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Stem Cell Research Center
In August 2001, federal funding for research involving human embryonic stem cells (hESCs) was restricted to but a few previously established cell lines. This development necessitated creation at Burnham of a safe-haven for generating and conducting research on non-approved hESCs and for human embryo research. To accomplish this goal, a wholly-owned subsidiary was created, called the Stem Cell Research Center (SCRC), using this entity as a vehicle for accounting for non-federal revenue and relevant expenses associated with conducting research on ineligible hESCs. With community and private foundation support, a ~1,200 sq ft laboratory was assigned to the SCRC and was supplied and equipped for deriving, maintaining, and performing experiments on non-approved hESCs. Stem Cell Program Director, Evan Snyder, serves as the Director of the SCRC in conjunction with Jeanne Loring (who oversees day-to-day operations).
The recent passage of Proposition 71 created the California Institute for Regenerative Medicine (CIRM), a granting agency with a mandate to provide $3B over 10 years in research and related support to California scientists engaged in stem cell research. Although those monies have been slow to materialize pending the resolution of lawsuits, sufficient funds have been generated by CIRM to launch a Training Program at the Burnham and sufficient enthusiasm has been engendered in the La Jolla biomedical community for the Burnham to join the 3 other institutions on the mesa (UCSD, Salk, and Scripps) in creating the San Diego Institute for Regenerative Medicine, catalyzed by philanthropic support from the region. Ultimately a jointly-occupied building serving as a “hub” for this Institute may house some of the functions of SCRC.
The SCRC is presently comprised of 4 cores:
- The hESC Culture & Derivation Core
- The hESC Characterization Core
- The hESC High-Throughput Analysis & New Technology Development Core
- The hESC Data Sharing & Training Core
The goals and accomplishments of each Core is discussed below. These Cores service the scientific projects of the Stem Cell Program, including projects funded this year by NIH as part of its grant to the Burnham to establish one of just 6 Exploratory Stem Cell Research Centers in the country. (Of course, only those aspects of the Cores compatible with NIH-guidelines for the use of hESCs are used for those projects or supported by federal funds).
GOALS The goal of this Core facility is to provide adequate amounts of high quality hESCs for routine use, as well as to generate innovative protocols for their improved maintenance, use, and differentiation. Because all of the hESC lines approved for federal funding were generated using methods that involved admixing human cells with mouse “feeder” cells and/or incorporated the use of animal serum or conditioned medium, they are poorly suited for clinical use and are likely to have undergone modifications with unpredictable consequences. Given the dearth of hESC lines that have been generated using defined medium, and recognizing that cells passaged for long periods of time in culture tend to become genetically unstable, it is necessary to have the means of establishing new hESC lines going forward. To meet this need, we established the Stem Cell Resource (SCR), a service to accept donations of left-over blastocysts from in vitro fertilization (IVF) clinics for research purposes, including the derivation of new hESC lines. The SCR, the first academic non-profit facility of its type in the world, has received a great deal of positive attention – including referrals from around the country -- and is designed to serve the needs of the broader international stem cell community as well, placing this Burnham core at the hub of such activity. With numerous layers of ethical, medical, and financial oversight now in place, SCR has already received and catalogued ~1000 excess normal and disease-bearing blastocysts under informed consent from donors and IVF clinics from around the country.
PROGRESS Over the past 2 years, this Core, in devising a set of minimal essential culture requirement for preserving pluripotency, has developed a novel, efficient culture system for maintaining the long-term growth of undifferentiated hESCs under totally-defined, “biologics-free” (serum-, feeder-, and conditioned media-free) conditions. These cells have not only remained stable in terms of their molecular characteristics and karyotype for >2 years, but they can be directed selectively towards various specific lineages (including neural and cardiac, uncontaminated by other undesired lineages) with appropriate stimuli, including small molecules. Cells generated by this Core have been successfully transplanted into a mouse model of a neurodegenerative disease where, without the need for immunosuppression, they have preserved motor function and significantly prolonged life. It has also been discovered that under these defined conditions, a heretofore unrecognized clonally-related cell type constitutively emerges from the hESCs. We have identified these cells as primitive endoderm, suggesting that these culture conditions promote a cascade of steps in vitro that emulate in vivo embryogenesis. This primitive endoderm-like cell and the defined conditions under which it emerges (previously unappreciated) constitute new intellectual property, the patents for which are presently being prosecuted, and interest in which has generated licensing agreements. Interestingly, because under these improved culture conditions hESC clones can now be generated from single cells (not possible with previously published techniques), we should be able to passage dispersed single cells into multi-well plates for developing cell-image-based high throughput screening assays to test molecules that induce or inhibit differentiation, aiding in our understanding of the mechanisms underlying pluripotency (see below). Finally, this Core has generated new hESC lines under defined biologics-free clinically-compatible conditions. One line in particular has remained karyotypically stable for >7 months while retaining the ability to generate progeny of all 3 germ layers.
