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In vitro formation of β cell pseudoislets using islet-derived endothelial cells.

Spelios MG, Kenna LA, Wall B, Akirav EM - PLoS ONE (2013)

Bottom Line: β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration.These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers.Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Islet Biology, Winthrop-University Hospital, Mineola, New York, United States of America.

ABSTRACT
β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.

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Col-IV and laminin are detected in and around the PI.A. IF staining of MS1 cells. Blue-DAPI, Green-CD31, Red-BS1 and merge. White/Yellow represents double positive cells. B. RT-PCR for laminin β1 and col-IV in MS1, whole murine islet preps, and βTC3 cells. Laminin α1 and α2 were not detected (data not shown). C. IF staining of MS1 cells. Blue- DAPI, Green- col-IV, White-Laminin, and merge. D. 3-D reconstruction of z-stack imaging of an 8 d old PI. E. Non-consecutive z-stack confocal images of a PI. Blue- DAPI, Red- Insulin, Green- col-IV, White- laminin.
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pone-0072260-g003: Col-IV and laminin are detected in and around the PI.A. IF staining of MS1 cells. Blue-DAPI, Green-CD31, Red-BS1 and merge. White/Yellow represents double positive cells. B. RT-PCR for laminin β1 and col-IV in MS1, whole murine islet preps, and βTC3 cells. Laminin α1 and α2 were not detected (data not shown). C. IF staining of MS1 cells. Blue- DAPI, Green- col-IV, White-Laminin, and merge. D. 3-D reconstruction of z-stack imaging of an 8 d old PI. E. Non-consecutive z-stack confocal images of a PI. Blue- DAPI, Red- Insulin, Green- col-IV, White- laminin.

Mentions: ECM proteins, such as laminin and col-IV, are produced by iECs and are an integral part of the islet structure [16]. We tested whether MS1 cells express markers of native iECs by staining for CD31 (PECAM-1) and BS1 (lectin), as well as, laminin and col-IV. MS1 cells were highly positive for the endothelial cell markers, CD31 and BS1 (Figure 3A). RT-PCR analysis detected the expression of laminin-β1 and col-IV mRNAs in MS1 cells and purified primary murine islets, but not in βTC3 cells (Figure 3B). The expression of ECM proteins was further validated by immunofluorescence (IF) staining showing strong col-IV and laminin expression in MS1 cells (Figure 3C). The ability of MS1 cells to produce col-IV and laminin and the absence of ECM proteins in βTC3 cells cultured alone prompted us to examine whether laminin and col-IV are found in MS1-induced PIs. Z-stack confocal imaging showed clear staining of laminin and col-IV in and around the surface of the PIs (Figure 3D). Nonconsecutive z-stack images showed diffused col-IV staining, while laminin staining exhibited a more punctuated pattern (Figure 3E, Video S1). No staining was observed in PIs incubated with secondary antibodies only (data not shown).


In vitro formation of β cell pseudoislets using islet-derived endothelial cells.

Spelios MG, Kenna LA, Wall B, Akirav EM - PLoS ONE (2013)

Col-IV and laminin are detected in and around the PI.A. IF staining of MS1 cells. Blue-DAPI, Green-CD31, Red-BS1 and merge. White/Yellow represents double positive cells. B. RT-PCR for laminin β1 and col-IV in MS1, whole murine islet preps, and βTC3 cells. Laminin α1 and α2 were not detected (data not shown). C. IF staining of MS1 cells. Blue- DAPI, Green- col-IV, White-Laminin, and merge. D. 3-D reconstruction of z-stack imaging of an 8 d old PI. E. Non-consecutive z-stack confocal images of a PI. Blue- DAPI, Red- Insulin, Green- col-IV, White- laminin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756083&req=5

pone-0072260-g003: Col-IV and laminin are detected in and around the PI.A. IF staining of MS1 cells. Blue-DAPI, Green-CD31, Red-BS1 and merge. White/Yellow represents double positive cells. B. RT-PCR for laminin β1 and col-IV in MS1, whole murine islet preps, and βTC3 cells. Laminin α1 and α2 were not detected (data not shown). C. IF staining of MS1 cells. Blue- DAPI, Green- col-IV, White-Laminin, and merge. D. 3-D reconstruction of z-stack imaging of an 8 d old PI. E. Non-consecutive z-stack confocal images of a PI. Blue- DAPI, Red- Insulin, Green- col-IV, White- laminin.
Mentions: ECM proteins, such as laminin and col-IV, are produced by iECs and are an integral part of the islet structure [16]. We tested whether MS1 cells express markers of native iECs by staining for CD31 (PECAM-1) and BS1 (lectin), as well as, laminin and col-IV. MS1 cells were highly positive for the endothelial cell markers, CD31 and BS1 (Figure 3A). RT-PCR analysis detected the expression of laminin-β1 and col-IV mRNAs in MS1 cells and purified primary murine islets, but not in βTC3 cells (Figure 3B). The expression of ECM proteins was further validated by immunofluorescence (IF) staining showing strong col-IV and laminin expression in MS1 cells (Figure 3C). The ability of MS1 cells to produce col-IV and laminin and the absence of ECM proteins in βTC3 cells cultured alone prompted us to examine whether laminin and col-IV are found in MS1-induced PIs. Z-stack confocal imaging showed clear staining of laminin and col-IV in and around the surface of the PIs (Figure 3D). Nonconsecutive z-stack images showed diffused col-IV staining, while laminin staining exhibited a more punctuated pattern (Figure 3E, Video S1). No staining was observed in PIs incubated with secondary antibodies only (data not shown).

Bottom Line: β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration.These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers.Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Islet Biology, Winthrop-University Hospital, Mineola, New York, United States of America.

ABSTRACT
β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.

Show MeSH
Related in: MedlinePlus