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.