Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms

In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express ‘cooperative traits'', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.     

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Microbes can engage in social interactions ranging from cooperation to warfare. Biofilms are structured, cooperative microbial communities. Like all cooperative communities, they are susceptible to invasion by selfish individuals who benefit without contributing. However, biofilms are pervasive and ancient, representing the first fossilized life. One hypothesis for the stability of biofilms is spatial structure: Segregated patches of related cooperative cells are able to outcompete unrelated cells. These dynamics have been explored computationally and in bacteria; however, their relevance to eukaryotic microbes remains an open question. The complexity of eukaryotic cell signaling and communication suggests the possibility of different social dynamics. Using the tractable model yeast, Saccharomyces cerevisiae, which can form biofilms, we investigate the interactions of environmental isolates with different social phenotypes. We find that biofilm strains spatially exclude nonbiofilm strains and that biofilm spatial structure confers a consistent and robust fitness advantage in direct competition. Furthermore, biofilms may protect against killer toxin, a warfare phenotype. During biofilm formation, cells are susceptible to toxin from nearby competitors; however, increased spatial use may provide an escape from toxin producers. Our results suggest that yeast biofilms represent a competitive strategy and that principles elucidated for the evolution and stability of bacterial biofilms may apply to more complex eukaryotes.     

Wider access to genotypic space facilitates loss of cooperation in a bacterial mutator

Understanding the ecological, evolutionary and genetic factors that affect the expression of cooperative behaviours is a topic of wide biological significance. On a practical level, this field of research is useful because many pathogenic microbes rely on the cooperative production of public goods (such as nutrient scavenging molecules, toxins and biofilm matrix components) in order to exploit their hosts. Understanding the evolutionary dynamics of cooperation is particularly relevant when considering long-term, chronic infections where there is significant potential for intra-host evolution. The impact of responses to non-social selection pressures on social evolution is arguably an under-examined area. In this paper, we consider how the evolution of a non-social trait--hypermutability--affects the cooperative production of iron-scavenging siderophores by the opportunistic human pathogen Pseudomonas aeruginosa. We confirm an earlier prediction that hypermutability accelerates the breakdown of cooperation due to increased sampling of genotypic space, allowing mutator lineages to generate non-cooperative genotypes with the ability to persist at high frequency and dominate populations. This may represent a novel cost of hypermutability.     

The hard problem of cooperation

Based on individual variation in cooperative inclinations, we define the “hard problem of cooperation” as that of achieving high levels of cooperation in a group of non-cooperative types. Can the hard problem be solved by institutions with monitoring and sanctions? In a laboratory experiment we find that the answer is affirmative if the institution is imposed on the group but negative if development of the institution is left to the group to vote on. In the experiment, participants were divided into groups of either cooperative types or non-cooperative types depending on their behavior in a public goods game. In these homogeneous groups they repeatedly played a public goods game regulated by an institution that incorporated several of the key properties identified by Ostrom: operational rules, monitoring, rewards, punishments, and (in one condition) change of rules. When change of rules was not possible and punishments were set to be high, groups of both types generally abided by operational rules demanding high contributions to the common good, and thereby achieved high levels of payoffs. Under less severe rules, both types of groups did worse but non-cooperative types did worst. Thus, non-cooperative groups profited the most from being governed by an institution demanding high contributions and employing high punishments. Nevertheless, in a condition where change of rules through voting was made possible, development of the institution in this direction was more often voted down in groups of non-cooperative types. We discuss the relevance of the hard problem and fit our results into a bigger picture of institutional and individual determinants of cooperative behavior.     

Gravity Constraints Drive Biological Systems Toward Specific Organization Patterns

Different cell lineages growing in microgravity undergo a spontaneous transition leading to the emergence of two distinct phenotypes. By returning these populations in a normal gravitational field, the two phenotypes collapse, recovering their original configuration. In this review, we hypothesize that, once the gravitational constraint is removed, the system freely explores its phenotypic space, while, when in a gravitational field, cells are “constrained” to adopt only one favored configuration. We suggest that the genome allows for a wide range of “possibilities” but it is unable per se to choose among them: the emergence of a specific phenotype is enabled by physical constraints that drive the system toward a preferred solution. These findings may help in understanding how cells and tissues behave in both development and cancer.     

