and proven to be therapeutic toward various diseases. However, it has been reported that MSC may associate with a cell-infusion-related lethal pulmonary thromboembolism after being administered intravenously. This study investigated the procoagulant property of MSC. (Methods) Mouse adipose-derived MSCs (ADSCs) were obtained from the inguinal adipose tissue of adult C57BL/6 mouse. MSC-specific cell surface markers and their differentiation abilities to adipogenic and osteogenic lineages were confirmed. ADSCs-derived procoagulant properties were assessed with in vitro assays; (1) dynamic blood coagulation monitoring in citrated whole-blood mixed with ADSCs using rotation thromboelastometry (ROTEM), and (2) measuring clotting time in citrated plasma mixed with ADSCs using a conventional clotting assay (KC4). Also, immunostaining, flow cytometry, and gene expression analysis of ADSCs for mouse tissue factor (TF) were performed. For detailed analyses, clotting assays using ADSCs mixed with normal plasma or factor VII-deficient plasma were performed. Furthermore, clotting assays using ADSCs pretreated with anti-TF inhibitory antibody were performed. For further investigations, the same evaluations were conducted on human ADSCs. (Results) First, in vivo transplantation experiments were performed, and more than 80% of mice (11 out of 13) were found to die within 24 h after intravenous administration of syngeneic ADSCs (1.5 x 105 cells). Histological assessments revealed that the cause of death was mainly massive thrombus formations occurred in lungs. ROTEM analyses demonstrated significant shortenings in clotting time and clot formation time, and increased α-angle in ADSCs samples compared with the control, indicating that the initiation, amplification, and propagation of blood coagulation were significantly enhanced by adding ADSCs. Clotting assays also showed a shortening in plasma clotting time when ADSCs were added in a cell-number dependent manner. When factor VIIdeficient plasma was used, no shortening was observed, implicating the involvement of extrinsic coagulation cascade. ADSCs were found to express strongly TF mRNA and numerous TF proteins around the cell surface. Also, the procoagulant properties were significantly suppressed when ADSCs were preconditioned with anti-TF inhibiting antibody. Detailed investigation on human ADSC lines also revealed their strong procoagulant activity triggered by TF. (Conclusion) The results demonstrate that ADSCs expressed TF, which may trigger a strong activation in the extrinsic coagulation pathway, resulting in the formation of cell-mediated thromboembolism. In clinics, the assessment of TF expression of ADSCs prior to the cell infusion, as well as preventing the occurrence of cell-mediated hypercoagulation status, is highly recommended for securing the clinical safety.
743. Proliferation and Multilineage Differentiation Potential of Adipose-Derived Stem Cells Isolated from Dystrophin and Utrophin Knockout Mice
Jihee Sohn,1 Adam Kozemchak,1 Nick Oyster,1 Seth Thompson,1 Ying Tang,1 Aiping Lu,1,2 Burhan Gharaibeh,1,2 Bing Wang,1 Johnny Huard.1,2 1 Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, PA; 2Department of Bioengineering and McGowan Institute for Regenerative Medicine, University of Pittsburgh, PA. Adipose tissue serves as a viable source of stem cells due to its relative ease of collection and availability in patients. We have previously isolated adipose derived stem cells (ADSCs) from subcutaneous fat from C57BL mice and demonstrated the ability of ADSCs to regenerate musculoskeletal tissue. ADSCs hold potential in cell therapy for a variety of musculoskeletal injuries and diseases including Duchenne Muscular Dystrophy (DMD). DMD is a genetic disease characterized by progressive weakening of the skeletal, cardiac, and diaphragmatic muscles. Most of the current treatments have been focused on the restoration of dystrophin within the dystrophic muscle; however, people have faced limitations, such as immunological reactions. Therefore, it is critical to find a successful therapy that will improve the histopathology of the muscles of DMD patients and restore their normal function. Various animal models have been used for DMD but the utrophin/dystrophin double knockout mice (dKO) emulates the phenotype seen in DMD patients and has been widely used to develop therapies for DMD. Previously in our lab, we have shown that muscle derived stem cells (MDSCs) from dKO mice are defective in proliferation and fusion, which correlate with the muscle histopathology. In this study, we determine if ADSCs isolated from subcutaneous fat tissue of 6 week old dKO mice, which are in the late stage of the disease, are affected in their proliferation and differentiation capacities