Enhancing Facilities for Genetic Manipulation and Engineering of Human Embryonic Stem Cells at UCSD

Enhancing Facilities for Genetic Manipulation and Engineering of Human Embryonic Stem Cells at UCSD

Funding Type: 
Shared Labs
Grant Number: 
CL1-00522-1.2
Award Value: 
$1,737,694
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Status: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The UCSD Human Embryonic Stem Cell Core Facility (HESCCF, also referred to as “the Core”) provides the infrastructure for UCSD scientists to launch and expand their research projects with human pluripotent stem cells (hPSCs, which include embryonic and induced pluripotent stem cells). Over the past four years (2006-2010), the staff of the core facility has trained over 100 researchers in the basic methods of human embryonic stem cell growth and differentiation. The staff has also conducted advanced scientific projects that aim to create the specialized cells needed by researchers and investigators. The shared research lab has served as a staging area for research by over 45 labs and over 100 individual researchers. These researchers are from all areas of the university – biology, neuroscience, bioengineering, material sciences, medicine, just to name a few. Management and use of the laboratory The HESCCF provides biosafety cabinets, incubators, microscopes, -150°C freezers, mycoplasma testing, karyotyping, MEFs (supportive feeder cells for hPSCs), quarantine and other control practices, sharing of protocols & best practices and specialized technologies (flow cytometry, confocal microscopy, automated microscopy, electrophysiological equipment). During this reporting period, in which renovations were completed, we were able to provide all of these services and technologies without disruption, by using interim space and CIRM-supported personnel. Renovations in CMM East were completed on September 2, 2009, and HESCCF operations returned all activities to the renovated space. To provide optimal stem cell services and innovative products, the HESCCF received a Stem Cell Program subsidy from the UCSD Stem Cell Program in addition to the CIRM operating funds received from CIRM. This facility runs as a small non-profit business for the benefit of California researchers at UCSD and neighboring institutes. All services and technologies are available on a recharge mechanism with the longer-term goal of sustaining operations beyond the funding period of this grant. Coming Advances With developments in the political landscape since the Obama executive order of March 2009 and the fast pace of research and discovery, the mission of the HESCCF is shifting to enable novel technology development critical to stem cell research. Our present mission has the following three parts: • Provide access to specialized shared equipment. The HESCCF’s high value technologies, including cell sorting and analysis, confocal microscopy and electrophysiology, enable the research of stem cell scientists at UCSD. These technologies and the cell culture, molecular, and cell biological resources of the Core are available to all users at approved rates. • Offer a location for “fast start” and preliminary studies by newly recruited faculty and current faculty who are finding that using hESC offers advantages not met by their current methods. The Core offers expertise and courses targeting stem cell methods that allow scientists to quickly generate preliminary data essential for planning future research and obtaining funding. • Contributing to the development of novel technologies. HESCCF resources and staff are dedicated to maintaining state-of-the-art technology and training. Services are performed and developed in close collaboration with scientists at UCSD. Noteworthy outcomes resulting from this project. Human Non-Embryonic Pluripotent Stem Cell Lines Core facility staff research associates have established the basic methods for human iPSC generation. Both retro- and lenti-viral methods for gene transduction of the reprogramming factors been established, and a number of iPSC lines have been generated from human foreskin fibroblasts. With the assistance of the core facility staff, users of the core facility can quickly launch research programs in the iPSC field. Genetically Engineered Human Stem Cells Staff scientists of the Core have generated a human embryonic stem cell line, which permits the targeted insertion of genetic elements into a specific locus on Chromosome 13. This “entry cell line” and the technology to target this chromosomal locus with genes of interest are available to all users of the Core. Novel Tools or Technologies The Core has established the basic methodology for homologous recombination in hPSCs using recombinant adeno-associated viral vectors (rAAV). To date two genes have been targeted for gene replacement using this method. In collaboration with stem cell researchers, the Core will facilitate the further development of this technology and generate a panel of hPSC marker cell lines. Outreach Activities Members of the public visited the CIRM UCSD HESCCF during the following events: • Sweetwater and San Dieguito High schools visit for Science on the Mesa, April 1, 2009 • NAS High School teacher Stem Cell Workshop, April 25, 2009 • Donor Event Lab tours, May 21, 2009 • Stem Cell Awareness Day, Sept 23, 2009