The smell of cooperation: rats increase helpful behaviour when receiving odour cues of a conspecific performing a cooperative task

Reciprocity can explain cooperative behaviour among non-kin, where individuals help others depending on their experience in previous interactions. Norway rats (Rattus norvegicus) cooperate reciprocally according to direct and generalized reciprocity. In a sequence of four consecutive experiments, we show that odour cues from a cooperating conspecific are sufficient to induce the altruistic help of rats in a food-exchange task. When rats were enabled to help a non-cooperative partner while receiving olfactory information from a rat helping a conspecific in a different room, they helped their non-cooperative partner as if it was a cooperative one. We further show that the cues inducing altruistic behaviour are released during the act of cooperation and do not depend on the identity of the cue provider. Remarkably, olfactory cues seem to be more important for cooperation decisions than experiencing a cooperative act per se. This suggests that rats may signal their cooperation propensity to social partners, which increases their chances to receive help in return.     

Spatial dynamics of feedback and feedforward regulation in cell lineages

Feedback mechanisms within cell lineages are thought to be important for maintaining tissue homeostasis. Mathematical models that assume well-mixed cell populations, together with experimental data, have suggested that negative feedback from differentiated cells on the stem cell self-renewal probability can maintain a stable equilibrium and hence homeostasis. Cell lineage dynamics, however, are characterized by spatial structure, which can lead to different properties. Here, we investigate these dynamics using spatially explicit computational models, including cell division, differentiation, death, and migration / diffusion processes. According to these models, the negative feedback loop on stem cell self-renewal fails to maintain homeostasis, both under the assumption of strong spatial restrictions and fast migration / diffusion. Although homeostasis cannot be maintained, this feedback can regulate cell density and promote the formation of spatial structures in the model. Tissue homeostasis, however, can be achieved if spatially restricted negative feedback on self-renewal is combined with an experimentally documented spatial feedforward loop, in which stem cells regulate the fate of transit amplifying cells. This indicates that the dynamics of feedback regulation in tissue cell lineages are more complex than previously thought, and that combinations of spatially explicit control mechanisms are likely instrumental.     

Defining the role of cooperation in early tumor progression

Competition among cells has long been recognized as an important part of carcinogenesis. However, the role of cellular cooperation in cancer has been largely ignored. In this work, we investigated the role of cooperation in early tumor progression using a mathematical and agent-based modeling approach. We hoped to learn how the introduction of cooperative cells into a cell population would affect the dynamics of the system. We modeled the stem cell compartment of tissue using a spatial structure organized into cell patches, with stem cells able to replicate or leave the stem cell compartment through apoptosis or differentiation. The cells could also acquire mutations in three oncogenes and two tumor suppressor genes. Cooperative cells in our model provided a cooperative signal that increased the fitness of their immediate neighbors, but did not affect their own fitness. Running simulations of the model, we found that if cooperative cells are introduced into a cell population, they steadily proliferate and confer a growth advantage to the entire population. This leads us to conclude that providing a cooperative signal is likely to be under positive selective pressure. When cooperative ability and mutation are concurrently present in the same cells, the overall cell population experiences a significant growth advantage, much greater than with cooperation or mutation alone. This growth advantage is diminished if cells with only oncogene/tumor suppressor mutations are also present in the population, suggesting that the optimal scenario for tumor growth would be for cooperative cells to take over a cell population, and then for mutations in oncogenes and tumor suppressors to arise on a background of cooperation. We predict that cooperation is particularly important in the very early stages of carcinogenesis, when tissue is morphologically and histologically normal. Our results have implications for the screening and early diagnosis of cancer.     

