• iPSC Generation from Patient Samples

iPSC Generation from Patient Samples

Our scientists have extensive experience using patient-specific PBMCs or fibroblasts to generate induced pluripotent stem cell (iPSC) lines. We have perfected both footprint-free (episomal, mRNA and other methods) and retroviral reprogramming methods for iPSC generation.

The advantages of using our footprint-free Custom iPSC Generation Service:

  • Highly efficient reprogramming (0.2%-0.5%)

  • High quality and purity iPSCs

  • Footprint-free reprogramming of patient-derived samples: PBMCs (blood)/ fibroblast

  • Well-optimized feeder/ feeder-free protocols

  • Suitable for drug discovery and cell therapy applications

  • Comprehensive suite of iPSC characterization and differentiation to somatic lineages services (teratoma, embryoid body formation, PCR and antibody pluripotency markers detection)

Products and Services

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FAQs
What will customers need to provide for iPSC generation from patient fibroblasts?
What is the minimum numbers of cells needed for iPSC generation from PBMCs?
Publications
  • 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.
Technical Details
6-steps IPSC Generation Service - Stable Cell Line Generation Service
6-steps IPSC Generation Service - Stable Cell Line Generation Service

 
Service Time Deliverables
1. Cell recovery and Quarantine
 2-3 weeks A Report on cloning and validation
2. Transfection with Vectors 1 week  
3. Colony Formation
1 month  
4. Colony expansion
1 month  
5. Characterization 
  2 clones with two vials of each clone (>2 x 105 viable cells/ vial)

Service details:

  • 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 

Deliverables:

  • 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 also engineer CRISPR/Cas9 modified iPSCs using patient samples.
Case Studies

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).  

Case Study Figure 1 - Stable Cell Line Generation Service

Figure 1.Human iPSCs were generated from patient-derived dermal fibroblasts, using feeder-free, proprietary protocols. The reprogrammed iPSC clones were characterized by pluripotency marker staining for OCT4, SOX2, SSEA4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP).

Have Questions?

An Applied StemCell technical expert is happy to help, contact us today!