A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.     

Oxygen-mediated regulation of tumor cell invasiveness. Involvement of a nitric oxide signaling pathway

Tumor hypoxia is associated with a poor prognosis for patients with various cancers, often resulting in an increase in metastasis. Moreover, exposure to hypoxia increases the ability of breast carcinoma cells to invade the extracellular matrix, an important aspect of metastasis. Here, we demonstrate that the hypoxic up-regulation of invasiveness is linked to reduced nitric oxide signaling. Incubation of human breast carcinoma cells in 0.5% versus 20% oxygen increased their in vitro invasiveness and their expression of the urokinase receptor, an invasion-associated molecule. These effects of hypoxia were inhibited by nitric oxide-mimetic drugs; and in a manner similar to hypoxia, pharmacological inhibition of nitric oxide synthesis increased urokinase receptor expression. The nitric oxide signaling pathway involves activation of soluble guanylyl cyclase (sGC) and the subsequent activation of protein kinase G (PKG). Culture of tumor cells under hypoxic conditions (0.5% versus 20% oxygen) resulted in lower cGMP levels, an effect that could be prevented by incubation with glyceryl trinitrate. Inhibition of sGC activity with a selective blocker or with the heme biosynthesis inhibitor desferrioxamine increased urokinase receptor expression. These compounds also prevented the glyceryl trinitrate-mediated suppression of urokinase receptor expression in cells incubated under hypoxic conditions. In contrast, direct activation of PKG using 8-bromo-cGMP prevented the hypoxia- and desferrioxamine-induced increases in urokinase receptor expression as well as the hypoxia-mediated enhanced invasiveness. Further involvement of PKG in the regulation of invasion-associated phenotypes was established using a selective PKG inhibitor, which alone increased urokinase receptor expression. These findings reveal that an important mechanism by which hypoxia increases tumor cell invasiveness (and possibly metastasis) requires inhibition of the nitric oxide signaling pathway involving sGC and PKG activation.     

Oxygen as a regulator of cellular phenotypes in pregnancy and cancer

Cellular phenotype is determined by genetic and microenvironmental factors. There is evidence that tissue oxygenation status is one of the microenvironmental factors regulating cellular behaviour. Both normal and pathological processes such as blastocyst implantation in the uterus, placentation, and rapidly growing tumours occur under conditions characterized by relatively low oxygen levels. In this review, we address the effects of low oxygen concentrations on the phenotype of trophoblast and cancer cells. We provide evidence that oxygenation levels play an important role in the regulation of normal and pathological cellular invasiveness as it occurs during trophoblast invasion of the uterus and in tumour progression and metastasis, drug resistance in cancer, and antitumour activity of natural killer cells of the immune system.     

Proteomic analysis of extracellular matrices used in stem cell culture

Numerous matrices for the growth of human embryonic stem cells (hESC) in vitro have been described. However, their exact composition is typically unknown. Information on the components of these matrices will aid in the development of a fully defined growth surface for hESCs. These matrices typically consist of mixture of proteins present in a wide range of abundance making their characterization challenging. In this study, we performed the proteomic analysis of five previously uncharacterized matrices: CellStart, Human Basement Membrane Extract (Human BME), StemXVivo, Bridge Human Extracellular Matrix (BridgeECM), and mouse embryonic fibroblast conditioned matrix (MEF-CMTX). Based on a proteomics protocol optimized using lysates from HeLa cells, we undertook the analysis of the five complex extracellular matrix (ECM) samples using a combination of strong anion and cation exchange chromatography and SDS-PAGE. For each of these matrices, we identify numerous proteins, indicating their complex nature. We also compared these results with a similar proteomics analysis of the growth matrix, Matrigel?. From these analyses, we observed that fibronectin is a primary component of nearly all hESC supportive matrices. This observation led to the investigation of the suitability of fibronectin as a defined ECM for the growth of hESCs. We found that fibronectin promotes the maintenance of pluripotent H9 and CA1 hESCs in an undifferentiated state using mTeSR1 medium. This finding validates the utility of characterizing matrices used for hESC growth in revealing ECM components required for culturing hESCs in a universally applicable defined system.     

Role of nodal signaling and the microenvironment underlying melanoma plasticity

The incidence of melanoma has increased dramatically over the last 50 yr, and although melanoma accounts for only 10% of all skin cancers, it is responsible for over 80% of skin cancer deaths. Recent studies have uncovered critical molecular events underlying melanocytic transformation and melanomagenesis. Among these noteworthy observations are the acquisition of stem cell-associated proteins, such as the Notch receptors and Nodal, which have also been implicated in melanoma progression. For example, we have demonstrated that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. In addition, molecular cross-talk exists between the Notch and Nodal signaling pathways. Interestingly, the acquisition of stem cell-associated plasticity is often acquired via epigenetic mechanisms, and is therefore receptive to reprogramming in response to embryonic microenvironments. Here, we review the concept of melanoma plasticity, with an emphasis on the emerging role of Nodal as a regulator of melanoma tumorigenesis and progression, and present findings related to epigenetic reprogramming.     

