- Email: [email protected]
862. Hypoxia Inhibits Senescence and Maintains Mesenchymal Stem Cell Properties through Downregulation of E2A-p21 by HIF-TWIST
STEM CELL THERAPIES III fibroblasts on mitomycin-c treated mouse embryonic fibroblasts and cultured in the medium for CM ESCs, and found that they formed round structures 14 to 21 days after the transduction. These iPS-CIs expressed ES markers such as SSEA3, SSEA4 and TRA-1-60, and showed alkaline phosphatase activity, suggesting that these iPS-CIs have been reprogrammed. On the other hand, when these iPS-CIs were transplanted to the testis or the hypodermis in immune-deficient mice, they formed poorly differentiated malignant tumors very similar to human embryonal carcinoma, indicating that iPS-CIs had been transformed in the process of iPS generation. It should be carefully examined whether iPSCs established from patients with chromosome instability before they are applied to the regenerative medicine. The possibility to target Oct3/4 or c-Myc to treat patients who unfortunately had iPSC-derived embryonal carcinoma (iPS-EC) in clinical practice of regenerative medicine will be discussed.
862. Hypoxia Inhibits Senescence and Maintains Mesenchymal Stem Cell Properties through Downregulation of E2A-p21 by HIF-TWIST
Chih-Chien Tsai,1,3 Yann-Jang Chen,2 Tu-Lai Yew,3 Shih-Chieh Hung.1,3,4 1 Institute of Pharmacology, Faculty of Medicine, National YangMing University, Taipei, Taiwan; 2Institute of Genome Sciences, Department of Life Sciences, National Yang-Ming University, Taipei, Taiwan; 3Stem Cell Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; 4Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan. Low-density culture provides a method for rapid expansion of human mesenchymal stem cells (MSCs). However, MSCs enriched by this method underwent senescence and lost stem cell properties, whereas combined with hypoxic culture preserved the early stem cell properties. The mechanism was mediated through direct downregulation of E2A-p21 by HIF-1α-TWIST. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST upregulated E2A and p21 in hypoxic cells. Further, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanding by up to 100 population doublings under hypoxic conditions exhibit telomerase activity with maintained telomere length, have a normal karyotyping and genetic integrity, and do not form tumors. These results provide a method for efficiently expanding MSCs without losing stem cell properties and increasing tumorigenecity.
863. IPS Technology and It’s Implications for Gene and Cell Therapy Duanqing Pei.1 1 Guangzhou Institutes of Biomedicine and Health, CAS, Guangzhou, China.
The generation of induced pluripotent stem cells (iPSCs) from somatic cells by exogenous factors opens a new era of possibilities for regenerative medicine. Potentially, once safety concerns are overcome differentiated lineages derived from patient specific iPSCs could be used for transplantation purposes and this would change the way we perceive medicine nowadays. However, for patients with genetic diseases the mutation will need to be corrected as otherwise the functional defect will remain. And the same can be said for other acquired diseases as well in which viruses can access cells through a specific receptor, for instance AIDS. To overcome these issues there is growing interest in applying zinc finger technology for either correcting mutations or knocking out genes. But this technology has Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
limitations and is lengthy. In this regard, gene therapy of iPSCs or their differentiated progeny using clinically approved vectors could be a valuable possibility. Here we described the generation of iPSCs from a Chinese patient with Wilson’s disease that bears the R778L Chinese hotspot mutation in the ATP7B gene, whose product is a liver enzyme responsible for copper export into bile and blood. These iPSCs were pluripotent and could be readily differentiated into functional hepatocytes that display abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Importantly, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B could reverse the functional defect. In the future, hepatocytes from similarly genetically corrected iPSCs may also be an option for autologous transplantation in Wilson’s disease. Notably, we also provide proof of principle that our experimental model based on iPSCs can be used to screen compounds aimed to correct the abnormality.
864. A GMP-Compliant Process for the ScaleUp and Large Scale Banking of Human Embryonic Stem Cells
Lara J. Ausubel,1 Patricia Lopez,1 Rui-Lin Wu,1 Tania Aguilar,1 Jing Chai,1 Wei Dang,1 Patricia Huang,1 Rafat Khan,1 Derek Kong,1 Robin Wesselschmidt,1 David Hsu,1 Larry Couture.1 1 Center for Biomedicine and Genetics, City of Hope, Duarte, CA. Human pluripotent stem cells represent an important resource for the development of cellular therapies in the field of regenerative medicine. However, current culture methods do not allow for efficient scale-up and banking, thus presenting a major challenge to the field. Our objective is to apply our cGMP biologics and cell line process development expertise to the establishment of scalable cGMP-complaint banking processes for human embryonic stem cells (hESCs) and to generate cGMP hESC banks that could be used to support the development of cells for use in clinical trials. Though there are several well established research methods for the culture of hESCs, many use a co-culture method comprised of animal or human feeder cells and research-grade growth factors, all of which may contribute to an uncertain product profile containing unknown elements such as viruses with potential risk to human health or cell product integrity. We describe our process for large-scale banking which involves adapting hESC lines from standard research laboratory conditions to cGMP-compatible conditions and methods. This process allows for the scale-up culture and cryopreservation of 200-500 vial master cell banks under feeder-free conditions. Of particular importance was the development of qualifying assays to demonstrate the pluripotency, functionality, and purity of the hESCs that are needed to release these banks for clinical use. Using these methods, we have successfully banked hESCs at large scale under cGMP conditions.