CORE #2. hESC CHARACTERIZATION.
GOALS The goal of this Core facility is to provide advanced instrumentation and technical expertise needed for molecular characterization approved and non-approved hESCs, including immunocytochemistry, FACS, gene microarrays, quantitative PCR, proteomics (in conjunction with the Proteomics Center), and cell imaging (including high-resolution intracellular imaging).
PROGRESS: Using its advanced gene array technology, this Core has already been successful in imposing – based on gene expression patterns -- an overall organizational scheme upon the hESC, a task heretofore elusive in the field. These data -- which can provide guidance to others on how gene array data and fingerprinting should be gathered, interpreted, and employed -- have been published in the stem cell literature by Dr. Loring. The Core has also performed proteomic profiling on undifferentiated hESCs compared to their clonally-related progeny in order to better understand the inherent developmental processes that appear to constitutively unfold when hESCs are grown under defined conditions. The primitive endoderm-like cells (described above), which can constitute their own isolatable product, have been profiled independently. Taken together, the various databases being generated by this Core can be mined for many years to come.
CORE #3. hESC HIGH-THROUGHPUT ANALYSIS & NEW
GOALS The goal of this Core is to provide a bank of hESC lines carrying various reporter genes and high throughput screening (HTS) technologies for performing screens of chemical libraries, cDNA libraries, or shRNA/siRNA libraries. The principal read-out for screens will be based on automated high throughput (HT) microscopy. The Core is directed by Mark Mercola, with Drs. Jeff Price and Fred Levine serving as deputy directors. A HT microscope was purchased last year for this Core, which can be used in conjunction with the Institute’s robotic liquid handling systems in the chemical library screening facility. Such equipment provides opportunities for our scientists to use chemical biology approaches for studying stem cells, including the identification of chemical entities encouraging stem cells to differentiate toward desired lineages. These capabilities will be enhanced as a result of recent NIH funding for a screening program at the Burnham (although studies under those auspices will be limited to NIH-approved hESCs).
PROGRESS: This Core has generated a collection of reporter hESC lines, in which various developmental stage- and/or tissue-specific promoters drive expression of a GFP reporter gene. In addition to lines in which GFP is transcribed from ubiquitous promoters, several stably transfected hESC lines have been generated that contain GFP driven by promoters indicative of pancreatic and cardiac development. An Oct-4 GFP line, which signals the pluripotent state, has been quite useful for both FACS and for generating single cell clones from hESCs. The ability to grow isolated single hESCs (clones) that can express lentiviral-transduced transgenes should greatly improve these reporter gene assays, heretofore an obstacle to HTS. Furthermore, using this Core, members of the Stem Cell Program have optimized two high-content cell-based screening assays for molecules regulating the human insulin gene promoter, and ~24,000 compounds have been screened with each assay, resulting in identification of promising chemical leads.
CORE #4. hESC DATA SHARING & TRAINING.
GOALS The goal of this Core is to provide a database that contains useful information about hESCs, allowing researchers to select the optimal cell line for their particular experiments. A web-accessible relational database has been established with corporate support to serve as a repository for data about hESCs derived and characterized at the Burnham, as well as by other organizations. The database provides a repository for complex datasets derived from experiments using DNA microarray and chemical library screens performed at Burnham. Dr. Loring is the curator this Core. Another goal of this Core is to train scientists (particularly those newly entering the field of stem cell biology) in the technical, ethical, and logistical issues regarding research involving human embryos and hESCs. To this end, the Core has instituted several programs to help scientists within and beyond the Burnham with hESC research (including reaching out to, educating, and engaging the lay public). The projects launched thus far are described briefly in the next section.