Comparison of sample preparation techniques for large‐scale proteomics

Numerous workflows exist for large‐scale bottom‐up proteomics, many of which achieve exceptional proteome depth. Herein, we evaluated the performance of several commonly used sample preparation techniques for proteomic characterization of HeLa lysates [unfractionated in‐solution digests, SDS‐PAGE coupled with in‐gel digestion, gel‐eluted liquid fraction entrapment electrophoresis (GELFrEE) technology, SCX StageTips and high‐/low‐pH reversed phase fractionation (HpH)]. HpH fractionation was found to be superior in terms of proteome depth (>8400 proteins detected) and fractionation efficiency compared to other techniques. SCX StageTip fractionation required minimal sample handling and was also a substantial improvement over SDS‐PAGE separation and GELFrEE technology. Sequence coverage of the HeLa proteome increased to 38% when combining all workflows, however, total proteins detected improved only slightly to 8710. In summary, HpH fractionation and SCX StageTips are robust techniques and highly suited for complex proteome analysis.     

Mass Spectrometry–based Proteomic Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture

The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis.In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach.The two defining characteristics of human embryonic stem cells (hESCs),¹ self-renewal and pluripotency, are maintained by a delicate balance of intracellular and extracellular signaling processes. Extracellular regulation is primarily the result of changes in the microenvironment surrounding the cells during growth in vitro or in vivo. HESCs interact with this “niche ” through support cells, extracellular matrix (ECM) components, and autocrine/paracrine signaling (reviewed in Refs. 1–3). Modulation of any of these supportive elements individually or in combination has been used extensively to alter hESC behavior (1–3).The culture of hESCs, as well as that of human induced pluripotent stem cells (hiPSCs), is conventionally performed on a layer of irradiated mouse embryonic fibroblast cells (MEFs). These MEFs are believed to promote the maintenance of hESCs and hiPSCs through the secretion of beneficial support proteins and cytokines into the soluble microenvironment. A number of proteomic studies have been conducted that examine the secretome of feeder-cell layers in an attempt to elucidate proteins and pathways essential for hESC and hiPSC survival (4–7). Alternatively, hESCs and hiPSCs can be cultured in feeder-free conditions in the absence of support cells. In feeder-free conditions, hESCs and hiPSCs are most often grown on the basement membrane matrix Matrigel™ in medium that has been previously conditioned by MEFs (MEF-CM). Matrigel™ is a gelatinous mixture that is secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (8). Although recent studies have proposed that a variety of defined matrices can support the growth of hESCs and hiPSCs, few of these can maintain a wide range of stem cell lines and therefore are typically not used in place of Matrigel™. The properties of Matrigel™ that make it such an effective matrix for hESC and hiPSC culture remain poorly understood. Because of the complexity of matrices like Matrigel™, the majority of proteomic studies that examine the hESC and hiPSC microenvironment have focused on contributions from support cells and soluble extracellular factors.The ECM is typically a complex network of structural proteins and glycosaminoglycans that function to support cells through the regulation of processes such as adhesion and growth factor signaling (9). Thus, it is not surprising that the generation of a well-defined matrix capable of facilitating hESC and hiPSC self-renewal has remained difficult (10). Previous proteomic investigations of Matrigel™ and other matrices supportive of hESC maintenance in vitro have revealed the presence of numerous growth, binding, and signaling proteins (11, 12). Further examination of how hESCs and hiPSCs interact with these complex matrices would provide critical information about what role the ECM plays in the organization of processes involved in the regulation of self-renewal and pluripotency.A recent study has established the ability of hESC-derived matrix microenvironments to alter tumorigenic properties through the reprogramming of metastatic melanoma cells (13). Importantly, this effect was found to be dependent on the exposure of metastatic cells to hESC-derived conditioned Matrigel™. Culture of metastatic melanoma cells in hESC-conditioned medium did not promote the reprogramming effect. These data suggest that the proteins responsible for this effect were integrated in the matrix. With the use of immunochemical techniques, it was later found that the left-right determination (Lefty) proteins A and B that were deposited in the matrix by hESCs during conditioning were at least in part responsible for the cellular change observed in metastatic cells (14). The Lefty A and B proteins are antagonists of transforming growth factor (TGF)-β signaling that act directly on Nodal protein, a critical regulator of the stem cell phenotype (15, 16). Subsequent studies of conditioned matrix utilizing mESCs implicated the bone morphogenic protein (BMP) 4 antagonist Gremlin as a primary regulator of the observed changes in metastatic cells (17). Collectively, these studies were all biased by a targeted analysis of potential effectors of metastatic cells. A comprehensive proteomic analysis of conditioned matrix could potentially reveal other factors involved in metastatic cell reprogramming. Furthermore, proteomic examination of hESC and hiPSC conditioned matrix could expose factors important in the regulation of self-renewal and pluripotency by the microenvironment in vitro.To this end, we have analyzed both types of human pluripotent stem cells, hESCs and hiPSCs, via a mass spectrometry (MS)-based proteomics approach to identify proteins deposited during growth in feeder-free conditions in vitro on Matrigel™. To investigate the hESC- and hiPSC-derived matrix, the metabolic labeling technique known as stable isotope labeling with amino acids in cell culture (SILAC) was used (18). SILAC facilitates the identification of hESC- and hiPSC-derived proteins that would otherwise be confounded by the presence of mouse-derived protein background from Matrigel™. From the proteomic analysis of three cells lines, namely, the hESC lines H9 and CA1 and the hiPSC line BJ-1D, we identified a total of 621, 1355, and 1350 total unique proteins, respectively. This work represents the first analysis of a hESC- and hiPSC-derived conditioned matrix and resulted in the identification of at least one novel microenvironmental contributor responsible for the regulation of human pluripotent stem cells.