The Berkeley Human Embryonic Stem Cell Shared Research Laboratory

The Berkeley Human Embryonic Stem Cell Shared Research Laboratory

Funding Type: 
Shared Labs
Grant Number: 
CL1-00519-1.2
Award Value: 
$1,688,129
Status: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The central mission of our Berkeley CIRM Shared Stem Cell Facility (SSCF) is to provide our East Bay users with knowledge, expertise, training, and equipment to advance scientific knowledge of human embryonic and other stem cells. Our facility is designed to support human embryonic stem cell (hESC) culture, including high quality cell culture space equipped with biosafety cabinets, incubators, cryogenic storage, and a standard microscope. In addition, we have developed and focused state-of-the-art resources and expertise to meet a growing need for our users, imaging. In particular, we have a two photon and visible confocal fluorescence microscope and a high throughput fluorescence imager that are being increasingly used. In addition, our analytical and sorting flow cytometry capabilities complement the imaging equipment by providing high throughput cell fluorescence measurements. Finally, the facility is equipped with a laminar flow hood to conduct chemistry to create biomaterials and micropatterned surfaces for stem cell culture, as well as subsequent analysis by imaging and flow. We are very thankful to CIRM for enabling the construction and development of this state-of-the-art stem cell facility. Also, the generous resources of CIRM have been additionally leveraged, as the facility director has obtained additional campus funds to purchase additional equipment and further enhance the capabilities of the existing equipment. Our facility is directed by Prof. David Schaffer in collaboration with the management oversight committee. In addition, it is managed by Dr. Mary West, who has successfully overseen the installation of the equipment and resources described above, provides rigorous training to our users, and aids in the development of imaging strategies to enable numerous experiments. In greater scientific detail, 57 students and postdoctoral fellows from the laboratories of 11 PIs have approved access and are using the facility to date. Therefore, these resources are enabling and enhancing a large and growing number of research projects, which are described in greater detail in the full scientific progress report. As one example, the Robey and Winoto labs are developing and optimizing in vitro culture systems to induce T cell development from human embryonic stem cells and iPS cell lines. An important parameter is the choice of the starting cell line, and a number of non-registry human ES cells lines are being compared. Therefore the availability of lab space that is separate from NIH funded labs is key to being able to carry out these studies. As another example, the Healy lab is developing new synthetic substrates for culturing human embryonic stem cells under defined conditions. They have made significant progress with several surface formulations, and quantitative measurements on the maintenance of hESC proliferation and pluripotency are being aided by the imaging equipment in the CIRM facility. As another example, the Schaffer lab has engineered new gene delivery vehicles that can augment and edit the genome of hESCs and neural stem cells. Quantification of gene delivery efficiency and gene targeting has been greatly aided with the flow cytometry and high throughput imaging capabilities of the CIRM facility. As an additional example, the Li lab has been utilizing micropatterned surfaces of variable mechanical properties to study the effects of substrate mechanics on stem cell function, and these efforts have been enabled by the high throughput imager. Finally, the Kumar lab studies how the molecular and mechanical properties of the cellular cytoskeleton regulate cell function. For example, he uses laser ablation of subcellular features such as cytoskeletal filaments to provide real time information on the mechanical properties of the cytoskeleton. He is establishing laser ablation using the multiphoton microscope in the CIRM facility, a capability that will be applied to study cytoskeletal mechanics of stem cells. We thus anticipate that this valuable facility will thus be increasingly utilized to advance a growing number of projects.

