November 11, 2013. Less than a decade ago, Shinya Yamanaka first reported that somatic cells derived from easily accessible skin fibroblasts could be reprogrammed into stem cells, work for which he shared the 2012 Nobel Prize in Physiology or Medicine. The vision of a nearly unlimited supply of these induced pluripotent stem (iPS) cells and the prospect that they could be converted into neurons have been heady for neuroscientists studying brain disorders such as Alzheimer’s disease, autism, and schizophrenia. Technical challenges have slowed progress (see SRF related conference story), but the excitement has not gone away. Not surprisingly, iPS cells garnered much attention at the 2013 World Congress of Psychiatric Genetics. Although only a few schizophrenia studies were presented, researchers are looking to stem cells as venues for testing the effects of the growing list of genes implicated in the disorder (see SRF related conference story).
In his introduction to the plenary talk on Saturday morning, October 19, 2013, conference co-chair Jordan Smoller called the reprogramming of stem cells an important breakthrough. "It offers really transformative opportunities to dissect the functional effects of genetic variants and translate genetic discoveries into novel therapeutics," he said. Smoller then turned the microphone over to the featured speaker, Harvard University’s Kevin Eggan, who provided an overview of how iPS cells can be used to investigate the genetics of psychiatric disease and discussed several challenges for stem cell science.
Technical problems, some with available solutions
Given that psychiatric illnesses affect select populations of neurons, one concern is whether stem cells can be used to make the “right” cell types, Eggan said. Citing MRI evidence of gray matter loss in schizophrenia, he suggested that the two major neuronal cell types—projection neurons and interneurons—are both of interest and will need to be made in order to model the disease. While several methods to make projection neurons do exist, they are currently limited by prohibitively long culture times, so Eggan focused on recent advances in producing interneurons from iPSCs, a robust protocol that he characterized as “ready for prime time” (Maroof et al., 2013).
One difficulty with differentiating progenitor cells into specific populations is a lack of complete efficiency in making the desired cell type. “No matter how good the protocol that you have is, … the cell type you’re interested in [is] always embedded in the milieu of diverse cell types from the nervous system,” Eggan said. To overcome this problem, Asim M. Maroof of Sloan-Kettering Institute for Cancer Research, New York City, and colleagues (including Eggan) used a reporter gene expressing green fluorescent protein (GFP) to identify interneurons of interest. Interneuron differentiation was then confirmed using a migration assay, to examine whether the cells traveled to the cortex after transplantation into the medial ganglionic eminence. By adding the human GFP-positive cells to cultures of dissociated embryonic mouse cortex, the researchers mimicked the normal development of the interneurons (that occurs in the presence of excitatory cells), allowing them to confirm a GABAergic physiological phenotype.
Eggan also discussed work from Steve McCarroll’s lab that finds, via transcriptional approaches, that these GFP-positive cells consist of a few different classes of cells. Though one population expresses high levels of the progenitor marker nestin (indicative of immature neurons), another expresses somatostatin, a cell type implicated in schizophrenia. A smaller minority express parvalbumin, the interneuron class most strongly implicated in the disease (see SRF related news story). In the future, we need more differentiation and more specific interneuron fates, said Eggan. “Clearly, progress is being made … on making relevant human cell types,” he added, but additional advances are needed to generate more purified, neurochemically distinct cultures.
How to model psychiatric illness?
Eggan then asked the audience to “suspend reality” for the remainder of his talk and assume that the technical limitations have been solved so that iPS cells can be used to generate the particular classes of neurons that are affected in various psychiatric illnesses. How do we then model the effects of genetic variants? Skepticism about iPS cells has focused on how the reprogrammed cells differ from embryonic stem (ES) cells, said Eggan, leading to questions about whether the reprogramming process leaves behind a memory of the somatic state or produces mutations. In collaboration with Alex Meissner, Eggan produced several different lines of iPS and ES cells and found considerable variation in DNA methylation and gene expression between different ES cell lines, as well as between different iPS cell lines (Bock et al., 2011). These data suggest that focusing on differences between the two stem cell types, he said, “is missing a much broader point … that any two pluripotent cell lines are different from each other.”
This variation has real ramifications for the behavior of the cells, he added. When using the same strategy to differentiate each of the iPS and ES cell lines into neurons, each individual line performs differently, resulting in variable proportions of different cell types between lines—something he called “a major phenotypic driver” (Boulting et al., 2011). This variability will make detecting real case/control differences difficult.
Gene targeting is one potential way to “gain better traction” in this issue, said Eggan. By correcting or introducing a variant that has been identified as relevant to mental illness and then examining the cell’s phenotype, researchers will be able to see if the mutation is relevant. Eggan has successfully used this approach in amyotrophic lateral sclerosis, and similar approaches are underway in psychiatric disease. This comparison between isogenic control lines and genetically modified lines is “really where the stem cell field needs to go,” he concluded.
In an afternoon symposium the next day, moderator Jay Tischfield of Rutgers University, New Brunswick, New Jersey, also raised several questions about using iPS cells to model psychiatric illnesses. Like Eggan, he emphasized the need to produce the cell types relevant to each disorder, as well as homogeneous cultures. Another question, said Tischfield, is whether epigenetic changes in vivo will manifest in iPSC-derived cultures that have “synthetic” (in vitro) developmental histories. Finally, he asked what phenotypes should be examined in these cells, noting that the transcriptome, proteome, morphology, and electrophysiology of the cells have been popular choices to date.
In the same session, Flora Vaccarino of Yale University, New Haven, Connecticut, described her work addressing another concern that has been raised about iPS cells: Are they genetically and phenotypically stable across time? The process of creating iPSCs has been suspected of causing de novo copy number variations, but Vaccarino and colleagues have found otherwise when examining the genome and transcriptome of several iPSC lines (Abyzov et al., 2012). The researchers detected an average of two CNVs in each iPSC line that were not found in the specific skin fibroblasts from which the cells originated. About half of these CNVs, however, were found in the original fibroblast population, reflecting somatic mosaicism in the skin cells and letting the iPS cells mostly off the hook.
Moving into patients
In a morning session on October 21, Alexander Urban of Stanford University, California, presented a genomic characterization of iPSCs derived from people with 22q11 deletion syndrome, a disorder in which a quarter to a third of patients develop schizophrenia (see SRF related news story). Urban and colleagues generated 25 iPSC lines from seven patients and seven controls, and found that the iPSCs, for the most part, have stable genomes and good neuronal differentiation potential. He noted that more cell lines would, of course, be desirable, but that the labor-intensive process of creating them is currently the limiting factor.
On Friday afternoon, October 18, Kristen Brennand of Mount Sinai Hospital, New York City, recapped her previous work on neurons grown from iPSCs from people with schizophrenia (see SRF related news story) and presented new findings concerning iPSC-derived neural progenitor cells (NPCs), which give rise to neurons. Mass spectrometry revealed that NPCs derived from people with schizophrenia contained altered levels of proteins involved in synapses, such as NLGN3, and oxidative stress. Similarly, a dye-based assay revealed increased oxidative stress in these NPCs. Newly born neurons from NPCs derived from people with schizophrenia were also slow to migrate in vitro, as revealed by watching individual cells move away from clumps of NPCs called neurospheres. This seemed to reflect a problem inherent to the neuron itself and might mimic aspects of the disrupted brain development noted in schizophrenia.—Allison A. Curley.