Advances and Challenges in Cell Biology for Cultured Meat.

Cultured meat is an emerging biotechnology that aims to produce meat from animal cell culture, rather than from the raising and slaughtering of livestock, on environmental and animal welfare grounds. The detailed understanding and accurate manipulation of cell biology are critical to the design of cultured meat bioprocesses. Recent years have seen significant interest in this field, with numerous scientific and commercial breakthroughs. Nevertheless, these technologies remain at a nascent stage, and myriad challenges remain, spanning the entire bioprocess. From a cell biological perspective, these include the identification of suitable starting cell types, tuning of proliferation and differentiation conditions, and optimization of cell-biomaterial interactions to create nutritious, enticing foods. Here, we discuss the key advances and outstanding challenges in cultured meat, with a particular focus on cell biology, and argue that solving the remaining bottlenecks in a cost-effective, scalable fashion will require coordinated, concerted scientific efforts. Success will also require solutions to nonscientific challenges, including regulatory approval, consumer acceptance, and market feasibility. However, if these can be overcome, cultured meat technologies can revolutionize our approach to food.

Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells.

Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

Cell-cell communication through FGF4 generates and maintains robust proportions of differentiated cell types in embryonic stem cells.

During embryonic development and tissue homeostasis, reproducible proportions of differentiated cell types are specified from populations of multipotent precursor cells. Molecular mechanisms that enable both robust cell-type proportioning despite variable initial conditions in the precursor cells, and the re-establishment of these proportions upon perturbations in a developing tissue remain to be characterized. Here, we report that the differentiation of robust proportions of epiblast-like and primitive endoderm-like cells in mouse embryonic stem cell cultures emerges at the population level through cell-cell communication via a short-range fibroblast growth factor 4 (FGF4) signal. We characterize the molecular and dynamical properties of the communication mechanism and show how it controls both robust cell-type proportioning from a wide range of experimentally controlled initial conditions, as well as the autonomous re-establishment of these proportions following the isolation of one cell type. The generation and maintenance of reproducible proportions of discrete cell types is a new function for FGF signaling that might operate in a range of developing tissues.

Live Visualization of ERK Activity in the Mouse Blastocyst Reveals Lineage-Specific Signaling Dynamics.

FGF/ERK signaling is crucial for the patterning and proliferation of cell lineages that comprise the mouse blastocyst. However, ERK signaling dynamics have never been directly visualized in live embryos. To address whether differential signaling is associated with particular cell fates and states, we generated a targeted mouse line expressing an ERK-kinase translocation reporter (KTR) that enables live quantification of ERK activity at single-cell resolution. 3D time-lapse imaging of this biosensor in embryos revealed spatially graded ERK activity in the trophectoderm prior to overt polar versus mural differentiation. Within the inner cell mass (ICM), all cells relayed FGF/ERK signals with varying durations and magnitude. Primitive endoderm cells displayed higher overall levels of ERK activity, while pluripotent epiblast cells exhibited lower basal activity with sporadic pulses. These results constitute a direct visualization of signaling events during mammalian pre-implantation development and reveal the existence of spatial and temporal lineage-specific dynamics.

A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice.

The FGF/ERK signaling pathway is highly conserved throughout evolution and plays fundamental roles during embryonic development and in adult organisms. While a plethora of expression data exists for ligands, receptors and pathway regulators, we know little about the spatial organization or dynamics of signaling in individual cells within populations. To this end we developed a transcriptional readout of FGF/ERK activity by targeting a histone H2B-linked Venus fluorophore to the endogenous locus of Spry4, an early pathway target, and generated Spry4H2B-Venus embryonic stem cells (ESCs) and a derivative mouse line. The Spry4H2B-Venus reporter was heterogeneously expressed within ESC cultures and responded to FGF/ERK signaling manipulation. In vivo, the Spry4H2B-Venus reporter recapitulated the expression pattern of Spry4 and localized to sites of known FGF/ERK activity including the inner cell mass of the pre-implantation embryo and the limb buds, somites and isthmus of the post-implantation embryo. Additionally, we observed highly localized reporter expression within adult organs. Genetic and chemical disruption of FGF/ERK signaling, in vivo in pre- and post-implantation embryos, abrogated Venus expression establishing the reporter as an accurate signaling readout. This tool will provide new insights into the dynamics of the FGF/ERK signaling pathway during mammalian development.

Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling.

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.

Study of chemoresistant CD133+ cancer stem cells from human glioblastoma cell line U138MG using multiple assays.

Glioblastoma is one of the most common malignant tumours in adults, with an average life expectancy of less than 1 year. The high mortality of glioblastomas is attributed to its resistance to conventional chemotherapeutic agents. Numerous studies have established the presence of a cancer stem population within glioblastomas. These CSC (cancer stem cell) populations express the cell-surface marker, CD133, and are tumorigenic and chemoresistant. Hence, CSCs make a potential target for anticancer therapies. We have focused on techniques that can reliably identify and isolate a viable CSC population, and studied their chemoresistant attributes. We show the presence of a CSC population within a slowly proliferating glioblastoma cell line, U138MG. An improvised neurosphere enrichment culture technique was developed for the isolation of CSC population. Stem cell neurospheres obtained by this protocol maintained their viability for several weeks, and could be redispersed for deriving colony-forming units and secondary spheres from single-cell suspensions. RT-PCR (reverse transcription-PCR), cell surface localization by immunofluorescence and enumeration by FACS analysis showed that the sphere cultures of U138MG grown on agarose-coated plates had elevated CD133 levels. Drug sensitivity assays indicated that these enriched spheres were more resistant to drug treatment than their non-CSC controls. Drug-resistant CSC had an increased expression of ABC (ATP-binding-cassette) drug transporters, such as ABCC2, ABCC4, ABCG2 and p-glycoprotein, indicative of their role in the resistance mechanisms. These studies will facilitate the development of in vitro assays for the sparse CSC population and strategies for improved treatment regimens for glioblastomas.

An innovative methodology for analyzing digital visibility images in an urban environment.

A novel methodology combining digital imaging, conventional fixed visibility monitors, and solar radiation monitors has been developed to characterize the visual air quality of the El Paso and Ciudad Juarez urban vista. The authors have found that the digital image quality is reproducible and useful for quantitative analysis of visibility conditions. Regions of interest were selected in images along view paths of interest and values for a contrast variable of interest, typically the coefficient of variation or contrast ratio (CR) for discrete targets, were computed. Both of these indices are bounded at 0 and 1 and are scaled to the "clean day" maxima for a given date, time, and selected view paths. This produces a relative visibility index for various view paths. With the siting of a Belfort (6230A) visibility monitor at a central location, it has been possible to initiate contrast analysis of various targets in current and archived camera images obtained near this monitor. For uniformly "clean" days, as indicated by fine particulate matter observations and visual inspection, the authors have been able to use the extinction coefficient (Bext) derived from the 6230A to put the relative visibility index, based on CR, on an absolute basis in terms of an ideal target located at a given distance. This permits the generation of contrast extinction, Bext/C, for each view path that is independent of the actual target intrinsic contrast (within limits) and allows the comparison of Bext/C along different view paths with other air quality indices. Multiple linear regression was used to derive a relationship between the CR-based Bext/C value and air quality parameters. Visibility attenuation because of sulfate particles was found to have the highest correlation with Bext/C. In addition, solar radiation was observed to be a significant predictor of visibility in the urban region.