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WCPG 2013—iPS Cells: Transformative Opportunity With a Way to Go

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.

Comments on Related News


Related News: Researchers Model Susceptibility to Schizophrenia in a Petri Dish

Comment by:  Alan Mackay-Sim
Submitted 13 April 2011
Posted 13 April 2011

With a heritability of 50 percent, schizophrenia is very clearly a disease of disturbed biology, but to dissect the biological contribution to its etiology, researchers need relevant, patient-derived cell models. Ideally, we need cell models that can tell us how schizophrenia cell biology leads to an altered brain. Induced pluripotent stem (iPS) cells are genetically engineered cells, from a patient's cells (e.g., fibroblasts), that resemble embryonic stem cells, that can be used to generate neurons. There is much excitement that they will be useful as models for many brain disorders and diseases. Two new papers in Molecular Psychiatry and Nature report on applying iPS cell technology to schizophrenia by generating iPS cells from patients with a DISC1 mutation (Chiang et al., 2011) and from patients selected with a high likelihood of a genetic component to disease (Brennand et al., 2011).

When specific genes are implicated, then animal models can provide breakthroughs by determining the cellular functions of the implicated genes and their mutations. Although schizophrenia lacks single commonly mutated genes of large effect, some candidate genes, such as DISC1, are being identified in some families. This is now a very hot area for research that is identifying the role of this gene at the cellular level and in animal models. As such candidate genes are identified and their functions are ascertained, it will be essential to demonstrate their direct relevance in schizophrenia through patient-derived cellular models. In this regard, a new tool has emerged in the recent letter to Molecular Psychiatry reporting the generation of induced pluripotent cells from two patients with DISC1 mutation (Chiang et al., 2011). This preliminary study did not report a disease-associated phenotype in these iPS cells.

A disease-associated phenotype is best identified by comparing iPS cells from patients and controls, as now demonstrated by Brennand et al. (2011). This work is a significant new contribution to the field because it has demonstrated differences in the biology of neurons derived from patients and controls. As proof of principle, they have identified differences in the way patient neurons branch (they have fewer branches) and connect with each other (they connect to fewer other neurons). Most importantly, the patient neurons had normal physiological properties. That is to say, their physiology was not different from controls. These are interesting and important distinctions that are a reassuring proof of principle for this model, suggesting that the etiology of schizophrenia derives from altered connectivity of neuronal circuits and not from basic neuronal functions. This fits with the postulated “neurodevelopmental hypothesis” of schizophrenia. Patient neurons also had decreased levels of synaptic proteins (PSD95, glutamate receptor), which is consistent with “synaptic hypotheses” of schizophrenia. These are early days yet, but this cell model already demonstrates how a relevant cell model can provide a path for unifying etiological hypotheses.

Another aim for developing cell models of schizophrenia is to use them for drug discovery. Patient-control differences in cell functions can be the basis for screening chemical compounds that ameliorate this difference. Here, too, Brennand et al. (2011) demonstrate proof of principle by showing that loxapine treatment of the patient neurons increased their connectivity towards control levels. Only loxapine, of five antipsychotic drugs tested, had this effect, but the results are a clear sign of the utility of such cells for drug screening to find new potential drug candidates.

These two papers are a great start to using iPS cells as models of schizophrenia.

References:

Chiang CH, Su1Y, Wen Z, Yoritomo N, Ross CA, Margolis RL, Song H, Ming G-I. (2011) Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation. Molecular Psychiatry advance online publication, 22 February 2011. Abstract

Brennand KJ, Simone A, Jou1 J, Gelboin-Burkhart C, Tran N, Sangar S, Li Y, Mu Y, Chen G, Yu D, McCarthy S, Sebat J, Gage FH (2011). Modeling schizophrenia using human induced pluripotent stem cells. Nature.

View all comments by Alan Mackay-Sim

Related News: Researchers Model Susceptibility to Schizophrenia in a Petri Dish

Comment by:  Akira Sawa, SRF Advisor
Submitted 13 April 2011
Posted 13 April 2011

I fully appreciate the efforts of Brennand and colleagues as pioneers. Indeed, this is great work. Like any pioneering work, this paper will be both applauded and criticized. The strength of the paper is in providing ways for us to analyze iPS cells and derived neurons. The multifaceted approach taken in this study will be a great platform for many investigators.

Schizophrenia is, clinically, a very heterogeneous condition, but for the past several years, basic scientists have tended to oversimplify the disorder. It is also true that this trend makes the neurobiology of schizophrenia move productively forward in some ways. I believe that the new tools for studying the biology of schizophrenia, such as iPSC-derived neurons, will teach us how difficult it is to draw simplified pathways for the disorder. Nonetheless, some common pathway(s) may be identified in the future, I optimistically hope.

Based on the great experimental procedures that this paper provides, many other groups may need to address whether or not these data are reproducible or not in “general” cases of schizophrenia. In such studies, the most important issue is to examine detailed clinical information of the subjects in comparison with this study.

View all comments by Akira Sawa