Skip Navigation
Johns Hopkins Bloomberg School of Public HealthCAAT

CAAT Grants Program

Research Grants 2012-2013: Summaries

  • Thierry Dorval and Hye Young Shin
    Human embryoid bodies as 3D models for development toxicity prediction
  • Francesca Levi-Schaffer
    Human culture model as a replacement to the animal assays for assessing the potential of cosmetic ingredients to cause non-immunological contact urticaria (NICU)
  • Yusuke Marikara
    Novel axial elongation morphogenesis systems using embryonic stem cells to investigate teratogenic factors
  • Yuhei Nishimura, MD, PhD
    Development of zebrafish-based assays for the assessment of developmental neurotoxicity of chemicals at low doses
  • Marie-Gabrielle Zurich, PhD
    Brain aggregate cell cultures as an in vitro model for developmental neurotoxicity testing


Abstracts

Thierry Dorval and Hye Young Shin
Human embryoid bodies as 3D models for development toxicity prediction

For pharmaceutical companies, defining relevant and accurate protocols for toxicity prediction is a key step in order to avoid drug development failure. Among the different type of toxicity, developmental toxicity is particularly difficult to assess. The animal model experiments are limited and do not correlate well with the human response. In such a context, the recent advances in human embryonic stem cell (hESC) research provide an attractive opportunity to define new approaches for developmental toxicity prediction. The use of these cells makes it possible to recapitulate in vitro the early stage of development, and thus can help to identify compounds inducing failure in the development process. To improve the relevance of the model, we propose to set up a protocol based on human embryoid body (EB) culture. This three dimensional structure is more likely to mimic the early stage of development and physiological environment than a traditional 2D monolayer.  

When combined, Institut Pasteur Korea (IPK) expertise creates opportunities for defining complex and accurate protocols for developmental toxicity in a High Content Screening (HCS) context. In the “Cellular Differentiation and Toxicity” team, we possess the expertise in stem cell and EB culture, differentiation, toxicity and image analysis. In this project, we propose to combine those expertise by developing a large number of EBs, subjecting them to teratogen drugs, quantify the resulting phenotype, and performing data analysis to predict development toxicity. This approach can be used in a drug discovery pipeline to reduce the number of animal usually used in toxicity testing.


Francesca Levi-Schaffer
Human culture model as a replacement to the animal assays for assessing the potential of cosmetic ingredients to cause non-immunological contact urticaria (NICU)

It is well known that a growing number of ingredients present in fragrance mix, nail polish, hair dyes and emulsions can trigger skin reaction recognized by the medical community as contact urticaria. This reaction which is characterized by the appearance of wheal and flare in healthy skin within 30-60 min of the cosmetic product's application leads to significant discomfort. This can happen in both allergic individuals (previously exposed to the same cosmetic product) and in non allergic individuals (who have applied the product for the first time). We have hypothesized that this second reaction, also called non immunologic contact urticaria (NICU), is driven by a direct activation of mast cells, that are cells that reside in the skin. These cells release histamine and other proinflammatory mediators.

Our aim is to develop a reliable replacement of the current animal ear swelling method consisting only of human cells. During the first two years of research we developed a co-culture system consisting of human mast cells and skin fibroblasts. In this system we assessed mast cell activation/urticariogenic properties of different cosmetic ingredients by measuring their released mediators.

During the third year of research we will try to simplify the co-culture method by using instead of fibroblasts their membranes. We will validate this simplified model by using urticariogenic compounds and we will try to dissect the molecular mechanism of mast cell activation used by some of these compounds.


