Glowing green spots in these MG-63 bone cells (each blue dot is a nucleus) indicate that a fluorescent “beacon” molecule has bound to RNA produced by expression of the bone-specific ALPL gene. Credit: Darling Lab/Brown University |
In
a new study, Brown University researchers demonstrate a new tool for
visually tracking in real-time the transformation of a living population
of stem cells into cells of a specific tissue. The “molecular beacons,”
which could advance tissue engineering research, light up when certain
genes are expressed and don’t interfere with the development or
operation of the stem cells.
A
novel set of custom-designed “molecular beacons” allows scientists to
monitor gene expression in living populations of stem cells as they turn
into a specific tissue in real-time. The technology, which Brown
University researchers describe in a new study, provides tissue
engineers with a potentially powerful tool to discover what it may take
to make stem cells transform into desired tissue cells more often and
more quickly. That’s a key goal in improving regenerative medicine
treatments.
“We’re
not the inventors of molecular beacons but we have used it in a way
that hasn’t been done before, which is to do this in long-term culture
and watch the same population change in a reliable and harmless way,”
said graduate student Hetal Desai, lead author of the paper published
online Sept. 5, 2012, in the journal Tissue Engineering Part A.
In
their research, Desai and corresponding author Eric Darling, assistant
professor of biology in the Department of Molecular Pharmacology,
Physiology, and Biotechnology, designed their beacons to fluoresce when
they bind to mRNA from three specific genes in fat-derived stem cells
that are expressed only when the stem cells are transforming into bone
cells.
Throughout
21 days of their development, the cells in the experiments remained
alive and unfettered, Desai said, except that some populations received a
chemical inducement toward becoming bone and others did not. Over those
three weeks, the team watched the populations for the fluorescence of
the beacons to see how many stem cells within each population were
becoming bone and the timing of each gene expression milestone.
The
beacons’ fluorescence made it easy to see a distinct pattern in that
timing. Expression of the gene ALPL peaked first in more than 90 percent
of induced stem cells on day four, followed by about 85% expressing the
gene COL1A1 on day 14. The last few days of the experiments saw an
unmistakably sharp rise in expression of the gene BGLAP in more than 80
percent of the induced stem cells.
Each
successive episode of gene expression ramped up from zero to the peak
more quickly, the researchers noted, leading to a new hypothesis that
the pace of the stem cell transformation, or “differentiation” in stem
cell parlance, may become more synchronized in a population over time.
“If you could find a way to get them on this track earlier, you could get the differentiation faster,” Darling said.
Meanwhile
the stem cell populations that were not induced with bone-promoting
chemicals, showed virtually no beacon fluorescence or expression of the
genes, indicating that the beacons were truly indicators of steps along
the transformation from stem cell to bone.
Beacons don’t affect cells
Desai
said the team took extra care to design beacons that would not alter
the cells’ development or functioning in any way. While the beacons do
bind to messenger RNA produced in gene expression, for example, they do
not require adding any genes to the stem cells’ DNA, or expressing any
special proteins, as many other fluorescence techniques do.
A population of fat-derived stem cells expresses the bone-specific COL1A1 gene. Green fluorescence from a “molecular beacon” shows increasing expression from day 9, left, through day 11 and day 14. By day 16, right, expression begins to wane. Credit: Darling Lab/Brown University |
The
team performed several experiments using the beacons in conventionally
developing bone cells to make sure that they developed normally even as
the beacons were in operation. While some scientists design RNA-based
probes to purposely interfere with gene expression, this team had the
opposite intent.
“You
know that [.38 Special] song ‘Hold on Loosely but Don’t Let Go?’” Desai
said. “That’s sort of the theme song for this. There’s a set of rules
for interference RNA, and we essentially did the opposite of what those
rules said you should do.”
Toward quicker healing
Now
that the beacons’ performance in indicating milestones of stem cell
differentiation has been demonstrated, Darling said, the technology can
be applied to studying the process in a wide variety of cells and under a
variety of other experimental conditions.
In
the case of tissue engineering, he said, the beacons can aid
experiments seeking to determine what conditions (inducing chemicals or
otherwise) are most effective in converting the most stem cells to
desired tissues most quickly. They could help tissue engineers learn the
best timing for adding an inducing chemical. They might also provide a
way for tissue engineers to identify and harvest only those cells that
are converting to the desired tissue.
“They
are becoming bone cells and if you enrich for them and you get rid of
all the ones that aren’t becoming bone cells, it stands to reason that
you will have a better product at the end,” Darling said.
More broadly, Darling added, molecular beacons are proving useful in a wide variety of gene expression studies.
“The
reason we are looking at this technique is that we wanted something we
could use on any cell, look at any gene and not affect that cell while
we are looking at it,” Darling said. “If this is acting as we believe it
is, we can really look at any gene that we want. It seems like a very
versatile tool.”
In addition to Darling and Desai, the other authors are Indu Voruganti, Chathuraka Jayasuriya, and Qian Chen.
The National Institutes of Health provided funding for the research.
Source: Brown University
