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468. Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood
PLURIPOTENT STEM CELLS 468. Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood
Rui-Jun Su,1,2 David J. Baylink,1 Amanda Nieses,1 Xianmei Meng,1 Jason B. Kiroyan,1 Kimberly J. Payne,2 Yuyou Duan,3 Benjamin Tschudy-Seney,3 Mary Kearns-Jonker,2 K.-H William Lau,4 XiaoBing Zhang.1 1 Department of Medicine, Loma Linda University, Loma Linda, CA; 2Division of Anatomy, Loma Linda University, Loma Linda, CA; 3Department of Internal Medicine, Institute for Regenerative Cures, University of California Davis Medical Center, Sacramento, CA; 4Jerry L. Pettis Memorial VA Medical Center, Loma Linda, CA. The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from adult peripheral blood instead of skin fibroblasts or other cell types will substantially advance the field of regenerative medicine. Many technological breakthroughs have been reported recently. Sendai virus and other vectors can be used to generate integration-free iPSCs from T or B cells. However, iPSCs from T or B cells affect downstream applications, likely due to their genomic rearrangement. Thus far, episomal vector is the simplest approach for generating integration-free iPSCs. Recently, we published an improved episomal vector that leads to a 20-fold increase in the efficiency of generating integration-free iPSCs from cord blood hematopoietic progenitors or CD34+ cells (Meng et al. Molecular Therapy, 2012). To extend this finding to adult peripheral blood (PB) cells, we cultured PB mononuclear cells (MNCs) for 4-6 days in hematopoietic culture conditions. In contrast to CB CD34+ cells, the use of episomal vectors that express OCT4 and SOX2 (OS) and MYC and KLF4 (MK) only led to the generation of 0-1 iPSC colonies per 1x106 cultured PB MNCs (from 1 ml PB) after 3 weeks of reprogramming culture. To further increase the reprogramming efficiency of PB MNCs, we screened several reprogramming enhancing factors and identified a novel factor BCL-XL that increases OSMK-mediated reprogramming by up to 20-fold. In addition, we found that depletion of CD3/CD19+ cells or T/B cells led to a further increase in reprogramming efficiency by 2-5 fold. From 6 independent donors, we were able to generate 10-100 iPSC colonies from 1 ml PB. The generated iPSCs expressed typical pluripotency markers such as NANOG and TRA-1-60 and form teratomas after subcutaneous injection into immunodeficient mice. The presence of episomal plasmids in iPSCs was undetectable after 15 passages. PB iPSCs maintained normal karyotype after long-term in vitro culture. In vitro direct differentiation studies showed that adult PB iPSCs can be efficiently differentiated into mesenchymal stem cells, cardiomyocytes, neural cells and hepatocytes in several weeks. In addition, we found that even without the use of MYC, integrationfree PB iPSCs can also be efficiently generated with OCT4, SOX2, KLF4 and BCL-XL. This is the first report that without the use of strong oncogenes like MYC, LIN28 and SV40 Large T antigen or knockdown of genome guardian p53, integration-free iPSCs can be highly efficiently generated. In conclusion, we have identified a novel combination of reprogramming factors that can generate up to 100 integration-free iPSC colonies from 1 ml PB, an efficiency that is up to 10-fold higher than previously reported. Our discovery should have important applications for iPSC banking and disease modeling.
