- Email: [email protected]
Will Not Be Presented
22nd Annual ISCT Meeting
supports the isolation and expansion of MSCs from various tissues. The medium is xeno-free and can be used without additional coating of cell culture vessels. We compared MSC-Brew GMP Medium with other xeno-free MSC media as well as FCS- and platelet lysate (PL)-containing formulations. Method: MSCs were isolated from human adipose tissue, bone marrow and umbilical cord using different cultivation media. Proliferation behaviour was monitored for up to 5 passages followed by karyotyping. In parallel, the clonogenic potential was assessed using CFU-F assays. Differentiation potential was tested at passage 3, representing the passage usually used in autologous clinical treatments. The immunomodulatory properties of MSCs are the main reason for their great clinical potential. However, interaction of MSCs with cells of the innate- and adaptive-immune system are not completely understood. Therefore, we analyzed the expression of surface molecules as potential candidates for immune cell interaction using a library of >300 monoclonal antibodies. To this end, we compared MSC-Brew expanded MSCs with or without TNF-alpha/INFgamma or Poly-IC induced licensing. The same cells were tested for their immunosuppressive properties using a standardized in vitro T-cell suppression assay. Conclusion: We demonstrate that MSC-Brew GMP Medium supports efficient isolation and expansion of bona fide MSCs at relevant scale suited for cell therapy. Furthermore, we present a comprehensive characterization of expressed surface molecules which will contribute to deciphering the MSCs’ immunological mode of action, and development of quality control assays for MSC cell products.
207 WILL NOT BE PRESENTED
S71
immune-modulation may benefit to testicular torsion-induced infertility. We investigate the therapeutic efficacy and the mechanisms of MSCs in testicular torsion-induced germ cell injury when injected locally. Methods: Six to eight-week-old Sprague-Dawley rats received surgical 720 degree torsion for 3 hours, followed by detorsion on the left testis. 20 μl of phosphate-buffered saline without or with 3 × 10^4 MSCs from human orbital fat tissues (OFSCs) were given for 10 rats, respectively, via local injection into the left testis 30 minutes before detorsion. 20 μl of PBS injection for 6 rats with surgical exposure without torsion served as sham control. Histopathology with Johnsen’s score analysis, Western blot analysis for superoxide dismutase 2, Bax, Caspase-3, human insulin growth factor-1 and human stem cell factor, malondialdehyde (MDA) assay in testis and plasma, hormones level including testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone by ELISA Kits, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and fluorescence staining for P450, Sox-9 and VASA were performed. Results: Animals were sacrificed and bilateral orchiectomy was performed 7 days after torsion-detorsion. Local injections of OFSCs prevented torsioninduced infertility judging from Johnsen’s score. TUNEL assay and Western blot analysis on caspase 3 and Bax demonstrated that OFSCs prevented ischemic/ reperfusion induced intrinsic apoptosis. MDA assay revealed that OFSCs significantly reduced the oxidative stress in the damaged testicular tissues. After the OFSC injection, serum testosterone secretion was increased, while the elevation of FSH triggered by testicular injury was balanced. OFSCs also produced stem cell factor in the damaged testis. Immunofluorescence staining revealed that most transplanted cells surrounded the Leydig cells. Some of transplanted cells differentiated into p450 expressing cells within 7 days. Conclusions: Local injection of allogenic MSCs before surgical detorsion is a simple, clinical friendly procedure to rescue torsion-induced infertility.
