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288. Systematic Development and Qualification of Assays for the Characterization of Human Embryonic Stem Cells and Their Differentiated Progeny
PRESIDENTIAL SYMPOSIUM 288. Systematic Development and Qualification of Assays for the Characterization of Human Embryonic Stem Cells and Their Differentiated Progeny
Wei Dang,1 Patricia Huang,1 Aparna Krishnan,1 Derek Kong,1 Jing Chai,1 Katie Payte,1 Patricia Lopez,1 Tania Aguilar,1 Lara Ausubel,1 Sylvana Couture,1 Christine Hall,1 Robin Wesselschmidt,1 David Hsu,1 Larry Couture.1 1 Center for Applied Technology Development, Center for Biomedicine and Genetics, Beckman Research Institute of City of Hope, Duarte, CA. Stem cell therapy holds the promise to treat, perhaps cure many diseases, injuries and degenerative conditions of aging. Human embryonic stem cells (hESCs) are the starting cell population for many of these potential therapies since they are capable of both seemingly indefinite proliferation in the pluripotent state and possess the ability to differentiate into all of the cell types found in the adult. It is vital that the cellular therapeutics derived from these highly proliferative, highly plastic cells are well characterized prior to clinical use and the assays used to characterize these cellular populations are well developed. Flow cytometric and real-time quantitative polymerase chain reaction (RT-qPCR) assays are often used to detect and quantify the expression of cell specific markers. We describe a systematic method for the development and qualification of such assays for pluripotent cells and their differentiated progeny. This method is comprised of five activities: 1) selection and testing of reagents; 2) selection and banking of positive control and negative control cells; 3) assessing assay reproducibility (including both intra- and inter-assays); 4) performing qualification runs; 5) writing assay qualification technical reports and standard operating procedures (SOPs). We describe, with specific examples, how we evaluate assay parameters such as precision, accuracy, sensitivity, specificity, and dynamic range (linearity) during assay qualification. As one example, RT-qPCR assay sensitivity was evaluated by spiking a small percentage of hESCs into differentiated cells and the expression level of Oct4 and Nanog genes was evaluated by RT-qPCR. Our results suggest that as low as 0.002% of hESCs spiked into the differentiated cell population could be reliably detected. We describe the application of our systematic approach to assay development to the generation of qualified flow cytometric assays for markers including Oct3/4, SSEA1, SSEA4, Tra-1-60, and Tra-1-81.
289. Generation of Footprint Free iPSCs from CD34+ and PBMCs Using Sendai Virus Vector and Application of Alkaline Phosphatase Live Stainning Dye
Andrew Fontes D. Fontes,1 Rene Quintanilla,1 Uma Lakshmipathy,1 Pauline Lieu.1 1 Primary and Stem Cell Systems, Life Technologies, Carlsbad, CA. Induced pluripotent stem cells (iPSCs) provide unprecedented access to patient-specific iPSCs for in -vitro disease modeling and downstream clinical applications. However, a number of obstacles exist for the derivation of iPSC for research and therapeutic applications. First is the lack of an efficient method for the generation of footprint-free iPSCs. Current viral methods have the potential of genomic modifications caused by proviral integrations while synthetic methods remain cumbersome and are limited by cell type. Second is the ability to identify iPSCs using specific and non toxic methods while maintaining the integrity of live cells. Current methods utilize expensive and non sterile antibodies for live staining or chromogenic and fluorogenic compounds requiring cell fixation. Here we report the generation of zero-footprint iPSCs by an RNA Sendai virus (CytoTuneTM iPS Reprogramming Kit) to reprogram cells from multiple blood sources including Periphery Blood Mononuclear S114
cells (PBMCs) at high efficiency. PBMCs are readily available through routine venipuncture harvesting methods and eliminate invasive biopsies and extensive culturing and passaging necessary with fibroblasts. Sendai virus is an RNA virus that replicates in the cytoplasm and does not integrate in the cellular genome, thus enabling the generation of footprint-free iPSCs. The iPSCs generated in these systems are able to proliferate, display pluripotent markers, and give rise to EBs that can differentiate to all the three lineages. To further ease the identification of iPSCs we have introduced a new Alkaline Phosphatase Live Stain Dye, a novel flourogenic substrate for alkaline phosphotase that shows robust labeling of iPSC colonies 20-30 minutes after addition to basal culture media. Generation of footprintfree iPSCs using CytoTune iPS reprogramming kit in combination with AP Live Stain will simplify reprogramming workflow and enhance the quality of iPSCs.
