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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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Population growth of cultured MM.Growth curve of MM cells cultured in the presence of Bmp7 and Fgf2 for the following times: days 1–4 (P1, black), days 5–8 (P2, blue), days 9–12 (P3, red). The MM cell population expanded by >12-fold during the P1 and P2 culture periods, but ceased expanding during P3. Data are expressed as mean ± SD; n = 6 for P1 and P2; n = 5 for P3.
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pone-0062953-g004: Population growth of cultured MM.Growth curve of MM cells cultured in the presence of Bmp7 and Fgf2 for the following times: days 1–4 (P1, black), days 5–8 (P2, blue), days 9–12 (P3, red). The MM cell population expanded by >12-fold during the P1 and P2 culture periods, but ceased expanding during P3. Data are expressed as mean ± SD; n = 6 for P1 and P2; n = 5 for P3.

Mentions: Using Wt1-Cre/Rosa26R mice, we have previously shown that the mouse neonatal KSCs used here are derived from the MM and express several MM markers [34]. Given these similarities between KSCs and MM cells, we next investigated if the KSCs could integrate into developing nephrons to a similar extent as MM cells. To counter the possibility that any difference in KSC and MM integration potential could be due to the KSCs having been cultured extensively in vitro, we first established a population of cultured MM cells so that the KSCs could be compared with both freshly isolated and cultured MM. To this end, MM cells were isolated from E11.5 embryos and cultured in the presence of Fgf2 and Bmp7, which have been shown to support the survival and growth of MM in the short-term [14], [15]. As expected, in the absence of Bmp7 and Fgf2, the MM failed to grow (data not shown), whereas in the presence of these growth factors, the MM population expanded >12-fold during both the first (P1) and second (P2) 4 day culture periods. Population growth during the third culture period (P3) was limited, with no further growth occurring after day 10 (Figure 4). Thus, the growth kinetics of cultured KSCs and MM cells are very different, with KSCs displaying unlimited self-renewal [34], and MM cells ceasing to grow following 10 days of in vitro culture. To investigate if the expression of key MM markers was maintained during the in vitro culture period, immunostaining for Wt1 and Pax2, and semi-quantitative RT-PCR for Wt1, Pax2, Osr1, Sall1 and Gdnf were performed. MM cells expressed Wt1 throughout the culture period, although from day 8, there were noticeably fewer Wt1+ cells (Figure 5A, B, S1). Pax2 protein was detected in some cells during the initial 24-hour culture period, but neither Pax2 protein (not shown) nor mRNA were detectable from 48 h onwards (Fig. 5A, B, S1). In contrast, expression of Wt1, Osr1, Sall1 and Gdnf mRNA was maintained over the first 8 days of in vitro culture, and did not decrease until the third culture period (days 8–12) (Figure 5B, S1).


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)

Population growth of cultured MM.Growth curve of MM cells cultured in the presence of Bmp7 and Fgf2 for the following times: days 1–4 (P1, black), days 5–8 (P2, blue), days 9–12 (P3, red). The MM cell population expanded by >12-fold during the P1 and P2 culture periods, but ceased expanding during P3. Data are expressed as mean ± SD; n = 6 for P1 and P2; n = 5 for P3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646983&req=5

pone-0062953-g004: Population growth of cultured MM.Growth curve of MM cells cultured in the presence of Bmp7 and Fgf2 for the following times: days 1–4 (P1, black), days 5–8 (P2, blue), days 9–12 (P3, red). The MM cell population expanded by >12-fold during the P1 and P2 culture periods, but ceased expanding during P3. Data are expressed as mean ± SD; n = 6 for P1 and P2; n = 5 for P3.
Mentions: Using Wt1-Cre/Rosa26R mice, we have previously shown that the mouse neonatal KSCs used here are derived from the MM and express several MM markers [34]. Given these similarities between KSCs and MM cells, we next investigated if the KSCs could integrate into developing nephrons to a similar extent as MM cells. To counter the possibility that any difference in KSC and MM integration potential could be due to the KSCs having been cultured extensively in vitro, we first established a population of cultured MM cells so that the KSCs could be compared with both freshly isolated and cultured MM. To this end, MM cells were isolated from E11.5 embryos and cultured in the presence of Fgf2 and Bmp7, which have been shown to support the survival and growth of MM in the short-term [14], [15]. As expected, in the absence of Bmp7 and Fgf2, the MM failed to grow (data not shown), whereas in the presence of these growth factors, the MM population expanded >12-fold during both the first (P1) and second (P2) 4 day culture periods. Population growth during the third culture period (P3) was limited, with no further growth occurring after day 10 (Figure 4). Thus, the growth kinetics of cultured KSCs and MM cells are very different, with KSCs displaying unlimited self-renewal [34], and MM cells ceasing to grow following 10 days of in vitro culture. To investigate if the expression of key MM markers was maintained during the in vitro culture period, immunostaining for Wt1 and Pax2, and semi-quantitative RT-PCR for Wt1, Pax2, Osr1, Sall1 and Gdnf were performed. MM cells expressed Wt1 throughout the culture period, although from day 8, there were noticeably fewer Wt1+ cells (Figure 5A, B, S1). Pax2 protein was detected in some cells during the initial 24-hour culture period, but neither Pax2 protein (not shown) nor mRNA were detectable from 48 h onwards (Fig. 5A, B, S1). In contrast, expression of Wt1, Osr1, Sall1 and Gdnf mRNA was maintained over the first 8 days of in vitro culture, and did not decrease until the third culture period (days 8–12) (Figure 5B, S1).

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