Year 2

The UCSD Human Embryonic Stem Cell Core Facility (HESCCF) provides the infrastructure for UCSD scientists to launch and expand their research projects with human pluripotent stem cells (hPSCs, including embryonic and induced pluripotent stem cells). Over the past four years (2006-2010), the staff of the core facility has trained a cadre of researchers (over 100 scientists representing over 45 labs) in the basic methodologies of human embryonic stem cell growth and differentiation. During this reporting period, November 2009-October 2010, the staff has also conducted scientific projects to generate targeted transgene insertions. A wide range of projects on cardiovascular regeneration, cell microenvironments, childhood neurological disease, neurodegenerative disorders, and pancreas development, have been initiated and in some instances have been completed in the shared research lab. The large number of scientists actively using the services and resources available in the HESCCF has fostered multiple collaborations across campus. Under the direction of Dr. Willert, staff members of the HESCCF have developed two novel technologies for gene targeting in human embryonic stem cells. The first technology sought to establish a cell line with an integration site that can be readily targeted for gene insertion and is not subject to epigenetic silencing as is often times observed when genes are inserted into the genome at random. This cell line, which is karyotypically normal and able to differentiate into all derivative cell types of the major germ layers, serves as an important reagent cell line to insert and express transgenes of interest. The cell line is available to users of the shared research lab. The second technology initiated in the HESCCF aims to insert reporter genes into specific genes of interest with the goal of tagging genes that are activated or inactivated during differentiation. Our goal is to continue this tool and technology development and make it readily available to the UCSD research community. In addition to these projects, the HESCCF provides these technologies: • Characterization of hPSC lines. Basic protocols to ensure cell lines are pluripotent, karyotypically normal and free of bacterial contamination are available. Researchers and students have the option to be trained in these methods or have these characterizations performed by the HESCCF staff on a recharge basis. • Flow cytometry analysis and cell sorting: With the acquisition of a BD FACS Aria 2 and FACS Canto 2, the HESCCF is now providing critical tools for the analysis and isolation of specific sub-populations of undifferentiated and differentiated hPSCs. This technology is available to all UCSD stem cell scientists on a recharge basis. • Lenti- and retro-virus technology: The core provides expertise and resources to generate non-replicating lenti viruses to introduce genetic elements into hPSC lines. This technology is also available for induced pluripotent cell lines. • Cell imaging technologies. Confocal microscopy and live cell imaging capabilities are available at the Core. In addition, a High Content Analysis Microscope is available and managed by the UCSD Microscopy Core Facility (under direction of J. Meerloo). During this reporting period, the renovations to the satellite lab in EBU1 were completed. Full operations in EBU1 satellite will be described in the next reporting period. Management and use of the laboratory The HESCCF provides a stem cell culture laboratory available to all campus researchers. The HESCCF includes biosafety cabinets, quarantine and other control practices, -150 freezers, incubators, microscopes, mycoplasma testing, karyotyping, viable cell line services, MEFs, sharing of protocols & best practices. We also provide these services and technologies: Cell sorting BD FACS Aria2, Flow Cytometry BD FACS Canto 2, Olympus FV1000 Confocal Microscopy; Cellomics Automated Microscopy; Electrophysiological equipment; real time PCR; hESC cell-culture training in basic and advanced lab techniques; Reagents and special plastics; gene transduction using Amaxa Electroporator; Perkin Elmer Plate reader. To provide optimal stem cell services and innovative products, the HESCCF received a Stem Cell Program subsidy from the UCSD Stem Cell Program in addition to the CIRM operating funds received under award CL1-00522. Activities of the Oversight Committee The Shared Facilities Steering Committee meets at least annually to make decisions about future goals and needs. Their principal topic is equipment priorities. The Shared Facilities Steering committee has considered, for example, the purchase of a plasmid library, a possible role for the core in hESC ‘banking,’ implementation of new methods in advanced technologies like electrophysiology, and the management of the hESC satellite in Bioengineering. The Shared Facilities Steering committee is made up of 12 faculty members from all UCSD Schools and Divisions.

