Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Development and characterization of a scaffold-free 3D spheroid model of iPSC-derived human cardiomyocytes

Purpose: Cardiomyocytes are terminally differentiated cells in the adult heart and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSC) from human origin was developed and characterized. Methods: Human cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. 2D cultures were examined using immunofluorescence microscopy and Western blotting while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. Results: iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to pro-hypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived cardiomyocytes formed spheroidal MTs within 4 days showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature, and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium-transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca2+-release, extracellular calcium levels. Conclusions: 3D culture using iPSC-derived human cardiomyocytes provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

The SasS -SasR two-component signal transduction system required for early Myxococcus xanthus multicellular *development

Initiation of Myxococcus xanthus multicellular development requires both nutrient limitation and high cell density. The extracellular signal, A signal, which consists of a set of amino acids at specific concentrations, serves as a cell density signal in M. xanthus early development. A reporter gene, designated 4521, that requires both starvation and A signal for developmental expression was used to identify mutations in the signal transduction pathways. A group of point mutations located in the chromosomal sasB locus that bypasses both requirements was previously isolated. One of these point mutations, sasB7, was mapped to the sasS gene, which is predicted to encode a transmembrane histidine protein kinase required for normal development. SasS is a positive regulator of 4521 and a candidate A signal sensor. This dissertation continues the characterization of the sasB locus, focusing on the sasR gene and the functional relationship of SasS and SasR. ^ The sasR gene is located 2.2-kb downstream of sasS. It is predicted to encode an NtrC-like response regulator, which belongs to the family of sigma54 transcriptional activators. SasR is a positive regulator of 4521 gene and is required for normal development. The sasR mutant displays phenotypes similar to that of sasS mutant. Both SasS and SasR are required for the A-signal-dependent 4521 expression. Genetic epistasis analysis indicates that SasR functions downstream of SasS. Biochemical studies show that SasS has autokinase activity, and phosphorylated SasS is able to transfer its phosphate to SasR. We propose that SasS and SasR form a two-component signal transduction system in the A signal transduction pathway. ^ To search for the genes regulated by SasS and SasR, expression patterns of a group of developmental genes were compared in wild-type and sasS null mutant backgrounds. SasS and SasR were found to positively regulate sasN and 4521. The sasN gene was previously identified as a negative regulator of 4521, located at about 170-bp downstream of sasR. It is required for normal fruiting body development. Based on the above data, a regulatory network consisting of sasS, sasR, sasN, and 4521 is hypothesized, and the interactions of the components in this network can now be further studied. ^

The LPS O -antigen is required for Myxococcus xanthus social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when starved at high density. All of the identified M. xanthus lipopolysaccharide (LPS) O-antigen biosynthesis mutants exhibit defective motility and fruiting-body development. To determine the cause of these phenotypes, the cell-surface properties of the LPS O-antigen mutants were compared to wild-type cells. The binding characteristics of wild-type and LPS O-antigen-defective strains to cationic resin indicate that the mutant cell surfaces are more electronegative. Antibiotic sensitivity and hexadecane adhesion assays indicate that the wild-type M. xanthus cell surface is hydrophobic, supporting the idea that phospholipids are present in the outer leaflet of the outer membrane. The absence of the LPS O-antigen appears to expose charges associated with phospholipids and LPS core/lipid A, resulting in a dramatic alteration of the cell-surface organization and charge. These differences may affect the interaction of the LPS O-antigen mutants with their substratum and neighboring cells, leading to defects in social and single-cell gliding motility and thus, deficiencies in fruiting body formation. ^ The LPS O-antigen biosynthetic mutations also bypass the requirement of 4521 gene expression for the cell-density signal, A signal. The 4521 gene is overexpressed in these mutants. This 4521 overexpression is dependent on the sensor kinase SasS. Co-development with wild-type cells, or the addition of crude polysaccharides or membrane vesicles restores the ability of LPS O-antigen mutants to form fruiting bodies and lowers 4521 developmental gene expression to wild-type levels. Wild-type vesicles may attach or incorporate into the outer membrane of the mutants that lack LPS O-antigen, restoring a wild-type periplasmic status and allowing for normal levels of 4521 activity and fruiting body formation. We propose that the LPS composition and the configuration of the outer membrane are important elements for the complex behavioral response of M. xanthus fruiting body development. ^

Gene replacement by homologous recombination in the multicellular green alga Volvox carteri

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G → A nucleotide exchange in a 5′-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.