208 WILL NOT BE PRESENTED
209 RAPID PURIFICATION OF ADIPOSE-DERIVED MSC FOR USE IN CELLULAR THERAPIES BY SHORT-TERM PANNING D.T. Harris, A. Muise, L. White, M. Badowski Immunobiology, Univ. of Arizona, Tucson, Arizona, USA Mesenchymal stromal cells (MSC) can be isolated from many tissues and are the subject of multiple clinical investigations. Adipose tissue is a rich source of MSC, but often needs enrichment to increase potency and decrease volumes prior to use in cellular therapy. Prolonged in vitro culture enriches and expands MSC but may also induce functional changes, as well as senescence. A method was developed that utilized a method that employed plating followed by a 24h re-plating of digested adipose tissue (stromal vascular fraction obtained from lipoaspirate) in culture flasks to enrich MSC > 90% within 3–5 days, without cellular expansion. FACS analysis demonstrated expression of typical MSC phenotypic markers (CD73, CD105, CD90) along with maintenance of functional capabilities (e.g., CFU-F and directed differentiation), but minimal (less than one doubling) or no cell expansion. After enrichment MSC can then be harvested and resuspended at high concentrations in minimal volumes for clinical use. This panning approach represents a novel methodology that can even be used in a closed culture system to rapidly isolate and purify MSC for clinical therapies, with minimal costs and time.
210 LOCAL INJECTION OF MESENCHYMAL STEM CELLS PROTECTS TESTICULAR TORSION-INDUCED GERM CELL INJURY C. Hsiao1,2, A. Ji2, C. Chang1, C. Cheng1, L. Lee2, J. Ho1,2 1 Taipei Medical University, Taipei, Taiwan, 2Wan Fang Medical Center, Taipei, Taiwan Introduction: Testicular torsion is a urological emergency and infertility is a sequelae of ischemic injury. Surgical reduction/orchiopexy is indicated, but to date there is no effective method for restoration of spermatogenesis. The effects of mesenchymal stem cells (MSCs) on acute tissue injury have been demonstrated, and the abilities of paracrine support, differentiation and
211 NICHE-DEPENDENT REGULATIONS OF METABOLIC BALANCE IN HIGH-FAT DIET INDUCED DIABETIC MICE BY MESENCHYMAL STROMAL CELLS A. Ji2, Y. Chang2, Y. Fu2, O. Lee3, J. Ho1,2 1 Taipei Medical University, Taipei, Taiwan, 2Wan Fang Medical Center, Taipei, Taiwan, 3National Yang-Ming University, Taipei, Taiwan Mesenchymal stromal cells (MSCs) have great potentials to maintain glucose homeostasis and metabolic balance. Here, we demonstrate that, in mice continuously feed with high-fat diet (HFD) and developed non-insulin dependent diabetes, two episodes of systemic MSC transplantations effectively improve the glucose tolerance, blood sugar homeostasis and reduce the body weight through targeting pancreas and insulin-sensitive tissues and organs via site-specific mechanisms. MSCs support pancreatic islet growth by direct differentiation into insulinproducing cells and by mitigating the cytotoxicity of interleukin (IL)-1 and tumor necrosis factor alpha (TNF-α) in the pancreas. Localization of MSCs in the liver and skeletal muscles in diabetic animals is also enhanced and therefore improves glucose tolerance, although long-term engraftment is not observed. MSCs prevent HFD-induced fatty liver development and restore glycogen storage in hepatocytes. Increased expression of IL-1 receptor antagonist and Glut4 in skeletal muscles after MSC transplantation result in better blood sugar homeostasis. Intriguingly, systemic MSC transplantation does not alter adipocyte number, but it decreases HFD-induced cell infiltration in adipose tissues and reduces serum levels of adipokines, including leptin and TNF-α. Taken together, systemic MSC transplantation ameliorates HFD-induced obesity and restores metabolic balance through multi-systemic regulations which are nichedependent. Such findings have supported systemic transplantation of MSCs to correct metabolic imbalance.