compared to wild type (WT) ADSCs. ADSCs were isolated from dKO-homo (dys-/-utro-/-), dKO-hetero (dys-/-utro+/-) and C57BL WT mice and flow cytometry was used to evaluate the expression of CD45, Sca-1, CD34, CD44, and CD29 cell-surface antigens on the ADSCs obtained from dKO-hetero and dKO-homo, and WT mice. The ADSCs from all mouse strains were negative for CD45, a hematopoietic cell surface marker and positive for Sca-1, a stem cell marker, and CD29 and CD44, mesenchymal stem cell and progenitor cell surface markers. ADSCs were partially positive for CD34 antigen. Cell proliferation and apoptotic rates investigated using a Live Cell Imaging (LCI) system showed ADSCs from dKO mice have a higher proliferation rate than WT cells. This may explain the gradual muscle weakness and pseudohypertrophy observed in DMD. In term of multipotent differentiation, our result revealed that myogenesis, adipogenesis and chondrogenesis are impaired in ADSCs from the dKO mice. Impaired myogenesis observed with dKO cells may be a secondary effect of the increased proliferation observed with those cells. These results suggest a dysfunction in the ADSCs isolated from dKO homo mice and that blocking the exhaustion of ADSCs and stem cell therapy could be used to modulate adipose tissue formation in the muscles of DMD patients to restore structure and function.
744. Recipient MSCs Induced Differential Cytokine Gene Expression in the Small Intestine Allograft of Recipient Rats Liwei Zhu,1 Wei Wang,1 Fuxin Li,1 Shanni Li,1 Xianghui He.1 Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China.
1
Mesenchymal stem cells (MSCs) have been used to alleviate the immune rejection and induce tolerance in organ transplantation. To Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
S287
(Objectives) Mesenchymal stem cells (MSCs) have been highlighted in the field of regenerative medicine for their availabilities and multipotencies, and proven to be therapeutic toward various diseases. However, it has been reported that MSC may associate with a cell-infusion-related lethal pulmonary thromboembolism after being administered intravenously. This study investigated the procoagulant property of MSC. (Methods) Mouse adipose-derived MSCs (ADSCs) were obtained from the inguinal adipose tissue of adult C57BL/6 mouse. MSC-specific cell surface markers and their differentiation abilities to adipogenic and osteogenic lineages were confirmed. ADSCs-derived procoagulant properties were assessed with in vitro assays; (1) dynamic blood coagulation monitoring in citrated whole-blood mixed with ADSCs using rotation thromboelastometry (ROTEM), and (2) measuring clotting time in citrated plasma mixed with ADSCs using a conventional clotting assay (KC4). Also, immunostaining, flow cytometry, and gene expression analysis of ADSCs for mouse tissue factor (TF) were performed. For detailed analyses, clotting assays using ADSCs mixed with normal plasma or factor VII-deficient plasma were performed. Furthermore, clotting assays using ADSCs pretreated with anti-TF inhibitory antibody were performed. For further investigations, the same evaluations were conducted on human ADSCs. (Results) First, in vivo transplantation experiments were performed, and more than 80% of mice (11 out of 13) were found to die within 24 h after intravenous administration of syngeneic ADSCs (1.5 x 105 cells). Histological assessments revealed that the cause of death was mainly massive thrombus formations occurred in lungs. ROTEM analyses demonstrated significant shortenings in clotting time and clot formation time, and increased α-angle in ADSCs samples compared with the control, indicating that the initiation, amplification, and propagation of blood coagulation were significantly enhanced by adding ADSCs. Clotting assays also showed a shortening in plasma clotting time when ADSCs were added in a cell-number dependent manner. When factor VIIdeficient plasma was used, no shortening was observed, implicating the involvement of extrinsic coagulation cascade. ADSCs were found to express strongly TF mRNA and numerous TF proteins around the cell surface. Also, the procoagulant properties were significantly suppressed when ADSCs were preconditioned with anti-TF inhibiting antibody. Detailed investigation on human ADSC lines also revealed their strong procoagulant activity triggered by TF. (Conclusion) The results demonstrate that ADSCs expressed TF, which may trigger a strong activation in the extrinsic coagulation pathway, resulting in the formation of cell-mediated thromboembolism. In clinics, the assessment of TF expression of ADSCs prior to the cell infusion, as well as preventing the occurrence of cell-mediated hypercoagulation status, is highly recommended for securing the clinical safety.
