- Email: [email protected]
713. Fetal Renal Stem Cells Attenuate Ischemic Injury in the Rat Kidney
SOMATIC STEM CELLS 712. Human Skin Regeneration From Single Gene-Edited Epidermal Stem Cell Clones
Blanca Duarte,1 Francesca Miselli,2 Rodolfo Murillas,1 Alessandra Recchia,2 Marcela Del Río,1,3 Fernando Larcher.1,3 1 Epithelial Biomedicine, CIEMAT-CIBERER, Madrid, Spain; 2 Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Department of Bioengineering, University Carlos III de Madrid (UC3M), Madrid, Spain. Ex vivo gene therapy represents one of the various current strategies suitable to treat genodermatoses such as Epidermolysis Bullosa (EB).Efforts are being put on the development of safer and efficient approaches minimizing genotoxic effects due to transgene-induced insertional mutagenesis. Gene-editing through nuclease-facilitated homologous recombination (HR) has been recently proven feasible on skin cells. We have recently reported the ZFN-facilitated, HRmediated insertion of a marker gene into the AAVS1 locus in a limited number of human epidermal cells that, upon grafting, persisted as small foci in the skin regenerated in immunodeficient mice consistent with a stem cell behavior (Coluccio et al., 2013). However, since these foci comprised less than 1% of the total graft surface, the actual capacity of regeneration of a whole epidermal surface has not been proven. In this study we report the engraftment and persistent skin regeneration achieved with a single stem cells clone isolated from HR-mediated gene targeted human keratinocytes. GFP positive keratinocytes (less than 1%) present in culture after co-infection with IDLV-GFP and Ad-ZFN (designed for the AAVS1 locus) were sorted and expanded in culture. Although previous attempts of FACS-selecting viable cells containing less than 5% of GFP positive human keratinocytes failed, in this case supplementation of the culture medium with a ROCK inhibitor allowed the expansion of sorted keratinocytes displaying stem features. Only one of the very few GFP-positive keratinocyte colonies maintained an undifferentiated phenotype and grew larger than two-centimeters in diameter, consisting with the characteristics of a holoclone. The expanded population displayed a highly homogeneous morphology and a sharp peak of fluorescence. PCR analysis of DNA isolated from the clone showed bands consistent with “on target” integration (of the HR-cassette containing the GFP gene) on the AAVS1 locus. Expanded keratinocytes were used to prepare bioengineered skin equivalents for grafting to immunodeficient mice. Four weeks after grafting, human skin regeneration was confirmed through GFP epifluorescence. Histological analysis revealed the regeneration of a human skin with normal architecture and epidermal differentiation markers. Molecular analysis of regenerated skin biopsies is ongoing to ruling out selection of putative off-target or rearrangement events. Our results demonstrate that gene-edited epidermal stem cells preserve their plentiful regenerative capacity without harm and confirm that a strategy based on skin regeneration from very limited number of gene corrected stem cell clones ex vivo is achievable.
713. Fetal Renal Stem Cells Attenuate Ischemic Injury in the Rat Kidney Marina G. M. C. M. da Cunha,1 Silvia Zia,1 Fanny O. Arcolino,1 Elena Levtchenko,1 Jan Deprest,1 Jaan Toelen.1 1 Development and Regeneration, KULeuven, Leuven, Belgium.
Introduction: Despite extensive research into an effective treatment for acute renal injury (AKI), the mortality rate still remains high. Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, as a subtype of these cells is derived from the fetal renal tract. We hypothesize that hAFSCs delivered in rats with renal ischemia and reperfusion (I/R) injury, have a nephroprotective effect due to their immunomodulatory and anti-inflammatory effect, preventing extensive fibrosis as a late complication. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Materials and Methods: In the first phase of this study, we isolated hAFSCs from human amniotic fluid samples and characterized their immunophenotypic and differentiation properties. Out of 120 clones a cell line with a high expression of CD24 and CD117 and the renal progenitor markers Six2 and Lin1 was selected. In the second phase, I/R injury was performed in thirty male 8week-old Wistar rats and treatment via direct arterial injection was performed 6 hours later. The control group (n=10) received 800ml of vehicle, the treated group (n =10) received 1x106 hAFSCs in 800ml of vehicle. Serum creatinine and proteinuria levels were measured at 24 hours, 48 hours and 2 months after I\R injury. Histological analysis was performed to quantify tubular necrosis and hyaline cast formation at 48 hours, and interstitial fibrosis by Masson’s Trichrome and PAS staining at 2 months. Additionally, the expression of Ki-67, ED-1 and α-SMA was assessed using immunohistochemistry at 48 hours. Results: A subtype of hAFSCs with renal marker expression has been isolated and characterized. These cells showed a nephroprotective effect in the rat ischemia model. There was a significant lower creatinine and proteinuria level at 24 and48 hours. We observed significantly less tubulointerstitial injury, which was characterized by a higher tubular proliferation, lower inflammatory response and an inhibition of myofibroblast expression. The treated group also showed less fibrosis at 2 months. Conclusion: We conclude a subset of cells from hAFSCs with renal phenotype is nephroprotective when delivered in rats with renal ischemia and reperfusion injury.
