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Epithelial-mesenchymal transition in cells expanded in vitro from lineage-traced adult human pancreatic beta cells.

Russ HA, Ravassard P, Kerr-Conte J, Pattou F, Efrat S - PLoS ONE (2009)

Bottom Line: These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC).However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.

ABSTRACT

Background: In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential.

Methodology/principal findings: Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.

Conclusions/significance: These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.

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Related in: MedlinePlus

Expression of mesenchymal genes in eGFP+ cells.Cells at passage 4 were stained with antibodies to eGFP and the indicated mesenchymal marker. Nuclei were stained blue with DAPI. Bar = 20 µm. The percentages indicate the fraction of cells positive for each mesenchymal marker among eGFP+ cells (green digits) and among all the cells (white digits). Data are mean±SD of >400 cells scored from each donor (n = 3 donors).
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pone-0006417-g004: Expression of mesenchymal genes in eGFP+ cells.Cells at passage 4 were stained with antibodies to eGFP and the indicated mesenchymal marker. Nuclei were stained blue with DAPI. Bar = 20 µm. The percentages indicate the fraction of cells positive for each mesenchymal marker among eGFP+ cells (green digits) and among all the cells (white digits). Data are mean±SD of >400 cells scored from each donor (n = 3 donors).

Mentions: These analyses suggested that expression of the mesenchymal and MSC markers was greatly elevated in cells derived from beta cells. To directly verify this possibility, labeled cells at passage 4 were stained with antibodies to eGFP and 5 mesenchymal and MSC markers (Fig. 4). Scoring of >400 cells stained for each antigen in cells derived from each of 3 donors revealed that 94.1±9.3% and 93.6±4.5% of eGFP+ cells were positive for CD90 and CD105, respectively. Similarly, 99.7±0.5% and 95.9±5.5% of eGFP+ cells were positive for N-cadherin and vimentin, respectively. In contrast, in freshly isolated islets, and in islet cells during the first 2 days of culture, only very rare C-peptide+ cells were positive for these markers (data not shown). The mesenchymal and MSC markers were also highly abundant in eGFP− cells (Fig. 4), confirming the lack of significant differences in transcript levels for these markers between sorted eGFP+ and eGFP− cells (Fig. 3B). Taken together, these findings directly demonstrate the activation of mesenchymal and MSC markers in cells derived from beta cells, thus supporting the occurrence of EMT in these cells.


Epithelial-mesenchymal transition in cells expanded in vitro from lineage-traced adult human pancreatic beta cells.

Russ HA, Ravassard P, Kerr-Conte J, Pattou F, Efrat S - PLoS ONE (2009)

Expression of mesenchymal genes in eGFP+ cells.Cells at passage 4 were stained with antibodies to eGFP and the indicated mesenchymal marker. Nuclei were stained blue with DAPI. Bar = 20 µm. The percentages indicate the fraction of cells positive for each mesenchymal marker among eGFP+ cells (green digits) and among all the cells (white digits). Data are mean±SD of >400 cells scored from each donor (n = 3 donors).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712769&req=5

pone-0006417-g004: Expression of mesenchymal genes in eGFP+ cells.Cells at passage 4 were stained with antibodies to eGFP and the indicated mesenchymal marker. Nuclei were stained blue with DAPI. Bar = 20 µm. The percentages indicate the fraction of cells positive for each mesenchymal marker among eGFP+ cells (green digits) and among all the cells (white digits). Data are mean±SD of >400 cells scored from each donor (n = 3 donors).
Mentions: These analyses suggested that expression of the mesenchymal and MSC markers was greatly elevated in cells derived from beta cells. To directly verify this possibility, labeled cells at passage 4 were stained with antibodies to eGFP and 5 mesenchymal and MSC markers (Fig. 4). Scoring of >400 cells stained for each antigen in cells derived from each of 3 donors revealed that 94.1±9.3% and 93.6±4.5% of eGFP+ cells were positive for CD90 and CD105, respectively. Similarly, 99.7±0.5% and 95.9±5.5% of eGFP+ cells were positive for N-cadherin and vimentin, respectively. In contrast, in freshly isolated islets, and in islet cells during the first 2 days of culture, only very rare C-peptide+ cells were positive for these markers (data not shown). The mesenchymal and MSC markers were also highly abundant in eGFP− cells (Fig. 4), confirming the lack of significant differences in transcript levels for these markers between sorted eGFP+ and eGFP− cells (Fig. 3B). Taken together, these findings directly demonstrate the activation of mesenchymal and MSC markers in cells derived from beta cells, thus supporting the occurrence of EMT in these cells.

Bottom Line: These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC).However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.

ABSTRACT

Background: In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential.

Methodology/principal findings: Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.

Conclusions/significance: These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.

Show MeSH
Related in: MedlinePlus