290. Comparison of Enzymatic and NonEnzymatic Passaging of Human Embryonic Stem Cells
Patricia M. Lopez,1 Lara J. Ausubel,1 Rui-Lin Wu,1 Tania F. Aguilar,1 Jing Chai,1 Katie J. Payte,1 Wei Dang,1 Patricia Huang,1 Derek Kong,1 Aparna Krishnan,1 Robin Wesselschmidt,1 David Hsu,1 Larry Couture.1 1 Center for Biomedicine and Genetics, Center for Applied Technology Development, Beckman Research Institute of City of Hope, Duarte, CA. Human embryonic stem cells (hESCs) have enormous potential as a model for early human development as well as starting material for the development of cell replacement therapies. As such, methods need to be developed that can exploit the highly proliferative capability of these cells under conditions amenable to standardization and using defined non-xenogenic reagents. In order to propagate these cells at large scale while maintaining a stable expression of appropriate markers and normal karyotype, optimal culture conditions need to be determined. Here, we present results comparing the propagation of stem cells using non-enzymatic and enzymatic passaging methods. We examined numerous combinations of enzymes and commercially available enzyme-free dissociating reagents to develop an optimal protocol that allows for a reliable method for bulk passage of hESCs in support of large scale culture. We report the propagation efficiency and characterization of several hESC lines as well as results showing that non-enzymatic passaging of stem cells allows the cells to retain good viability as well as to expand as expected. Moreover, hESCs expressed pluripotency markers and maintained their normal karyotype under these conditions. These results provide valuable information that will assist in achieving the goal of the large scale expansion of hESCs that is required if they are to serve as the starting material for the development of cellular therapies.
Presidential Symposium 291. Human Visual Cortex Response to Retinal Gene Therapy Re-Administration Manzar Ashtari,1 Laura Cyckowski,1 Kathleen Marshall,1 Daniel Chung,1 Albert Maguire,1 Kenneth Shindler,1 Jean Bennett.1 1 Children’s Hospital of Philadelphia, Philadelphia, PA.
Background: Previously, we showed that in humans with Leber’s congenital amaurosis due to RPE65 mutations (LCA2), the visual cortex can be made responsive to visual input through unilateral ocular gene therapy. Recently, our team performed a 2nd gene administration to the contralateral eyes of 5 previously treated patients. In addition to clinical tests, functional magnetic resonance imaging (fMRI) was performed to evaluate cortical response and determine if prior exposure to RPE65 genes would have any immunologic effects. fMRI Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
Wei Dang,1 Patricia Huang,1 Aparna Krishnan,1 Derek Kong,1 Jing Chai,1 Katie Payte,1 Patricia Lopez,1 Tania Aguilar,1 Lara Ausubel,1 Sylvana Couture,1 Christine Hall,1 Robin Wesselschmidt,1 David Hsu,1 Larry Couture.1 1 Center for Applied Technology Development, Center for Biomedicine and Genetics, Beckman Research Institute of City of Hope, Duarte, CA. Stem cell therapy holds the promise to treat, perhaps cure many diseases, injuries and degenerative conditions of aging. Human embryonic stem cells (hESCs) are the starting cell population for many of these potential therapies since they are capable of both seemingly indefinite proliferation in the pluripotent state and possess the ability to differentiate into all of the cell types found in the adult. It is vital that the cellular therapeutics derived from these highly proliferative, highly plastic cells are well characterized prior to clinical use and the assays used to characterize these cellular populations are well developed. Flow cytometric and real-time quantitative polymerase chain reaction (RT-qPCR) assays are often used to detect and quantify the expression of cell specific markers. We describe a systematic method for the development and qualification of such assays for pluripotent cells and their differentiated progeny. This method is comprised of five activities: 1) selection and testing of reagents; 2) selection and banking of positive control and negative control cells; 3) assessing assay reproducibility (including both intra- and inter-assays); 4) performing qualification runs; 5) writing assay qualification technical reports and standard operating procedures (SOPs). We describe, with specific examples, how we evaluate assay parameters such as precision, accuracy, sensitivity, specificity, and dynamic range (linearity) during assay qualification. As one example, RT-qPCR assay sensitivity was evaluated by spiking a small percentage of hESCs into differentiated cells and the expression level of Oct4 and Nanog genes was evaluated by RT-qPCR. Our results suggest that as low as 0.002% of hESCs spiked into the differentiated cell population could be reliably detected. We describe the application of our systematic approach to assay development to the generation of qualified flow cytometric assays for markers including Oct3/4, SSEA1, SSEA4, Tra-1-60, and Tra-1-81.
