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New Cell Culture, Analysis Approach Helps Explain Differences in Zika Virus Susceptibility

NEW YORK – A team led by researchers at the Broad Institute has demonstrated the potential of using so-called cell villages from several donors to characterize expression variability in neural progenitor cells (NPC), along with related genetic features that appear to influence brain function and vulnerability to Zika virus infection.

"Our findings establish an integrated experimental format that uses natural and synthetic perturbations to identify genes and genetic variants that change a cell's phenotype in a meaningful way," co-corresponding authors Steven McCarroll and Kevin Eggan, both affiliated with the Broad Institute, and their colleagues wrote.

As they reported in Cell Stem Cell on Wednesday, the researchers first showed that they could distinguish individual donors within a larger "cell village" — a pool of cells from different donors in a shared environment — based on SNP patterns found in single-cell RNA sequence data, making it possible to profile genetic features, phenotypes, expression variability, and expression quantitative trait loci (eQTL) of individuals within a larger sample pool rather than assessing cells from each donor separately.

"The cell village strategy yields data that are much more comparable and uniform from donor to donor and cell line to cell line," McCarroll said in an email. "This is because the cells are grown in a shared culture environment and assayed at the exact same time and in the exact same way. We find that this greatly strengthens genetic and biological signals relative to noise and variability in the data. In addition to this rigor, the cell village approach is also scalable and cost-efficient."

From there, they used an algorithm known as Dropulation ("droplet-based sequencing of populations") to distinguish and characterize individual cells by donor using single-cell RNA sequencing profiles on the pooled cells. Another algorithm known as Census-seq enabled them to assess genetic variation in the cells based on low-coverage whole-genome sequencing profiles.

The team also turned to CRISPR-Cas9-based genetic screens to assess the functional consequences of a series of genetic alterations affecting distinct NPC genes, including a screen for NPC factors contributing to Zika virus response.

After validating the strategies in a handful of human embryonic stem cell lines, the investigators relied on their Dropulation and Census-seq algorithms to assess cell villages of "stem cell-derived NGN2-accelerated progenitors" (SNaPs) generated by exposing induced pluripotent stem cells to the transcription factor neurogenin-2 (NGN2). One cell village of SNaPs contained cells from 44 donors and another one cells from 21 donors.

Together, the profiles provided a look at expression quantitative trait loci in the cells, the researchers explained, including eQTLs at sites previously implicated in brain traits or neurodevelopmental conditions through genome-wide association studies.

Genes with known or suspected roles in neurodevelopment turned up in a list of "eGenes" with expression profiles influenced by genetic variants in NPCs, for example, while the team's subsequent CRISPR-based screening experiments led to a gene called CACHD1 with apparent ties to progenitor cell differentiation and proliferation.

By tracking Zika virus responses in the NPC villages, meanwhile, the team got a glimpse at the expression of immune and other genes in SNaP cells with or without Zika virus susceptibility. In combination with CRISPR-based screening data, the expression patterns highlighted the importance of an interferon-responsive gene called IFITM3 — and a SNP regulating IFITM3 expression — for Zika virus susceptibility.

While higher-than-usual IFITM3 levels dialed down viral replication in follow-up cell line experiments, the researchers explained, Zika virus infection rates ramped up in the absence of the gene.

More generally, the authors suggested that "[p]opulation-scale in vitro culture systems provide promising ways to capture the influence of genetic variation on a wide range of cell types and phenotypes."

"We hope that these and other new approaches open opportunities to find and characterize the many genetic and environmental factors that shape human development," they wrote.