Cornell Induced Pluripotent Stem Cell Core Laboratory
About the iPS Cell Core
| Reprogramming |
|---|
| ES and iPS Cell Culture |
| Characterization and Differentiation |
Established in August 2009, the Induced Pluripotent Stem Cell Core Laboratory (iPS Core) is located in the Veterinary Research Tower T9-010, and is part of the NYSTEM funded Mammalian Cell Reprogramming Core. Since the discovery of iPS technology (1), there has been a great deal of interest in the use of iPS cells as a source of stem cells for clinical therapy in a variety of animal models, and as a tool for understanding the maintenance of the pluripotent state and mechanisms of differentiation. There are, however, significant barriers to entry in stem cell culture, necessitating the establishment of a core facility which can perform services for researchers from different disciplines while minimizing variance and maintaining a level of quality control which is necessary for the successful culture and maintenance of stem cells.
The goal of the iPS Core is to provide researchers within the Cornell Community and the field with a resource for the generation and utilization of iPS cells. To this end the iPS Core offers a variety of services on a cost recovery basis including vectors for reprogramming somatic cells to pluripotent stem cells, established iPS cell lines, and a variety of services related to the characterization of the pluripotent state. Additionally, we are actively pursuing non-integrating reprogramming methods and the creation of human, canine and equine iPS cell lines.
Contact the iPS Core for more information.
(1) K. Takahashi, S. Yamanka. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126, 663-676 (August 2006).
Reprogramming
At this time, the iPS Core employs a lentiviral transduction method which can be used to routinely reprogram murine embryonic fibroblasts (MEFs) to a stem cell like state. The lentiviral vectors were created by the Jaenisch lab at the Whitehead Institute (1), and when transfected along with viral packaging constructs into a cell line such as 293T, produce a virus that is capable of expressing the reprogramming factors Oct 4, Sox 2, Klf 4, and c-Myc in a doxycyline inducible fashion. When introduced to MEFs, colonies of cells exhibiting stem cell like morphologies can be produced in as little as 2 weeks. These cells can be expanded into cell lines that maintain ES cell-like attributes and turn on the endogenous expression of these factors even after doxycyline has been removed from the culture media. At this time, the iPS Core is also pursuing the use of lentiviral reprogramming methods for the transformation of canine, equine and human cells to the pluripotent state as well.
In addition to lentiviral transduction, the iPS Core is interested in optimizing methods for non-integrating reprogramming as well. One potential drawback of lentiviral reprogramming strategies is that the transgenes are randomly inserted into the genomes of the target cells. Using alternative reprogramming systems such as the PiggyBac system (2), adenoviral methods (3), and transient plasmid transfection (4) can provide an alternative in situations where downstream applications require genetically unmodified iPS cells.
At this time, the iPS Core offers both ready to use lentiviral particles and in-house reprogramming.
Contact the iPS Core for more information.
(1) T. Bambrink, R. Foreman, G. G. Welstead, C. J. Lengner, M. Wernig, H. Suh, R. Jaenisch. Sequential Expression of Pluripotency Markers during Direct Reprogramming of Mouse Somatic Cells. Cell Stem Cell 2, 151-159 (February 2008).
(2) K. Woltjen, L. P. Michael, P. Mosheni, R. Desai, M. Mileikovsky, R. Hamalainen, R. Cowling, W. Wang, P. Liu, M. Gerstenstein, K. Kaji, H. Sung, A. Nagy. piggyBac Transposition Reprograms Fibroblasts to Induced Pluripotent Stem Cells. Nature 458, 766-770 (April 2009).
(3) W. Zhou, C. R. Freed. Adenoviral Gene Delivery can Reprogram Human Fibroblasts to Induced Pluripotent Stem Cells. Stem Cells 27, 2667-2674 (2009)
(4) K. Okita, H. Hong, K. Takahashi, S. Yamanaka. Generation of Mouse-Induced Pluripotent Stem Cells with Plasmid Vectors. Nature Protocols 5, 418-428 (2010).
Culturing and maintaining ES and iPS cells requires a great commitment of time and resources which may represent a significant challenge to researchers interested in utilizing ES and iPS cells in their experiments. The iPS Core routinely maintains ES, iPS, and other mammalian cell lines in culture, and is uniquely positioned to assist researchers by serving as a resource for tissue culture. At this time the iPS Core provides murine derived embryonic fibroblasts, iPS and ES cell lines, and is working towards becoming an established source for canine, equine and human derived cell lines.
Contact the iPS Core for more information.
Characterization and Differentiation
True ES cells and completely reprogrammed iPS cells are capable of differentiating into tissues of all three germ lineages. Validating the pluripotent state, however, is a daunting task requiring numerous assays. One goal of the iPS Core is to establish methods to evaluate the potential of iPS cells, and determine if they are truly comparable to true ES cells. Directed in-vitro differentiation, as well as a variety of protein and gene expression assays can be used to determine the potential of iPS cells. In addition, the iPS Core plans to utilize in-vivo techniques (teratoma formation) in order to validate the iPS cells.
Contact the iPS Core for more information.
Contact Information
Christian Abratte – Technician
Induced Pluripotent Stem Cell Core Laboratory
Veterinary Research Tower T9-010
Telephone: (607) 253-4189
Email: ca258@cornell.edu