Descripton of iPSC Core Services
The iPSC Core facilitates the derivation of iPS cells from mouse and human somatic cells and serves as a resource for sharing iPSC lines and iPSC technology within the UPenn and the broader scientific community. The following services are offered by the Core. Please contact the Core Director to set up a strategy meeting before beginning any service.
Expansion and quality control of primary cells
The Core will accept primary cells (fibroblasts, PBMCs) for iPSC derivation. Cells should be at low passage and are proliferating as non-dividing cells are not likely to generate iPSCs. We will assess cell growth and determine optimal media conditions; mycoplasma testing will also be performed. Parental cell lines will also be cryopreserved to serve as back-up stock. This service will take about 2-3 weeks to complete.
Derivation of iPSCs
After the primary cell culture has passed quality control, the Core staff will proceed with reprogramming as follows:
- Plate parental cells in 6-well plates
- Transduce cells with 4-factor lentiviral vector
- Transfer transduced cells onto feeders
- Pick iPSC colonies after 3-4 weeks
- Expand 3-6 clones and freeze ~6 vials per clonally expanded iPSC line
- Perform mycoplasma testing on each frozen iPSC line
Inherent to iPSC reprogramming, a certain percentage of cells will fail to produce iPSCs that is likely cell specific and not due to technical reasons. If we do not obtain stable colonies after the first round of reprogramming, we will repeat the process. However, if the second round fails, we will not repeat again and will only charge 50% of the service fee. This service will require 3-6 months to complete. At this point, established iPSC lines can be transferred to the investigator without further characterization or they can be expanded and characterized at the Core.
Characterization of iPS cells
The Core offers two services for characterization of iPSC lines derived at the Core: basic characterization for expression of pluripotency markers and differentiation by embryoid body (EB) formation. Up to three clonally expanded iPS cell lines derived from one parental cell line will be expanded and tested. Other quality control services including karyotyping and DNA finger printing are also offered.
Each iPSC line (up to three lines per parental cell line) will be subjected to alkaline phosphatase staining; immunostaining with antibodies against Oct4, Nanog, SSEA4, and TRA1-60; and qPCR analysis for endogenous Oct4, endogenous Sox2, Nanog, Rex1 and Dnmt3b gene expression.
Each iPSC line (up to three per parental cell line) will be differentiated in media without bFGF (human) or LIF (mouse) for 2 weeks and harvested. RNA will be prepared and qPCR will be performed to assay for the expression of differentiation markers for the three cell lineages.
iPSC culture will be expanded and live culture will be sent to Cell Line Genetics for karyotyping.
DNA Finger Printing
Feeder-free iPSC culture will be expanded and cells will be harvested. Genomic DNA will be isolated from iPSCs and the corresponding parental cell line. DNA will be sent to Cell Line Genetics for analysis for orginating vs. resulting cell vertification.
The Core does not currently offer teratoma formation assays. However, we can facilitate this service with the UPenn Stem Cell and Xenograft Core Facility and the MCRC Histology Core Facility.
Hands-on training option for iPSC derivation
For researchers who would like to train on a one-on-one basis for iPSC reprogramming, the Core accepts a limited number of reseachers (1-2 at the same time) with cell culture experience to derive iPSCs in our facility. With the help of the Core staff, the researcher will be responsible for all work involving cell expansion and quality control and derivation. All reagents will be provided by the Core. The training period will last up to 4 months, and up to 4 parental cell lines may be used for reprogramming during this time period.
iPS cell line distribution
The Core has several established mouse and human iPSC lines for purchase for research purposes by non-profit institutions. For outside purchases, an MTA between UPenn and your institution is required.
The Core offers technical consulting for iPSC related research projects. Each session typically lasts 1-2 hours. Please contact the Director for more information.
Hands-on training course for ES/iPS cell culture
Training by the Core staff on ES/iPS cell culture including thawing, expansion, manual and enzymatic passaging, feeder-free culture, and EB formation. This training typically lasts 1 month.
|Services||Rates (per unit)|
|Cell expansion and quality control*||$300|
|iPSC derivation (hands-on training option)*||$4,000|
|Basic iPSC characterization *||$600|
|iPSC DNA finger printing||$650|
|Distribution of iPSC lines||$600|
* Special pricing may be available for more than 4 units. Please contact the Core Director.