UCSF researchers use microfluidic
technology to probe human brain development
UC San Francisco researchers have
identified cells' unique features within the developing human brain,
using the latest technologies for analyzing gene activity in
individual cells, and have demonstrated that large-scale cell surveys
can be done much more efficiently and cheaply than was previously
thought possible.
"We have identified novel
molecular features in diverse cell types using a new strategy of
analyzing hundreds of cells individually," said Arnold
Kriegstein, MD, PhD, director of the Eli and Edythe Broad Center of
Regeneration Medicine and Stem Cell Research at UCSF. "We expect
to use this approach to help us better understand how the complexity
of the human cortex arises from cells that are spun off through cell
division from stem cells in the germinal region of the brain."
The research team used technology
focused on a "microfluidic" device in which individual
cells are captured and flow into nano-scale chambers, where they
efficiently and accurately undergo the chemical reactions needed for
DNA sequencing. The research showed that the number of reading steps
needed to identify and spell out unique sequences and to successfully
identify cell types is 100 times fewer than had previously been
assumed. The technology, developed by Fluidigm Corporation, can be
used to individually process 96 cells simultaneously.
"The routine capture of single
cells and accurate sampling of their molecular features now is
possible," said Alex Pollen, PhD, who along with fellow
Kriegstein-lab postdoctoral fellow Tomasz Nowakowski, PhD, conducted
the key experiments, in which they analyzed the activation of genes
in 301 cells from across the developing human brain. Their results
were published online August 3 in Nature Biotechnology.
Kriegstein said the identification of
hundreds of novel biomarkers for diverse cell types will improve
scientists' understanding of the emergence of specialized neuronal
subtypes. Ultimately, the combination of this new method of focusing
on gene activity in single cells with other single-cell techniques
involving microscopic imaging is likely to reveal the origins of
developmental disorders of the brain, he added.
The process could shed light on several
brain disorders, including lissencephaly, in which the folds in the
brain's cortex fail to develop, as well as maladies diagnosed later
in development, such as autism and schizophrenia, Kriegstein said.
According to the Nature Biotechnology
study co-authors, this strategy of analyzing molecules in single
cells is likely to find favor not only among researchers who explore
how specialized cells arise at specific times and locations within
the developing organism, but also among those who monitor cell
characteristics in stem cells engineered for tissue replacement, and
those who probe the diversity of cells within tumors to identify
those responsible for survival and spread of cancerous cells.
No matter how pure, in any unprocessed
biological sample there are a variety of cells representing various
tissue types. Researchers have been sequencing the combined genetic
material within these samples. To study which genes are active and
which are dormant, they use the brute repetition of sequencing steps
to capture an adequate number of messenger RNA sequences, which are
transcribed from switched-on genes. However, it is difficult to
conclude from mixed tissue samples which genes are expressed by
particular cell types.
Pollen and Nowakowski showed that fewer
steps – and less time and money – are needed to distinguish
different cell types through single-cell analysis than had previously
been thought.
"We are studying an ecosystem of
different, but related, cell types in the brain," Pollen said.
"We are breaking that community down into the different
populations of cells with the goal of understanding their functional
parts and components so we can accurately predict how they will
develop."
Joe Shuga, PhD, co-author on the paper
and a senior scientist at Fluidigm Corporation, said the system
developed by the company to routinely capture and prepare the cells
for messenger-RNA sequencing yields more accurate reading of the
sequences.
The research was funded by the Damon
Runyon Cancer Research Foundation, the California Institute for
Regenerative Medicine, and the National Institutes of Health.
UCSF is the nation's leading university
exclusively focused on health. Now celebrating the 150th anniversary
of its founding as a medical college, UCSF is dedicated to
transforming health worldwide through advanced biomedical research,
graduate-level education in the life sciences and health professions,
and excellence in patient care. It includes top-ranked graduate
schools of dentistry, medicine, nursing and pharmacy; a graduate
division with world-renowned programs in the biological sciences, a
preeminent biomedical research enterprise and two top-tier hospitals,
UCSF Medical Center and UCSF Benioff Children's Hospital San
Francisco. Please visit http://www.ucsf.edu.
Contact: Jeffrey Norris
415-502-6397
University of California - San
Francisco
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