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Johns Hopkins Bloomberg School of Public HealthCAAT

CAAT Grants Program

Research Grants 2013-2014: Summaries

  • Sabra Klein
    Differentiated human respiratory epithelial cell cultures as a surrogate system for assessing the effects of estrogenic
    compounds on pulmonary disease pathogenesis
  • Yusuke Marikawa
    Novel axial elongation morphogenesis systems using embryonic stem cells to investigate teratogenic factors
  • Jeffery Morgan
    A new 3D in vitro model of chemical transport across the human placenta
  • Walter Petroll
    A novel anterior corneal construct for ocular toxicity testing in vitro
  • Nicole zur Nieden
    Skeletal teratogenicity of environmental chemicals predicted with human induced pluripotent stem cells in vitro

Abstracts

Sabra Klein
Differentiated human respiratory epithelial cell cultures as a surrogate system for assessing the effects of estrogenic compounds on pulmonary disease pathogenesis

The beneficial and harmful effects of natural estrogen-based therapies, alternative plant-derived estrogen therapies, and inadvertent exposure to xenoestrogens in the environment are debated, with consideration limited to the effects on reproductive tissues and cells. An in vitro system for screening the impact of hormonal and environmental substances on human cells is needed, with attention paid to cells and tissues outside of the reproductive system. The development of pulmonary diseases differs between the sexes and is affected by estrogen exposure. Females tend to suffer more severe pulmonary disease than males, which is partly caused by heightened inflammation. Severe pulmonary inflammation in females can be reversed by treatment with estrogen—a potent anti-inflammatory agent. This proposal will characterize how estrogens affect influenza virus replication, cell function, and inflammation, using an in vitro differentiated human respiratory epithelial cells (hREC). Respiratory epithelial cells are the primary cell type infected with influenza viruses and provide an alternative model for testing the therapeutic or harmful effects of natural estrogens, phytoestrogens (e.g. genistein from soy), and xenoestrogens (e.g. Bisphenol A from plastics) on responses to infection. The long-term goal of this proposal is to replace whole animal testing with an alternative - in vitro hREC cultures. This in vitro system for screening the impact of hormonal and environmental substances on human respiratory epithelial cell function could be expanded to include cellular responses to toxins, allergens, pathogens, and other environmental contaminants.

Epithelix Sarl has provided a line of credit to Dr. Klein for these studies. Epithelix produces standardized in vitro human lung tissue. We offer our sincere appreciation to Epithelix for this contribution.

Yusuke Marikawa
Novel axial elongation morphogenesis systems using embryonic stem cells to investigate teratogenic factors

The objective of our research is to generate culture systems for mouse and human embryonic stem cells, which can be used to identify drugs and gene mutations that potentially induce birth defects in human. We have established culture systems using mouse cells, which display a highly unique morphological change, i.e., transformation from spherical to elongated cell aggregates. We have shown that this elongation morphogenesis has molecular characteristics similar to the cranial-caudal, or head-to-toe, body axis of normal embryos. Thus, our culture system can mimic the body elongation process that normally occurs in the embryo during early gestation period. Disturbance in axial elongation morphogenesis could cause severe birth defects, such as caudal dysgenesis 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 culture systems can serve as valuable tools to investigate the mechanisms of birth defects, the use of model animals for birth defect research should be substantially minimized. The proposed project focuses on molecular profiling of our elongation systems to obtain information on what kind of genes are involved in this specific morphogenesis. We are also conducting chemical screening to identify drug compounds that interfere with the elongation morphogenesis as substances to cause birth defects, i.e., teratogens.

Jeffery Morgan
A new 3D in vitro model of chemical transport across the human placenta

Toxicity testing seeks to determine the effects of exposure to environmental chemicals and drugs and one window of vulnerability is the fetus and its developing immune system. New and reliable methods that don’t rely on animals are needed to determine if a chemical or drug crosses the placenta and poses a risk to the developing fetus. Current methods to make these kinds of measurements have significant limitations. Some of these methods include the use of discarded human placental tissue tested in the Petri dish and the use of selected cell lines grown as a sheet of single cells in two dimensions (2D). However, these methods are difficult, cumbersome, or they don’t adequately mimic the functions of the normal placenta. It’s well known that cells grown in three dimensions (3D) are better mimics of natural tissues and organs than the same cells grown as a sheet of cells in 2D. In 3D culture, cell shape, cell-to-cell contacts and cell function are closer to normal tissues and organs. Using a new scaffold-free
technology that maximizes cell-to-cell contact (developed in our lab), we’ve shown that human TCL1 cells will self-assemble and aggregate to form 3D multi-cellular and multi-layered structures with enhanced cellular junctions and function similar to human placenta. Our goal is to develop and validate a new 3D in vitro model of the human placenta that can be used to quantify the transport of chemicals and drugs and be used to replace/reduce the use of animals.

Walter Petroll
A novel anterior corneal construct for ocular toxicity testing in vitro

Products to which human eyes may be exposed must be tested to establish their potential to cause ocular irritation. In vivo ocular irritation tests provide useful information, but can produce variable responses and require the use of animals such as rabbits. Because corneal damage is the most important factor in determining overall ocular toxicity, an in vitro model of the cornea would have great potential as an alternative to animal testing. A key element to successful tissue engineering of a realistic corneal construct is the development of a 3-D extracellular matrix (artificial stroma) of similar thickness and collagen density to the in vivo tissue, which also supports maintenance of normal stromal cells (corneal keratocytes). In addition, such a tissue should support the differentiation of corneal epithelial cells, which make up the front surface of the cornea and are thus the first cells exposed to ocular irritants.

We have developed a model in which cell-seeded 3-D collagen matrices are compressed to achieve high stiffness stromal tissue equivalents that support differentiation of corneal keratocytes. We also have developed a unique human telomerase-immortalized corneal epithelial cell line (hTCEpi), which expresses key differentiation markers under stratified, air-lifted culture conditions. Our pilot data suggest that compressed collagen matrices support the differentiation and stratification of hTCEpi under serum-free air-lifted conditions in vitro. The purpose of the proposed research is to further develop and characterize these epithelial-stromal constructs, and to investigate the feasibility of using them as a novel platform for ocular toxicity testing in vitro.

Nicole zur Nieden
Skeletal teratogenicity of environmental chemicals predicted with human induced pluripotent stem cells in vitro

Birth defects that affect musculoskeletal tissues account for 5% of all infant deaths. They may be caused by chemical by-products released into the environment or chemical ingredients in pesticides, fungicides, and paints imposing substantial burdens on the affected individuals and families. Evaluating the safety of such chemicals using a suitable prenatal model of human embryos is therefore an essential scientific and societal goal. Screening is needed to uncover which chemicals might potentially cause such devastating birth defects. However, traditional in vitro screening tests still require the killing of animals or the in vitro use of the ethically challenged embryonic stem cells. This project aims to provide a reliable in vitro developmental screening assay that will replace traditional animal methods and improve in vitro screening by utilizing differentiating human induced pluripotent stem cells to model the developing skeleton of the embryo. These cells are artificially made from adult cells and reverted back into an unspecialized state from which they may develop into all possible cell types, among them bone-forming cells. With this ethically acceptable model, tolerable exposure levels of environmental chemicals could then be based on the individuals in the population that are most vulnerable, namely the developing embryo.