Year 3

The facility, both the main location and the Bioengineering satellite, provides the infrastructure for UCSD scientists to launch and expand their research projects with human pluripotent stem cells (hPSCs). The staff of the core facility has trained over 100 scientists in over 45 labs in the basic methods of hESC growth and differentiation. A wide range of projects on cardiovascular regeneration, cell microenvironments, childhood neurological disease, neurodegenerative disorders, and pancreas development have been initiated and in some instances have been completed in the shared research lab. The large number of scientists actively using the HESCCF has fostered multiple collaborations across campus. Existence of these well-designed and well-equipt facilities attract has attracted substantial federal funding. The HESCCF has become a center for training, experimental design, and conduct of cell sorting and flow cytometry analysis. With the acquisition of a BD FACS Aria 2 and FACS Canto 2, the analysis and isolation of specific sub-populations of undifferentiated and differentiated hPSCs is conducted. This technology will soon be available to all resident scientists in the Sanford Consortium building. Our goals are to continue the training and research function of the HESCCF, to support the development of the satellite CERC, and make tools and solutions readily available to the UCSD research community and our partner institutions. The HESCCF provides these technologies: • Characterization of hPSC lines. Basic protocols to ensure cell lines are pluripotent, karyotypically normal and free of bacterial contamination are available. Researchers and students have the option to be trained in these methods or have these characterizations performed by the HESCCF staff on a recharge basis. • Lenti- and retro-virus technology: The core provides expertise and resources to generate non-replicating lenti viruses to introduce genetic elements into hPSC lines. This technology is also available for induced pluripotent cell lines. • Cell imaging technologies. Confocal microscopy and live cell imaging capabilities are available at the Core. In addition, a High Content Analysis Microscope is available and managed by the UCSD Microscopy Core Facility (under direction of J. Meerloo). During this period, the Cell Engineering Research Center (CERC) satellite became fully operational, offering core laboratory facilities and services to bioengineering researchers engaged with scientists and physicians to bring therapeutic advances to California. Located in the Jacobs School of Engineering, the CERC provides novel engineering expertise from the faculty of Bioengineering in important technological areas including nanotechnology, biomaterials, instrumentation, and tissue engineering for regeneration. http://www-bioeng.ucsd.edu/cerc/. In January – March, 2011 a Student Tissue Engineering Laboratory was conducted in the CERC. The students gained hands on experience with experimental techniques, protocol design, data analysis, and journal-quality report writing. The students fabricated a collagen gel and synthetic polymers, cultured cells, performed differentiation assays and completed team projects. Management and use of the laboratory The HESCCF provides a stem cell culture laboratory available to all campus researchers. The HESCCF includes biosafety cabinets, quarantine and other control practices, -150 freezers, incubators, microscopes, mycoplasma testing, karyotyping, viable cell line services, MEFs, sharing of protocols & best practices. We also provide these services and technologies: Cell sorting BD FACS Aria2, Flow Cytometry BD FACS Canto 2, Olympus FV1000 Confocal Microscopy; Cellomics Automated Microscopy; Electrophysiological equipment; real time PCR; hESC cell-culture training in basic and advanced lab techniques; Reagents and special plastics; gene transduction using Amaxa Electroporator; Perkin Elmer Plate reader. Activities of the Oversight Committee The Shared Facilities Steering Committee meets at least annually to make decisions about future goals and needs. Their principal topic is equipment priorities, for example, new equipment to enhance the facility when the HESCCF moves to the Sanford Consortium for Regenerative Medicine, a possible role for the core in banking hESC, iPS, and other cells, how to offer advanced technologies like electrophysiology, and the management of the CERC satellite facility in Bioengineering.