289. Generation of Footprint Free iPSCs from CD34+ and PBMCs Using Sendai Virus Vector and Application of Alkaline Phosphatase Live Stainning Dye
Andrew Fontes D. Fontes,1 Rene Quintanilla,1 Uma Lakshmipathy,1 Pauline Lieu.1 1 Primary and Stem Cell Systems, Life Technologies, Carlsbad, CA. Induced pluripotent stem cells (iPSCs) provide unprecedented access to patient-specific iPSCs for in -vitro disease modeling and downstream clinical applications. However, a number of obstacles exist for the derivation of iPSC for research and therapeutic applications. First is the lack of an efficient method for the generation of footprint-free iPSCs. Current viral methods have the potential of genomic modifications caused by proviral integrations while synthetic methods remain cumbersome and are limited by cell type. Second is the ability to identify iPSCs using specific and non toxic methods while maintaining the integrity of live cells. Current methods utilize expensive and non sterile antibodies for live staining or chromogenic and fluorogenic compounds requiring cell fixation. Here we report the generation of zero-footprint iPSCs by an RNA Sendai virus (CytoTuneTM iPS Reprogramming Kit) to reprogram cells from multiple blood sources including Periphery Blood Mononuclear S114
cells (PBMCs) at high efficiency. PBMCs are readily available through routine venipuncture harvesting methods and eliminate invasive biopsies and extensive culturing and passaging necessary with fibroblasts. Sendai virus is an RNA virus that replicates in the cytoplasm and does not integrate in the cellular genome, thus enabling the generation of footprint-free iPSCs. The iPSCs generated in these systems are able to proliferate, display pluripotent markers, and give rise to EBs that can differentiate to all the three lineages. To further ease the identification of iPSCs we have introduced a new Alkaline Phosphatase Live Stain Dye, a novel flourogenic substrate for alkaline phosphotase that shows robust labeling of iPSC colonies 20-30 minutes after addition to basal culture media. Generation of footprintfree iPSCs using CytoTune iPS reprogramming kit in combination with AP Live Stain will simplify reprogramming workflow and enhance the quality of iPSCs.
290. Comparison of Enzymatic and NonEnzymatic Passaging of Human Embryonic Stem Cells
Patricia M. Lopez,1 Lara J. Ausubel,1 Rui-Lin Wu,1 Tania F. Aguilar,1 Jing Chai,1 Katie J. Payte,1 Wei Dang,1 Patricia Huang,1 Derek Kong,1 Aparna Krishnan,1 Robin Wesselschmidt,1 David Hsu,1 Larry Couture.1 1 Center for Biomedicine and Genetics, Center for Applied Technology Development, Beckman Research Institute of City of Hope, Duarte, CA. Human embryonic stem cells (hESCs) have enormous potential as a model for early human development as well as starting material for the development of cell replacement therapies. As such, methods need to be developed that can exploit the highly proliferative capability of these cells under conditions amenable to standardization and using defined non-xenogenic reagents. In order to propagate these cells at large scale while maintaining a stable expression of appropriate markers and normal karyotype, optimal culture conditions need to be determined. Here, we present results comparing the propagation of stem cells using non-enzymatic and enzymatic passaging methods. We examined numerous combinations of enzymes and commercially available enzyme-free dissociating reagents to develop an optimal protocol that allows for a reliable method for bulk passage of hESCs in support of large scale culture. We report the propagation efficiency and characterization of several hESC lines as well as results showing that non-enzymatic passaging of stem cells allows the cells to retain good viability as well as to expand as expected. Moreover, hESCs expressed pluripotency markers and maintained their normal karyotype under these conditions. These results provide valuable information that will assist in achieving the goal of the large scale expansion of hESCs that is required if they are to serve as the starting material for the development of cellular therapies.
Presidential Symposium 291. Human Visual Cortex Response to Retinal Gene Therapy Re-Administration Manzar Ashtari,1 Laura Cyckowski,1 Kathleen Marshall,1 Daniel Chung,1 Albert Maguire,1 Kenneth Shindler,1 Jean Bennett.1 1 Children’s Hospital of Philadelphia, Philadelphia, PA.
Background: Previously, we showed that in humans with Leber’s congenital amaurosis due to RPE65 mutations (LCA2), the visual cortex can be made responsive to visual input through unilateral ocular gene therapy. Recently, our team performed a 2nd gene administration to the contralateral eyes of 5 previously treated patients. In addition to clinical tests, functional magnetic resonance imaging (fMRI) was performed to evaluate cortical response and determine if prior exposure to RPE65 genes would have any immunologic effects. fMRI Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy