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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

Differentiation of cells expanded from human islet cells into adipocytes and osteocytes.A, Islet cells at the indicated passage number were incubated in Lonza induction medium and stained with Oil Red O for adipocytes and Alizarin Red for osteocytes. Human BM-MSC served as positive control. Bar = 100 µm. B, Islet cells at passage 5 and BM-MSC were incubated in Lonza adipogenesis induction medium and stained with antibodies to eGFP and FABP4. Nuclei were stained blue with DAPI. Bar = 30 µm. The eGFP+ cells shown do not stain for FABP4. The single FABP4+ cell shown is not eGFP+. C, Quantitation of the staining in B, based on counting >500 cells in cultures derived from each donor. Data represent percent of FABP4+ among eGFP+ cells (green bars) and among all the cells (black bars) and are mean±SD (n = 6 donors for islet cells and 2 donors for BM-MSC). p = 4.18E-14.
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pone-0006417-g005: Differentiation of cells expanded from human islet cells into adipocytes and osteocytes.A, Islet cells at the indicated passage number were incubated in Lonza induction medium and stained with Oil Red O for adipocytes and Alizarin Red for osteocytes. Human BM-MSC served as positive control. Bar = 100 µm. B, Islet cells at passage 5 and BM-MSC were incubated in Lonza adipogenesis induction medium and stained with antibodies to eGFP and FABP4. Nuclei were stained blue with DAPI. Bar = 30 µm. The eGFP+ cells shown do not stain for FABP4. The single FABP4+ cell shown is not eGFP+. C, Quantitation of the staining in B, based on counting >500 cells in cultures derived from each donor. Data represent percent of FABP4+ among eGFP+ cells (green bars) and among all the cells (black bars) and are mean±SD (n = 6 donors for islet cells and 2 donors for BM-MSC). p = 4.18E-14.

Mentions: To investigate whether the expanded islet cells, which acquired MSC markers, also developed multipotency, they were treated with inducers of adipocyte and osteocyte differentiation. As seen in Fig. 5A, these inducers resulted in efficient differentiation of adult human BM-MSC into adipocytes and osteocytes, as detected by staining with Oil Red O and Alizarin Red, respectively. In contrast, only very rare cells stained with these dyes were detected in cells expanded from adult human islets. To specifically assay the capacity to differentiate into adipocytes in cells derived from beta cells, eGFP+ cells were stained with antibodies for the adipocyte marker fatty acid binding protein 4 (FABP4). As seen in Fig. 5B and 5C, 73.1±13.8% of BM-MSC were stained for this marker following induction of differentiation into adipocytes. In contrast, only 5.5±2.9% of all cells expanded from adult human islet cells and induced to differentiate into adipocytes at passages 3–8 were positive for FABP4 (based on counting >500 cells each in cells derived from 6 donors). Among all cells positive for FABP4, only 1.4% were eGFP+ cells (representing <0.08% of the total population). When scoring specifically eGFP+ cells (a total of 2378 cells counted from 5 donors), 1.9±1.6% were found to be positive for FABP4. Similar results were obtained with 2 additional kits of reagents for induction of MSC differentiation into adipocytes (data not shown). These results demonstrate that cells derived from beta cells, which undergo EMT and acquire MSC markers, are not bona fide MSC, as judged by their inability to efficiently differentiate into mesodermal cell types. In addition, the adipocyte and osteocyte differentiation capacity of eGFP− cells in these cultures, the majority of which also express MSC markers and are derived from unknown non-beta-cell sources in the islet preparation, is also quite limited, compared with that of BM-MSC.


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)

Differentiation of cells expanded from human islet cells into adipocytes and osteocytes.A, Islet cells at the indicated passage number were incubated in Lonza induction medium and stained with Oil Red O for adipocytes and Alizarin Red for osteocytes. Human BM-MSC served as positive control. Bar = 100 µm. B, Islet cells at passage 5 and BM-MSC were incubated in Lonza adipogenesis induction medium and stained with antibodies to eGFP and FABP4. Nuclei were stained blue with DAPI. Bar = 30 µm. The eGFP+ cells shown do not stain for FABP4. The single FABP4+ cell shown is not eGFP+. C, Quantitation of the staining in B, based on counting >500 cells in cultures derived from each donor. Data represent percent of FABP4+ among eGFP+ cells (green bars) and among all the cells (black bars) and are mean±SD (n = 6 donors for islet cells and 2 donors for BM-MSC). p = 4.18E-14.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712769&req=5

pone-0006417-g005: Differentiation of cells expanded from human islet cells into adipocytes and osteocytes.A, Islet cells at the indicated passage number were incubated in Lonza induction medium and stained with Oil Red O for adipocytes and Alizarin Red for osteocytes. Human BM-MSC served as positive control. Bar = 100 µm. B, Islet cells at passage 5 and BM-MSC were incubated in Lonza adipogenesis induction medium and stained with antibodies to eGFP and FABP4. Nuclei were stained blue with DAPI. Bar = 30 µm. The eGFP+ cells shown do not stain for FABP4. The single FABP4+ cell shown is not eGFP+. C, Quantitation of the staining in B, based on counting >500 cells in cultures derived from each donor. Data represent percent of FABP4+ among eGFP+ cells (green bars) and among all the cells (black bars) and are mean±SD (n = 6 donors for islet cells and 2 donors for BM-MSC). p = 4.18E-14.
Mentions: To investigate whether the expanded islet cells, which acquired MSC markers, also developed multipotency, they were treated with inducers of adipocyte and osteocyte differentiation. As seen in Fig. 5A, these inducers resulted in efficient differentiation of adult human BM-MSC into adipocytes and osteocytes, as detected by staining with Oil Red O and Alizarin Red, respectively. In contrast, only very rare cells stained with these dyes were detected in cells expanded from adult human islets. To specifically assay the capacity to differentiate into adipocytes in cells derived from beta cells, eGFP+ cells were stained with antibodies for the adipocyte marker fatty acid binding protein 4 (FABP4). As seen in Fig. 5B and 5C, 73.1±13.8% of BM-MSC were stained for this marker following induction of differentiation into adipocytes. In contrast, only 5.5±2.9% of all cells expanded from adult human islet cells and induced to differentiate into adipocytes at passages 3–8 were positive for FABP4 (based on counting >500 cells each in cells derived from 6 donors). Among all cells positive for FABP4, only 1.4% were eGFP+ cells (representing <0.08% of the total population). When scoring specifically eGFP+ cells (a total of 2378 cells counted from 5 donors), 1.9±1.6% were found to be positive for FABP4. Similar results were obtained with 2 additional kits of reagents for induction of MSC differentiation into adipocytes (data not shown). These results demonstrate that cells derived from beta cells, which undergo EMT and acquire MSC markers, are not bona fide MSC, as judged by their inability to efficiently differentiate into mesodermal cell types. In addition, the adipocyte and osteocyte differentiation capacity of eGFP− cells in these cultures, the majority of which also express MSC markers and are derived from unknown non-beta-cell sources in the islet preparation, is also quite limited, compared with that of BM-MSC.

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