Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status
Jason P Awe, Patrick C Lee, Cyril Ramathal, Agustin Vega-Crespo, Jens Durruthy-Durruthy, Aaron Cooper, Saravanan Karumbayaram, William E Lowry, Amander T Clark, Jerome A Zack, Vittorio Sebastiano, Donald B Kohn, April D Pyle, Martin G Martin, Gerald S Lipshutz, Patricia E Phelps, Renee A Reijo Pera and James A Byrne
The reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic “stem cell cassette” (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming cassette influences those genes and whether these cells still have clinical potential are not yet known.
In this study, we used both microarray and sensitive real-time polymerase chain reaction to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using non-restrictive linear amplification polymerase chain reaction. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions.
We found that a STEMCCA derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared to post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions by using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status.
For the first time these studies provide proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.
List of human dermal derivatives with patient-specific therapeutic potential
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