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Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with traits of renal progenitors.

Swetha G, Chandra V, Phadnis S, Bhonde R - J. Cell. Mol. Med. (2011)

Bottom Line: When injected into E13.5 kidney rudiments, the cells incorporated into the developing kidney primordia and co-culture with E13.5 spinal cord resulted in branching and tubulogenesis in these cells.When implanted under renal capsule of unilaterally nephrectomized mice, these cells differentiated into immature glomeruli and vascular ducts.Our study demonstrates that EMT plays a major role in imparting plasticity to terminally differentiated GPECs by producing metastable cells with traits of kidney progenitors.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Pune, India.

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In vitro renal commitment assays prove the functional progenitor status of CD24+ cells. Nephron development assays were performed to assess the functional progenitor status of the cells. CD24+ cells were fluorescently tagged with CMFDA and injected into E13.5 kidney cultured in vitro (n = 6). Whole mount staining of E13.5 kidney after 5 days of co-culture with cells (A). Note that cells treated with 0.1 μM PGE2 failed to integrate into the developing kidney (A.a). Confocal optical section of the whole mount staining of E13.5 kidney with cells for tubular protein laminin (B). Co-culture of cells with E13.5 spinal cord, heterologous inducer of uteric buds branching (n= 6). Cells were cultured for 5 days, placed next to embryonic E13.5 spinal cord on a transwell filter. Cells aggregated and formed EBs by d2 which was then cultured for an additional 5 days on matrigel in E13.5 conditioned media. Tubulogenesis and branching morphogenesis were observed in the EBs after 5 day in culture (C). Cells at P1 treated with PGE2 showed poor aggregation and survival potential in SFM. No branching or tubulogenesis was observed when grown on matrigel (D). Matrigel tube formation assay on CD44+ CD24+ cells (n = 3). A total of 2 × 105 cells were seeded on a layer of matrigel, cells started aligning in tandem by 8 hrs and tube formation was observed by 16 hrs on matrigel (E). Morphological changes of the cells were observed at various time-points under phase contrast microscope and photographed (100× magnification). Endothelial differentiation was confirmed by the expression of endothelial specific marker PECAM (CD31) (92.2 ± 6.5%) in the cells after differentiation over undifferentiated cells (0.3 ± 1%) (F). (Scale bar = 50 μm). Abbreviations: CMFDA, 5, chloromethylfluorescein diacetate; SFM, serum-free media; E-embryonic day; d, day; F, field; EB, embryoid body.
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fig06: In vitro renal commitment assays prove the functional progenitor status of CD24+ cells. Nephron development assays were performed to assess the functional progenitor status of the cells. CD24+ cells were fluorescently tagged with CMFDA and injected into E13.5 kidney cultured in vitro (n = 6). Whole mount staining of E13.5 kidney after 5 days of co-culture with cells (A). Note that cells treated with 0.1 μM PGE2 failed to integrate into the developing kidney (A.a). Confocal optical section of the whole mount staining of E13.5 kidney with cells for tubular protein laminin (B). Co-culture of cells with E13.5 spinal cord, heterologous inducer of uteric buds branching (n= 6). Cells were cultured for 5 days, placed next to embryonic E13.5 spinal cord on a transwell filter. Cells aggregated and formed EBs by d2 which was then cultured for an additional 5 days on matrigel in E13.5 conditioned media. Tubulogenesis and branching morphogenesis were observed in the EBs after 5 day in culture (C). Cells at P1 treated with PGE2 showed poor aggregation and survival potential in SFM. No branching or tubulogenesis was observed when grown on matrigel (D). Matrigel tube formation assay on CD44+ CD24+ cells (n = 3). A total of 2 × 105 cells were seeded on a layer of matrigel, cells started aligning in tandem by 8 hrs and tube formation was observed by 16 hrs on matrigel (E). Morphological changes of the cells were observed at various time-points under phase contrast microscope and photographed (100× magnification). Endothelial differentiation was confirmed by the expression of endothelial specific marker PECAM (CD31) (92.2 ± 6.5%) in the cells after differentiation over undifferentiated cells (0.3 ± 1%) (F). (Scale bar = 50 μm). Abbreviations: CMFDA, 5, chloromethylfluorescein diacetate; SFM, serum-free media; E-embryonic day; d, day; F, field; EB, embryoid body.

Mentions: Here, we demonstrate the ability of CD24+ cells to subsist, proliferate and integrate into the developing E13.5 kidney in an in vitro system. The CD24+ cells (1000–1500 cells/tissue) tagged with green fluorescent dye marker CMFDA were injected into the kidney rudiments dissected from E13.5 kidney and cultured for 5 days on transwell filters without additional growth factors. Whole mount and antibody staining of the rudiments showed that the CD24+ cells have integrated into the developing kidney. Optical sectioning of the whole mount kidney rudiment showed clusters of CD24+ cells integrated with the 3D kidney primordia (Fig. 6A). Anti-laminin staining showed that the cells have integrated into the developing tubules and uteric-bud stalk [34] (Fig. 6B). PGE2 treated cells failed to integrate into the developing kidney. However, few cells remained attached to kidney surface (Fig. 6B).


Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with traits of renal progenitors.