The protein kinases of Caenorhabditis elegans: A model for signal transduction in multicellular organisms

Caenorhabditis elegans should soon be the first multicellular organism whose complete genomic sequence has been determined. This achievement provides a unique opportunity for a comprehensive assessment of the signal transduction molecules required for the existence of a multicellular animal. Although the worm C. elegans may not much resemble humans, the molecules that regulate signal transduction in these two organisms prove to be quite similar. We focus here on the content and diversity of protein kinases present in worms, together with an assessment of other classes of proteins that regulate protein phosphorylation. By systematic analysis of the 19,099 predicted C. elegans proteins, and thorough analysis of the finished and unfinished genomic sequences, we have identified 411 full length protein kinases and 21 partial kinase fragments. We also describe 82 additional proteins that are predicted to be structurally similar to conventional protein kinases even though they share minimal primary sequence identity. Finally, the richness of phosphorylation-dependent signaling pathways in worms is further supported with the identification of 185 protein phosphatases and 128 phosphoprotein-binding domains (SH2, PTB, STYX, SBF, 14-3-3, FHA, and WW) in the worm genome.

How amoeboids self-organize into a fruiting body: Multicellular coordination in Dictyostelium discoideum

When individual amoebae of the cellular slime mold Dictyostelium discoideum are starving, they aggregate to form a multicellular migrating slug, which moves toward a region suitable for culmination. The culmination of the morphogenesis involves complex cell movements that transform a mound of cells into a globule of spores on a slender stalk. The movement has been likened to a “reverse fountain,” whereby prestalk cells in the upper part form a stalk that moves downwards and anchors to the substratum, while prespore cells in the lower part move upwards to form the spore head. So far, however, no satisfactory explanation has been produced for this process. Using a computer simulation that we developed, we now demonstrate that the processes that are essential during the earlier stages of the morphogenesis are in fact sufficient to produce the dynamics of the culmination stage. These processes are cAMP signaling, differential adhesion, cell differentiation, and production of extracellular matrix. Our model clarifies the processes that generate the observed cell movements. More specifically, we show that periodic upward movements, caused by chemotactic motion, are essential for successful culmination, because the pressure waves they induce squeeze the stalk downwards through the cell mass. The mechanisms revealed by our model have a number of self-organizing and self-correcting properties and can account for many previously unconnected and unexplained experimental observations.

Development of a multicellular co-culture model of normal and cystic fibrosis human airways in vitro

Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models

Solid tumours display a complex drug resistance phenotype that involves inherent and acquired mechanisms. Multicellular resistance is an inherent feature of solid tumours and is known to present significant barriers to drug permeation in tumours. Given this barrier, do acquired resistance mechanisms such as P-glycoprotein (P-gp) contribute significantly to resistance? To address this question, the multicellular tumour spheroid (MCTS) model was used to examine the influence of P-gp on drug distribution in solid tissue. Tumour spheroids (TS) were generated from either drug-sensitive MCF7WT cells or a drug-resistant, P-gp-expressing derivative MCF7Adr. Confocal microscopy was used to measure time courses and distribution patterns of three fluorescent compounds; calcein-AM, rhodamine123 and BODIPY-taxol. These compounds were chosen because they are all substrates for P-gp-mediated transport, exhibit high fluorescence and are chemically dissimilar. For example, BODIPY-taxol and rhodamine 123 showed high accumulation and distributed extensively throughout the TSWT, whereas calcein-AM accumulation was restricted to the outermost layers. The presence of P-gp in TSAdr resulted in negligible accumulation, regardless of the compound. Moreover, the inhibition of P-gp by nicardipine restored intracellular accumulation and distribution patterns to levels observed in TSWT. The results demonstrate the effectiveness of P-gp in modulating drug distribution in solid tumour models. However, the penetration of agents throughout the tissue is strongly determined by the physico-chemical properties of the individual compounds.

Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms

The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, in a multicellular organism, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. In this thesis, we describe methods that enable the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. We first engineered a eukaryotic phenylalanyl-tRNA synthetase (PheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). Using Caenorhabditis elegans, we expressed the engineered PheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells -- and only those cells -- to be labeled with azides. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affnity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins -- including proteins with previously unknown expression patterns -- expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; spatiotemporal selectivity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we next developed a two-component system to drive overlapping -- but not identical -- patterns of expression of engineered PheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-PheRS system for highly efficient PheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells at specific developmental stages.