Myeloid cell differentiation in normal bone marrow and acute myeloid leukemia assessed by multi-dimensional flow cytometry.

A detailed analysis of normal myeloid differentiation was performed using mutlidimensional flow cytometry. Based on two light scattering and three color immunofluorescence signals, the normal maturation pathways of both the monocyte and neutrophil lineages could be elucidated. Gradual changes of light scattering properties and cell surface antigen expression defined the pathways of each of the lineages. The consistency of the location of these lineage specific pathways found in normal individuals provided the basis for the discrimination between normal and leukemic cells in acute myeloid leukemia (ANLL). The position of leukemic cells in patients with ANLL in a five-dimensional space was compared with the position of the maturation tracks in normal individuals. The expression of normal antigens on leukemic cells provided the tools to: (1) distinguish normal from clonal populations of leukemic cells in all 15 patients; (2) detect a lineage predominance, either monocytic or neutrophilic, in all 15 patients; (3) detect maturation heterogeneity in all 15 patients. Although maturation pathways of the monocytic and the neutrophilic lineages were analogous to the normal patterns they were distinct in several ways. The expression of normal antigens on leukemic cells may provide the tools to: (1) obtain a new frame-work for classification of leukemia based on the ability to quantify the aberrant antigen expression and to define a 'distance from normal' based on the characteristics studied (the maturation heterogeneity of the leukemic cells also can be correlated with the clinical outcome of the patients); (2) detect minimal residual disease using the difference in locations of the leukemic cells in the multidimensional space from the normal maturation pathways (3) monitor relapse and changes in phenotypes which may accompany chemotherapy, suggesting the appearance of variant or new clones.     

Clonal analysis of the differentiation potential of human adipose-derived adult stem cells

Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.     

Microenvironment driven invasion: a multiscale multimodel investigation

Cancer is a complex, multiscale process, in which genetic mutations occurring at a subcellular level manifest themselves as functional and morphological changes at the cellular and tissue scale. The importance of interactions between tumour cells and their microenvironment is currently of great interest in experimental as well as computational modelling. Both the immediate microenvironment (e.g. cell-cell signalling or cell-matrix interactions) and the extended microenvironment (e.g. nutrient supply or a host tissue structure) are thought to play crucial roles in both tumour progression and suppression. In this paper we focus on tumour invasion, as defined by the emergence of a fingering morphology, which has previously been shown to be dependent upon harsh microenvironmental conditions. Using three different modelling approaches at two different spatial scales we examine the impact of nutrient availability as a driving force for invasion. Specifically we investigate how cell metabolism (the intrinsic rate of nutrient consumption and cell resistance to starvation) influences the growing tumour. We also discuss how dynamical changes in genetic makeup and morphological characteristics, of the tumour population, are driven by extreme changes in nutrient supply during tumour development. The simulation results indicate that aggressive phenotypes produce tumour fingering in poor nutrient, but not rich, microenvironments. The implication of these results is that an invasive outcome appears to be co-dependent upon the evolutionary dynamics of the tumour population driven by the microenvironment.     

Cooperation in defence against a predator

The origin and the evolutionary stability of cooperation between unrelated individuals is one of the key problems of evolutionary biology. In this paper, a cooperative defence game against a predator is introduced which is based on Hamilton's selfish herd theory and Eshel's survival game models. Cooperation is altruistic in the sense that the individual, which is not the target of the predator, helps the members of the group attacked by the predator and during defensive action the helper individual may also die in any attack. In order to decrease the long term predation risk, this individual has to carry out a high risk action. Here I show that this kind of cooperative behaviour can evolve in small groups. The reason for the emergence of cooperation is that if the predator does not kill a mate of a cooperative individual, then the survival probability of the cooperative individual will increase in two cases. If the mate is non-cooperative, then—according to the dilution effect, the predator confusion effect and the higher predator vigilance—the survival probability of the cooperative individual increases. The second case is when the mate is cooperative, because a cooperative individual has a further gain, the active help in defence during further predator attacks. Thus, if an individual can increase the survival rate of its mates (no matter whether the mate is cooperative or not), then its own predation risk will decrease.     

Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.     

The evolution of competition and policing: opposing selection within and among groups

Background  

Although selection favors exploitative competition within groups, a group of hypercompetitive individuals may be less productive than a cooperative group. When competition is costly for group fitness, among-group selection can favor groups with 'policing' individuals who reduce within-group competition at a cost to their own fitness, or groups of individuals who restrain their competitive intensity ('self policing'). We examine these possibilities in a series of explicit population-genetic models.     

Cooperative and non-cooperative processes of apparent movement of random-dot cinematograms

In this study, we investigated the cooperative and non-cooperative models of stereopsis on apparent movement of the short-range process using spatial frequency filtered random-dot cinematograms. Our results showed that when spatial frequencies were below 4 cycles/degree, maximum displacement (dmax) was decreasing (linearly) with increasing mean frequencies, but at 4 cycles/degree and above dmax stayed constant. For low frequencies, non-cooperative models such as Marr and Poggio's could explain these findings, but not for frequencies above 4 cycles/degree. However, in a previous study we found that the average cooperative neighbourhood for apparent movement of the short-range process is 15 arc min. This fortuitous agreement on 4 cycles/degree could suggest that dmax being constant at frequencies above 4 cycles is related to a cooperative process.     

Murine Adipose Tissue-Derived Stromal Cell Apoptosis and Susceptibility to Oxidative Stress In Vitro Are Regulated by Genetic Background

Adipose tissue-derived stromal cells (ADSCs) are of interest for regenerative medicine as they are isolated easily and can differentiate into multiple cell lineages. Studies of their in vitro proliferation, survival, and differentiation are common; however, genetic effects on these phenotypes remain unknown. To test if these phenotypes are genetically regulated, ADSCs were isolated from three genetically diverse inbred mouse strains- C57BL/6J (B6), BALB/cByJ (BALB), and DBA/2J (D2)- in which genetic regulation of hematopoietic stem function is well known. ADSCs from all three strains differentiated into osteogenic and chondrogenic lineages in vitro. ADSCs from BALB grew least well in vitro, probably due to apoptotic cell death after several days in culture. BALB ADSCs were also the most susceptible to the free radical inducers menadione and H₂O₂. ADSCs from the three possible F1 hybrids were employed to further define genetic regulation of ADSC phenotypes. D2, but not B6, alleles stimulated ADSC expansion in BALB cells. In contrast, B6, but not D2, alleles rescued BALB H₂O₂ resistance. We conclude that low oxidative stress resistance does not limit BALB ADSC growth in vitro, as these phenotypes are genetically regulated independently. In addition, ADSCs from these strains are an appropriate model system to investigate genetic regulation of ADSC apoptosis and stress resistance in future studies. Such investigations are essential to optimize cell expansion and differentiation and thus, potential for regenerative medicine.     

Iterated prisoner's dilemma in an asocial world dominated by loners, not by defectors

Cooperation among genetically unrelated individuals can arise when pairs of individuals interact repeatedly in the Prisoner’s Dilemma. However, the conditions allowing the evolution of reciprocal cooperation become extremely restrictive as the size of the cooperative group increases, because defectors can exploit cooperators more efficiently in larger groups. Here we consider three strategies: Tit for Tat, defector, and loner. Loner beats defector in a non-cooperative world. However, a cooperative strategy Tit for Tat (TFT₀) that stops cooperation after the first iteration when there is at least one defector in the group, can invade a world of loners, even in sizable groups, if both the TFT₀ and the defector strategies arise at the same frequency by mutation.     