Year 2

The central mission of our Berkeley CIRM Shared Stem Cell Facility (SSCF) is to provide our East Bay users with knowledge, expertise, training, and equipment to advance scientific knowledge of human embryonic and other stem cells. Our facility is designed to support human embryonic stem cell (hESC) culture, including high quality cell culture space equipped with biosafety cabinets, incubators, cryogenic storage, and a standard microscope. In addition, we have developed and focused state-of-the-art resources and expertise to meet a growing need for our users, imaging. In particular, we have a two photon and visible confocal fluorescence microscope and a high throughput fluorescence imager (ImageXpress) that are being increasingly used. Our analytical and sorting flow cytometry capabilities complement the imaging equipment by providing high throughput cell fluorescence measurements. Finally, the facility is equipped with a laminar flow hood to conduct chemistry to create biomaterials and micropatterned surfaces for stem cell culture, as well as subsequent analysis by imaging and flow. We are very thankful to CIRM for enabling the construction and development of this state-of-the-art stem cell facility. Also, the generous resources of CIRM have been additionally leveraged, as the facility director has obtained additional campus funds to purchase additional equipment and further enhance the capabilities of the existing equipment. Our facility is directed by Prof. David Schaffer in collaboration with the management oversight committee. In addition, it is managed by Dr. Mary West, who has successfully overseen the installation of the equipment and resources described above, provides rigorous training to our users, and aids in the development of imaging strategies to enable numerous experiments. In greater scientific detail, 75 students and postdoctoral fellows from the laboratories of 21 PIs have approved access and are using the facility to date. Therefore, these resources are enabling and enhancing a large and growing number of research projects, which are described in greater detail in the full scientific progress report. The goal of a project in the Song Li lab that has made great progress this year is to derive functional neural crest stem cells (NCSCs) from embryonic stem cells and induced pluripotent stem cells. The derived NCSCs were used in nerve and blood vessel regeneration. In addition, other projects aimed at identification and characterization of adult stem cells in blood vessels. The marker expression and multipotency were determined by using ImageXpress and flow cytometry at CIRM SSCF. The next phase of this study focuses on the function of vascular stem cells in other diseases including arthrosclerosis. As another example, the Healy lab is developing new synthetic substrates for culturing human embryonic stem cells under defined conditions. Progress was made and published by the Healy lab in creating synthetic culture surfaces and materials for long-term hESC propagation, a project that has involved heavy usage of the ImageXpress. As a final example, the Schaffer lab has engineered new gene delivery vehicles that can augment and edit the genome of hESCs and neural stem cells. Quantification of gene delivery efficiency and gene targeting has been greatly aided with the flow cytometry and high throughput imaging capabilities of the CIRM facility. Finally, the Kumar lab studies how the molecular and mechanical properties of the cellular cytoskeleton regulate cell function. For example, he uses laser ablation of subcellular features such as cytoskeletal filaments to provide real time information on the mechanical properties of the cytoskeleton. He is establishing laser ablation using the multiphoton microscope in the CIRM facility, a capability that will be applied to study cytoskeletal mechanics of stem cells. We anticipate in the upcoming year that this valuable facility will thus be increasingly utilized to advance a growing number of projects.