Yusuke Marikara
Novel axial elongation morphogenesis systems using embryonic stem cells to investigate teratogenic factors
 
The long-term goal of our research is to establish in vitro culture systems of mouse and human embryonic stem cells, which can be used to screen drugs and gene mutations that potentially cause severe morphological birth defects in humans. We recently created in vitro culture systems using mouse cell lines, which exhibit a unique morphological change, i.e., transformation from spherical to elongated cell aggregates.  We have shown that this elongation morphogenesis have molecular characteristics similar to the cranial-caudal, or head-to-toe, body axis of normal embryos. Thus, our in vitro system appears to mimic the body elongation process that normally occurs during early gestation period.  Disturbance in axial elongation morphogenesis can cause severe birth defects, such as caudal dysgensis and neural tube closure defects.  However, their etiology and pathological mechanisms are still not well understood.  Most studies on birth defects have been conducted using model mammalian species, such as mice and rats, in which many animals are experimented and sacrificed. However, once we demonstrate that our in vitro systems can serve as critical tools to investigate the mechanisms of birth defects, the use of model animals for birth defect research should be substantially reduced. The proposed project focuses on molecular profiling of our in vitro elongation systems to obtain information on what kind of genes are involved in this specific morphogenesis. We are also conducting chemical screening to identify compounds that interfere with the elongation morphogenesis as potential substances to cause birth defects.


Yuhei Nishimura, MD, PhD
Development of zebrafish-based assays for the assessment of developmental neurotoxicity of chemicals at low doses

Our goal is to develop zebrafish-based assays that can be used to reliably detect developmental neurotoxicity (DNT) caused by exposure to chemicals at low doses. The developing brain is more sensitive to chemical toxicants than the adult brain and exposure during development has been implicated in neuropsychiatric and neurological diseases. The impairments observed in these disorders represent a continuum, from typical clinical syndromes at one extreme to small subclinical deficits in sensory, motor, and behavioral impairment at the other. However, it is often difficult to assess the subtle effects caused by exposure to low concentrations of chemicals that do not induce any external malformation. Large studies in which specific functional domains are assessed using objective instruments are required to detect these subtle changes. The use of non-mammalian animal models allows for testing large numbers of individuals while reducing expense and using fewer mammals. Zebrafish offer several advantages for DNT testing, including the low cost of husbandry, high fecundity, and rapid ex utero development. Furthermore, zebrafish are transparent, allowing for easy observation of morphological changes in the nervous system. Zebrafish larvae also exhibit a number of simple and complex behavioral programs. We propose to develop novel assays to quantify the morphological and behavioral changes in zebrafish following exposure to chemicals at low doses. The assays developed in this study can be performed in a relatively high-throughput manner, making it possible to undertake a systematic and objective analysis and to increase statistical power to detect subtle but significant levels of DNT.


Marie-Gabrielle Zurich, PhD
Brain aggregate cell cultures as an in vitro model for developmental neurotoxicity testing

Each brain cell type constitutes a potential target for xenobiotics. Interactions between the different types of cells may also be affected by xenobiotics. A suitable in vitro model for neurotoxicity testing should therefore contain all types of brain cells, and allow the multiple cell-cell interactions involved in brain physiology. Furthermore, for developmental neurotoxicity testing (DNT) it is crucial to use in vitro models that reproduce the critical maturational events allowing cells to reach their final level of differentiation. These characteristics are typical for aggregating brain cell cultures. The cells prepared from rat embryonic brain reaggregate spontaneously into even-sized spheroids, kept in suspension by continued gyratory agitation. All the brain cell types are found in the aggregates, where they interact by physical contacts and by exchange of soluble factors. The maturation process takes about one month. As in the adult brain, a discrete population of undifferentiated stem/precursor cells persists besides the highly differentiated cells in the mature aggregates. The interest of using this model for DNT has been revealed by previous studies. However, in depth characterization is required. The work of the second year will focus on signaling pathways acting on neural stem and progenitor cells. Perturbation of these pathways by valproic acid, a developmental toxicant, will be studied. Particular attention will be given to the population of immature cells which persist in highly differentiated cultures. This project will enable the detection of relevant endpoints to monitor the effects of toxicants on critical developmental events, such as proliferation and early differentiation.