Differentiating stem cells signal with multicolored beacons
Molecular beacons illuminate the differentiation of stem cells
LifeSciencesToday based on materials from Brown University: Beacons light up stem cell transformationNew specially developed molecular "beacons" allow scientists to observe the expression of genes in stem cell populations in real time during their differentiation into cells of certain tissues.
The method developed by scientists at Brown University gives tissue engineers a powerful tool to find what can make stem cells differentiate into cells of the right tissue more often and faster. This is one of the main problems in improving the methods of regenerative medicine.
"We are not the inventors of molecular beacons, but we have used them in a way that no one has done before us. Using them, we observe changes in one population in a long–lived culture using a reliable and cell-safe method," says graduate student Hetal Desai, lead author of an article published online in the journal Tissue Engineering Part A (Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells).
The beacons developed by Desai and her colleagues fluoresce when they bind to the matrix RNAs of three specific genes derived from adipose tissue stem cells, which are expressed only when these stem cells begin to differentiate into bone cells.
For 21 days of their development, these cells remained alive and did not fluoresce, with the exception of populations that received a chemical stimulus to differentiate into bone tissue cells. During these three weeks, scientists observed the fluorescence of beacons in these populations to determine how many stem cells in each population were transformed into bone cells and the timing of the start of expression of individual genes.
The first peak of the expression of the ALPL gene in more than 90 percent of induced stem cells occurred on the fourth day, after which the COL1A1 gene began to be expressed in 85 percent of cells on day 14. In the last few days of experiments, a sharp increase in the expression of the BGLAP gene was unmistakably observed in more than 80 percent of the induced stem cells.
Glowing green spots in MG-63 bone cells (each blue dot is a nucleus) show that the fluorescent "beacon" molecule has bound to RNA synthesized during the expression of the bone-specific gene ALPL. (Photo: Darling Lab/Brown University)
The researchers noted that each subsequent episode of gene expression developed from zero to peak faster. This allowed them to hypothesize that the transformation, or differentiation, of the stem cells that make up the population can synchronize over time.
"If we could find a way to get them to take this path earlier, differentiation would go faster," says the head of the study. Eric Darling, PhD, Associate Professor of Biology at BU.
At the same time, in stem cell populations that were not stimulated by substances directing their differentiation towards bone tissue cells, the fluorescence of beacons or the expression of these genes was practically not observed, indicating that beacons are indeed indicators of the stages of transformation of stem cells into bone cells.
According to Desai, her group has made every effort to develop such beacons that do not change the development and functioning of cells in any way. While beacons do bind to matrix RNAs synthesized during gene expression, they do not require the addition of any genes to the DNA of stem cells or the expression of any special proteins, which many other fluorescent methods require.
To make sure that cells develop normally even in the presence of beacons, scientists conducted a series of experiments using them in normally developing bone cells.
While some researchers are developing RNA-based probes for targeted intervention in gene expression, Dr. Darling's group faced completely opposite tasks.
"There is a set of rules for RNA interference, and we were essentially doing the opposite of what is written in these rules," Desai explains.
Now that the effectiveness of beacons in determining the stages of stem cell differentiation has been demonstrated, Dr. Darling says, this method can be used to study processes in a wide variety of cells and under a variety of other experimental conditions.
In tissue engineering, he continues, beacons can help experiments in which scientists try to determine which conditions (chemical or other) are most effective for the rapid differentiation of most stem cells into cells of the desired tissue. They will help to find out the optimal time for the addition of differentiation-inducing chemicals, as well as to identify and isolate only those cells that are transformed into the desired tissue.
"They become bone cells, and if you increase their number and get rid of all those that do not become bone cells, it goes without saying that you will have a better product at the exit," concludes Dr. Darling.
In a broader sense, he adds, molecular beacons have proven their usefulness in a wide variety of studies concerning gene expression.
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