212 A XENO-FREE FIBRINOGEN DEPLETION METHOD SUPPORT SCALABLE EXPANSION OF HUMAN MESENCHYMAL STEM CELL Y. Huang1, S. Tsai1, Y. Cheng1, C. Lai2, C. Ku2, Y. Lee1 1 R&D department, AventaCell Biomedical Co., Ltd., New Taipei City, Taiwan, 2Manufacture Department, AventaCell Biomedical Co., Ltd., New Taipei City, Taiwan
supports the isolation and expansion of MSCs from various tissues. The medium is xeno-free and can be used without additional coating of cell culture vessels. We compared MSC-Brew GMP Medium with other xeno-free MSC media as well as FCS- and platelet lysate (PL)-containing formulations. Method: MSCs were isolated from human adipose tissue, bone marrow and umbilical cord using different cultivation media. Proliferation behaviour was monitored for up to 5 passages followed by karyotyping. In parallel, the clonogenic potential was assessed using CFU-F assays. Differentiation potential was tested at passage 3, representing the passage usually used in autologous clinical treatments. The immunomodulatory properties of MSCs are the main reason for their great clinical potential. However, interaction of MSCs with cells of the innate- and adaptive-immune system are not completely understood. Therefore, we analyzed the expression of surface molecules as potential candidates for immune cell interaction using a library of >300 monoclonal antibodies. To this end, we compared MSC-Brew expanded MSCs with or without TNF-alpha/INFgamma or Poly-IC induced licensing. The same cells were tested for their immunosuppressive properties using a standardized in vitro T-cell suppression assay. Conclusion: We demonstrate that MSC-Brew GMP Medium supports efficient isolation and expansion of bona fide MSCs at relevant scale suited for cell therapy. Furthermore, we present a comprehensive characterization of expressed surface molecules which will contribute to deciphering the MSCs’ immunological mode of action, and development of quality control assays for MSC cell products.
207 WILL NOT BE PRESENTED
S71
immune-modulation may benefit to testicular torsion-induced infertility. We investigate the therapeutic efficacy and the mechanisms of MSCs in testicular torsion-induced germ cell injury when injected locally. Methods: Six to eight-week-old Sprague-Dawley rats received surgical 720 degree torsion for 3 hours, followed by detorsion on the left testis. 20 μl of phosphate-buffered saline without or with 3 × 10^4 MSCs from human orbital fat tissues (OFSCs) were given for 10 rats, respectively, via local injection into the left testis 30 minutes before detorsion. 20 μl of PBS injection for 6 rats with surgical exposure without torsion served as sham control. Histopathology with Johnsen’s score analysis, Western blot analysis for superoxide dismutase 2, Bax, Caspase-3, human insulin growth factor-1 and human stem cell factor, malondialdehyde (MDA) assay in testis and plasma, hormones level including testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone by ELISA Kits, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and fluorescence staining for P450, Sox-9 and VASA were performed. Results: Animals were sacrificed and bilateral orchiectomy was performed 7 days after torsion-detorsion. Local injections of OFSCs prevented torsioninduced infertility judging from Johnsen’s score. TUNEL assay and Western blot analysis on caspase 3 and Bax demonstrated that OFSCs prevented ischemic/ reperfusion induced intrinsic apoptosis. MDA assay revealed that OFSCs significantly reduced the oxidative stress in the damaged testicular tissues. After the OFSC injection, serum testosterone secretion was increased, while the elevation of FSH triggered by testicular injury was balanced. OFSCs also produced stem cell factor in the damaged testis. Immunofluorescence staining revealed that most transplanted cells surrounded the Leydig cells. Some of transplanted cells differentiated into p450 expressing cells within 7 days. Conclusions: Local injection of allogenic MSCs before surgical detorsion is a simple, clinical friendly procedure to rescue torsion-induced infertility.