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469. iPSC-Based Strategy To Correct the Bleeding Phenotype in Hemophilia A Targeting FVIII Expression to Endothelial Cells
Maria Talmon,1 Gabriella Ranaldo,1 Chantal Grosso,1 Angelo Lombardo,2 Simone Merlin,1 Stefania E. Cannizzo,1 Angel Raya,3 Luigi Naldini,2 Piercarla Schinco,4 Antonia Follenzi.1 1 Dept Health Sciences, Università del Piemonte Orientale, Novara, Italy; 2Tiget, H San Raffaele, Milan, Italy; 3Inst. for Bioengineering of Catalonia, Barcelona, Spain; 4Az. Osp/Univ S.Giovanni Battista-Molinette, Torino, Italy. Hemophilia A (HA) is a X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. Current therapy includes infusion of recombinant or plasma-derived FVIII. However, the main complication related to the treatment is the development of neutralizing antibodies anti-FVIII, common in 20-40% of the treated patients. Reprogramming of genetically corrected somatic cells can be used to generate high amount of autologous, disease-free induced Pluripotent Stem Cells (iPSCs), which can be then differentiated into progenitor cells relevant for gene and cell therapy applications. In hemophilic patients, to harvest fibroblasts from skin biopsies is risky; for this reason, we utilized peripheral blood cells as an easyto-access source of cells and reprogrammed mononuclear cells from donors and hemophilic patients. Moreover, to limit the risk of insertional mutagenesis, we reprogramed mononuclear blood cells with a Cre-excisable lentiviral vector (LV) expressing Oct4, Sox2 and Kl4. Using this method we successfully obtained iPSCs both from healthy and hemophilic donors. Cells displayed hESC-like morphology: colonies were compact, uniform and with defined borders when grown on feeder cells. Moreover, iPSCs are positive at alkaline phosphatase staining and expressed specific stem cell markers at RNA and protein level: Nanog, Oct3/4, Sox2, TRA-1-60 and SSEA 3/4. RT-PCR showed activation of the endogenous reprogramming factors in iPSC. Nevertheless, these cells showed demethylation at CpG islands at the core of Oct4 promoter, epigenetic marker of complete reprogramming to a pluripotent state. iPSCs expressed the three germ layers markers and differentiated in adipose, osteogenic and condrogenic cell lineages. Moreover, before reprogramming, HA mononuclear cells were corrected with a LV expressing human B-domain-deleted FVIII (hBDD-FVIII) under control of the PGK promoter and gave rise to iPS colonies. RT-PCR showed that corrected HA-iPSCs expressed hBDD-FVIII for 2 months but later analysis showed that in HA corrected-iPSCs PGK promoter was silenced and hBDD-FVIII was no longer expressed. Correction with LV carrying the hBDD-FVIII under the control of the VEC promoter showed long term expression. Importantly, the obtained iPSCs differentiated into endothelial cells (EC). Indeed, iPSCs during differentiation acquired a typical endothelial-like morphology with expression of EC markers. Then, iPSC-derived EC were transduced with LV expressing GFP under the control of endothelial-specific promoter, Flk-1. Flk-1-GFP+ cells were transplanted in NOD-SCID HA mice. GFP+ cells were detected in liver up to 1m post transplantation. Overall, these data will be instrumental to assess the engraftment, the proliferation and the levels of FVIII expression from differentiated, gene corrected and reprogramming factor free iPSC to confirm the suitability of this approach for hemophilia A gene-cell-therapy.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
Rui-Jun Su,1,2 David J. Baylink,1 Amanda Nieses,1 Xianmei Meng,1 Jason B. Kiroyan,1 Kimberly J. Payne,2 Yuyou Duan,3 Benjamin Tschudy-Seney,3 Mary Kearns-Jonker,2 K.-H William Lau,4 XiaoBing Zhang.1 1 Department of Medicine, Loma Linda University, Loma Linda, CA; 2Division of Anatomy, Loma Linda University, Loma Linda, CA; 3Department of Internal Medicine, Institute for Regenerative Cures, University of California Davis Medical Center, Sacramento, CA; 4Jerry L. Pettis Memorial VA Medical Center, Loma Linda, CA. The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from adult peripheral blood instead of skin fibroblasts or other cell types will substantially advance the field of regenerative medicine. Many technological breakthroughs have been reported recently. Sendai virus and other vectors can be used to generate integration-free iPSCs from T or B cells. However, iPSCs from T or B cells affect downstream applications, likely due to their genomic rearrangement. Thus far, episomal vector is the simplest approach for generating integration-free iPSCs. Recently, we published an improved episomal vector that leads to a 20-fold increase in the efficiency of generating integration-free iPSCs from cord blood hematopoietic progenitors or CD34+ cells (Meng et al. Molecular Therapy, 2012). To extend this finding to adult peripheral blood (PB) cells, we cultured PB mononuclear cells (MNCs) for 4-6 days in hematopoietic culture conditions. In contrast to CB CD34+ cells, the use of episomal vectors that express OCT4 and SOX2 (OS) and MYC and KLF4 (MK) only led to the generation of 0-1 iPSC colonies per 1x106 cultured PB MNCs (from 1 ml PB) after 3 weeks of reprogramming culture. To further increase the reprogramming efficiency of PB MNCs, we screened several reprogramming enhancing factors and identified a novel factor BCL-XL that increases OSMK-mediated reprogramming by up to 20-fold. In addition, we found that depletion of CD3/CD19+ cells or T/B cells led to a further increase in reprogramming efficiency by 2-5 fold. From 6 independent donors, we were able to generate 10-100 iPSC colonies from 1 ml PB. The generated iPSCs expressed typical pluripotency markers such as NANOG and TRA-1-60 and form teratomas after subcutaneous injection into immunodeficient mice. The presence of episomal plasmids in iPSCs was undetectable after 15 passages. PB iPSCs maintained normal karyotype after long-term in vitro culture. In vitro direct differentiation studies showed that adult PB iPSCs can be efficiently differentiated into mesenchymal stem cells, cardiomyocytes, neural cells and hepatocytes in several weeks. In addition, we found that even without the use of MYC, integrationfree PB iPSCs can also be efficiently generated with OCT4, SOX2, KLF4 and BCL-XL. This is the first report that without the use of strong oncogenes like MYC, LIN28 and SV40 Large T antigen or knockdown of genome guardian p53, integration-free iPSCs can be highly efficiently generated. In conclusion, we have identified a novel combination of reprogramming factors that can generate up to 100 integration-free iPSC colonies from 1 ml PB, an efficiency that is up to 10-fold higher than previously reported. Our discovery should have important applications for iPSC banking and disease modeling.