714. Combined Preconditioning and In Vivo Chemoselection With 6-Thioguanine for Selection of Genetically Modified Hematopoietic Stem Cells
Katrin Hacke,1 Janet A. Treger,1 Brooke T. Bogan,1 Valerie Rezek,1 Munetoshi Narukawa,2 Saki Shimizu,2 Pei-Qi Liu,3 Andreas Reik,3 André M. Lieber,4 Gay M. Crooks,5 Donald B. Kohn,6 Dong Sung An,2 Scott G. Kitchen,1 Philip D. Gregory,3 Gregory J. Cost,3 Michael C. Holmes,3 Noriyuki Kasahara.1 1 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles; 2School of Nursing, University of California, Los Angeles; 3Sangamo BioSciences, Richmond; 4Division of Medical Genetics, Department of Medicine, University of Washington, Seattle; 5Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles; 6Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles. We have recently developed a novel and highly efficient strategy that uses the purine analog 6-thioguanine (6TG) as a single agent for both preconditioning and in vivo chemoselection of hypoxanthine/ guanine-phosphoribosyltransferase (HPRT)-deficient hematopoietic stem and progenitor cells (HSPC). In a murine model of bone marrow (BM) transplantation, combined 6TG conditioning and post-transplantation chemoselection consistently achieved ~95% engraftment of HPRT-deficient donor BM in the absence of any other cytotoxic conditioning regimen. Moreover, long-term reconstitution of histologically and immunophenotypically normal hematopoiesis was observed in both primary and secondary recipients, without significant toxicity. To translate this strategy for combined 6TG preconditioning and chemoselection into a clinically feasible approach, we are testing two different approaches for genetic modification of normal HSPC to render them HPRT-deficient and thus 6TG-resistant: (1) We have constructed and validated a lentiviral vector expressing short-hairpin RNA (shRNA) that targets a consensus sequence present in both human and murine HPRT genes, and which achieves effective longterm reduction of HPRT expression in cells from either species in vitro. In both primary murine BM cells and human CD34+ HSPC S275
Blanca Duarte,1 Francesca Miselli,2 Rodolfo Murillas,1 Alessandra Recchia,2 Marcela Del Río,1,3 Fernando Larcher.1,3 1 Epithelial Biomedicine, CIEMAT-CIBERER, Madrid, Spain; 2 Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Department of Bioengineering, University Carlos III de Madrid (UC3M), Madrid, Spain. Ex vivo gene therapy represents one of the various current strategies suitable to treat genodermatoses such as Epidermolysis Bullosa (EB).Efforts are being put on the development of safer and efficient approaches minimizing genotoxic effects due to transgene-induced insertional mutagenesis. Gene-editing through nuclease-facilitated homologous recombination (HR) has been recently proven feasible on skin cells. We have recently reported the ZFN-facilitated, HRmediated insertion of a marker gene into the AAVS1 locus in a limited number of human epidermal cells that, upon grafting, persisted as small foci in the skin regenerated in immunodeficient mice consistent with a stem cell behavior (Coluccio et al., 2013). However, since these foci comprised less than 1% of the total graft surface, the actual capacity of regeneration of a whole epidermal surface has not been proven. In this study we report the engraftment and persistent skin regeneration achieved with a single stem cells clone isolated from HR-mediated gene targeted human keratinocytes. GFP positive keratinocytes (less than 1%) present in culture after co-infection with IDLV-GFP and Ad-ZFN (designed for the AAVS1 locus) were sorted and expanded in culture. Although previous attempts of FACS-selecting viable cells containing less than 5% of GFP positive human keratinocytes failed, in this case supplementation of the culture medium with a ROCK inhibitor allowed the expansion of sorted keratinocytes displaying stem features. Only one of the very few GFP-positive keratinocyte colonies maintained an undifferentiated phenotype and grew larger than two-centimeters in diameter, consisting with the characteristics of a holoclone. The expanded population displayed a highly homogeneous morphology and a sharp peak of fluorescence. PCR analysis of DNA isolated from the clone showed bands consistent with “on target” integration (of the HR-cassette containing the GFP gene) on the AAVS1 locus. Expanded keratinocytes were used to prepare bioengineered skin equivalents for grafting to immunodeficient mice. Four weeks after grafting, human skin regeneration was confirmed through GFP epifluorescence. Histological analysis revealed the regeneration of a human skin with normal architecture and epidermal differentiation markers. Molecular analysis of regenerated skin biopsies is ongoing to ruling out selection of putative off-target or rearrangement events. Our results demonstrate that gene-edited epidermal stem cells preserve their plentiful regenerative capacity without harm and confirm that a strategy based on skin regeneration from very limited number of gene corrected stem cell clones ex vivo is achievable.
