iPSC Generation from Patient Samples
Our scientists are experts in the generation of induced pluripotent stem cell (iPSC) lines from patient specific fibroblasts or blood cells. We have three methods for iPSC generation: mRNA, episomal (both genomic footprint-free), or retroviral.
Genomic footprint-free iPSC Generation Service - our custom cell line generation service.
No genomic footprint - the non-integrating system is safe in drug discovery and cell therapy applications.
Two methods available: Episomal DNA vectors containing the reprogramming factors or mRNA.
High reprogramming efficiency (0.2%-0.5%).
The reprogramming vectors/mRNA are removed naturally during the cell cycle.
Safe to handle - no viral particles.
1. Cell recovery and Quarantine
|2-3 weeks||A Report on cloning and validation|
|2. Transfection with Vectors||1 week|
3. Colony Formation
4. Colony expansion
|2 clones with two vials of each clone (>2 x 105 viable cells/ vial)|
- For iPSC generation from fibroblasts, you will need to provide 1 x 10^6 cells from patient fibroblasts.
- For iPSC generation from PBMCs, you will need to provide 2 vials of 2-3 x 10^6 cells from your patient samples
- At least 2 clones with two vials of each clone (>2 x 105 viable cells/ vial)
- Clones are characterized by pluripotency markers (OCT4, SOX2, SSEA4, TRA-1-60, TRA-1-80)
- Embryoid body formation (optional)
- Teratoma analysis (optional)
Applied StemCell, Inc. can create CRISPR/Cas9 modified iPSCs using patient samples.
1. What will customers need to provide for iPSC generation from patient fibroblasts?
For iPSC generation from patient fibroblasts, customers will need to provide 1 x 10^6 cells.
2. What is the minimum numbers of cells needed for iPSC generation from PBMCs?
For iPSC generation from PBMCs, we will need 2 vials of 2-3 x 10^6 cells.
CASE STUDY – Patient derived iPSC Generation
ASC has successfully generated iPSCs from several disease specific patient samples. Here is one such project:
Case Study: Generation of Human iPSCs from Patient-derived Skin Fibroblasts
Goal: To generate an iPSC line from patient derived dermal fibroblasts.
The dermal fibroblasts were obtained by skin biopsy from a patient with a rare disorder. The fibroblasts were plated, transfected via electroporation and cultured with ASC’s proprietary iPSC generation media until iPSC colonies appeared. Five high quality candidate colonies were expanded and characterized by immunocytochemical staining using primary antibodies for: OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81) and alkaline phosphatase staining. All five clones stained positive for various pluripotency markers tests as well as for alkaline phosphatase (Figure1).
Figure 1. Representative iPSC clones reprogrammed from patient derived fibroblasts. The clones tested positive for pluripotency markers: OCT4, SOX2, SSEA4, TRA-160, TRA-1-81 and for alkaline phosphatase (AP).
- Allende, M. L., Cook, E. K., Larman, B. C., Nugent, A., Brady, J. M., Golebiowski, D., ... & Proia, R. L. (2018). Cerebral organoids derived from Sandhoff disease induced pluripotent stem cells exhibit impaired neurodifferentiation. Journal of Lipid Research, jlr-M081323.
- Field, A. R., Jacobs, F. M., Fiddes, I. T., Phillips, A. P., Reyes-Ortiz, A. M., LaMontagne, E., ... & Katzman, S. (2017). Structurally conserved primate lncRNAs are transiently expressed during human cortical differentiation and influence cell type specific genes. bioRxiv, 232553.