The GEiC supports investigators in all aspects of the generation, maintenance, and differentiation of induced pluripotent stem cells.
Induced pluripotent stem technology (iPSC): The ability to re-program cells back to a pluripotent state has altered biology and medicine in profound ways. The GEiC has established robust methodologies to enable investigators to leverage the potential of iPSC technology. These include all of the methods needed to generate, maintain, genetically manipulate, and differentiate iPSCs. The GEiC works directly with investigators to obtain patient fibroblasts or renal tubular epithelial cells. These cells can be converted into induced pluripotent stem cells (iPSCs), which can then be studied directly or differentiated into a variety of cell types. The capabilities of the GEiC to produce targeted mutations in pluripotent stem cells or iPSCs via genome engineering further enhances the utility of these cells for functional analysis of genomic variants.
Patient Sample Processing
Skin biopsy samples must be stored at 4°C in DMEM + antibiotics. Samples from off-site should be shipped on ice as soon as possible after collection, as the yield of viable cells deteriorates with time. We have successfully processed samples from skin biopsies that were processed up to 7 days after collection. We have also implemented a protocol to isolate renal tubular epithelial cells from urine for subsequent reprogramming and generation of iPSCs from patients. This non-invasive procedure simplifies the IRB process and enables studies that might not otherwise be allowed, particularly with children or critically ill patients. To obtain viable renal tubular epithelial cells from the sample, the urine must be transported on ice and arrive at the GEiC within 4 hr of collection. Please use our patient sample submission form when dropping off samples.
- Fibroblast Expansion – Skin biopsies are processed using the dry plate method to generate a homogenous population of low passage fibroblasts. We freeze 10 vials per sample and each vial contains ~500,000 fibroblasts. The remaining skin tissue is also banked along with the derived fibroblasts, as it is often possible to re-dissociate and produce additional fibroblasts if necessary.
- Renal Epithelial Cell Expansion – The cells are collected by centrifugation and cultured for ~2-3 weeks prior to splitting the culture for expansion and cryopreservation. Ten vials containing ~500,000 renal tubular epithelial cells are frozen down for each patient sample. We are currently developing a protocol to improve the viability so that samples collected off-site can be processed in the GEiC.
Reprogramming to generate patient-derived iPSCs
After generating sufficient starting material (fibroblasts or renal tubular epithelial cells), the cells are transduced with Sendai virus, a non-integrating virus, that transiently expresses Oct4, c-Myc, Sox-2, and Klf4, the four transcription factions originally discovered to reprogram cells into iPSCs (Takahashi et al., 2007). In contrast to most other reprogramming protocols, which rely on viral vectors that integrate into the host genome, the Sendai virus is eventually lost from the cell. The loss of expression of the four transcription factors after reprogramming is beneficial because there are fewer alterations to the host genome from viral integration and subsequent iPSC differentiation is unimpeded by continued expression of pluripotency factors. The cells are converted to iPSCs over an extended period during which the cells are continually monitored and maintained. After 3-4 weeks (on average), experienced GEiC scientists identify colonies with cells that have a morphology consistent with the ‘pluripotent state’. These cells are ‘picked’ for further expansion utilizing a feeder-free culture system. After expansion, the cells are cryopreserved (1 million iPSCs/vial) in liquid nitrogen. In the event that the reprogramming event fails to produce colonies with a pluripotent appearance, one additional attempt will be made with the sample. If appropriate pluripotent colonies are still not produced, the line is considered refractory to reprogramming, and we recommend submitting another sample.
Custom iPSC Line Creation – Prices vary depending on project. Please contact us to get a quote.
CRISPR nucleases can be used to create iPSC lines with specific genomic modifications (as above). The capability to genetically incorporate (or correct) disease-causing point mutations in patient-derived iPSC lines will be invaluable for elucidating disease mechanisms through functional genomics, for identifying therapeutic agents and for the development of new cell-based therapies. The GEC has now been merged with the Induced Pluripotent Stem cell (iPSC) facility, which banks patient fibroblasts, generates iPSCs, and develops iPSC differentiation schemes. This allows for a seamless transition from patient derived iPSC generation to the engineering of modified iPSC lines. The GEP will have access to all the capabilities of this newly combined Center.
Characterization of iPSCs
Using iPSCs generated as above, the cells are characterized for expression of pluripotent markers and their ability to generate germ layers. The cells are stained for multiple markers including OCT4, SOX2, TRA1-60/81, NANOG, and SSEA4. In addition, germ layer formation via spontaneous differentiation into embryoid bodies (EBs) is assessed using antibody staining for markers of each germ layer: ectodermal (TUJ1 or GFAP), endodermal (FOXA2 or AFP), or mesodermal (SMA or DESMIN). Cells that appear pluripotent by these assays can then be karyotyped for an additional fee to ensure that gross chromosomal rearrangements have not occurred during the culturing process. iPSCs that pass these criteria are then delivered to the investigator or used in downstream differentiation assays as requested by the investigator.
Stem Cell Training Course
Under the sponsorship of STEMCELL Technologies and in collaboration with the hESC facility, the GEiC offers a two-day training courseon how to maintain hESC/iPSC cultures using feeder-free systems and induce their differentiation into selected cell types. The course consists of technical lectures each day and an instructional booklet is provided to each participant. On day 1, the trainees gain hands-on experience in passaging hES and iPS cells. On day 2, the participants learn to generate embryoid bodies and discuss protocols for differentiating pluripotent cells into specific lineages. Most importantly, the training course offers hands-on practical sessions and in-depth discussions of topics and questions raised by the participants.