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Stem cells derived from neonatal mouse kidney generate functional proximal tubule-like cells and integrate into developing nephrons in vitro.

Ranghini E, Fuente Mora C, Mora CF, Edgar D, Kenny SE, Murray P, Wilm B - PLoS ONE (2013)

Bottom Line: Finally, we compared the ability of KSCs to integrate into developing kidneys ex vivo with that of metanephric mesenchyme cells.We found that KSCs integrated into nascent nephrons to a similar extent as metanephric mesenchyme cells while both were excluded from ureteric bud branches.Our analysis of the behavior of the two cell types shows that some, but not all KSC characteristics are similar to those of the MM.

View Article: PubMed Central - PubMed

Affiliation: Institute of Translational Medicine, Faculty of Health and Life Sciences, The University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
We have recently shown that kidney-derived stem cells (KSCs) isolated from the mouse newborn kidney differentiate into a range of kidney-specific cell types. However, the functionality and integration capacity of these mouse KSCs remain unknown. Therefore, the main objectives of this study were (1) to determine if proximal tubule-like cells, generated in vitro from KSCs, displayed absorptive function typical of proximal tubule cells in vivo, and (2) to establish whether the ability of KSCs to integrate into developing nephrons was comparable with that of metanephric mesenchyme (MM), a transient population of progenitor cells that gives rise to the nephrons during kidney organogenesis. We found that proximal tubule-like cells generated in vitro from mouse KSCs displayed megalin-dependent absorptive function. Subsequently, we used a chimeric kidney rudiment culture system to show that the KSCs could generate proximal tubule cells and podocytes that were appropriately located within the developing nephrons. Finally, we compared the ability of KSCs to integrate into developing kidneys ex vivo with that of metanephric mesenchyme cells. We found that KSCs integrated into nascent nephrons to a similar extent as metanephric mesenchyme cells while both were excluded from ureteric bud branches. Our analysis of the behavior of the two cell types shows that some, but not all KSC characteristics are similar to those of the MM.

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E11.5 re-aggregated kidneys form organotypic renal structures.(A, B) Immunostaining for Wt1 (red) and Pax2 (green) (A), or Wt1 (green) and laminin-111 (red) (B) shows the presence of condensed MM (pink arrowheads) surrounding the UB (white arrowheads) after 3 days of culture, thus demonstrating that the induction of the mesenchyme had taken place. (C) Immunostaining for megalin (green) and Pax2 (blue) at day 3 of rudiment culture reveals the presence of proximal tubules (open arrowheads). (D) Immunostaining for synaptopodin (green) demonstrates the presence of nascent podocytes within developing glomerular structures (open arrowheads) at 5 days of culture. Scale bars are 75 µm (A), 150 µm (B, C) and 100 µm (D).
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pone-0062953-g002: E11.5 re-aggregated kidneys form organotypic renal structures.(A, B) Immunostaining for Wt1 (red) and Pax2 (green) (A), or Wt1 (green) and laminin-111 (red) (B) shows the presence of condensed MM (pink arrowheads) surrounding the UB (white arrowheads) after 3 days of culture, thus demonstrating that the induction of the mesenchyme had taken place. (C) Immunostaining for megalin (green) and Pax2 (blue) at day 3 of rudiment culture reveals the presence of proximal tubules (open arrowheads). (D) Immunostaining for synaptopodin (green) demonstrates the presence of nascent podocytes within developing glomerular structures (open arrowheads) at 5 days of culture. Scale bars are 75 µm (A), 150 µm (B, C) and 100 µm (D).

Mentions: To investigate if the KSCs could integrate into developing kidney rudiments and give rise to renal cell types that were located appropriately within the nascent nephrons, we made use of an ex vivo culture system, in which E11.5 kidney rudiments are disaggregated into single cells, then re-aggregated to form organoids capable of developing renal-like structures [36]. After three days of culture, re-aggregated organoids contained regions of induced MM, as evidenced by the presence of Pax2+ Wt1+ cellular aggregates surrounding Pax2+ Wt1− UB epithelium (Figure 2A, B). At the same time point, proximal tubule cells expressing apical megalin had also started to develop in the organoids (Figure 2C), and after five days, cells expressing the podocyte-specific marker, synaptopodin [39], could be observed within developing glomeruli (Figure 2D). This ex vivo culture system has previously been used to test the nephrogenic potential of various types of exogenous cells, such as embryonic stem cells (ESCs) [40], mesenchymal stem cells [41], and amniotic fluid stem cells [42] using a protocol that involves recombining the exogenous cell type under test with disaggregated kidney rudiments to generate a chimeric organoid. Using this system, chimeras were generated with the neonatal KSCs that had been labelled with quantum dots (QDs), fluorescent nanocrystals that are very effective for tracking cells over short time periods [43]. Following ex vivo culture, the chimeric rudiments were immunostained for megalin and synaptopodin. QD+ KSCs were found within both megalin+ proximal tubules and synaptopodin+ nascent glomeruli, indicating that the cells could differentiate to appropriately located renal cell types within the environment of the developing kidney (Figure 3).