Quorum sensing provides a molecular mechanism for evolution to tune and maintain investment in cooperation

As selection frequently favors noncooperating defectors in mixed populations with cooperators, mechanisms that promote cooperation stability clearly exist. One potential mechanism is bacterial cell-to-cell communication, quorum sensing (QS), which can allow cooperators to prevent invasion by defectors. However, the impact of QS on widespread maintenance of cooperation in well-mixed conditions has not been experimentally demonstrated over extended evolutionary timescales. Here, we use wild-type (WT) Vibrio campbellii that regulates cooperation with QS and an unconditional cooperating (UC) mutant to examine the evolutionary origins and dynamics of novel defectors during a long-term evolution experiment. We found that UC lineages were completely outcompeted by defectors, whereas functioning QS enabled the maintenance of cooperative variants in most WT populations. Sequencing evolved populations revealed multiple luxR mutations that swept the UC lineages. However, the evolution of mutant lineages with reduced levels of bioluminescence (dims) occurred in many WT lineages. These dim variants also decreased other cooperative phenotypes regulated by QS, including protease production, indicating they result from changes to QS regulation. This diminished investment phenotype optimizes a tradeoff between cooperative input and growth output and suggests that decreasing the cost of QS could be a favorable strategy for maintaining the cooperative behaviors it regulates.Subject terms: Bacterial evolution, Microbial ecology, Molecular evolution     

Magnifying Stem Cell Lineages: The Stop-EGFP Mouse

Cell fate mapping techniques which can label clonal cell lineages are of importance because they allow one to investigate the distribution and types of daughter cells arising from single precursor cells. Thus, the potential of precursor cells to generate various types of descendent cells can be studied at the single-cell level. The stop-EGFP transgenic mouse carries a premature stop codon-containing enhanced green fluorescent protein (EGFP) gene as a target gene for mutations. A cell having undergone a mutation at the premature stop codon and its descendant cell lineage will express EGFP, thus a clonal cell lineage can be traced in vivo using a fluorescent microscope. Using the stop-EGFP mouse, stem cell clonal lineages in the mouse dorsal epidermis can be investigated in vivo and repeated analyses of the same cell lineages can be performed over time. In vivo imaging studies possible with the stop-EGFP mouse provide new insights into the structure of epidermal proliferative units (EPUs). The stop-EGFP system provides a novel tool for investigating clonal cell lineages in developmental studies as well as in stem cell biology.     

Enhancement of cell growth in tissue-engineering constructs under direct perfusion: Modeling and simulation

Perfusion bioreactors improve mass transfer in cell-scaffold constructs. We developed a mathematical model to simulate nutrient flow through cellular constructs. Interactions among cell proliferation, nutrient consumption, and culture medium circulation were investigated. The model incorporated modified Contois cell-growth kinetics that includes effects of nutrient saturation and limited cell growth. Nutrient uptake was depicted through the Michaelis-Menton kinetics. To describe the culture medium convection, the fluid flow outside the cell-scaffold construct was described by the Navier-Stokes equations, while the fluid dynamics within the construct was modeled by Brinkman's equation for porous media flow. Effects of the media perfusion were examined by including time-dependant porosity and permeability changes due to cell growth. The overall cell volume was considered to consist of cells and extracellular matrices (ECM) as a whole without treating ECM separately. Numerical simulations show when cells were cultured subjected to direct perfusion, they penetrated to a greater extent into the scaffold and resulted in a more uniform spatial distribution. The cell amount was increased by perfusion and ultimately approached an asymptotic value as the perfusion rates increased in terms of the dimensionless Peclet number that accounts for the ratio of nutrient perfusion to diffusion. In addition to enhancing the nutrient delivery, perfusion simultaneously imposes flow-mediated shear stress to the engineered cells. Shear stresses were found to increase with cell growth as the scaffold void space was occupied by the cell and ECM volumes. The macro average stresses increased from 0.2 mPa to 1 mPa at a perfusion rate of 20 microm/s with the overall cell volume fraction growing from 0.4 to 0.7, which made the overall permeability value decrease from 1.35 x 10(-2)cm(2) to 5.51 x 10(-4)cm(2). Relating the simulation results with perfusion experiments in literature, the average shear stresses were below the critical value that would induce the chondrocyte necrosis.