Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

Inhibition of FGF and TGF-ß Pathways in hESCs Identify STOX2 as a Novel SMAD2/4 Cofactor.

The fibroblast growth factor (FGF) and the transforming growth factor-ß (TGF-ß) pathways are both involved in the maintenance of human embryonic stem cells (hESCs) and regulate the onset of their differentiation. Their converging functions have suggested that these pathways might share a wide range of overlapping targets. Published studies have focused on the long-term effects (24-48 h) of FGF and TGF-ß inhibition in hESCs, identifying direct and indirect target genes. In this study, we focused on the earliest transcriptome changes occurring between 3 and 9 h after FGF and TGF-ß inhibition to identify direct target genes only. Our analysis clearly shows that only a handful of target transcripts are common to both pathways. This is surprising in light of the previous literature, and has implications for models of cell signaling in human pluripotent cells. In addition, we identified STOX2 as a novel primary target of the TGF-ß signaling pathway. We show that STOX2 might act as a novel SMAD2/4 cofactor. Taken together, our results provide insights into the effect of cell signaling on the transcription profile of human pluripotent cells.

Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology.

Modifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC), and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed. Here, we combined molecular and cellular biology with CRISPR/Cas9-mediated genome engineering to pinpoint the function of serine 12 of OCT4 in ESCs. Using chemical inhibitors and an antibody specific to OCT4 phosphorylated on S12, we identified cyclin-dependent kinase (CDK) 7 as upstream kinase. Surprisingly, generation of isogenic mESCs that endogenously ablate S12 revealed no effects on pluripotency and self-renewal, potentially due to compensation by other phosphorylation events. Our approach reveals that modification of distinct amino acids by precise genome engineering can help to clarify the functions of post-translational modifications on proteins encoded by essential gene in an endogenous context.

Comparative analysis of differential gene expression tools for RNA sequencing time course data.

RNA sequencing (RNA-seq) has become a standard procedure to investigate transcriptional changes between conditions and is routinely used in research and clinics. While standard differential expression (DE) analysis between two conditions has been extensively studied, and improved over the past decades, RNA-seq time course (TC) DE analysis algorithms are still in their early stages. In this study, we compare, for the first time, existing TC RNA-seq tools on an extensive simulation data set and validated the best performing tools on published data. Surprisingly, TC tools were outperformed by the classical pairwise comparison approach on short time series (<8 time points) in terms of overall performance and robustness to noise, mostly because of high number of false positives, with the exception of ImpulseDE2. Overlapping of candidate lists between tools improved this shortcoming, as the majority of false-positive, but not true-positive, candidates were unique for each method. On longer time series, pairwise approach was less efficient on the overall performance compared with splineTC and maSigPro, which did not identify any false-positive candidate.

A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).

Switch enhancers interpret TGF-ß and Hippo signaling to control cell fate in human embryonic stem cells.

A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the transforming growth factor ß (TGF-ß) pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex composed of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGF-ß pathway (SMAD2/3), and the pluripotency regulator OCT4 (TSO). TSO collaborates with NuRD repressor complexes to buffer pluripotency gene expression while suppressing ME genes. Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements, and upon fate specification we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch-enhancer elements and provide a framework to understand how cellular context dictates interpretation of the same morphogen signal in development.

The TGFß superfamily in stem cell biology and early mammalian embryonic development.

BACKGROUND: Members of the Transforming Growth Factor-beta (TGFß) superfamily of cytokines are essential for early embryonic development and play crucial roles in pluripotency and differentiation of embryonic stem cells in vitro. SCOPE OF REVIEW: In this review, we discuss how TGFß family signals are read by cells and how they are modulated by the cellular context. Furthermore, we review recent advances in our understanding of TGFß function in embryonic stem cells and point out hot topics at the intersection of TGFß signaling and stem cell biology fields. MAJOR CONCLUSION: TGFß family signals are essential for early mammalian development and the importance of this pathway is reflected in pluripotent stem cells derived from the mammalian embryo. GENERAL SIGNIFICANCE: Understanding signaling pathways underlying pluripotency and cell fate specification holds promises for the advent of personalized regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. PAPERCLIP:

The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis.

The liver is frequently exposed to insults, including toxic chemicals and alcohol, viral infection or metabolic overload. Although it can fully regenerate after acute injury, chronic liver damage causes liver fibrosis and cirrhosis, which can result in complete liver failure. In this study, we demonstrate that the NF-E2-related factor 2 (Nrf2) transcription factor protects the liver from acute and chronic toxin-mediated damage. Repair of the liver injury that occurs after a single treatment with the hepatotoxin carbon tetrachloride (CCl(4)) was severely delayed in Nrf2-deficient mice. The defect in repair was accompanied by an enhanced and prolonged inflammatory and profibrotic response. After long-term CCl(4) treatment, liver fibrosis was strongly aggravated in the Nrf2 knockout mice and inflammation was enhanced. We demonstrate that these abnormalities are at least in part due to the reduced expression of known and novel Nrf2 target genes in hepatocytes, which encode enzymes involved in the detoxification of CCl(4) and its metabolites. These results suggest that activation of Nrf2 may be a novel strategy to prevent or ameliorate toxin-induced liver injury and fibrosis.

The cytoprotective Nrf2 transcription factor controls insulin receptor signaling in the regenerating liver.