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469. iPSC-Based Strategy To Correct the Bleeding Phenotype in Hemophilia A Targeting FVIII Expression to Endothelial Cells
Maria Talmon,1 Gabriella Ranaldo,1 Chantal Grosso,1 Angelo Lombardo,2 Simone Merlin,1 Stefania E. Cannizzo,1 Angel Raya,3 Luigi Naldini,2 Piercarla Schinco,4 Antonia Follenzi.1 1 Dept Health Sciences, Università del Piemonte Orientale, Novara, Italy; 2Tiget, H San Raffaele, Milan, Italy; 3Inst. for Bioengineering of Catalonia, Barcelona, Spain; 4Az. Osp/Univ S.Giovanni Battista-Molinette, Torino, Italy. Hemophilia A (HA) is a X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. Current therapy includes infusion of recombinant or plasma-derived FVIII. However, the main complication related to the treatment is the development of neutralizing antibodies anti-FVIII, common in 20-40% of the treated patients. Reprogramming of genetically corrected somatic cells can be used to generate high amount of autologous, disease-free induced Pluripotent Stem Cells (iPSCs), which can be then differentiated into progenitor cells relevant for gene and cell therapy applications. In hemophilic patients, to harvest fibroblasts from skin biopsies is risky; for this reason, we utilized peripheral blood cells as an easyto-access source of cells and reprogrammed mononuclear cells from donors and hemophilic patients. Moreover, to limit the risk of insertional mutagenesis, we reprogramed mononuclear blood cells with a Cre-excisable lentiviral vector (LV) expressing Oct4, Sox2 and Kl4. Using this method we successfully obtained iPSCs both from healthy and hemophilic donors. Cells displayed hESC-like morphology: colonies were compact, uniform and with defined borders when grown on feeder cells. Moreover, iPSCs are positive at alkaline phosphatase staining and expressed specific stem cell markers at RNA and protein level: Nanog, Oct3/4, Sox2, TRA-1-60 and SSEA 3/4. RT-PCR showed activation of the endogenous reprogramming factors in iPSC. Nevertheless, these cells showed demethylation at CpG islands at the core of Oct4 promoter, epigenetic marker of complete reprogramming to a pluripotent state. iPSCs expressed the three germ layers markers and differentiated in adipose, osteogenic and condrogenic cell lineages. Moreover, before reprogramming, HA mononuclear cells were corrected with a LV expressing human B-domain-deleted FVIII (hBDD-FVIII) under control of the PGK promoter and gave rise to iPS colonies. RT-PCR showed that corrected HA-iPSCs expressed hBDD-FVIII for 2 months but later analysis showed that in HA corrected-iPSCs PGK promoter was silenced and hBDD-FVIII was no longer expressed. Correction with LV carrying the hBDD-FVIII under the control of the VEC promoter showed long term expression. Importantly, the obtained iPSCs differentiated into endothelial cells (EC). Indeed, iPSCs during differentiation acquired a typical endothelial-like morphology with expression of EC markers. Then, iPSC-derived EC were transduced with LV expressing GFP under the control of endothelial-specific promoter, Flk-1. Flk-1-GFP+ cells were transplanted in NOD-SCID HA mice. GFP+ cells were detected in liver up to 1m post transplantation. Overall, these data will be instrumental to assess the engraftment, the proliferation and the levels of FVIII expression from differentiated, gene corrected and reprogramming factor free iPSC to confirm the suitability of this approach for hemophilia A gene-cell-therapy.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy