Collaborative Laboratory for Human Embryonic Stem Cell Research at Sanford-Burnham Medical Research Institute

Collaborative Laboratory for Human Embryonic Stem Cell Research at Sanford-Burnham Medical Research Institute

Funding Type: 
Shared Labs
Grant Number: 
CL1-00511-1.2
Award Value: 
$1,805,884
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
Status: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The Sanford-Burnham Medical Research Institute (SBMRI) Stem Cell Research Center (SCRC) supports research that (a) involves a range of pluripotent & somatic stem & progenitor cell types; (b) examines the behavior & therapeutic use of exogenous & endogenous stem/progenitor cells in animal models of a range of diseases & organ systems with an eye towards clinical translation; (c) leverages the unique strengths & resources of SBMRI, for example, in (i) chemical genomics & high-throughput screening (HTS) (for which stem cells can serve as models of diseases or biosensors for the onset of various biological processes); (ii) genomic, epigenomic, proteomic, microRNA, & metabolomic profiling; & (iii) bioinformatics. In addition, SCRC has emerged as an epicenter for education, data exchange, & collaborations in San Diego. SCRC is organized into 6 integrated sub-cores, each with its own goals & research tasks which nevertheless are interdependent & complementary, reflecting the increasingly diverse & multidisciplinary nature of the stem cell field. •Sub-Core 1: Human Stem Cell Culture & Derivation: This Sub-Core’s goal is to provide adequate amounts of high-quality pathogen-free human embryonic stem cells (hESCs) for the routine use of SBMRI investigators as well as to generate innovative protocols for their improved derivation, maintenance, storage, differentiation, & application. This core has developed unique strategies for turning hESCs & human induced pluripotent stem cells (hIPSCs) into pure neurons that can model disease states that are then compatible with drug discovery instrumentation. The Sub-Core is also tasked with generating sufficient numbers of cells for pre-clinical translational studies that could lead to IND filings with the FDA. •Sub-Core 2: Human Stem Cell Characterization. In addition to such routine procedures as teratoma formation & embryoid body analysis to affirm pluripotence, & the initiation of an in-house karyotyping service, characterization via this Sub-Core also, importantly, includes the use of gene and protein expression profiling. Novel insights into potential drug targets have been gleaned in this manner. In collaboration with the Proteomics Core, this Sub-Core has amassed the largest, most comprehensive comparative proteomic dataset to date of any biological system, comparing undifferentiated hESCs with their pure neurectodermal derivatives. •Sub-Core 3. High-Throughput Analysis: This Sub-Core’s goal is to provide a bank of hESC & hIPSC lines that either bear disease-relevant assayable phenotypes or carry reporter genes that will enable high-throughput (HT) technologies for performing screens of chemical & gene libraries. Such capabilities enable users to identify molecules that (a) encourage hESCs to maintain, or hIPSCs to acquire, their pluripotent state; (b) to differentiate toward desired lineages, or (c) to block or reverse the appearance of an informative disease marker or process. The Viral Vector Core is now housed in SCRC given that the genetic modification of stem/progenitor cells has become its largest demand. This sub-core has generated a collection of reporter stem cell lines, in which a reporter gene is transcribed from various developmental stage- &/or tissue-specific promoters, making them “biosensors”. Stem cells have also been used in this manner not only to discover new candidate drugs but also to screen for drug toxicities. •Sub-Core 4. Human Stem Cell Data Sharing & Training: One goal of this Sub-Core is to facilitate data management, analysis, & recovery. A web-accessible relational database is being upgraded to serve as a repository for complex datasets derived from experiments using gene microarray, differential proteomic profiling, & chemical library screens. Another goal is to train scientists & the lay community in the technical, ethical, & logistical issues regarding research involving embryos, stem cells, & regenerative medicine. •Sub-Core 5. Advanced & Automated Cell Imaging: Although this sub-core consolidates the SCRC’s routine microscopic functions, it is largely built around the unique sophisticated high-resolution high-content intra- & inter-cellular imaging & HT microscopy developed at SBMRI. It can monitor phenotypes that require sophisticated, quantitative, functional, real-time, long-term readouts of subcellular locations, morphology, & physiology in living cells. We have determined that this instrumentation can be applied to hESCS & HIPSCs for HT drug discovery. •Sub-Core 6: Human Induced Stem Cell Generation: This recently-established Sub-Core has been creating disease-modeling hIPSC lines through the reprogramming of fibroblasts from patients with a range of diseases. To date, >50 such lines have been created modeling a wide range of diseases. Such “disease-in-a-dish” technology will be used to identify mechanisms of disease, novel drug targets, & the therapeutic drugs themselves.

Year 2

The Sanford-Burnham Medical Research Institute (SBRMI) Stem Cell Research Center (SCRC) has been able to support stem cell research in San Diego by (1) serving as an epicenter for education, training, data exchange, & collaborations; (2) providing cutting-edge stem cell technology-based services; (3) providing comprehensive systems biology tools; (4) supporting & accelerating translational research; (5) bridging basic stem cell research & drug development & screening. SCRC has been ably fulfilling its missions: (1) to study stem cell as one component in a series of intrinsic developmental programs; (2) to establish disease models using stem cells by understanding how the perturbations of the stem cell biology contribute to disease; (3) to translate potential stem cell-mediated therapies rationally to clinical practice. Under the directorship of Dr. Evan Snyder, the SCRC has obtained CIRM funding to expand to 3000 ft2 in 2 locations & engage > 60 labs across the Mesa in aspects of stem cell research, involve >150 investigators, & service many academic & private sector entities in San Diego. >60 peer-reviewed stem cell relevant papers were published in 2011 alone at SBMRI, many of which were the outcome of the collaborations between labs & appeared in the top-tier journals. Most gratifyingly, the SCRC has become a “harbor” for collaboration between labs & even institutions. To reflect the increasingly diverse & multidisciplinary nature of the stem cell field, SCRC is organized into 4 integrated sub-cores, each with its own goals & research tasks. Sub-Core 1: Cell generation, culture, manipulation & supply. The goal for this sub-core is to provide support for stem cell related research including facility, reagents, human embryonic stem cell (hESC) & induced pluripotent Stem Cell (hiPSC) lines, & advanced tools & services. It has become the main source for scientists to obtain high-quality & application tested stem cell culture reagents. The advanced hiPSC reprogramming service has attracted hundreds of projects from research institutes across the Mesa as well as from biotech/pharmaceutical companies. The expansion of the shared hESC, hiPSC (& their starting cells) & somatic stem cell lines housed at SCRC has provided a large pool from which researchers may choose. The Sub-Core is also developing proprietary methods for generating specific types of cells from human stem cells, e.g., neurons & pancreatic ß-cells, which will serve to facilitate disease modeling for drug screening as well as accelerating pre-clinical translational studies that might lead to the filing of INDs. A cryopreservation service also operates within this sub-core. Sub-Core 2: Cell characterization. To affirm pluripotence, the SCRC routinely performs pluripotency marker immunodetection assays as well as in vitro differentiation capability assays & genetic profiling (the PluriTest). An in vivo teratoma formation service has also been established. Collaborating with other core in SBMRI, SCRC performs more systematic analysis on the stem cell DNA, mRNA, proteins & their modification as well as its metabolomics & pharmacogenomics. Currently, we have compiled the largest, most comprehensive comparative proteomic dataset on stem cell & its pure neurectodermal derivatives. Recently, SCRC introduced microRNA screening & analysis, a new powerful tool. Novel insights into potential drug targets have been gleaned in this manner. Sub-Core 3: High-Throughput (HT) technologies. Interfacing with the Institute’s Prebys Chemical Genomics Center & utilizing state-of-the-art high-resolution, high-content, HT cellular imaging microscopy developed at SBMRI, the SCRC is able to (1) provide adequate numbers of human pluripotent stem cells with specific reporters as ‘biosensors’ or iPSC lines that bear disease relevant mutations; (2) differentiate hESCs/hiPSCs toward desired lineages; (3) identify tightly disease-associated assayable phenotypes; (4) develop cell activity or phenotype-based assays for large compound library screening. In this manner, users can not only discover new candidate drugs but also screen for drug toxicities. Sub-Core 4: Training, Education & Data Sharing. Being a training facility, SCRC provides various free ad hoc short training courses including hESC/hiPSC maintenance, hiPSC generation, lineage-specific differentiation, etc. SCRC hosts the monthly Southern California Stem Cell Consortium, a monthly stem cell User’s meeting; a monthly Journal Club – all designed to engage researchers & trainees throughout the region. On the easy-to-use SCRC website, new protocols for stem cell differentiation & somatic cell reprogramming have been added to the previously posted protocols for hESC maintenance & characterization. Equally important, SCRC opens its door to the public at any time. SCRC conducts numerous tours to non-stem cell scientists, patient advocates, journalists, donors, high school students, & residents from the local communities.

