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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

Changes in expression of epithelial and mesenchymal genes in human islet cells during the first 3 weeks in culture.RNA was extracted from cells at the indicated passage number (each passage is equivalent to one week) and analyzed by qRT-PCR. A, analysis of epithelial genes. B, analysis of mesenchymal genes. Data represent relative quantification (RQ) (compared to passage 0) and are mean±SE (n = 6 donors). *p<0.05; ** p<0.005; ***p<0.0005 (compared to passage 0).
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pone-0006417-g002: Changes in expression of epithelial and mesenchymal genes in human islet cells during the first 3 weeks in culture.RNA was extracted from cells at the indicated passage number (each passage is equivalent to one week) and analyzed by qRT-PCR. A, analysis of epithelial genes. B, analysis of mesenchymal genes. Data represent relative quantification (RQ) (compared to passage 0) and are mean±SE (n = 6 donors). *p<0.05; ** p<0.005; ***p<0.0005 (compared to passage 0).

Mentions: Human islets isolated from both male and female donors (age 44.9±12.8; BMI 27.6±5.8) were dissociated into single cells and cultured as described [8]. One day after infection with the two lentiviruses constituting the labeling system cultures were treated with tamoxifen overnight to label the beta cells. They were then expanded and split 1∶2 once a week. Analysis of gene expression in the cultured cells revealed a rapid loss of beta-cell and epithelial phenotypes, as manifested by a large decrease in the levels of insulin and E-cadherin mRNAs (Fig. 2A). In parallel, a pronounced increase in levels of transcripts encoding mesenchymal and MSC markers was detected. These included the mesenchymal markers smooth-muscle actin (SMA), N-cadherin, and vimentin, and the MSC markers CD73, CD90, and CD105 (Fig. 2B). These results were reproducible in cells derived from 6 donors.


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)

Changes in expression of epithelial and mesenchymal genes in human islet cells during the first 3 weeks in culture.RNA was extracted from cells at the indicated passage number (each passage is equivalent to one week) and analyzed by qRT-PCR. A, analysis of epithelial genes. B, analysis of mesenchymal genes. Data represent relative quantification (RQ) (compared to passage 0) and are mean±SE (n = 6 donors). *p<0.05; ** p<0.005; ***p<0.0005 (compared to passage 0).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006417-g002: Changes in expression of epithelial and mesenchymal genes in human islet cells during the first 3 weeks in culture.RNA was extracted from cells at the indicated passage number (each passage is equivalent to one week) and analyzed by qRT-PCR. A, analysis of epithelial genes. B, analysis of mesenchymal genes. Data represent relative quantification (RQ) (compared to passage 0) and are mean±SE (n = 6 donors). *p<0.05; ** p<0.005; ***p<0.0005 (compared to passage 0).
Mentions: Human islets isolated from both male and female donors (age 44.9±12.8; BMI 27.6±5.8) were dissociated into single cells and cultured as described [8]. One day after infection with the two lentiviruses constituting the labeling system cultures were treated with tamoxifen overnight to label the beta cells. They were then expanded and split 1∶2 once a week. Analysis of gene expression in the cultured cells revealed a rapid loss of beta-cell and epithelial phenotypes, as manifested by a large decrease in the levels of insulin and E-cadherin mRNAs (Fig. 2A). In parallel, a pronounced increase in levels of transcripts encoding mesenchymal and MSC markers was detected. These included the mesenchymal markers smooth-muscle actin (SMA), N-cadherin, and vimentin, and the MSC markers CD73, CD90, and CD105 (Fig. 2B). These results were reproducible in cells derived from 6 donors.

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