Historically, effects of environmental toxicants on human development have been deduced via epidemiological studies because direct experimental analysis has not been possible. However, in recent years, the derivation of human pluripotent stem cells has provided a potential experimental system to directly probe human development. Here, we used human embryonic stem cells (hESCs) to study the effect of environmental toxicants on human germ cell development, with a focus on differentiation of the founding population of primordial germ cells (PGCs), which will go on to form the oocytes of the adult. We demonstrate that human PGC numbers are specifically reduced by exposure to polycyclic aromatic hydrocarbons (PAHs), a group of toxicants common in air pollutants released from gasoline combustion or tobacco smoke. Further, we demonstrate that the adverse effects of PAH exposure are mediated through the aromatic hydrocarbon receptor (AHR) and BAX pathway. This study demonstrates the utility of hESCs as a model system for direct examination of the molecular and genetic pathways of environmental toxicants on human germ cell development.

Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15-590 fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process. Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular characterization of enhancers of somatic cell reprogramming.

Abstract Introduction 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. Methods 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. Results 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. Conclusion 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.

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

Introduction

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.

Methods

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.

Results

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.

Conclusion

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.