Year 3

Sanford-Burnham Medical Research Institute (SBRMI) Stem Cell Research Center (SCRC) is comprised of 4 integrated sub-cores: (1) Human Stem Cell Generation, Culture, Manipulation, & Supply; (2) Human Stem Cell Characterization; (3) High-Throughput (HT) Technologies (including such advanced & automated image analysis as high-content screening); & (4) Human Stem Cell Data-Sharing, Training, & Education. The Viral Vector Core is also housed in the SCRC. Two years ago, the Human Stem Cell Generation Sub-Core set its goal to become the largest bank of well-characterized patient starting cells & disease-modeling hiPSCs in academia. To this end, SCC has been in the midst of establishing relationships with clinical entities in San Diego (UCSD, VA Hospital, Rady Children’s Hospital) to create a pipeline for somatic cell samples. In addition, SCRC has formed relationships with investigators abroad who have banks of cells from well-characterized patients. Furthermore, to help validate potential pathophysiological pathways & novel drug targets, SCRC has partnered with large hospital-based banks of well-archived clinical specimens from patients with similar disorders. This sub-core also established the integration free reprogramming methods on human skin fibroblasts. Recently, the sub-core successfully reprogrammed immortalized B lymphocytes. This achievement is significant because (1) reprogramming immortalized B lymphocyte is considered a very difficult process (2) a large number of the patient biopsies particularly for the mental illness are preserved in the format of EBV immortalized B lymphocyte. This will enable the researchers to generate relevant disease models from these samples, which was impossible in the past. The Sub-core also formed collaborations with Intrexon to develop a high throughput reprogramming method using a computer controlled laser system. The Characterization Sub-Core profiles hESCs, hIPSCs, & somatic stem cells using of immunocytochemistry &cytometry; gene microarrays & deep sequencing; quantitative PCR, methylation, SNP, & microRNA/SiRNA screens; large-scale comprehensive (phospho)proteomics, metabolomics, &pharmacogenetics; & high-resolution cellular imaging – all with an eye to identifying biomarkers for the diagnosis, prognosis, progression, & therapeutic options for such disorders. Via the SCRC website, the largest comparative phosphoproteomic dataset in any biological system, comparing human pluripotent stem cells with their pure neurectodermal derivatives can accessed publicly. Advanced gene microarrays, quantitative PCR, microRNA screens, & proteomics were used to provide genomic fingerprinting of an array of human stem cell lines & their differentiated progeny. “PluriTest”, a web-based method for determining whether a given cell with a particular profile of gene expression does or does not comport to being “pluripotent”, a tool that is coming to replace the use of teratoma formation as a proof of pluripotence. One of the principal achievements of the HT Technologies Sub-Core was creating read-outs for its chemical, cDNA, or shRNA/siRNA screens based on automated HT high-content microscopy used in conjunction with the SBMRI’s robotic liquid handling systems in the chemical library screening facility of the Prebys Center for Chemical Genomics (PCCG). Such efforts have been enhanced by NIH funding of a Comprehensive Screening Center at SBMRI as well as a recent partnership with Johnson & Johnson. Being a training facility, SCRC also provides various hands-on training courses on hESC/hiPSC maintenance, hiPSC generation, lineage-specific differentiation, etc. Newly updated protocols for stem cell differentiation & somatic cell reprogramming have been posted to the SCRC website. More recently, a Standard Operating Procude (SOP) booklet covering almost all the experiments related to the stem cell culture, manipulation, and characterization was published on the SCRC website for the training course. Currently, SCRC hosts the monthly Southern California Stem Cell Consortium, a monthly stem cell User’s meeting; a monthly Journal Club – all designed to engage researchers and & trainees throughout the region. SCRC conducts numerous tours to non-stem cell scientists, non-scientists, patient advocates, journalists, donors, high school students, & residents from the local communities who are interested in learning about stem cell biology.

