Mouse adult hematopoietic stem cells actively synthesize ribosomal RNA.

The contribution of basal cellular processes to the regulation of tissue homeostasis has just started to be appreciated. However, our knowledge of the modulation of ribosome biogenesis activity in situ within specific lineages remains very limited. This is largely due to the lack of assays that enable quantitation of ribosome biogenesis in small numbers of cells in vivo. We used a technique, named Flow-FISH, combining cell surface antibody staining and flow cytometry with intracellular ribosomal RNA (rRNA) FISH, to measure the levels of pre-rRNAs of hematopoietic cells in vivo. Here, we show that Flow-FISH reports and quantifies ribosome biogenesis activity in hematopoietic cell populations, thereby providing original data on this fundamental process notably in rare populations such as hematopoietic stem and progenitor cells. We unravel variations in pre-rRNA levels between different hematopoietic progenitor compartments and during erythroid differentiation. In particular, our data indicate that, contrary to what may be anticipated from their quiescent state, hematopoietic stem cells have significant ribosome biogenesis activity. Moreover, variations in pre-rRNA levels do not correlate with proliferation rates, suggesting that cell type-specific mechanisms might regulate ribosome biogenesis in hematopoietic stem cells and progenitors. Our study contributes to a better understanding of the cellular physiology of the hematopoietic system in vivo in unperturbed situations.

Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells.

UNLABELLED: Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder-free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, interleukin-6, and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG-derived hepatoblasts. hESC- and HepaRG-derived cholangiocyte-like cells expressed markers of cholangiocytes including cytokeratin 7 and osteopontin, and the transcription factors SOX9 and hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor, and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca(2+) . We demonstrated by integrative genomics that the expression of genes, which signed hESC- or HepaRG-cholangiocytes, separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte-like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins. CONCLUSION: We have developed a robust and efficient method for differentiating pluripotent stem cells into cholangiocyte-like cells, which display structural and functional similarities to bile duct cells in normal liver. These cells will be useful for the in vitro study of the molecular mechanisms of bile duct development and have important potential for therapeutic strategies, including bioengineered liver approaches.

Compensation between Wnt-driven tumorigenesis and cellular responses to ribosome biogenesis inhibition in the murine intestinal epithelium.

Ribosome biogenesis inhibition causes cell cycle arrest and apoptosis through the activation of tumor suppressor-dependent surveillance pathways. These responses are exacerbated in cancer cells, suggesting that targeting ribosome synthesis may be beneficial to patients. Here, we characterize the effect of the loss-of-function of Notchless (Nle), an essential actor of ribosome biogenesis, on the intestinal epithelium undergoing tumor initiation due to acute Apc loss-of-function. We show that ribosome biogenesis dysfunction strongly alleviates Wnt-driven tumor initiation by restoring cell cycle exit and differentiation in Apc-deficient progenitors. Conversely Wnt hyperactivation attenuates the cellular responses to surveillance pathways activation induced by ribosome biogenesis dysfunction, as proliferation was maintained at control-like levels in the stem cells and progenitors of double mutants. Thus, our data indicate that, while ribosome biogenesis inhibition efficiently reduces cancer cell proliferation in the intestinal epithelium, enhanced resistance of Apc-deficient stem and progenitor cells to ribosome biogenesis defects may be an important concern when using a therapeutic strategy targeting ribosome production for the treatment of Wnt-dependent tumorigenesis.

Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote.

Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection. Our results also indicated that injection into the pronucleus compared to the cytoplasm is preferable to generate knock-in alleles with an oligonucleotide or a circular plasmid. Finally, we showed that Cas9D10A nickase variant was less efficient than wild-type Cas9 for generating knock-in alleles and caused a higher rate of mosaicism. Thus, our study provides valuable information that will help to improve the future production of precise genetic modifications in mice.