Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status

Abstract

IntroductionThe 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 vector influences those genes and whether these cells still have clinical potential are not yet known.MethodsIn this study, we used both microarray and sensitive real-time PCR 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 nonrestrictive linear amplification PCR. 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.ResultsWe 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 with 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 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.ConclusionFor the first time, these studies provide a 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.

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

“…This technique was chosen in large part due to its reproducibility, relatively high reprogramming efficiency (0.02%), and robust ability to reprogram across many different cell types 10 . This technique is superior to other reprogramming methodologies such as sendai virus, episomal plasmids, and synthetic mRNA based reprogramming that suffer from lower reprogramming efficiencies and reproducibility 10 .…”

“…This technique was chosen in large part due to its reproducibility, relatively high reprogramming efficiency (0.02%), and robust ability to reprogram across many different cell types 10 . This technique is superior to other reprogramming methodologies such as sendai virus, episomal plasmids, and synthetic mRNA based reprogramming that suffer from lower reprogramming efficiencies and reproducibility 10 . Although there is a correlation between HIV-based vectors integrating into increased gene activity hotspots 15 , there is no guarantee that they will integrate into a gene, much less into a safer area of a gene, the intron.…”

“…Analyze the STEMCCA integration site by non-restrictive linear amplification PCR as described 10 . 2.…”

“…We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMPgrade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions 10 .…”

“…We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions 10 . Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus.…”