Year 4

The CIRM funded shared research laboratories, including the Human Embryonic Stem Cell Core Facility (HESCCF) located in the Sanford Consortium for Regenerative Medicine (SCRM) and the Cell Engineering Research Center (CERC) located in the Jacobs School of Engineering, have been the corner stones of the UCSD Stem Cell Program and have offered scientists, from early career to established, a unique environment to launch, sustain and expand their stem cell research programs. In December 2011, the HESCCF moved from its original location on the UCSD Health Sciences campus to the SCRM, the CIRM Major Facility that provides research space to the Consortium member institutes: UCSD, Salk Institute, The Scripps Research Institute, Sanford-Burnham Institute and the La Jolla Institute for Allergy and Immunology. Since its inception, the HESCCF has provided a fast start for researchers at and around UCSD to work with human embryonic stem cells (hESCs). With the support of the CIRM-funded grant, the HESCCF was able to expand significantly, adding several enabling technologies and establishing a satellite laboratory in the School of Engineering, now called the CERC, where novel technologies, such as nanotechnologies, biomaterials, and tissue engineering are explored and applied to the study of stem cells and regenerative medicine. The combined staff of the HESCCF and CERC has interacted with over 200 scientists in over 50 labs at UCSD and its neighboring institutes. Research projects initiated and elaborated in these core facilities touch on a broad spectrum of topics, including cancer stem cells, early human embryonic patterning and development, blood, skin, liver and pancreas development, and stem cell interactions with their microenvironments/niches. Investigators operating in the core facility address a variety of human malignancies, such as childhood neurological diseases, cardiovascular problems, neurodegenerative disorders (including ALS, Alzheimer’s and Parkinson’s Diseases), and Type 1 diabetes. The large number of scientists actively using the HESCCF and CERC has fostered multiple collaborations across campus and the Torrey Pines Mesa. Existence of these well-designed and well-equipped facilities has attracted substantial funding beyond CIRM, including from the National Institutes of Health, the American Cancer Society, the Juvenile Diabetes Research Foundation, to name a few. The HESCCF and CERC have become centers for training where scientists can acquire many of the critical techniques and skills to become competent and competitive stem cell researchers. In recent years, the HESCCF has emphasized the expansion of flow cytometry and its application to human pluripotent stem cells and their derivatives. Now operating four flow cytometers (2 cell sorters and 2 analyzers) the HESCCF is now able to meet the critical need for single cell analysis and isolation for the entire stem cell community. The HESCCF provides the following resources and technologies: Cell culture facilities: 7 biosafety cabinets and 11 incubators provide ample capacity for over 60 scientists to conduct stem cell research. Characterization of hPSC lines: Basic protocols to ensure cell lines are pluripotent, karyotypically normal and free of bacterial contamination are available. Training: Researchers and students are trained in essential hESC methods. Lenti- and retro-virus technology: In coordination with the adjacent Viral Vector Core Facility of the SCRM, the HESCCF provides expertise and resources to generate non-replicating lenti viruses to introduce genetic elements into hPSC lines. This technology is also available for induced pluripotent cell lines. Cell imaging technologies. Confocal microscopy, live cell imaging capabilities and high content imaging are available. The Cell Engineering Research Center (CERC), which became fully operational in 2011, offers the following laboratory services to bioengineering researchers: Cell culture facilities: Biosafety cabinets, incubators, microscopes, freezers and refrigerators provide the essentials for stem cell culture. Training: In the Student Tissue Engineering Laboratory students have gained hands-on experience with experimental techniques, such as collagen gel and synthetic polymers fabrication. Specialized technologies: The CERC offers FACS, GPC, Rheometer, Dielectrophoresis capability, and a Cyntellect LEAP.

Year 5

The CIRM funded shared research laboratories, including the Human Embryonic Stem Cell Core Facility (HESCCF) located in the Sanford Consortium for Regenerative Medicine (SCRM) and the Cell Engineering Research Center (CERC) located in the Jacobs School of Engineering, have been the corner stones of the UCSD Stem Cell Program and have offered scientists, from early career to established, a unique environment to launch, sustain and expand their stem cell research programs. In December 2011, the HESCCF moved from its original location on the UCSD Health Sciences campus to the SCRM, the CIRM Major Facility that provides research space to the Consortium member institutes: UCSD, Salk Institute, The Scripps Research Institute, Sanford-Burnham Institute and the La Jolla Institute for Allergy and Immunology. Since its inception, the HESCCF has provided a fast start for researchers at and around UCSD to work with human embryonic stem cells (hESCs). With the support of the CIRM-funded grant, the HESCCF was able to expand significantly, adding several enabling technologies and establishing a satellite laboratory in the School of Engineering, now called the CERC, where novel technologies, such as nanotechnologies, biomaterials, and tissue engineering are explored and applied to the study of stem cells and regenerative medicine. The combined staff of the HESCCF and CERC has interacted with over 200 scientists in over 50 labs at UCSD and its neighboring institutes. Research projects initiated and elaborated in these core facilities touch on a broad spectrum of topics, including cancer stem cells, early human embryonic patterning and development, blood, skin, liver and pancreas development, and stem cell interactions with their microenvironments/niches. Investigators operating in the core facility address a variety of human malignancies, such as childhood neurological diseases, cardiovascular problems, neurodegenerative disorders (including ALS, Alzheimer’s and Parkinson’s Diseases), and Type 1 diabetes. The large number of scientists actively using the HESCCF and CERC has fostered multiple collaborations across campus and the Torrey Pines Mesa. Existence of these well-designed and well-equipped facilities has attracted substantial funding beyond CIRM, including from the National Institutes of Health, the American Cancer Society, the Juvenile Diabetes Research Foundation, the National Foundation for Ectodermal Dysplasias, to name a few. The HESCCF and CERC have become centers for training where scientists can acquire many of the critical techniques and skills to become competent and competitive stem cell researchers. In recent years, the HESCCF has emphasized the expansion of flow cytometry and its application to human pluripotent stem cells and their derivatives. Now operating four flow cytometers (2 cell sorters and 2 analyzers) the HESCCF is now able to meet the critical need for single cell analysis and isolation for the entire stem cell community. The HESCCF provides the following resources and technologies: Cell culture facilities: 7 biosafety cabinets and 11 incubators provide ample capacity for over 60 scientists to conduct stem cell research. Characterization of hPSC lines: Basic protocols to ensure cell lines are pluripotent, karyotypically normal and free of bacterial contamination are available. Training: Researchers and students are trained in essential hESC methods. Lenti- and retro-virus technology: In coordination with the adjacent Viral Vector Core Facility of the SCRM, the HESCCF provides expertise and resources to generate non-replicating lenti viruses to introduce genetic elements into hPSC lines. This technology is also available for induced pluripotent cell lines. Cell imaging technologies. Confocal microscopy, live cell imaging capabilities and high content imaging are available. The Cell Engineering Research Center (CERC), which became fully operational in 2011, offers the following laboratory services to bioengineering researchers: Cell culture facilities: Biosafety cabinets, incubators, microscopes, freezers and refrigerators provide the essentials for stem cell culture. Training: In the Student Tissue Engineering Laboratory students have gained hands-on experience with experimental techniques, such as collagen gel and synthetic polymers fabrication. Specialized technologies: The CERC offers FACS, GPC, Rheometer, Dielectrophoresis capability, and a Cyntellect LEAP.

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