Year 4

The Sanford-Burnham Medical Research Institute (SBMRI) Stem Cell Research Center (SCRC) and Core Facility (SCC) supports research that (a) involves a range of pluripotent & somatic stem & progenitor cell types; (b) examines the behavior & therapeutic use of exogenous & endogenous stem/progenitor cells in animal models of a range of diseases & organ systems with an eye towards clinical translation; (c) leverages the unique strengths & resources of SBMRI, including high-throughput screening, & epigenomic, proteomic, microRNA, & metabolomics. In addition, SCRC has become an epicenter for education, data exchange, & collaborations on the Mesa. As the goal for the SCRC SCC is to be the top stem cell facility in the nation, new technology development has been the major focus besides the service projects. Since generating induced pluripotent stem cell (iPSC) is the major service provided by the SCC, further improvement in the reprogramming technology was carried out, including identifying better reagents and optimal condition for each step, adjusting the protocol for different tissue samples, increasing the sample throughput to reduce the cost, etc. In collaboration with Intrexon, a 96-well format reprogramming method was established using a computer controlled laser system. 8-12 samples can be performed simultaneously on one plate. The throughput was increase 10 folds and its reagent cost is only 1/10 of the previous method. In addition to the successful reprogramming on the Epstein-Barr virus immortalized lymphoblastoids, the SCC successfully reprogrammed the umbilical cord blood cells. This achievement along with the high throughput reprogramming method makes it feasible to establish a library of iPSCs from the healthy individuals with an enormous diversified genetic background. Because of this broad range of normal individuals with different immunocompatability markers (called “human leukocyte antigens” or “HLA types”), it would be easy to locate the cell line that has the closest matching HLA type to the patient. The specialized cells can be derived and are immediately suitable for transplantation into the patient without the need for immunosuppression. This enormous potential in future cell therapeutics has been recognized by many cord blood banks. Partnerships with national and international cord blood banks on iPSC generation from healthy donors are expected to be established in 2014. . The Characterization Sub-Core profiles hESCs, hiPSCs, & somatic stem cells using of immunocytochemistry &cytometry; gene microarrays & deep sequencing; quantitative PCR, methylation, SNP, & microRNA/SiRNA screens; large-scale comprehensive (phospho)proteomics, metabolomics, & pharmacogenetics; & high-resolution cellular imaging – all with an eye to identifying biomarkers for the diagnosis, prognosis, progression, & therapeutic options for such disorders. Via the SCRC website, the largest comparative phosphoproteomic dataset in any biological system, comparing human pluripotent stem cells with their pure neurectodermal derivatives can accessed publicly. Advanced gene microarrays, quantitative PCR, microRNA screens, & proteomics were used to provide genomic fingerprinting of an array of human stem cell lines & their differentiated progeny. The SCRC’s successful service program has been facilitating local stem cell related research. Many of these projects produced high quality papers published in the first tier scientific journals. In 2013, the researchers in SCRC published 63 stem cell research papers in the areas of degenerative disease, stem cells & regenerative biology and development & aging. To further enhance the collaboration among the stem cell labs, SCRC hosts the monthly Southern California Stem Cell Consortium and invites top stem cell researchers to present their exciting new discoveries. A monthly stem cell User’s meeting and a monthly Journal Club organized by the SCC are all designed to engage researchers & trainees throughout the region. Being a training facility, SCRC also provides various hands-on training courses on hESC/hiPSC maintenance, hiPSC generation, lineage-specific differentiation, etc. Newly updated protocols for stem cell differentiation & somatic cell reprogramming have been posted to the SCRC website. SCRC also conducts numerous tours to non-stem cell scientists, non-scientists, patient advocates, journalists, donors, high school students, & residents from the local communities who are interested in learning about stem cell biology.

© 2013 California Institute for Regenerative Medicine