Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell–derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.

The tightly controlled replication of hepatocytes in liver regeneration and uncontrolled proliferation of tumor cells in hepatocellular carcinoma (HCC) are often modulated by common regulatory pathways. Several microRNAs (miRNAs) are involved in HCC progression by modulating posttranscriptional expression of multiple target genes. miR-221, which is frequently up-regulated in HCCs, delays fulminant liver failure in mice by inhibiting apoptosis, indicating a pleiotropic role of miR-221 in hepatocytes. Here, we hypothesize that modulation of miR-221 targets in primary hepatocytes enhances proliferation, providing novel clues for enhanced liver regeneration. We demonstrate that miR-221 enhances proliferation of in vitro cultivated primary hepatocytes. Furthermore, applying two-thirds partial hepatectomy as a surgically induced liver regeneration model we show that adeno-associated virus-mediated overexpression of miR-221 in the mouse liver also accelerates hepatocyte proliferation in vivo. miR-221 overexpression leads to rapid S-phase entry of hepatocytes during liver regeneration. In addition to the known targets p27 and p57, we identify Aryl hydrocarbon nuclear translocator (Arnt) messenger RNA (mRNA) as a novel target of miR-221, which contributes to the pro-proliferative activity of miR-221. Conclusion: miR-221 overexpression accelerates hepatocyte proliferation. Pharmacological intervention targeting miR-221 may thus be therapeutically beneficial in liver failure by preventing apoptosis and by inducing liver regeneration. (Hepatology 2013;)

The POU-domain transcription factor OCT4 is associated with the pluripotent state of cells comprising the inner cell mass of pre-implantation embryos and has been known to play a critical role in the maintenance of pluripotency of embryonic stem cells. Reactivation of OCT4 expression is postulated to occur in differentiated cells that have undergone carcinogenesis, or tumor formation. In contrast to earlier studies, recent reports describe OCT4 expression in several human tumor cell lines. To resolve the apparent discrepancy in OCT4 expression between earlier and recent studies, we determined OCT4 expression in the cervical carcinoma cell line HeLa and the breast cancer cell line MCF7 in comparison with the human teratoma cell line nTera by immunofluorescence, Western blot, and RT-PCR analyses. We were unable to detect staining of the OCT4 transcription factor in the nucleus of HeLa and MCF7 cells by immunofluorescence using two different monoclonal antibodies. Faint cytoplasmic staining in HeLa and MCF7 cells was observed; however, no OCT4 signal could be detected by Western blot analysis. In addition, we were unable to detect significant levels of OCT4 mRNA in HeLa and in MCF7 cells by RT-PCR. Furthermore, the OCT4 promoter region is highly methylated in HeLa and MCF7 cells. We argue that recent reports of OCT4 expression in these and other cancer cell lines could actually be attributed to OCT4 pseudogene expression or misinterpretation of background signals in immunofluorescence experiments. In conclusion, we emphasize the need for adequate controls in investigations of OCT4 expression in somatic cell lines by immunofluorescence and RT-PCR. Disclosure of potential conflicts of interest is found at the end of this article.