Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled "Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application" was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.

Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of morphology-based approaches. Copyright © 2012 John Wiley & Sons, Ltd.

Assessing The Role Of Multi-protein Complexes In Determining Phenotype

Understanding regulatory mechanisms in complex biological systems is an important challenge, in particular to understand disease mechanisms, and to discover new therapies and drugs. In this paper, we consider the important question of cellular regulation of phenotype. Using single gene deletion data, we address the problem of linking a phenotype to underlying functional roles in the organism and provide a sound computational and statistical paradigm that can be extended to address more complex experimental settings such as multiple deletions. We apply the proposed approaches to publicly available data sets to demonstrate strong evidence for the involvement of multi-protein complexes in the phenotypes studied.

LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells

BACKGROUND: Stem cells with the ability to form clonal floating colonies (spheres) were recently isolated from the neonatal murine spiral ganglion. To further examine the features of inner ear-derived neural stem cells and their derivatives, we investigated the effects of leukemia inhibitory factor (LIF), a neurokine that has been shown to promote self-renewal of other neural stem cells and to affect neural and glial cell differentiation. RESULTS: LIF-treatment led to a dose-dependent increase of the number of neurons and glial cells in cultures of sphere-derived cells. Based on the detection of developmental and progenitor cell markers that are maintained in LIF-treated cultures and the increase of cycling nestin-positive progenitors, we propose that LIF maintains a pool of neural progenitor cells. We further provide evidence that LIF increases the number of nestin-positive progenitor cells directly in a cell cycle-independent fashion, which we interpret as an acceleration of neurogenesis in sphere-derived progenitors. This effect is further enhanced by an anti-apoptotic action of LIF. Finally, LIF and the neurotrophins BDNF and NT3 additively promote survival of stem cell-derived neurons. CONCLUSION: Our results implicate LIF as a powerful tool to control neural differentiation and maintenance of stem cell-derived murine spiral ganglion neuron precursors. This finding could be relevant in cell replacement studies with animal models featuring spiral ganglion neuron degeneration. The additive effect of the combination of LIF and BDNF/NT3 on stem cell-derived neuronal survival is similar to their effect on primary spiral ganglion neurons, which puts forward spiral ganglion-derived neurospheres as an in vitro model system to study aspects of auditory neuron development.

Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer.

Anticancer therapies currently used in the clinic often can neither eradicate the tumor nor prevent disease recurrence due to tumor resistance. In this study, we showed that chemoresistance to pemetrexed, a multi-target anti-folate (MTA) chemotherapeutic agent for non-small cell lung cancer (NSCLC), is associated with a stem cell-like phenotype characterized by an enriched stem cell gene signature, augmented aldehyde dehydrogenase activity and greater clonogenic potential. Mechanistically, chemoresistance to MTA requires activation of epithelial-to-mesenchymal transition (EMT) pathway in that an experimentally induced EMT per se promotes chemoresistance in NSCLC and inhibition of EMT signaling by kaempferol renders the otherwise chemoresistant cancer cells susceptible to MTA. Relevant to the clinical setting, human primary NSCLC cells with an elevated EMT signaling feature a significantly enhanced potential to resist MTA, whereas concomitant administration of kaempferol abrogates MTA chemoresistance, regardless of whether it is due to an intrinsic or induced activation of the EMT pathway. Collectively, our findings reveal that a bona fide activation of EMT pathway is required and sufficient for chemoresistance to MTA and that kaempferol potently regresses this chemotherapy refractory phenotype, highlighting the potential of EMT pathway inhibition to enhance chemotherapeutic response of lung cancer.

Analysis of Wnt/beta-catenin signaling in kidney tubulogenesis

The β-catenin/Lef/Tcf-mediated Wnt pathway is central to the developmental of all animals, stem cell renewal, and cancer progression. Prior studies in frogs and mice have indicated that the ligand Wnt-4 is essential for the mesenchyme to epithelial transition that generates tubules in the context of kidney organogenesis. More recently, Wnt-9b in mice, was likewise found to be required. Yet despite the importance of Wnt signals in renal development, the corresponding Frizzled receptor(s) and downstream signaling mechanim(s) are unclear. My work addresses these knowledge gaps using in vitro (Madin-Darby Canine Kidney cells) and in vivo (Xenopus laevis and zebrafish pronephros) tubulogenic kidney model systems. Employing established reporter constructs of Wnt/β-catenin pathway activity, I have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions. I have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/β-catenin pathway using β-Engrailed and dnTCF-4, constructs that suppress this pathway. I have further found that MDCK cells express the Frizzled-6 receptor, and that Wnt-4 forms a biochemical complex with Frizzled-6, yet does not appear to transduce Wnt-4's canonical signal. Additionally, I demonstrate that standard Hepatocyte Growth Factor (HGF)-mediated (non-physiologic) induction of MDCK tubulogenesis in collagen matrices is not altered by activation or suppression of β-catenin signaling activity; however, β-catenin signaling maintains cell survival in this in vitro system. Using a Wnt/β-catenin signaling reporter in Xenopus laevis, I detect β-catenin signaling activity in the early pronephric epithelial kidney tubules. By inhibiting the Wnt/β-catenin signaling pathway in both zebrafish and Xenopus , a significant loss of kidney tubulogenesis is observed with little or no effect on adjoining axis or somite development. This inhibition also leads to the appearance of severe edema that phenocopies embryos depleted for Wnt-4. Tubulogenic loss does not appear to be caused by increased cell death in the Xenopus pronephric field, but rather by lessened expression of tubule epithelium genes associated with cellular differentiation. Together, my results show that Wnt/β-catenin signaling is required for renal tubule development and that Wnt-4 is a strong candidate for activating this pathway. ^

Agent-based modelling for cement hydration

The Agent-Based Modelling and simulation (ABM) is a rather new approach for studying complex systems withinteracting autonomous agents that has lately undergone great growth in various fields such as biology, physics, social science, economics and business. Efforts to model and simulate the highly complex cement hydration process have been made over the past 40 years, with the aim of predicting the performance of concrete and designing innovative and enhanced cementitious materials. The ABM presented here - based on previous work - focuses on the early stages of cement hydration by modelling the physical-chemical processes at the particle level. The model considers the cement hydration process as a time and 3D space system, involving multiple diffusing and reacting species of spherical particles. Chemical reactions are simulated by adaptively selecting discrete stochastic simulation for the appropriate reaction, whenever that is necessary. Interactions between particles are also considered. The model has been inspired by reported cellular automata?s approach which provides detailed predictions of cement microstructure at the expense of significant computational difficulty. The ABM approach herein seeks to bring about an optimal balance between accuracy and computational efficiency.

Application of Agent Technology for Fault Diagnosis of Telecommunication Networks

La presente tesis doctoral contribuye al problema del diagnóstico autonómico de fallos en redes de telecomunicación. En las redes de telecomunicación actuales, las operadoras realizan tareas de diagnóstico de forma manual. Dichas operaciones deben ser llevadas a cabo por ingenieros altamente cualificados que cada vez tienen más dificultades a la hora de gestionar debidamente el crecimiento exponencial de la red tanto en tamaño, complejidad y heterogeneidad. Además, el advenimiento del Internet del Futuro hace que la demanda de sistemas que simplifiquen y automaticen la gestión de las redes de telecomunicación se haya incrementado en los últimos años. Para extraer el conocimiento necesario para desarrollar las soluciones propuestas y facilitar su adopción por los operadores de red, se propone una metodología de pruebas de aceptación para sistemas multi-agente enfocada en simplificar la comunicación entre los diferentes grupos de trabajo involucrados en todo proyecto de desarrollo software: clientes y desarrolladores. Para contribuir a la solución del problema del diagnóstico autonómico de fallos, se propone una arquitectura de agente capaz de diagnosticar fallos en redes de telecomunicación de manera autónoma. Dicha arquitectura extiende el modelo de agente Belief-Desire- Intention (BDI) con diferentes modelos de diagnóstico que gestionan las diferentes sub-tareas del proceso. La arquitectura propuesta combina diferentes técnicas de razonamiento para alcanzar su propósito gracias a un modelo estructural de la red, que usa razonamiento basado en ontologías, y un modelo causal de fallos, que usa razonamiento Bayesiano para gestionar debidamente la incertidumbre del proceso de diagnóstico. Para asegurar la adecuación de la arquitectura propuesta en situaciones de gran complejidad y heterogeneidad, se propone un marco de argumentación que permite diagnosticar a agentes que estén ejecutando en dominios federados. Para la aplicación de este marco en un sistema multi-agente, se propone un protocolo de coordinación en el que los agentes dialogan hasta alcanzar una conclusión para un caso de diagnóstico concreto. Como trabajos futuros, se consideran la extensión de la arquitectura para abordar otros problemas de gestión como el auto-descubrimiento o la auto-optimización, el uso de técnicas de reputación dentro del marco de argumentación para mejorar la extensibilidad del sistema de diagnóstico en entornos federados y la aplicación de las arquitecturas propuestas en las arquitecturas de red emergentes, como SDN, que ofrecen mayor capacidad de interacción con la red. ABSTRACT This PhD thesis contributes to the problem of autonomic fault diagnosis of telecommunication networks. Nowadays, in telecommunication networks, operators perform manual diagnosis tasks. Those operations must be carried out by high skilled network engineers which have increasing difficulties to properly manage the growing of those networks, both in size, complexity and heterogeneity. Moreover, the advent of the Future Internet makes the demand of solutions which simplifies and automates the telecommunication network management has been increased in recent years. To collect the domain knowledge required to developed the proposed solutions and to simplify its adoption by the operators, an agile testing methodology is defined for multiagent systems. This methodology is focused on the communication gap between the different work groups involved in any software development project, stakeholders and developers. To contribute to overcoming the problem of autonomic fault diagnosis, an agent architecture for fault diagnosis of telecommunication networks is defined. That architecture extends the Belief-Desire-Intention (BDI) agent model with different diagnostic models which handle the different subtasks of the process. The proposed architecture combines different reasoning techniques to achieve its objective using a structural model of the network, which uses ontology-based reasoning, and a causal model, which uses Bayesian reasoning to properly handle the uncertainty of the diagnosis process. To ensure the suitability of the proposed architecture in complex and heterogeneous environments, an argumentation framework is defined. This framework allows agents to perform fault diagnosis in federated domains. To apply this framework in a multi-agent system, a coordination protocol is defined. This protocol is used by agents to dialogue until a reliable conclusion for a specific diagnosis case is reached. Future work comprises the further extension of the agent architecture to approach other managements problems, such as self-discovery or self-optimisation; the application of reputation techniques in the argumentation framework to improve the extensibility of the diagnostic system in federated domains; and the application of the proposed agent architecture in emergent networking architectures, such as SDN, which offers new capabilities of control for the network.

