Cellular Neurobiology

The Stem Cell groups at the Stanley Center for Psychiatric Research are developing stem cell–derived neuronal models and using genome engineering in human pluripotent stem cells (hPSCs) to investigate in vitro phenotypes associated with genetic variants underlying psychiatric disease. The Stem Cell groups are focused on the following projects:

The Human EnCELLopedia
Stem Cell Genome Engineering
Human Brain Organoid Models
Neuronal Spheroids for Screening and Therapeutic Target Studies
The Brain Interaction Network
A Human Stem Cell Genome Project
Massively Mosaic Experimental Systems

The Human EnCELLopedia

Biobanking efforts at the Stanley Center are uniquely focused on collection and generation of hPSC lines from patients with a spectrum of psychiatric conditions as well as from ancestrally matched controls. Efforts to include sample collection for stem cell derivation, as part of Stanley Center–wide global genetic collections, are ongoing. The Stanley Center also recently partnered with California Institute of Regenerative Medicine (CIRM) to perform whole genome sequencing (WGS) of hundreds of lines from the CIRM iPSC repository. The overall aim of the Human EnCELLopedia project is to establish a rich community resource of curated and whole-genome sequenced stem cell lines, serving as a foundation for fruitful collaborations investigating cellular biology mechanisms underlying psychiatric disease. Many of our biobanked lines, selected for genetic variants of interest and/or absence of culture-acquired variants, are now being actively deployed into ongoing research projects. To learn more about the Human EnCELLopedia, contact Kiki Lilliehook or Ralda Nehme.

Stem Cell Genome Engineering

The Lindy Barrett lab has developed a CRISPR-Cas9–based genome engineering pipeline, described in Stem Cell Reports, for editing hPSCs at scale. The lab is now generating hPSCs with gene-disrupting mutations to address questions of basic gene function, fluorescent reporters to probe brain regions and cell identity, epitope tags to interrogate protein-protein interactions, and point mutations to study variants identified through whole-exome and whole-genome sequencing of psychiatric cohorts. These cellular resources are utilized in research projects throughout the Stanley Center community.

Human Brain Organoid Models

Developing improved cellular systems that model complex human genetic traits, while accounting for non-disease-relevant variation in genetic background, is an important goal in the Paola Arlotta lab. Drawing from foundational work on cortical neuronal development and circuitry in the mouse cortex, the group has optimized a protocol, published in Nature, that supports long-term development of self-organizing whole brain organoids derived from human stem cells. These human organoids generate a large diversity of cell classes from distinct regions of the brain and display robust neuronal networks, presence of synaptic spines, and light sensitivity. Human brain organoids present promises and challenges for gaining insight into human neurodevelopmental and neuropsychiatric pathologies.

Neuronal Spheroids for Screening and Therapeutic Target Studies

Developing screening assays relevant to psychiatric disease using human neurons is the focus of the Lee Rubin lab. The group recently reported a robust spheroid culture protocol in Stem Cell Reports, noteworthy for large-scale production of hPSC-derived neurons with mature features. The group now seeks to “close the gap between spheroid and organoids” with continued technical improvements to increase human brain-like mature features, in order to eventually implement the method in their wide studies of psychiatric disease mechanisms. In collaboration with Beth Stephens and Jeff Cottrell, some of these Stanley Center–wide efforts include following up on the Nature paper linking the human leukocyte antigen (HLA) locus, a schizophrenia GWAS hit, with mechanisms of synaptic pruning downstream of the complement 4 (C4) protein.

The Brain Interaction Network

In collaboration with Kasper Lage, the Eggan group is using proteomics approaches to investigate networks in human cultured neurons. The approaches hinge on optimized NGN2-driven reprogramming of hPSCs to rapidly generate synaptically mature cortical neurons at scale. From a refined schizophrenia GWAS “hit” list of index proteins, the team pursues immunoprecipitations followed by liquid chromatography/tandem mass spectrometry, which enables identification of interaction partners. Subsequent computational analysis, described in Nature Methods, is then used to resolve pathway relationships.

A Human Stem Cell Genome Project

The ability of hPSCs to renew indefinitely enables limitless propagation in culture, but also raises concerns about the long-term genomic integrity of hPSCs. To gain a holistic view of the genetic landscape of hPSCs, Kevin Eggan and team performed whole exome sequencing (WES) and WGS of 114 publically available human embryonic stem cell lines, acquired and banked at the Stanley Center. The report on stem cell exomes, published in Nature, showed that hPSCs in culture can acquire and expand cancer-associated mutations that would not have been detected by methods currently used for quality control (such as karyotyping and SNP genotyping). These findings point to the importance of sequence-based approaches for genetic screening of hPSCs and their differentiated derivatives, both in experimental models of disease and cell replacement therapies, across the stem cell field.

Massively Mosaic Experimental Systems

In collaboration with Steve McCarroll, the Eggan lab is harnessing WGS data to generate unique fingerprints — composed of genetic variants used as an intrinsic barcode — of large numbers of hPSC lines. This bioinformatics framework serves as the foundation for development of mosaic experimental systems, in which large numbers of hPSC lines are co-cultured in the same dish and analyzed using Drop-seq, and then have their donor identity inferred after the experiment. Efforts underway aim to develop Drop-seq to measure gene expression levels at a cellular level. These approaches enable scaling of stem cell disease models in a way that is internally controlled, pointing towards applications for mapping eQTLs as a function of psychiatric disease.