Development of an impedance based biosensor for studies of spheroids formation

Three-dimensional (3D) multicellular spheroids are exceptional in vitro cell models for their ability to accurately mimic real cell-cell interaction processes. However, the challenges in producing well-defined spheroids with controlled size together with the deficiency of techniques to monitor them significantly restrict their use. Herein, a novel device to study spheroid formation in real time is presented. By exploiting electrochemical impedance spectroscopy, a multi-electrode array (MEA) attached to a calcium alginate scaffold is able to monitor the behaviour of 36 different hydrogel wells. The scaffold contains inverted shape pyramidal microwells, which guide the aggregation of cells into spheroids with controlled dimensions. Preliminar studies on calcium alginate, optimisation of fabrication strategy are shown, together with testing of the device in the presence and the absence of the hydrogel. Lastly, the device was tested for its intended aim, i.e. to monitor the formation of a spheroid, proving its potential as an impedance biosensor.

In vitro Microfluidic Model For The Study Of Vaso-occlusive Processes.

Vaso-occlusion, responsible for much of the morbidity of sickle-cell disease, is a complex multicellular process, apparently triggered by leukocyte adhesion to the vessel wall. The microcirculation represents a major site of leukocyte-endothelial interactions and vaso-occlusive processes. We have developed a biochip with subdividing interconnecting microchannels that decrease in size (40 μm to 10 μm in width), for use in conjunction with a precise microfluidic device, to mimic cell flow and adhesion through channels of sizes that approach those of the microcirculation. The biochips were utilized to observe the dynamics of the passage of neutrophils and red blood cells, isolated from healthy and sickle-cell anemia (SCA) individuals, through laminin or endothelial adhesion molecule-coated microchannels at physiologically relevant rates of flow and shear stress. Obstruction of E-selectin/intercellular adhesion molecule 1-coated biochip microchannels by SCA neutrophils was significantly greater than that observed for healthy neutrophils, particularly in the microchannels of 40-15 μm in width. Whereas SCA red blood cells alone did not significantly adhere to, or obstruct, microchannels, mixed suspensions of SCA neutrophils and red blood cells significantly adhered to and obstructed laminin-coated channels. Results from this in vitro microfluidic model support a primary role for leukocytes in the initiation of SCA occlusive processes in the microcirculation. This assay represents an easy-to-use and reproducible in vitro technique for understanding molecular mechanisms and cellular interactions occurring in subdividing microchannels of widths approaching those observed in the microvasculature. The assay could hold potential for testing drugs developed to inhibit occlusive mechanisms such as those observed in SCA and thrombotic diseases.

Insights on the Milky Way bulge formation from the correlations between kinematics and metallicity

Context. Two main scenarios for the formation of the Galactic bulge are invoked, the first one through gravitational collapse or hierarchical merging of subclumps, the second through secular evolution of the Galactic disc. Aims. We aim to constrain the formation of the Galactic bulge through studies of the correlation between kinematics and metallicities in Baade's Window (l = 1 degrees, b = -4 degrees) and two other fields along the bulge minor axis (l = 0 degrees, b = -6 degrees and b = -12 degrees). Methods. We combine the radial velocity and the [Fe/H] measurements obtained with FLAMES/GIRAFFE at the VLT with a spectral resolution of R = 20 000, plus for the Baade's Window field the OGLE-II proper motions, and compare these with published N-body simulations of the Galactic bulge. Results. We confirm the presence of two distinct populations in Baade's Window found in Hill et al. (2010, A&A, submitted): the metal-rich population presents bar-like kinematics while the metal-poor population shows kinematics corresponding to an old spheroid or a thick disc. In this context the metallicity gradient along the bulge minor axis observed by Zoccali et al. (2008, A&A, 486, 177), visible also in the kinematics, can be related to a varying mix of these two populations as one moves away from the Galactic plane, alleviating the apparent contradiction between the kinematic evidence of a bar and the existence of a metallicity gradient. Conclusions. We show evidence that the two main scenarios for the bulge formation co-exist within the Milky Way bulge.