Swetha G, Chandra V, Phadnis S, Bhonde R - J. Cell. Mol. Med. (2011)

In vitro renal commitment assays prove the functional progenitor status of CD24+ cells. Nephron development assays were performed to assess the functional progenitor status of the cells. CD24+ cells were fluorescently tagged with CMFDA and injected into E13.5 kidney cultured in vitro (n = 6). Whole mount staining of E13.5 kidney after 5 days of co-culture with cells (A). Note that cells treated with 0.1 μM PGE2 failed to integrate into the developing kidney (A.a). Confocal optical section of the whole mount staining of E13.5 kidney with cells for tubular protein laminin (B). Co-culture of cells with E13.5 spinal cord, heterologous inducer of uteric buds branching (n= 6). Cells were cultured for 5 days, placed next to embryonic E13.5 spinal cord on a transwell filter. Cells aggregated and formed EBs by d2 which was then cultured for an additional 5 days on matrigel in E13.5 conditioned media. Tubulogenesis and branching morphogenesis were observed in the EBs after 5 day in culture (C). Cells at P1 treated with PGE2 showed poor aggregation and survival potential in SFM. No branching or tubulogenesis was observed when grown on matrigel (D). Matrigel tube formation assay on CD44+ CD24+ cells (n = 3). A total of 2 × 105 cells were seeded on a layer of matrigel, cells started aligning in tandem by 8 hrs and tube formation was observed by 16 hrs on matrigel (E). Morphological changes of the cells were observed at various time-points under phase contrast microscope and photographed (100× magnification). Endothelial differentiation was confirmed by the expression of endothelial specific marker PECAM (CD31) (92.2 ± 6.5%) in the cells after differentiation over undifferentiated cells (0.3 ± 1%) (F). (Scale bar = 50 μm). Abbreviations: CMFDA, 5, chloromethylfluorescein diacetate; SFM, serum-free media; E-embryonic day; d, day; F, field; EB, embryoid body.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822804&req=5

fig06: In vitro renal commitment assays prove the functional progenitor status of CD24+ cells. Nephron development assays were performed to assess the functional progenitor status of the cells. CD24+ cells were fluorescently tagged with CMFDA and injected into E13.5 kidney cultured in vitro (n = 6). Whole mount staining of E13.5 kidney after 5 days of co-culture with cells (A). Note that cells treated with 0.1 μM PGE2 failed to integrate into the developing kidney (A.a). Confocal optical section of the whole mount staining of E13.5 kidney with cells for tubular protein laminin (B). Co-culture of cells with E13.5 spinal cord, heterologous inducer of uteric buds branching (n= 6). Cells were cultured for 5 days, placed next to embryonic E13.5 spinal cord on a transwell filter. Cells aggregated and formed EBs by d2 which was then cultured for an additional 5 days on matrigel in E13.5 conditioned media. Tubulogenesis and branching morphogenesis were observed in the EBs after 5 day in culture (C). Cells at P1 treated with PGE2 showed poor aggregation and survival potential in SFM. No branching or tubulogenesis was observed when grown on matrigel (D). Matrigel tube formation assay on CD44+ CD24+ cells (n = 3). A total of 2 × 105 cells were seeded on a layer of matrigel, cells started aligning in tandem by 8 hrs and tube formation was observed by 16 hrs on matrigel (E). Morphological changes of the cells were observed at various time-points under phase contrast microscope and photographed (100× magnification). Endothelial differentiation was confirmed by the expression of endothelial specific marker PECAM (CD31) (92.2 ± 6.5%) in the cells after differentiation over undifferentiated cells (0.3 ± 1%) (F). (Scale bar = 50 μm). Abbreviations: CMFDA, 5, chloromethylfluorescein diacetate; SFM, serum-free media; E-embryonic day; d, day; F, field; EB, embryoid body.
Mentions: Here, we demonstrate the ability of CD24+ cells to subsist, proliferate and integrate into the developing E13.5 kidney in an in vitro system. The CD24+ cells (1000–1500 cells/tissue) tagged with green fluorescent dye marker CMFDA were injected into the kidney rudiments dissected from E13.5 kidney and cultured for 5 days on transwell filters without additional growth factors. Whole mount and antibody staining of the rudiments showed that the CD24+ cells have integrated into the developing kidney. Optical sectioning of the whole mount kidney rudiment showed clusters of CD24+ cells integrated with the 3D kidney primordia (Fig. 6A). Anti-laminin staining showed that the cells have integrated into the developing tubules and uteric-bud stalk [34] (Fig. 6B). PGE2 treated cells failed to integrate into the developing kidney. However, few cells remained attached to kidney surface (Fig. 6B).

Bottom Line: When injected into E13.5 kidney rudiments, the cells incorporated into the developing kidney primordia and co-culture with E13.5 spinal cord resulted in branching and tubulogenesis in these cells.When implanted under renal capsule of unilaterally nephrectomized mice, these cells differentiated into immature glomeruli and vascular ducts.Our study demonstrates that EMT plays a major role in imparting plasticity to terminally differentiated GPECs by producing metastable cells with traits of kidney progenitors.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Pune, India.

Show MeSH
Related in: MedlinePlus