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Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.

Zhao Y, Zhao H, Zhang Y, Tsatralis T, Cao Q, Wang Y, Wang Y, Wang YM, Alexander SI, Harris DC, Zheng G - BMC Cell Biol. (2014)

Bottom Line: The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%.Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yzha7726@uni.sydney.edu.au.

ABSTRACT

Background: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.

Results: Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.

Conclusion: In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

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Primary mouse renal peritubular endothelial cell performance in mono- and co-culture with mouse proximal tubular epithelial cell over 6 days. (A) Co-localization of VE-cadherin (green) and α-SMA (red) positive cells cultured in MPRECs mono-cultures without VEGF. (B) Peritubular endothelial cell and proximal tubular epithelial cell co-culture. In the co-culture system, isolated peritubular endothelial cells were placed in the bottom chamber of fibronectin pre-coated Transwell plates and proximal tubular epithelial cells were seeded onto the polyester inserts (pore size 0.4 μm). Endothelial cell medium was used for the co-culture. (C) After 6 days, primary mouse renal peritubular endothelial cell performance in mono- and co-culture in the presence or absence of VEGF was assessed by FSP-1(green, top panels) staining and co-localization of α-SMA (red) and VE-cadherin (green, bottom panels). Orange represents α-SMA and VE-cadherin double positive areas. (D) Statistical analysis of primary mouse renal peritubular endothelial cell performance after cells were cultured for 6 days. FSP-1 (green) and α-SMA (red) expression in MRPECs was quantified and expressed as a percentage of positive stained cells against total cells. Images presented are representative of at least 5 independent replicate experiments. Data are expressed as mean ± SEM with N = 5 for each group. *P < 0.05, **P < 0.01 vs. respective control. Original magnification was x 200. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).
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Fig4: Primary mouse renal peritubular endothelial cell performance in mono- and co-culture with mouse proximal tubular epithelial cell over 6 days. (A) Co-localization of VE-cadherin (green) and α-SMA (red) positive cells cultured in MPRECs mono-cultures without VEGF. (B) Peritubular endothelial cell and proximal tubular epithelial cell co-culture. In the co-culture system, isolated peritubular endothelial cells were placed in the bottom chamber of fibronectin pre-coated Transwell plates and proximal tubular epithelial cells were seeded onto the polyester inserts (pore size 0.4 μm). Endothelial cell medium was used for the co-culture. (C) After 6 days, primary mouse renal peritubular endothelial cell performance in mono- and co-culture in the presence or absence of VEGF was assessed by FSP-1(green, top panels) staining and co-localization of α-SMA (red) and VE-cadherin (green, bottom panels). Orange represents α-SMA and VE-cadherin double positive areas. (D) Statistical analysis of primary mouse renal peritubular endothelial cell performance after cells were cultured for 6 days. FSP-1 (green) and α-SMA (red) expression in MRPECs was quantified and expressed as a percentage of positive stained cells against total cells. Images presented are representative of at least 5 independent replicate experiments. Data are expressed as mean ± SEM with N = 5 for each group. *P < 0.05, **P < 0.01 vs. respective control. Original magnification was x 200. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).

Mentions: Immunofluorescence staining of MRPECs cultured for 2, 4 and 6 days showed VE-cadherin positive staining. However, a large number of cells were also α-SMA positive after 2 days of culture, suggesting that the cells were undergoing phenotypic change (Figure 4A). To maintain MRPEC endothelial phenotype, vascular endothelial growth factor (VEGF) was added to the culture medium and MRPECs were co-cultured with mouse proximal tubular epithelial cells (MPTECs) which were obtained via direct culture of tubule fractions (Figure 4B). FSP-1 and α-SMA were used as markers for fibroblasts [4]. While FSP-1 (Figure 4C, top panels) staining was low in all groups, α-SMA staining co-localized with VE-cadherin (Figure 4C, bottom panels) significantly diminished in culture of peritubular endothelial cells after treatment with VEGF and especially when combined in co-culture with proximal tubular epithelial cells (Figure 4C, D). Statistical analysis showed the effect of co-culture of MRPECs with MPTECs was minimal as compared to mono-culture of MRPECs without any treatment (Figure 4D). However, α-SMA expression decreased significantly when the cells were treated with VEGF (from 35.0 ± 3.6 to 14.3 ± 3.0, P <0.05). When the cells were co-cultured with MPTECs and treated with VEGF, the number of α-SMA positive cells was the lowest among all groups (from 35.0 ± 3.6 to 5.3 ± 1.5, P < 0.001) (Figure 4D). This result demonstrated that co-culture with MPTECs and VEGF treatment is an effective method for maintaining isolated MRPECs.Figure 4


Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.