743. Proliferation and Multilineage Differentiation Potential of Adipose-Derived Stem Cells Isolated from Dystrophin and Utrophin Knockout Mice
Jihee Sohn,1 Adam Kozemchak,1 Nick Oyster,1 Seth Thompson,1 Ying Tang,1 Aiping Lu,1,2 Burhan Gharaibeh,1,2 Bing Wang,1 Johnny Huard.1,2 1 Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, PA; 2Department of Bioengineering and McGowan Institute for Regenerative Medicine, University of Pittsburgh, PA. Adipose tissue serves as a viable source of stem cells due to its relative ease of collection and availability in patients. We have previously isolated adipose derived stem cells (ADSCs) from subcutaneous fat from C57BL mice and demonstrated the ability of ADSCs to regenerate musculoskeletal tissue. ADSCs hold potential in cell therapy for a variety of musculoskeletal injuries and diseases including Duchenne Muscular Dystrophy (DMD). DMD is a genetic disease characterized by progressive weakening of the skeletal, cardiac, and diaphragmatic muscles. Most of the current treatments have been focused on the restoration of dystrophin within the dystrophic muscle; however, people have faced limitations, such as immunological reactions. Therefore, it is critical to find a successful therapy that will improve the histopathology of the muscles of DMD patients and restore their normal function. Various animal models have been used for DMD but the utrophin/dystrophin double knockout mice (dKO) emulates the phenotype seen in DMD patients and has been widely used to develop therapies for DMD. Previously in our lab, we have shown that muscle derived stem cells (MDSCs) from dKO mice are defective in proliferation and fusion, which correlate with the muscle histopathology. In this study, we determine if ADSCs isolated from subcutaneous fat tissue of 6 week old dKO mice, which are in the late stage of the disease, are affected in their proliferation and differentiation capacities compared to wild type (WT) ADSCs. ADSCs were isolated from dKO-homo (dys-/-utro-/-), dKO-hetero (dys-/-utro+/-) and C57BL WT mice and flow cytometry was used to evaluate the expression of CD45, Sca-1, CD34, CD44, and CD29 cell-surface antigens on the ADSCs obtained from dKO-hetero and dKO-homo, and WT mice. The ADSCs from all mouse strains were negative for CD45, a hematopoietic cell surface marker and positive for Sca-1, a stem cell marker, and CD29 and CD44, mesenchymal stem cell and progenitor cell surface markers. ADSCs were partially positive for CD34 antigen. Cell proliferation and apoptotic rates investigated using a Live Cell Imaging (LCI) system showed ADSCs from dKO mice have a higher proliferation rate than WT cells. This may explain the gradual muscle weakness and pseudohypertrophy observed in DMD. In term of multipotent differentiation, our result revealed that myogenesis, adipogenesis and chondrogenesis are impaired in ADSCs from the dKO mice. Impaired myogenesis observed with dKO cells may be a secondary effect of the increased proliferation observed with those cells. These results suggest a dysfunction in the ADSCs isolated from dKO homo mice and that blocking the exhaustion of ADSCs and stem cell therapy could be used to modulate adipose tissue formation in the muscles of DMD patients to restore structure and function.
744. Recipient MSCs Induced Differential Cytokine Gene Expression in the Small Intestine Allograft of Recipient Rats Liwei Zhu,1 Wei Wang,1 Fuxin Li,1 Shanni Li,1 Xianghui He.1 Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China.
1
Mesenchymal stem cells (MSCs) have been used to alleviate the immune rejection and induce tolerance in organ transplantation. To Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
S287