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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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KSC-derived proximal tubule cells display normal absorptive function.(A) Immunofluorescence staining shows that some KSC-derived cells expressed the proximal tubule marker megalin (green). Nuclei are stained with DAPI (blue). (B) Immunostaining shows that FBSA (red) was uptaken by megalin+ KSCs (green); nuclei are stained with DAPI (blue). Asterisks indicate cells co-stained for megalin and FBSA. (C) In vitro functionality assay demonstrates that in the presence of either receptor-associated protein (RAP), or an excess of unlabeled BSA, the uptake of FBSA (red) was almost completely blocked. Nuclei are stained with Hoechst 33342 (blue). Scale bars are 50 µm (A), 25 µm (B) and 100 µm (C).
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pone-0062953-g001: KSC-derived proximal tubule cells display normal absorptive function.(A) Immunofluorescence staining shows that some KSC-derived cells expressed the proximal tubule marker megalin (green). Nuclei are stained with DAPI (blue). (B) Immunostaining shows that FBSA (red) was uptaken by megalin+ KSCs (green); nuclei are stained with DAPI (blue). Asterisks indicate cells co-stained for megalin and FBSA. (C) In vitro functionality assay demonstrates that in the presence of either receptor-associated protein (RAP), or an excess of unlabeled BSA, the uptake of FBSA (red) was almost completely blocked. Nuclei are stained with Hoechst 33342 (blue). Scale bars are 50 µm (A), 25 µm (B) and 100 µm (C).

Mentions: Previous studies have shown that stem/progenitor cells derived from mouse and human kidney can generate cells that express markers of proximal tubule cells [25], [26], [34] including megalin (Figure 1A), an endocytic receptor that plays a key role in mediating the reabsorption of proteins from the glomerular filtrate [38]. We have previously shown that KSCs derived from mouse neonates can spontaneously generate proximal tubule-like cells when cultured under routine conditions [34], but as yet, the functionality of these cells has not been demonstrated. To test the functionality of mouse KSC-derived megalin+ cells, we investigated their ability to internalize fluorescent bovine serum albumin (FBSA). Our results showed that cells within the population were able to internalize FBSA, as indicated by punctate cytoplasmic staining. Double immunofluorescence staining demonstrated that only cells expressing megalin displayed uptake of FBSA (Figure 1B), suggesting that FBSA uptake was mediated through the megalin receptor. To confirm this, the cells were incubated with FBSA in the presence of either excess unlabeled BSA, or receptor-associated protein (RAP), a megalin ligand that competitively blocks megalin-mediated protein uptake [37], [38]. Under both conditions, FBSA uptake was almost completely blocked, thus indicating that the uptake of BSA by KSC-derived proximal tubule cells is both saturable, and megalin-dependent (Figure 1C). These findings demonstrate that megalin+ KSC-derived proximal tubule-like cells display normal absorptive function in vitro.


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)

KSC-derived proximal tubule cells display normal absorptive function.(A) Immunofluorescence staining shows that some KSC-derived cells expressed the proximal tubule marker megalin (green). Nuclei are stained with DAPI (blue). (B) Immunostaining shows that FBSA (red) was uptaken by megalin+ KSCs (green); nuclei are stained with DAPI (blue). Asterisks indicate cells co-stained for megalin and FBSA. (C) In vitro functionality assay demonstrates that in the presence of either receptor-associated protein (RAP), or an excess of unlabeled BSA, the uptake of FBSA (red) was almost completely blocked. Nuclei are stained with Hoechst 33342 (blue). Scale bars are 50 µm (A), 25 µm (B) and 100 µm (C).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646983&req=5

pone-0062953-g001: KSC-derived proximal tubule cells display normal absorptive function.(A) Immunofluorescence staining shows that some KSC-derived cells expressed the proximal tubule marker megalin (green). Nuclei are stained with DAPI (blue). (B) Immunostaining shows that FBSA (red) was uptaken by megalin+ KSCs (green); nuclei are stained with DAPI (blue). Asterisks indicate cells co-stained for megalin and FBSA. (C) In vitro functionality assay demonstrates that in the presence of either receptor-associated protein (RAP), or an excess of unlabeled BSA, the uptake of FBSA (red) was almost completely blocked. Nuclei are stained with Hoechst 33342 (blue). Scale bars are 50 µm (A), 25 µm (B) and 100 µm (C).
Mentions: Previous studies have shown that stem/progenitor cells derived from mouse and human kidney can generate cells that express markers of proximal tubule cells [25], [26], [34] including megalin (Figure 1A), an endocytic receptor that plays a key role in mediating the reabsorption of proteins from the glomerular filtrate [38]. We have previously shown that KSCs derived from mouse neonates can spontaneously generate proximal tubule-like cells when cultured under routine conditions [34], but as yet, the functionality of these cells has not been demonstrated. To test the functionality of mouse KSC-derived megalin+ cells, we investigated their ability to internalize fluorescent bovine serum albumin (FBSA). Our results showed that cells within the population were able to internalize FBSA, as indicated by punctate cytoplasmic staining. Double immunofluorescence staining demonstrated that only cells expressing megalin displayed uptake of FBSA (Figure 1B), suggesting that FBSA uptake was mediated through the megalin receptor. To confirm this, the cells were incubated with FBSA in the presence of either excess unlabeled BSA, or receptor-associated protein (RAP), a megalin ligand that competitively blocks megalin-mediated protein uptake [37], [38]. Under both conditions, FBSA uptake was almost completely blocked, thus indicating that the uptake of BSA by KSC-derived proximal tubule cells is both saturable, and megalin-dependent (Figure 1C). These findings demonstrate that megalin+ KSC-derived proximal tubule-like cells display normal absorptive function in vitro.

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
Related in: MedlinePlus