Stability threshold approach for complex dynamical systems

Acknowledgments This paper was developed within the scope of the IRTG 1740/TRP 2011/50151-0, funded by the DFG/FAPESP, and supported by the Government of the Russian Federation (Agreement No. 14.Z50.31.0033 with the Institute of Applied Physics RAS). The first author thanks Dr Roman Ovsyannikov for valuable discussions regarding estimation of the mistake probability.

Purified hematopoietic stem cells without facilitating cells can repopulate fully allogeneic recipients across entire major histocompatibility complex transplantation barrier in mice

We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105 HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104 HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104 HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.

Long-term culture of lymphohematopoietic stem cells.

Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy.

Splitting complex temporal questions for question answering systems

This paper presents a multi-layered Question Answering (Q.A.) architecture suitable for enhancing current Q.A. capabilities with the possibility of processing complex questions. That is, questions whose answer needs to be gathered from pieces of factual information scattered in different documents. Specifically, we have designed a layer oriented to process the different types of temporal questions. Complex temporal questions are first decomposed into simpler ones, according to the temporal relationships expressed in the original question. In the same way, the answers of each simple question are re-composed, fulfilling the temporal restrictions of the original complex question. Using this architecture, a Temporal Q.A. system has been developed. In this paper, we focus on explaining the first part of the process: the decomposition of the complex questions. Furthermore, it has been evaluated with the TERQAS question corpus of 112 temporal questions. For the task of question splitting our system has performed, in terms of precision and recall, 85% and 71%, respectively.

T-cell epitope prediction and immune complex simulation using molecular dynamics:state of the art and persisting challenges

Atomistic Molecular Dynamics provides powerful and flexible tools for the prediction and analysis of molecular and macromolecular systems. Specifically, it provides a means by which we can measure theoretically that which cannot be measured experimentally: the dynamic time-evolution of complex systems comprising atoms and molecules. It is particularly suitable for the simulation and analysis of the otherwise inaccessible details of MHC-peptide interaction and, on a larger scale, the simulation of the immune synapse. Progress has been relatively tentative yet the emergence of truly high-performance computing and the development of coarse-grained simulation now offers us the hope of accurately predicting thermodynamic parameters and of simulating not merely a handful of proteins but larger, longer simulations comprising thousands of protein molecules and the cellular scale structures they form. We exemplify this within the context of immunoinformatics.

Developing Agent Interaction Protocols with PRALU

The purpose of the paper is to explore the possibility of applying existing formal theories of description and design of distributed and concurrent systems to interaction protocols for real-time multi-agent systems. In particular it is shown how the language PRALU, proposed for description of parallel logical control algorithms and rooted in the Petri net formalism, can be used for the modeling of complex concurrent conversations between agents in a multi-agent system. It is demonstrated with a known example of English auction on how to specify an agent interaction protocol using considered means.

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.