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  • Fluorescence method may advance cell-replacement treatment strategies for diabetes

Mar 2007
Technique isolates the necessary cells through sorting

Gary Boas

Treatment strategies for diabetes have recently focused on generating islet replacement cells in the pancreas. However, isolating and characterizing progenitor cells from the fetal pancreas — which would help to deepen understanding of pancreatic development —have proved difficult in animal models and haven’t yet been reported in humans.

In the Jan. 2 issue of PNAS, investigators at Stanford University School of Medicine in California described a study in which they identified markers of fetal pancreatic epithelium and used them to isolate islet progenitor cells from the pancreas of fetal mice with fluorescence-activated cell sorting. Discovery of the markers could help to advance studies of islet development and replacement and, in this way, contribute to better strategies for the treatment of diabetes.

Previous attempts to isolate progenitor cells from the fetal pancreas have met with challenges. Studies using transgenic marking methodologies in mice, for example, have produced heterogeneous populations with incomplete separation of the progenitor and other cells. Furthermore, human tissue samples simply lack the surface markers that are needed to isolate progenitor cells.

The researchers demonstrated that they could separate progenitor and other cells from the fetal pancreas without relying on genetic modification. The study was several years in the making, said principal investigator Seung K. Kim, and it involved laborious empirical work. A particular challenge was determining the necessary conditions for keeping the cells alive. “The pancreas produces a lot of digestive — or hydrolytic — enzymes that are destructive to cells,” he said. “That’s why we focused on the embryonic pancreas: The older it gets, the more of these things it makes.”

Once they had established the necessary conditions, the researchers — including Kim and first author Takuya Sugiyama — systematically tested more than 30 stem cell markers to determine which enabled them to isolate pancreatic cells expressing neurogenin 3, an established marker of islet progenitors. (They were encouraged by previous studies in which stem cells had been isolated from bone marrow and other organs; in certain organs, stem and progenitor cells express the same sets of cell-surface markers.) Using immunohistology, they identified two stem cell markers that would allow isolation of the cells by fluorescence-assisted cell sorting (FACS): CD133 and CD49f.

A method based on fluorescence-activated cell sorting enables isolation of islet progenitor cells from the pancreas in fetal mice. A FACS plot of sorted cells after exposure to an anti-CD133 antibody — CD133 is one of the markers used to isolate the cells. The percentages of CD133- and CD133+ are noted (left). The fluorescence image on the right shows cells expressing glucagon and insulin, two types of endocrine cells in the pancreas. Reprinted with permission of PNAS.

They used FACS to isolate these progenitor cell populations. After acquiring single cells from the mouse dorsal pancreas, they treated the cells for 15 minutes in a blocker solution containing the FACS buffer. They performed FACS in the Stanford facility, using a modified triple-laser instrument and software from either Dako in Fort Collins, Colo., or BD Biosciences in San Jose, Calif. They sorted the cells twice to remove contamination from other fractions.

Next, they tested whether they could separate hormone-expressing cells from cells expressing neurogenin 3 from enhanced GFP transgenic mice using FACS. They observed a 110-fold enrichment and a high purification of the neurogenin 3-expressing cells. Experiments with yellow fluorescent protein transgenic mice yielded similar results, thus demonstrating that the strategy enabled purification of the cells to near-homogeneity.

Finally, using new in vitro cell culture methods, they allowed the cells isolated by FACS to develop into hormone-expressing endocrine cells — the cells destroyed by type I diabetes — that are the focus of islet replacement treatment strategies. They surveyed a number of culture conditions and found that glucagon- and insulin-expressing cells — two types of endocrine cells in the pancreas — developed when the cells at low density were cocultured either with mouse embryonic fibroblasts treated with mitomycin C or with PA6 mouse stromal cells.

With other members of the laboratory, including Ryan T. Rodriguez and Graeme W. McLean, Kim and Sugiyama applied the FACS methods to isolating progenitor cells from the human fetal pancreas. Kim noted that they are working to improve the FACS-based isolation to the human fetal pancreas. Once they successfully address the challenges, they expect the cell culture methods will help to shed light on the role of neurogenin 3 in developing islet cells in the human pancreas.

The researchers suggested that the FACS isolation method also could contribute to the purification and study of islet progenitor cells other than those derived from the fetal pancreas — those derived from the adult pancreas, for example. Knowledge gleaned from such studies could help to advance cell-replacement treatment strategies for diabetes.

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