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Generation of transgene-free porcine intermediate type induced pluripotent stem cells.

ABSTRACT: Physiologically and anatomically, humans and pigs share many similarities, which make porcine induced pluripotent stem cells (piPSCs) very attractive for modeling human cell therapy as well as for testing safety of iPSC based cell replacement therapies. To date, several integrative and non-integrative strategies have been reported to successfully generate piPSCs, but all resulting piPSCs had integration of transgenes. The use of integrative methods has the disadvantage of potential lack of silencing or inappropriate re-activation of these genes during differentiation, as well as uncertainty regarding disruption of important genomic regions caused by integration. In our study, we performed a non-integrative vector based reprogramming approach using porcine fetal fibroblasts. The resulting four piPSC lines were positive for pluripotency marker and when subjected to in vitro and in vivo differentiation assays, all four lines formed embryoid bodies, capable to differentiate into all three germ layers, and three out of the four cell lines formed teratomas. PCR analysis on genomic and plasmid DNA revealed that the episomal vectors were undetectable in six out of eight subclones derived from one of the piPSC lines (piPSC1) above passage 20. These piPSCs could potentially be ideal cell lines for the generation of porcine in vitro and in vivo models. Furthermore, subsequent analyses of our new transgene independent piPSCs could provide novel insights on the genetic and epigenetic necessities to achieve and maintain piPSCs.

SUBMITTER: Li D

PROVIDER: S-EPMC6300098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 vector 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 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.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 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.For 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.

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Generation of transgene-free porcine intermediate type induced pluripotent stem cells.

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