Induced Pluripotent Stem Cell Generation
The Tom and Sue Ellison Stem Cell Core offers reprogramming of cell lines into Induced Pluripotent Cells (iPSC). There are two non-integrating options available. The first is an Episomal method. This method requires the transfection of 1×106 cells with three vectors (pCXLE-hsk, pCXLE-hul, pCXLS-h). (Addgene; https://www.addgene.org/ submitted by J A Thomson; Yu et al., 2009, Science 324:797-801).
The other available option is utilizing a non-integrating Sendai viral kit available from Thermofisher. https://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf. The Cytotune 2.0 kit is especially good for reprogramming peripheral blood mononuclear cells isolated from whole blood and requires only 3×105 cells, though there is an increased charge for the expense of the kit.
The IPSC generation service takes approximately 30 days from scheduled start day. We generate a minimum of 3 clones* for you and provide you with frozen stock. A human fetal fibroblast line is run in parallel as a control.
IPS training includes reprograming one line with episomal factors as well as providing a control line for practicing techniques during the training. Training is one-on-one and includes isolation of clones*, pluripotent stem cell culture, and freezing.
Before we can schedule the training/reprograming, all incoming, non-peripheral blood somatic cell lines must test negative for mycoplasma. We recommend using the Mycoplasma Kit- Lonza #LT07-118. Any additional reagents or growth factors needed to grow the primary cell line before reprograming must be provided at the time of reprograming. It is essential to provide a low passage/low doubling, healthy adherent line for optimal fibroblast reprogramming.
Contact Chris Cavanaugh at ccava@uw.edu for further information and scheduling.
*The iPS clones generated and frozen are not characterized or stained for pluripotent factors. It is the responsibility of the receiving lab to confirm the cells are indeed pluripotent through additional screening.