865. Safeguarding Nonhuman Primate iPS Cells with Suicide Genes
Bonan Zhong,1 Korashon L. Watts,1 Martin E. Wohlfahrt,1 Jennifer E. Adair,1 Joerg Enssle,1 Hans-Peter Kiem.1,2,3 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Departments of Medicine, University of Washington School of Medicine, Seattle, WA; 3Departments of Pathology, University of Washington School of Medicine, Seattle, WA.
The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, S331
862. Hypoxia Inhibits Senescence and Maintains Mesenchymal Stem Cell Properties through Downregulation of E2A-p21 by HIF-TWIST
Chih-Chien Tsai,1,3 Yann-Jang Chen,2 Tu-Lai Yew,3 Shih-Chieh Hung.1,3,4 1 Institute of Pharmacology, Faculty of Medicine, National YangMing University, Taipei, Taiwan; 2Institute of Genome Sciences, Department of Life Sciences, National Yang-Ming University, Taipei, Taiwan; 3Stem Cell Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; 4Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan. Low-density culture provides a method for rapid expansion of human mesenchymal stem cells (MSCs). However, MSCs enriched by this method underwent senescence and lost stem cell properties, whereas combined with hypoxic culture preserved the early stem cell properties. The mechanism was mediated through direct downregulation of E2A-p21 by HIF-1α-TWIST. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST upregulated E2A and p21 in hypoxic cells. Further, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanding by up to 100 population doublings under hypoxic conditions exhibit telomerase activity with maintained telomere length, have a normal karyotyping and genetic integrity, and do not form tumors. These results provide a method for efficiently expanding MSCs without losing stem cell properties and increasing tumorigenecity.
863. IPS Technology and It’s Implications for Gene and Cell Therapy Duanqing Pei.1 1 Guangzhou Institutes of Biomedicine and Health, CAS, Guangzhou, China.
The generation of induced pluripotent stem cells (iPSCs) from somatic cells by exogenous factors opens a new era of possibilities for regenerative medicine. Potentially, once safety concerns are overcome differentiated lineages derived from patient specific iPSCs could be used for transplantation purposes and this would change the way we perceive medicine nowadays. However, for patients with genetic diseases the mutation will need to be corrected as otherwise the functional defect will remain. And the same can be said for other acquired diseases as well in which viruses can access cells through a specific receptor, for instance AIDS. To overcome these issues there is growing interest in applying zinc finger technology for either correcting mutations or knocking out genes. But this technology has Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
limitations and is lengthy. In this regard, gene therapy of iPSCs or their differentiated progeny using clinically approved vectors could be a valuable possibility. Here we described the generation of iPSCs from a Chinese patient with Wilson’s disease that bears the R778L Chinese hotspot mutation in the ATP7B gene, whose product is a liver enzyme responsible for copper export into bile and blood. These iPSCs were pluripotent and could be readily differentiated into functional hepatocytes that display abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Importantly, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B could reverse the functional defect. In the future, hepatocytes from similarly genetically corrected iPSCs may also be an option for autologous transplantation in Wilson’s disease. Notably, we also provide proof of principle that our experimental model based on iPSCs can be used to screen compounds aimed to correct the abnormality.
864. A GMP-Compliant Process for the ScaleUp and Large Scale Banking of Human Embryonic Stem Cells
Lara J. Ausubel,1 Patricia Lopez,1 Rui-Lin Wu,1 Tania Aguilar,1 Jing Chai,1 Wei Dang,1 Patricia Huang,1 Rafat Khan,1 Derek Kong,1 Robin Wesselschmidt,1 David Hsu,1 Larry Couture.1 1 Center for Biomedicine and Genetics, City of Hope, Duarte, CA. Human pluripotent stem cells represent an important resource for the development of cellular therapies in the field of regenerative medicine. However, current culture methods do not allow for efficient scale-up and banking, thus presenting a major challenge to the field. Our objective is to apply our cGMP biologics and cell line process development expertise to the establishment of scalable cGMP-complaint banking processes for human embryonic stem cells (hESCs) and to generate cGMP hESC banks that could be used to support the development of cells for use in clinical trials. Though there are several well established research methods for the culture of hESCs, many use a co-culture method comprised of animal or human feeder cells and research-grade growth factors, all of which may contribute to an uncertain product profile containing unknown elements such as viruses with potential risk to human health or cell product integrity. We describe our process for large-scale banking which involves adapting hESC lines from standard research laboratory conditions to cGMP-compatible conditions and methods. This process allows for the scale-up culture and cryopreservation of 200-500 vial master cell banks under feeder-free conditions. Of particular importance was the development of qualifying assays to demonstrate the pluripotency, functionality, and purity of the hESCs that are needed to release these banks for clinical use. Using these methods, we have successfully banked hESCs at large scale under cGMP conditions.
865. Safeguarding Nonhuman Primate iPS Cells with Suicide Genes
Bonan Zhong,1 Korashon L. Watts,1 Martin E. Wohlfahrt,1 Jennifer E. Adair,1 Joerg Enssle,1 Hans-Peter Kiem.1,2,3 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Departments of Medicine, University of Washington School of Medicine, Seattle, WA; 3Departments of Pathology, University of Washington School of Medicine, Seattle, WA.
The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, S331