PROGRESS: This core has made a great deal of progress in its sundry charges by launching a series of programs:
1. Stem cell website. The “Community of Stem Cell Scientists Website” (www.stemcellcommunity.org), which is an evolving and expanding novel resource accessed worldwide by scientists via the internet, features protocols, hESC line characterizations, and practical information, as well as news, provocative columns and discussions. The content, providing a heretofore unmet need for the international stem cell community, is growing rapidly and is routinely updated. The site was recently profiled in Science. It is being expanded to constitute the first comprehensive searchable index of gene expression data on hESCs.
2. NIH-sponsored stem cell training course. The 3rd annual NIH-sponsored Human Embryonic Stem Cell Training Course, conducted by the Burnham Institute for Medical Research in collaboration with Children’s Hospital of Orange County, was held on March 7-16, 2006. The course serves the international community (the only such NIH-funded course in the West). Approximately a dozen persons (with 1:2 instructor-to-student ratio) are trained annually through this course, which features both didactic and hands-on laboratory training in methods of hESC experimentation.
3. Short laboratory courses. In addition to the NIH-sponsored training course, the Center has organized mini-courses providing 2-3 day hand-on experience with hESC culture techniques. The launch of a training program funded by CIRM (to begin May, 2006) and the inauguration of the Chemicon funded training program (described in the next section) will accelerate our training capability.
4. Training updates. The Burnham Stem Cell Alumni Network keeps graduates of the short or long courses abreast of advances. Communication is accomplished by electronic communication, the website, and, occasionally by “refresher” sessions at the Stem Cell Center.
5. Specialized training experiences. Members of the Center have accommodated investigators from non-stem cell disciplines wishing to learn human stem cell techniques or to apply their expertise to questions in stem cell biology though “rotations” or “sabbaticals” at the SCRC. These have included: (a) investigators from other academic institutions; (b) researchers from the private sector wishing to learn proper stem cell analysis; (c) rotating graduate students; (e) non-scientists wishing to acquire first-hand familiarity with stem cell biology (e.g., ethicists, foundation directors, policy-makers).
6. Laboratory manual. The "Human Stem Cell Manual: A Laboratory Guide", the lab manual from the SCRC training courses, will be published by Elsevier in Fall, 2006.
7. Ethics workshops, white papers, & publications. Stem cell ethics workshops are organized annually by the SCRC. The 2nd Ethics Workshop was held on June 6-7, 2005. A white paper generated from this and last year’s workshop (June 14, 2004) was recently published in Nature Biotechnology. In addition, a series of peer-reviewed papers from the 2nd workshop will be published as a compendium in a dedicated issue of Stem Cells Review. Participating scholars are of international stature. The SCRC has effectively become the “Division for Stem Cell Studies” for the newly created inter-institutional Center for Ethics in Science and Technology (CEST) based in San Diego. Drs. Snyder and Loring recently authored an invited editorial in the New England Journal of Medicine on the ethical and scientific ramifications of the recent South Korean scientific fraud scandal.
8. Scientific symposia. The 2nd annual “Hot Topics in Stem Cell Research” Symposium, organized by The Burnham, was again conducted as a satellite to the Society for Neuroscience annual meeting. Adopted by the Christopher Reeve Paralysis Research Foundation to honor the life of Mr. Reeve, this symposium has become a regular feature at the Society’s annual meeting.
9. Southern California Stem Cell Consortium. Now its third year, the consortium is comprised of a multidisciplinary group of investigators from multiple Southern California institutions (extending to Los Angeles), who meet monthly at the Burnham for presentations, dialog, and idea exchanges, emulating a “regional lab meeting”. Serving as the “glue” for stem cell and developmental biologists in the state’s southern region as well as for the newly born San Diego Institute for Regenerative Medicine, the consortium deals not only with a broad range of scientific topics, typically focusing on updates from individual laboratories, but also topics in public policy and ethics.
10. Community education. Stem Cell Program members participate in many educational activities for the lay, clinical, and business community, including public lectures, media sessions, and science pieces for the lay and medical press. For example, Dr. Loring recently authored a Perspective piece in Science detailing the intellectual property landscape surrounding hESC. The Snyder Lab has contributed chapters to the Handbook of Stem Cells, Essentials of Stem Cell Biology, Neural Development and Stem Cells, and Methods in Neuroscience. We see these efforts to encourage dialogue about stem cell biology as an important aspect of the Program’s mission, since public and private support for this research is crucial for success.
