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RNA Sequencing Study Yields Clues about Neural Differentiation

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – Using a combination of high throughput RNA sequencing approaches, an international research team has tracked the transcriptional changes that occur as human embryonic stem cells differentiate into brain cells.

Researchers from the US and UK used short-, long-, and paired-end RNA-Seq approaches to compare the transcriptomes of undifferentiated human embryonic stem cells with cells at three stages of neural differentiation. The study, which appeared online last night in the Proceedings of the National Academy of Sciences, not only identified pathways associated with each cell stage, but also reveals a link between differentiation and diminished slicing isoform diversity — a pattern that the researchers dubbed "isoform specialization."

"These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of [human embryonic stem cells]," senior author Michael Snyder, genetics chair at Stanford University and director of the Stanford Center for Genomics and Personalized Medicine, and his colleagues wrote.

Snyder and his co-workers coaxed human embryonic stem cells into becoming differentiated neural cells through two processes — one involving human embryonic stem cells, initiation stage cells, neural progenitor cells, and neuron and glial cells and another involving the production of neural progenitor cells from so-called embryoid body-like neurospheres created from the H1 stem cells.

After verifying and characterizing the differentiated cell stages, the team sequenced messenger RNA libraries from the various stages of neural differentiation using short- and paired-end reads generated with Illumina sequencing and long reads generated with the Roche 454 FLX and Titanium platforms.

From the more than 150 million sequence reads generated, the researchers were able to detect both known and previously unannotated transcriptionally active regions, identifying stage-specific transcription and splicing patterns coinciding with differentiation.

For instance, the team noted, the expression of nervous system, neuron differentiation, and brain development-related genes was ramped up in neural progenitor cells compared with the stem cells.

Meanwhile, both the initiation and neural progenitor cell stages showed increased expression of receptor genes compared with even more differentiated cells, the researchers explained, suggesting it should be possible to direct these intermediate cells into becoming a wide variety of neuronal cell types.

In addition, they noted, between 25 and 65 percent of the unannotated transcripts detected were stage specific, suggesting these uncharacterized transcripts play an undetermined functional role in each cell stage.

The researchers also saw differences in the level of splicing isoform diversity during progressive stages of cellular differentiation, with stem cells showing greater diversity than the three more differentiated cell stages tested.

Based on these patterns, the team proposed that neural differentiation is characterized by what they call isoform specialization, with transcripts becoming increasingly specialized as cells edge closer to a differentiated state.

"[O]ur study demonstrated that greater splice junction diversity is present in [human embryonic stem cells] relative to cells undergoing neural differentiation," they wrote. "We suggest that this high transcript diversity contributes to the pluripotency of [human embryonic stem cells]."

Along with the insights into neuronal differentiation, the team noted, the new study highlights the potential of using RNA sequencing technology to track transcriptional patterns and dynamics over time.

"Future improvements of sequencing technologies, including longer reads, higher throughput, and reduced cost will aid in the definition of transcriptomes and alternative splicing in specific temporal and spatial contexts," they concluded.

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