The Nrf2 transcription factor is a crucial regulator of the cellular redox homeostasis through its capacity to induce the expression of enzymes, which detoxify reactive oxygen species, and of other antioxidant proteins. Therefore, it plays an important role in the protection from carcinogenesis induced by various insults. In addition, recent results identified a novel role of Nrf2 in tissue repair. In the liver, regeneration after partial hepatectomy was strongly delayed in the absence of Nrf2. This defect was shown to result from transient resistance to insulin and insulin-like growth factor 1 that was caused by chronic oxidative stress in hepatocytes. These results demonstrate a link between Nrf2 deficiency, oxidative stress and insulin resistance, and suggest that activation of this transcription factor could be a novel strategy to improve liver regeneration in patients with acute or chronic liver injury. In addition, it may help to alleviate oxidative stress-induced insulin resistance in the liver and potentially also in other organs.

Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance.

The liver is frequently challenged by surgery-induced metabolic overload, viruses or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signaling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress. After partial hepatectomy, liver regeneration was significantly delayed. Using in vitro and in vivo studies, we identified oxidative stress-mediated insulin/insulin-like growth factor resistance as an underlying mechanism. This deficiency impaired the activation of p38 mitogen-activated kinase, Akt kinase and downstream targets after hepatectomy, resulting in enhanced death and delayed proliferation of hepatocytes. Our results reveal novel roles of Nrf2 in the regulation of growth factor signaling and in tissue repair. In addition, they provide new insight into the mechanisms underlying oxidative stress-induced defects in liver regeneration. These findings may provide the basis for the development of new strategies to improve regeneration in patients with acute or chronic liver damage.

Electrophilic chemicals but not UV irradiation or reactive oxygen species activate Nrf2 in keratinocytes in vitro and in vivo.

The NF-E2-related factor 2 (Nrf2) transcription factor is a potent inducer of cytoprotective genes, which encode--among others--enzymes that detoxify reactive oxygen species (ROS). As we demonstrated a crucial role of Nrf2 in the prevention of skin carcinogenesis, it is of interest to identify Nrf2-activating factors in keratinocytes. For this purpose, keratinocytes from mice transgenic for an Nrf2-responsive reporter gene were analyzed. Electrophilic compounds activated the reporter in keratinocytes, and induced nuclear translocation of Nrf2 and the expression of known Nrf2 target genes. This is biologically relevant, as we show that Nrf2-mediated gene expression protects keratinocytes from the toxicity of these substances. By contrast, hydrogen peroxide, glucose oxidase, UVA, and UVB irradiation had no effect, although these treatments strongly increased the levels of intracellular ROS. To verify these results in vivo, transgenic reporter mice with and without functional Nrf2 alleles were topically treated with electrophilic chemicals or irradiated with UVB. Electrophiles but not UVB activated the reporter in an Nrf2-dependent manner. These results provide the basis for the identification of novel Nrf2 activators in keratinocytes with therapeutic potential for skin tumor prevention.

Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing.

The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.

Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing.

Alternative splicing in the extracellular domain is a characteristic feature of members of the fibroblast growth factor receptor (FGFR) family. This splicing event generates receptor variants, which differ in their ligand binding specificities. A poorly characterized splice variant is FGFR1-IIIb, recently found to be a functional FGF receptor predominantly expressed in the skin. Here we show that FGFR1-IIIb is expressed in normal and wounded mouse skin. Reduced expression of this type of receptor was found in wounds of healing-impaired genetically diabetic mice, suggesting that downregulation of FGFR1-IIIb is associated with wound healing defects. To address this possibility, we deleted the IIIb exon of FGFR1 in mice. The lack of FGFR-IIIb did not alter the expression of either FGFR1-IIIc, other FGF receptor genes or of FGFR1-IIIb ligands in normal and wounded skin. Histological analysis of the skin of FGFR1-IIIb knockout animals did not reveal any obvious abnormalities. Furthermore, full-thickness excisional skin wounds in these mice healed normally and no defects could be observed at the macroscopic or histological level. Finally, several genes that encode key players in wound repair were normally expressed in these animals. These data demonstrate that FGFR1-IIIb is dispensable for skin development and wound repair.

Fibroblast growth factor 22 and its potential role during skin development and repair.

We have isolated, using RT-PCR, a cDNA from mouse skin wounds that encodes fibroblast growth factor (FGF) 22, a recently discovered member of the FGF family, which is closely related to FGF-7 and FGF-10. Transient expression of tagged FGF-22 protein in COS-1 and MCF-7 cells revealed that the protein was present within the cell and at the cell surface but was not apparently released from the cell. Analysis of RNA expression revealed that FGF-22 transcripts were not detected in the developing mouse embryo until day E16.5 and in the adult mouse it was expressed in the brain, tongue, and skin, but not in other tissues examined. After skin injury, FGF-22 mRNA levels were slightly down-regulated within the first 5 days after wounding, but expression increased strongly at the later stages of the repair process. In situ hybridization revealed the presence of FGF-22 mRNA throughout the epidermis and hair follicle keratinocytes of E16.5 embryos, as well as in adult skin and keratinocytes of the hyperthickened wound epithelium. This expression pattern suggests a potential role for FGF-22 in cutaneous development and repair.