208 WILL NOT BE PRESENTED
209 RAPID PURIFICATION OF ADIPOSE-DERIVED MSC FOR USE IN CELLULAR THERAPIES BY SHORT-TERM PANNING D.T. Harris, A. Muise, L. White, M. Badowski Immunobiology, Univ. of Arizona, Tucson, Arizona, USA Mesenchymal stromal cells (MSC) can be isolated from many tissues and are the subject of multiple clinical investigations. Adipose tissue is a rich source of MSC, but often needs enrichment to increase potency and decrease volumes prior to use in cellular therapy. Prolonged in vitro culture enriches and expands MSC but may also induce functional changes, as well as senescence. A method was developed that utilized a method that employed plating followed by a 24h re-plating of digested adipose tissue (stromal vascular fraction obtained from lipoaspirate) in culture flasks to enrich MSC > 90% within 3–5 days, without cellular expansion. FACS analysis demonstrated expression of typical MSC phenotypic markers (CD73, CD105, CD90) along with maintenance of functional capabilities (e.g., CFU-F and directed differentiation), but minimal (less than one doubling) or no cell expansion. After enrichment MSC can then be harvested and resuspended at high concentrations in minimal volumes for clinical use. This panning approach represents a novel methodology that can even be used in a closed culture system to rapidly isolate and purify MSC for clinical therapies, with minimal costs and time.
210 LOCAL INJECTION OF MESENCHYMAL STEM CELLS PROTECTS TESTICULAR TORSION-INDUCED GERM CELL INJURY C. Hsiao1,2, A. Ji2, C. Chang1, C. Cheng1, L. Lee2, J. Ho1,2 1 Taipei Medical University, Taipei, Taiwan, 2Wan Fang Medical Center, Taipei, Taiwan Introduction: Testicular torsion is a urological emergency and infertility is a sequelae of ischemic injury. Surgical reduction/orchiopexy is indicated, but to date there is no effective method for restoration of spermatogenesis. The effects of mesenchymal stem cells (MSCs) on acute tissue injury have been demonstrated, and the abilities of paracrine support, differentiation and
211 NICHE-DEPENDENT REGULATIONS OF METABOLIC BALANCE IN HIGH-FAT DIET INDUCED DIABETIC MICE BY MESENCHYMAL STROMAL CELLS A. Ji2, Y. Chang2, Y. Fu2, O. Lee3, J. Ho1,2 1 Taipei Medical University, Taipei, Taiwan, 2Wan Fang Medical Center, Taipei, Taiwan, 3National Yang-Ming University, Taipei, Taiwan Mesenchymal stromal cells (MSCs) have great potentials to maintain glucose homeostasis and metabolic balance. Here, we demonstrate that, in mice continuously feed with high-fat diet (HFD) and developed non-insulin dependent diabetes, two episodes of systemic MSC transplantations effectively improve the glucose tolerance, blood sugar homeostasis and reduce the body weight through targeting pancreas and insulin-sensitive tissues and organs via site-specific mechanisms. MSCs support pancreatic islet growth by direct differentiation into insulinproducing cells and by mitigating the cytotoxicity of interleukin (IL)-1 and tumor necrosis factor alpha (TNF-α) in the pancreas. Localization of MSCs in the liver and skeletal muscles in diabetic animals is also enhanced and therefore improves glucose tolerance, although long-term engraftment is not observed. MSCs prevent HFD-induced fatty liver development and restore glycogen storage in hepatocytes. Increased expression of IL-1 receptor antagonist and Glut4 in skeletal muscles after MSC transplantation result in better blood sugar homeostasis. Intriguingly, systemic MSC transplantation does not alter adipocyte number, but it decreases HFD-induced cell infiltration in adipose tissues and reduces serum levels of adipokines, including leptin and TNF-α. Taken together, systemic MSC transplantation ameliorates HFD-induced obesity and restores metabolic balance through multi-systemic regulations which are nichedependent. Such findings have supported systemic transplantation of MSCs to correct metabolic imbalance.
212 A XENO-FREE FIBRINOGEN DEPLETION METHOD SUPPORT SCALABLE EXPANSION OF HUMAN MESENCHYMAL STEM CELL Y. Huang1, S. Tsai1, Y. Cheng1, C. Lai2, C. Ku2, Y. Lee1 1 R&D department, AventaCell Biomedical Co., Ltd., New Taipei City, Taiwan, 2Manufacture Department, AventaCell Biomedical Co., Ltd., New Taipei City, Taiwan