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.
Beacons for bone
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
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 percent 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.
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.
Editors: Brown University has a fiber link television studio available for domestic and international live and taped interviews, and maintains an ISDN line for radio interviews. For more information, call (401) 863-2476.
David Orenstein | EurekAlert!
Chains of nanogold – forged with atomic precision
23.09.2016 | Suomen Akatemia (Academy of Finland)
Self-assembled nanostructures hit their target
23.09.2016 | King Abdullah University of Science and Technology
Wissenschaftler der Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) haben zusammen mit Kollegen der Freien Universität Berlin ein neues Molekül entdeckt: Die Eisenverbindung in der seltenen Oxidationsstufe +4 gehört zu den Ferrocenen und ist äußerst schwierig zu synthetisieren.
Metallocene werden umgangssprachlich auch als Sandwichverbindungen bezeichnet. Sie bestehen aus zwei organischen ringförmigen Verbindungen, den...
Kompetenzzentrum Ultrapräzise Oberflächenbearbeitung (CC UPOB) lädt zum Expertentreffen im März 2017 ein
Ob in Weltraumteleskopen, deren Optiken trotz großer Abmessungen nanometergenau gefertigt sein müssen, in Handykameras oder in Endoskopen − Asphären kommen in...
Das Fraunhofer-Institut für Organische Elektronik, Elektronenstrahl- und Plasmatechnik FEP arbeitet seit Jahren an verschiedenen Entwicklungen zu OLED-Mikrodisplays, die auf organischen Halbleitern basieren. Durch die Integration einer Bildsensorfunktion direkt im Mikrodisplay, lässt sich u.a. die Augenbewegung in Datenbrillen aufnehmen und zur Steuerung von Display-Inhalten nutzen. Das verbesserte Konzept wird erstmals auf der Augmented World Expo Europe (AWE), vom 18. – 19. Oktober 2016, in Berlin, Stand B25 vorgestellt.
„Augmented Reality“ (erweiterte Realität) und „Wearable Displays“ (tragbare Displays) sind Schlagworte, denen man mittlerweile fast täglich begegnet. Beide...
The Fraunhofer Institute for Organic Electronics, Electron Beam and Plasma Technology FEP has been developing various applications for OLED microdisplays based on organic semiconductors. By integrating the capabilities of an image sensor directly into the microdisplay, eye movements can be recorded by the smart glasses and utilized for guidance and control functions, as one example. The new design will be debuted at Augmented World Expo Europe (AWE) in Berlin at Booth B25, October 18th – 19th.
“Augmented-reality” and “wearables” have become terms we encounter almost daily. Both can make daily life a little simpler and provide valuable assistance for...
Mit Methoden der künstlichen Intelligenz haben Chemiker der Universität Basel die Eigenschaften von rund 2 Millionen Kristallen berechnet, die aus vier verschiedenen chemischen Elementen zusammengesetzt sind. Dabei konnten die Forscher 90 bisher unbekannte Kristalle identifizieren, die thermodynamisch stabil sind und als neuartige Werkstoffe in Betracht kommen. Das berichten sie in der Fachzeitschrift «Physical Review Letters».
Elpasolith ist ein glasiges, transparentes, glänzendes und weiches Mineral mit kubischer Kristallstruktur. Erstmals entdeckt im El Paso County (USA), kann man...
23.09.2016 | Veranstaltungen
23.09.2016 | Veranstaltungen
23.09.2016 | Veranstaltungen