Year 3

Grant Number: CL1-00519 PI Name: David V. Schaffer Email: schaffer@berkeley.edu Project Title: The Berkeley Human Embryonic Stem Cell Shared Research Laboratory a. Scope of Research Taking Place in the Facility The central mission of our Berkeley CIRM Shared Stem Cell Facility (SSCF) is to provide our East Bay users with knowledge, expertise, training, and equipment to advance scientific knowledge of human embryonic stem cells, as well as other stem cells. Our facility focuses on providing imaging, flow cytometry, and sorting. To date, 90 students and postdoctoral fellows from the laboratories of 21 PIs use the facility. We list some examples here: Irina Conboy (BioE). The Conboy lab studies the intersection of aging and stem cell science. If age-imposed decline in the regenerative capacity of stem cells was understood, the debilitating lack of organ maintenance in the old could be ameliorated and perhaps, even reversed. They used the SSCF confocal microscope and flow cytometer in their studies. Paliwal P, et al. (2012) Age dependent increase in the levels of osteopontin inhibits skeletal muscle regeneration. Aging 4(8):553. Paliwal P, Conboy IM. (2011) Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells. Chem Biol 18(9):1153. Sanjay Kumar (BioE). The Kumar Lab’s interests center around the macromolecular basis of cell shape, mechanics, and adhesion, with special emphasis on the nervous system. Specifically, they seek to understand how elements of the cytoskeleton and adhesion machinery physically interact to form a three-dimensional architecture that drives cell shape and shape-dependent behavior and transduces biochemical signals. The SSCF swept field confocal was used to document changes in soft ECMs (MacKay). This microscope was also used by researcher Amit Pathik, who developed paradigm for investigating matrix regulation of invasion (Pathak). J. L. MacKay, et al. (2012). A genetic strategy for the dynamic and graded control of cell mechanics, motility, and matrix remodeling. Biophysical Journal 102: 434. A. Pathak and S. Kumar (2012). Independent regulation of tumor cell migration by matrix stiffness and confinement. PNAS 109 (26): 10334. David V. Schaffer (ChemE, BioE, and Helen Wills Neuroscience Institute). One recently-completed CIRM-funded project involved directed evolution and engineering of new viral gene delivery vehicles capable of highly efficient delivery to human embryonic and induced pluripotent stem cells. This Adeno-associated virus (AAV) variant study involved heavy usage of the flow cytometer and the ImageXpress Micro to quantitate the number of successfully infected (and gene-targeted) cells and colonies (Asuri). In a current CIRM-funded project, we are engineering synthetic materials for the scaleable expansion and dopaminergic differentiation of human ESCs and iPSCs (Keung). Also, we are developing optogenetic tools to investigate signaling pathways involved in stem cell fate decisions (Bugaj). Finally, we discovered a novel signaling pathway that regulates the differentiation of adult neural stem cells into neurons (Ashton). Asuri, P., et al. (2012) “Directed Evolution of Adeno-Associated Virus for Enhanced Gene Delivery and Gene Targeting in Human Pluripotent Stem Cells.” Molecular Therapy 20:329. Bugaj, L.J., et al. (2013) “A Modular Optogenetic Platform for Inducible Protein Clustering and Signaling Activation in Mammalian Cells.” Nature Methods 10:249. Keung, A.J., et al. (2012) “Soft Microenvironments Promote the Early Neurogenic Differentiation but not Self-renewal of Human Pluripotent Stem Cells.” Integrative Biology 4:1049. Ashton, R.S., et al. (2012) “Astrocytes Regulate Adult Hippocampal Neurogenesis Through Ephrin-B Signaling.” Nature Neuroscience 15:1399. b. Management and Use of the Laboratory The overall management of the CIRM Shared Stem Cell Facility CIRM SSCF is coordinated by the management oversight committee.CIRM SSCF is managed by Dr. Mary West. The facility is open to qualified researchers from 9:30 am to 5 pm with controlled access afterhours. c. Activities of Hired Laboratory Personnel The CIRM Shared Stem Cell Facility is managed by Dr. Mary West who is responsible for training, equipment function and maintenance. CIRM is also funding an operator, Alma Faleros, for the BD FACS instrument, who works from 12:30 to 9:30pm. d. Activities of the Oversight Committee The oversight committee is composed of Facility Director David Schaffer and Profs. Kevin Healy, Song Li, Sanjay Kumar, Irina Conboy, and Astar Winoto. The committee meets regularly with the Facility Manager to obtain updates on the facility, discuss prioritization for new equipment purchases, facility finances, and policies for access. e. Plans and Any Expected Changes for the Next Reporting Period In the next year we anticipate busy operation of the facility with no major changes.