713. Fetal Renal Stem Cells Attenuate Ischemic Injury in the Rat Kidney Marina G. M. C. M. da Cunha,1 Silvia Zia,1 Fanny O. Arcolino,1 Elena Levtchenko,1 Jan Deprest,1 Jaan Toelen.1 1 Development and Regeneration, KULeuven, Leuven, Belgium.
Introduction: Despite extensive research into an effective treatment for acute renal injury (AKI), the mortality rate still remains high. Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, as a subtype of these cells is derived from the fetal renal tract. We hypothesize that hAFSCs delivered in rats with renal ischemia and reperfusion (I/R) injury, have a nephroprotective effect due to their immunomodulatory and anti-inflammatory effect, preventing extensive fibrosis as a late complication. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Materials and Methods: In the first phase of this study, we isolated hAFSCs from human amniotic fluid samples and characterized their immunophenotypic and differentiation properties. Out of 120 clones a cell line with a high expression of CD24 and CD117 and the renal progenitor markers Six2 and Lin1 was selected. In the second phase, I/R injury was performed in thirty male 8week-old Wistar rats and treatment via direct arterial injection was performed 6 hours later. The control group (n=10) received 800ml of vehicle, the treated group (n =10) received 1x106 hAFSCs in 800ml of vehicle. Serum creatinine and proteinuria levels were measured at 24 hours, 48 hours and 2 months after I\R injury. Histological analysis was performed to quantify tubular necrosis and hyaline cast formation at 48 hours, and interstitial fibrosis by Masson’s Trichrome and PAS staining at 2 months. Additionally, the expression of Ki-67, ED-1 and α-SMA was assessed using immunohistochemistry at 48 hours. Results: A subtype of hAFSCs with renal marker expression has been isolated and characterized. These cells showed a nephroprotective effect in the rat ischemia model. There was a significant lower creatinine and proteinuria level at 24 and48 hours. We observed significantly less tubulointerstitial injury, which was characterized by a higher tubular proliferation, lower inflammatory response and an inhibition of myofibroblast expression. The treated group also showed less fibrosis at 2 months. Conclusion: We conclude a subset of cells from hAFSCs with renal phenotype is nephroprotective when delivered in rats with renal ischemia and reperfusion injury.
714. Combined Preconditioning and In Vivo Chemoselection With 6-Thioguanine for Selection of Genetically Modified Hematopoietic Stem Cells
Katrin Hacke,1 Janet A. Treger,1 Brooke T. Bogan,1 Valerie Rezek,1 Munetoshi Narukawa,2 Saki Shimizu,2 Pei-Qi Liu,3 Andreas Reik,3 André M. Lieber,4 Gay M. Crooks,5 Donald B. Kohn,6 Dong Sung An,2 Scott G. Kitchen,1 Philip D. Gregory,3 Gregory J. Cost,3 Michael C. Holmes,3 Noriyuki Kasahara.1 1 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles; 2School of Nursing, University of California, Los Angeles; 3Sangamo BioSciences, Richmond; 4Division of Medical Genetics, Department of Medicine, University of Washington, Seattle; 5Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles; 6Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles. We have recently developed a novel and highly efficient strategy that uses the purine analog 6-thioguanine (6TG) as a single agent for both preconditioning and in vivo chemoselection of hypoxanthine/ guanine-phosphoribosyltransferase (HPRT)-deficient hematopoietic stem and progenitor cells (HSPC). In a murine model of bone marrow (BM) transplantation, combined 6TG conditioning and post-transplantation chemoselection consistently achieved ~95% engraftment of HPRT-deficient donor BM in the absence of any other cytotoxic conditioning regimen. Moreover, long-term reconstitution of histologically and immunophenotypically normal hematopoiesis was observed in both primary and secondary recipients, without significant toxicity. To translate this strategy for combined 6TG preconditioning and chemoselection into a clinically feasible approach, we are testing two different approaches for genetic modification of normal HSPC to render them HPRT-deficient and thus 6TG-resistant: (1) We have constructed and validated a lentiviral vector expressing short-hairpin RNA (shRNA) that targets a consensus sequence present in both human and murine HPRT genes, and which achieves effective longterm reduction of HPRT expression in cells from either species in vitro. In both primary murine BM cells and human CD34+ HSPC S275