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.

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.Flp recombinase | murine leukemia virus | pluripotent cells | protein transfer

Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

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;)

There has been recent progress in the isolation and characterisation of stem/progenitor cells that may differentiate towards the hepatic lineage. This has raised expectations that therapy of genetic or acquired liver disease might be possible by transplanting stem/progenitor cells or their liver-committed progeny. However, it is currently impossible to determine from the many documented studies which of the stem/progenitor cell populations are the best for therapy of a given disease. This is largely because of the great variability in methods used to characterise cells and their differentiation ability, variability in transplantation models and inconsistent methods to determine the effect of cell grafting in vivo. This manuscript represents a first proposal, created by a group of investigators ranging from basic biologists to clinical hepatologists. It aims to define standardised methods to assess stem/progenitor cells or their hepatic lineage-committed progeny that could be used for cell therapy in liver disease. Furthermore standardisation is suggested both for preclinical animal models to evaluate the ability of such cells to repopulate the liver functionally, and for the ongoing clinical trials using mature hepatocytes. Only when these measures have been put in place will the promise of stem/progenitor-derived hepatocyte-based therapies become reality.

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. STEM CELLS 2008;26:692-697 Disclosure of potential conflicts of interest is found at the end of this article.

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR–Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps). However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK) dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic.