With the holiday season upon us, many of us tend to put on a few extra pounds of fat. However, fat also is a reservoir of mesenchymal stem cells (MSCs) that can make cartilage, bone or even more fat.
A new study by scientists at Brown University demonstrate the efficacy of a new method for extracting potential bone-making cells from human fat. To perfect their technique, the Brown University team, led by senior author Eric Darling, developed a fluorescent tag that identified any cell that expressed the ALPL (alkaline phosphatase liver/bone/kidney) gene. This probe is a fluorescent, oligodeoxynucleotide molecular beacon probe specific for ALPL mRNA. This probe hybridizes to the ALPL mRNA and fluoresces strongly as a result of the hydridization. Fluorescence-activated cell sorting ten isolates the ALPL-expressing cells from the non-expression cells effectively and easily.
Molecular beacon probes are single-stranded nucleic acid molecules that have a fluorescent dye attached to one end and a quenching molecule that prevents the dye from fluorescing at the other end. A short, complementary sequence at either end of the probe causes the probe to self-hybridize, thus bringing the glowing molecule and its inhibitor close together. Under these conditions, the probe does not glow. However, if the probe hybridizes to a specific sequence, then the glowing molecule and its inhibitor are far apart and the glowing molecule fluoresces.
The level of ALPL gene expression strongly correlates with the ability of cells to form bone. According to Darling and his team, their technique more than doubled the quantity of bone-making cells that could be extracted from fat. Brown University has applied for a patent on this method.
In culture, ALPL-expressing cells produced, on average, twice as much bony matrix as other cells. ALPL-expressing cells also were better at making cartilage or even fat. Even though other groups have isolated cells on the basis of gene expression, none of the available published techniques, to date, have used gene expression-based isolation to enrich for cell populations of a specific tissue, such as bone.
The lead author of this work, Hetal Marble, said that targeting gene expression rather than cell surface proteins is a relatively new paradigm for cell isolation and purification. Because using cell surface proteins always requires accepting certain assumptions about the cell types that are being isolated, gene expression-based isolation is a more pragmatic way to approach the problem. Consider this: cells that are in certain tissues express certain genes characteristic of that tissue. Therefore, using those gene expression patterns to isolate such cells leaves doubt that the cells you will isolate are in the process of differentiating into the desired tissue. Using cell surface proteins, however, is less precise because particular cell populations rarely are the only cell types that express that protein.
“Approaches like this allow us to isolate all the cells that are capable of doing what we want, whether they fit the archetype of what a stem cell is or not,” said Marble. “The paradigm shift is thinking about populations that are able to achieve am end point rather than isolating populations that fit a strictly defined archetype.”
In this experiment, Darling’s group used a special culture medium designed to crank up bone-specific gene expression in sensitive cells. This so-called “priming step” took four days in culture before the ALPL gene expression levels were high enough to properly detect it. However, in the future, Darling believes that by targeting a gene whose levels of expression increase earlier in during the process of differentiation, he and his team should be able to dispense with the four-day delay. This way, their technique would be applicable in the operating room, since surgeons could isolate fat from the patient with a bone break and prime it (or not), and then isolate the bone-making cells from the fat, which they would use to treat their patient’ fracture.
“If you can take the patient into the OR, isolate a bunch of their cells, sort them, and put them back in that’s ideally where we’d like to go with this,” said Darling. “Theoretically we could do this with other genes that might upregulate very quickly or are innately expressed.”