CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.
The pig represents an ideal large-animal model, intermediate between rodents and humans, for the preclinical assessment of emerging cell therapies. As no validated pig embryonic stem (pES) cell lines have been derived so far, pig induced pluripotent stem cells (piPSCs) should offer an alternative source of undifferentiated cells to advance regenerative medicine research from bench to clinical trial. We report here for the first time the derivation of piPSCs from adult fibroblast with only three transcription factors: Sox2 (sex determining region Y-box 2), Klf4 (Krüppel-like factor 4), and c-Myc (avian myelocytomatosis viral oncogene homolog). We have been able to demonstrate that exogenous Pou5f1 (POU domain class 5 transcription factor 1; abbreviated as Octamer-4: Oct4) is dispensable to achieve and maintain pluripotency in the generation of piPSCs. To the best of our knowledge, this is also the first report of somatic reprogramming in any species without the overexpression, either directly or indirectly, of Oct4. Moreover, we were able to generate piPSCs without the use of feeder cells, approaching thus xeno-free conditions. Our work paves the way for the derivation of clinical grade piPSCs for regenerative medicine.
Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands, targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features, and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall, the approach described here allows for the rapid generation of human cardiac grafts from hPSCs, in a total of 24 days, providing a suitable platform for cardiac engineering and disease modeling in the human setting.
Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. STEM CELLS 2016;34:2418-2428 SIGNIFICANCE STATEMENTWe have characterized, for the first time, the epigenetic changes observed in early human gonadal PGCs. Although the relative timing might differ due to species specific developmental progression, our data show that key epigenetic changes observed in the germline are conserved between mouse, pig, and human. Our work further demonstrates that gonadal epigenetic reprogramming starts in human PGCs around 9 weeks of gestation. Last, but not least, our observations regarding the BLIMP/PRMT5 complex highlights potential differences between germline regulatory complexes in mouse and human and suggests a potential role for lysinespecific demethylase-1 as a new epigenetic regulator during gonadal reprogramming in humans.
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