Year 4

The central mission of our Berkeley CIRM Shared Stem Cell Facility (SSCF) is to provide our East Bay users with knowledge, expertise, training, and equipment to advance scientific knowledge of human embryonic, induced pluripotent, and adult stem cells. We have reached a steady state usage of ~90 students and postdoctoral fellows from the laboratories of 21 PIs at the SSCF, located in Stanley Hall located on the North east end of campus. In addition, cell sorting instrumentation remains in the campus flow facility. Based on the interests of the users, we have continued in Year 4 to provide informational seminars, conduct new instrument demonstrations, offer bulk discounted rates on expensive stem cell medias, and find creative ways to obtain additional instrumentation that will be useful to users. For instance, this year we added considerable automated equipment by incorporating a High-Throughput Screening Facility (HTSF) into our management and administrative structure. This facility includes cell seeding equipment within a BSL-2 cell culture hood, two automated liquid handlers with complementary capabilities, a multi-label plate reader, and automated imager (similar to the SSCF ImageXpress Micro) with a robotic servicing arm for screening of multi-well plates. Most automation equipment was retained at its originally location in Li Ka Shing, in the CIRM Center of Excellence facility, and some CIRM-funded faculty are currently are using the facility to screen for protein partners in pathways that are unique to stem cell biology. In Stanley Hall, the SSCF recently added a new room, B203 Stanley, for a state-of-the-art instrument that helps to detect levels of multiple proteins or growth factors simultaneously from a small sample of tissue or serum. In addition, the HTSF automated imaging equipment and accompanying computer server was moved to B203 Stanley so the facility could have direct management over training and use. These additions this year will further enhance the offerings to stem cell researchers to widen the breadth of the instrumentation that we could offer to researchers, who are grateful that we have these instruments in our facility because of the shared aspect of maintenance and training. The previously highlighted PIs (detailed in Years 2 and 3) continue to have success using SSCF instrumentation and we would like to highlight publications by researchers who have thus made significant advancements to their research. Publications: Downing T., J. Soto, C. Morez, T. Houssin, A. Fritz, F. Yuan, J. Chu, S. Patel, D.V. Schaffer, S. Li, (2013), "Biophysical regulation of epigenetic state and cell reprogramming". Nature Materials, 12:1154-62 (PMID: 24141451). Cousin, W., M. Ho, R. Desai, A. Tham, R. Chen, S. Kung, C. Elab, I. Conboy, (2013) "Regenerative Capacity of Old Muscle Stem Cells Declines without Significant Accumulation of DNA Damage" Public Library of Science (PLoS), 8:e63528 (PMID: 23704914). Yousef, H., M.J. Conboy, J. Li, M. Zeiderman, T. Vazin, C. Schlesinger, D.V. Schaffer, and I.M. Conboy (2013) “hESC-Secreted Proteins can be Enriched for Multiple Regenerative Therapies by Heparin-Binding.” Aging, 5:357-372 (PMID 23793469). Elabd, C., W. Cousin, R. Chen, M. Chooljian, J. Pham, I. Conboy, M. Conboy (2013) "DNA methyltransferase-3-dependent nonrandom template segregation in differentiating embryonic stem cells." The Journal of Cell Biology, 203:73-85 (PMID: 24127215). A. Pathak and S. Kumar (2013). “Transforming potential and matrix stiffness co-regulate confinement sensitivity of tumor cell migration.” Integrative Biology 5: 1067-1075 (PMID: 23832051). Ma, Z., S. Koo, M. Finnegan, P. Loskill, N. Huebsch, N. Marks, B. Conklin, C. Grigoropoulos, K. Healy K. (2013) “Three-dimensional filamentous human diseased cardiac tissue model”, Biomaterials. (5):1367-1377 (PMID: 24268663). Halkias, J., H. Melichar, K. Taylor, J. Ross, B. Yen, S. Cooper, A. Winoto, and E. Robey (2013) "Opposing chemokine gradients control human thymocyte migration in situ" The Journal of Clinical Investigation, (5):2131-42 (PMID: 23585474). Bugaj, L.J., A.T. Choksi, C.K. Mesuda, R.S. Kane, and D.V. Schaffer (2013) “A Modular Optogenetic Platform for Inducible Protein Clustering and Signaling Activation in Mammalian Cells.” Nature Methods, 10:249-252 (PMID 23377377). Conway, A., T. Vazin, D.P. Spelke, N.A. Rode, K.E. Healy, R.S. Kane, and D.V. Schaffer (2013) “Multivalent Ligands to Control Stem Cell Behaviour in Vitro and in Vivo.” Nature Nanotechnology, 8:831-838 (PMID 24141540). Keung, A.J., M. Dong, D.V. Schaffer (co-corresponding author), and S. Kumar (2013) “Pan-neuronal Maturation But Not Neuronal Subtype Differentiation of Adult Neural Stem Cells is Mechanosensitive.” Scientific Reports, 3:1817 (PMID 23660869). Lei, Y. and D.V. Schaffer “A Fully Defined and Scalable 3D Culture System for Human Pluripotent Stem Cell Expansion and Differentiation.” Proceedings of the National Academy of Sciences USA (in press).

© 2013 California Institute for Regenerative Medicine