Stem cells derived from neonatal mouse kidney generate functional proximal tubule-like cells and integrate into developing nephrons in vitro.

Ranghini E, Fuente Mora C, Mora CF, Edgar D, Kenny SE, Murray P, Wilm B - PLoS ONE (2013)

E11.5 re-aggregated kidneys form organotypic renal structures.(A, B) Immunostaining for Wt1 (red) and Pax2 (green) (A), or Wt1 (green) and laminin-111 (red) (B) shows the presence of condensed MM (pink arrowheads) surrounding the UB (white arrowheads) after 3 days of culture, thus demonstrating that the induction of the mesenchyme had taken place. (C) Immunostaining for megalin (green) and Pax2 (blue) at day 3 of rudiment culture reveals the presence of proximal tubules (open arrowheads). (D) Immunostaining for synaptopodin (green) demonstrates the presence of nascent podocytes within developing glomerular structures (open arrowheads) at 5 days of culture. Scale bars are 75 µm (A), 150 µm (B, C) and 100 µm (D).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646983&req=5

pone-0062953-g002: E11.5 re-aggregated kidneys form organotypic renal structures.(A, B) Immunostaining for Wt1 (red) and Pax2 (green) (A), or Wt1 (green) and laminin-111 (red) (B) shows the presence of condensed MM (pink arrowheads) surrounding the UB (white arrowheads) after 3 days of culture, thus demonstrating that the induction of the mesenchyme had taken place. (C) Immunostaining for megalin (green) and Pax2 (blue) at day 3 of rudiment culture reveals the presence of proximal tubules (open arrowheads). (D) Immunostaining for synaptopodin (green) demonstrates the presence of nascent podocytes within developing glomerular structures (open arrowheads) at 5 days of culture. Scale bars are 75 µm (A), 150 µm (B, C) and 100 µm (D).
Mentions: To investigate if the KSCs could integrate into developing kidney rudiments and give rise to renal cell types that were located appropriately within the nascent nephrons, we made use of an ex vivo culture system, in which E11.5 kidney rudiments are disaggregated into single cells, then re-aggregated to form organoids capable of developing renal-like structures [36]. After three days of culture, re-aggregated organoids contained regions of induced MM, as evidenced by the presence of Pax2+ Wt1+ cellular aggregates surrounding Pax2+ Wt1− UB epithelium (Figure 2A, B). At the same time point, proximal tubule cells expressing apical megalin had also started to develop in the organoids (Figure 2C), and after five days, cells expressing the podocyte-specific marker, synaptopodin [39], could be observed within developing glomeruli (Figure 2D). This ex vivo culture system has previously been used to test the nephrogenic potential of various types of exogenous cells, such as embryonic stem cells (ESCs) [40], mesenchymal stem cells [41], and amniotic fluid stem cells [42] using a protocol that involves recombining the exogenous cell type under test with disaggregated kidney rudiments to generate a chimeric organoid. Using this system, chimeras were generated with the neonatal KSCs that had been labelled with quantum dots (QDs), fluorescent nanocrystals that are very effective for tracking cells over short time periods [43]. Following ex vivo culture, the chimeric rudiments were immunostained for megalin and synaptopodin. QD+ KSCs were found within both megalin+ proximal tubules and synaptopodin+ nascent glomeruli, indicating that the cells could differentiate to appropriately located renal cell types within the environment of the developing kidney (Figure 3).

Bottom Line: Finally, we compared the ability of KSCs to integrate into developing kidneys ex vivo with that of metanephric mesenchyme cells.We found that KSCs integrated into nascent nephrons to a similar extent as metanephric mesenchyme cells while both were excluded from ureteric bud branches.Our analysis of the behavior of the two cell types shows that some, but not all KSC characteristics are similar to those of the MM.

View Article: PubMed Central - PubMed

Affiliation: Institute of Translational Medicine, Faculty of Health and Life Sciences, The University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
We have recently shown that kidney-derived stem cells (KSCs) isolated from the mouse newborn kidney differentiate into a range of kidney-specific cell types. However, the functionality and integration capacity of these mouse KSCs remain unknown. Therefore, the main objectives of this study were (1) to determine if proximal tubule-like cells, generated in vitro from KSCs, displayed absorptive function typical of proximal tubule cells in vivo, and (2) to establish whether the ability of KSCs to integrate into developing nephrons was comparable with that of metanephric mesenchyme (MM), a transient population of progenitor cells that gives rise to the nephrons during kidney organogenesis. We found that proximal tubule-like cells generated in vitro from mouse KSCs displayed megalin-dependent absorptive function. Subsequently, we used a chimeric kidney rudiment culture system to show that the KSCs could generate proximal tubule cells and podocytes that were appropriately located within the developing nephrons. Finally, we compared the ability of KSCs to integrate into developing kidneys ex vivo with that of metanephric mesenchyme cells. We found that KSCs integrated into nascent nephrons to a similar extent as metanephric mesenchyme cells while both were excluded from ureteric bud branches. Our analysis of the behavior of the two cell types shows that some, but not all KSC characteristics are similar to those of the MM.

Show MeSH