A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells.
The generation of patient-specific induced pluripotent stem cells (iPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCs can be obtained in 4-5 months.
Center for Regenerative Medicine in Barcelona, Barcelona, Spain.
Angel
A
Raya
Ignasi
I
Rodríguez-Pizà
Susana
S
Navarro
Yvonne
Y
Richaud-Patin
Guillermo
G
Guenechea
Adriana
A
Sánchez-Danés
Antonella
A
Consiglio
Juan
J
Bueren
Juan Carlos
JC
Izpisúa Belmonte
D018929:Cell Culture Techniques; D054337:Cell Dedifferentiation; D002478:Cells, Cultured; D005199:Fanconi Anemia / Q000235:genetics / Q000628:therapy; D052217:Fanconi Anemia Complementation Group A Protein / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation; D014161:Transduction, Genetic; D014182:Transplantation, Autologous
C498471:FANCA protein, human; D052217:Fanconi Anemia Complementation Group A Protein; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4