Zhao Y, Zhao H, Zhang Y, Tsatralis T, Cao Q, Wang Y, Wang Y, Wang YM, Alexander SI, Harris DC, Zheng G - BMC Cell Biol. (2014)

Primary mouse renal peritubular endothelial cell performance in mono- and co-culture with mouse proximal tubular epithelial cell over 6 days. (A) Co-localization of VE-cadherin (green) and α-SMA (red) positive cells cultured in MPRECs mono-cultures without VEGF. (B) Peritubular endothelial cell and proximal tubular epithelial cell co-culture. In the co-culture system, isolated peritubular endothelial cells were placed in the bottom chamber of fibronectin pre-coated Transwell plates and proximal tubular epithelial cells were seeded onto the polyester inserts (pore size 0.4 μm). Endothelial cell medium was used for the co-culture. (C) After 6 days, primary mouse renal peritubular endothelial cell performance in mono- and co-culture in the presence or absence of VEGF was assessed by FSP-1(green, top panels) staining and co-localization of α-SMA (red) and VE-cadherin (green, bottom panels). Orange represents α-SMA and VE-cadherin double positive areas. (D) Statistical analysis of primary mouse renal peritubular endothelial cell performance after cells were cultured for 6 days. FSP-1 (green) and α-SMA (red) expression in MRPECs was quantified and expressed as a percentage of positive stained cells against total cells. Images presented are representative of at least 5 independent replicate experiments. Data are expressed as mean ± SEM with N = 5 for each group. *P < 0.05, **P < 0.01 vs. respective control. Original magnification was x 200. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Primary mouse renal peritubular endothelial cell performance in mono- and co-culture with mouse proximal tubular epithelial cell over 6 days. (A) Co-localization of VE-cadherin (green) and α-SMA (red) positive cells cultured in MPRECs mono-cultures without VEGF. (B) Peritubular endothelial cell and proximal tubular epithelial cell co-culture. In the co-culture system, isolated peritubular endothelial cells were placed in the bottom chamber of fibronectin pre-coated Transwell plates and proximal tubular epithelial cells were seeded onto the polyester inserts (pore size 0.4 μm). Endothelial cell medium was used for the co-culture. (C) After 6 days, primary mouse renal peritubular endothelial cell performance in mono- and co-culture in the presence or absence of VEGF was assessed by FSP-1(green, top panels) staining and co-localization of α-SMA (red) and VE-cadherin (green, bottom panels). Orange represents α-SMA and VE-cadherin double positive areas. (D) Statistical analysis of primary mouse renal peritubular endothelial cell performance after cells were cultured for 6 days. FSP-1 (green) and α-SMA (red) expression in MRPECs was quantified and expressed as a percentage of positive stained cells against total cells. Images presented are representative of at least 5 independent replicate experiments. Data are expressed as mean ± SEM with N = 5 for each group. *P < 0.05, **P < 0.01 vs. respective control. Original magnification was x 200. Cells in this figure were counterstained with DAPI to visualize nuclei (blue).
Mentions: Immunofluorescence staining of MRPECs cultured for 2, 4 and 6 days showed VE-cadherin positive staining. However, a large number of cells were also α-SMA positive after 2 days of culture, suggesting that the cells were undergoing phenotypic change (Figure 4A). To maintain MRPEC endothelial phenotype, vascular endothelial growth factor (VEGF) was added to the culture medium and MRPECs were co-cultured with mouse proximal tubular epithelial cells (MPTECs) which were obtained via direct culture of tubule fractions (Figure 4B). FSP-1 and α-SMA were used as markers for fibroblasts [4]. While FSP-1 (Figure 4C, top panels) staining was low in all groups, α-SMA staining co-localized with VE-cadherin (Figure 4C, bottom panels) significantly diminished in culture of peritubular endothelial cells after treatment with VEGF and especially when combined in co-culture with proximal tubular epithelial cells (Figure 4C, D). Statistical analysis showed the effect of co-culture of MRPECs with MPTECs was minimal as compared to mono-culture of MRPECs without any treatment (Figure 4D). However, α-SMA expression decreased significantly when the cells were treated with VEGF (from 35.0 ± 3.6 to 14.3 ± 3.0, P <0.05). When the cells were co-cultured with MPTECs and treated with VEGF, the number of α-SMA positive cells was the lowest among all groups (from 35.0 ± 3.6 to 5.3 ± 1.5, P < 0.001) (Figure 4D). This result demonstrated that co-culture with MPTECs and VEGF treatment is an effective method for maintaining isolated MRPECs.Figure 4

Bottom Line: The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%.Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yzha7726@uni.sydney.edu.au.

ABSTRACT

Background: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.

Results: Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.

Conclusion: In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

Show MeSH
Related in: MedlinePlus