Growing Knowledge: Using Stem Cells to Study Developmental Neurotoxicity

What the Future Holds

Both Buzanska and Fritsche believe their approaches to developmental neurotoxicity testing platforms will be speedier than conventional animal testing, but whether Buzanska's platform will ultimately be capable of high-throughput testing is still an open question. Fritsche says neurospheres may not deliver more than what she calls "medium-throughput screening." But not every compound has to be tested for developmental neurotoxicity, she points out; for instance, "the ones that don't pass the placenta are not of interest [in this context]." The compounds that should be a priority for testing are endocrine disruptors and those that show some potential for develop-mental toxicity based on animal screening, she says. Therefore, even though it takes four weeks to complete developmental neuro-toxicity testing with neurospheres, they could easily be used to test around 100 compounds per year with an automated setup, she says.

Shafer's project under way at the EPA is following an approach similar to the ones initiated in Europe to use neuroprogenitor cells to evaluate how chemicals may affect human neuro-development. Shafer says his team's goal is to develop what he calls a "first-tier" approach to identify chemicals that merit more detailed developmental neurotoxicity testing, perhaps following up with an alternative species model such as zebrafish and then a rodent or other mammalian species.

The neurodevelopmental processes for which Shafer and his colleagues are devising assays include proliferation, differentiation, neurite growth, the creation of synapse gaps (synapto-genesis), migration, myelination, and apoptosis. As the group works to develop assays to test how chemicals affect these processes, one objective is to ensure the assays are amenable to high-throughput testing, Shafer says. They are using monolayers of the commercial ReNcell CX model, derived from a human fetus. This will enable others to easily conduct the same tests, he says, and the two-dimensional mono-layers also are more amenable to high-throughput testing than the 3-D neurospheres.

Of the neurotoxicity suite that Shafer's group is developing, the proliferation assay is one of the furthest along. Neuroprogenitor proliferation is crucial to early brain development, when the neural tube expands rapidly and its anterior portion eventually gives rise to the brain. The proliferation assay initially proved itself in a small test with a group of chemicals documented to affect proliferation.^[25] More recently, the researchers tested the assay's performance with the 309 biologically active chemicals being evaluated by the ToxCast™ program run by the EPA National Center for Computational Toxicology. The Shafer group's assay showed that 125 ToxCast chemicals had a significant impact on neural proliferation.^[26] (Further unpublished work put that number at 112.) Participation in the program will allow these results to be compared with those from other screening and testing efforts, Shafer says.

At press time it was unclear how the ultimate fate of the August ruling on stem cells would affect projects of some of the researchers interviewed for this article. For instance, Smith is collaborating with Steve Stice, director of the University of Georgia Regenerative Bioscience Center, to develop a way to use "germ-like" cells derived from human embryonic stem cells to produce an in vitro system for investigating the developmental and reproductive effects of compounds that affect endocrine-system functioning. Early in human development, Smith explains, cells that eventually become part of the male or female reproductive system express two proteins, DDX4 and POU5f1. Smith and Stice had been measuring changes in metabolite levels in cells that express these proteins to determine the biochemical consequences of exposure to environmental chemicals. Smith says in time they also would like to look at impacts on neuronal cells.

In other human embryonic stem cell work, Woodruff and Mike McMaster of the UCSF departments of Cell and Tissue Biology and Obstetrics, Gynecology, and Reproductive Sciences are exposing stem cells to bisphenol A at levels similar to those measured in pregnant women and their fetuses. The team will examine how these exposures affect the cells' gene expression profiles and developmental potential.

One possible alternative to human embryonic stem cells that was discussed at the June NRC meeting is what are known as induced pluripotent stem cells. In the past few years, scientists have begun to recognize that the somatic cells that compose the vast majority of the adult human body can be "reprogrammed" to create these induced pluripotent stem cells, says M. William Lensch of Harvard University's Children's Hospital and the Harvard Stem Cell Institute. This is possible because all of an individual's cells contain the same DNA required to produce the entire body, he explains. An individual's functional cells, such as skin cells and liver cells, differ from one another because they express different parts of the DNA. The reprogramming process involves the cells' chromatin, which controls which genes are expressed.

Induced pluripotent stem cells are being used to produce a variety of human cell types, and researchers believe that, like embryonic stem cells, they can give rise to all of the body's cell types. In the future, human induced pluri-potent cells may prove useful for developmental neurotoxicity testing, Shafer says. They have the potential to help researchers represent a greater degree of genetic variability, he says.

Cite this: Growing Knowledge: Using Stem Cells to Study Developmental Neurotoxicity - Medscape - Oct 01, 2010.

Sidebar

Using Stem Cells to Investigate Autism

Ricardo Dolmetsch, a neurobiologist at Stanford University, is employing a very different approach to using stem cells in research related to children's environmental health. Dolmetsch's group is using human stem cells to investigate autism spectrum disorders (ASDs), and his group is laying the groundwork that may eventually allow them to create a system for screening environmental agents to assess whether they could play a role in these disorders, he says.

The reported incidence of ASDs has been rising, and although some circumstantial evidence points to environmental agents, good data are sorely lacking, Dolmetsch says. Tracking down links with conventional animal models is problematic because scientists believe some important classes of neurons exist only in primates, he points out.

Dolmetsch has succeeded in identifying some of the genes involved in ASDs by focusing on relatively rare disorders known to have a genetic link. Such groups of patients are likely to have similar "cellular molecular signatures," he explains.

He and his colleagues harvest cells from these patients' skin and reprogram them to become induced pluripotent stem cells, which are then converted into functional cells. The research team has successfully produced a wide variety of neurons and glial cells, including catecholaminergic cells, which control the secretion of hormones such as dopamine that have been implicated in both schizophrenia and autism.

In unpublished work presented at the NRC meeting in June,^[10] Dolmetsch's group reported success at identifying genes associated with the calcium signaling deficits and cardiac irregularities found in patients with Timothy syndrome, a rare disease characterized by webbed fingers and toes that is often associated with developmental delays and autism. They have also discovered a compound that appears to reverse the calcium signaling issues, which Dolmetsch says may ultimately help pharmacologists discover a drug that would help children with the syndrome.

The Stanford group is investigating links their work has turned up with other rare ASDs, too. They credit their successes thus far to what Dolmetsch calls the focused approach of narrowing in on genes they suspect to be involved with the diseases they are studying. Although Dolmetsch's group has not yet used induced pluripotent stem cells to study environmental triggers of ASDs, he says he believes "that at least in principle it is doable."

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