1. Volume 83, Issue 4, Pages 593-603 (April 2013)
Human embryonic stem cells differentiate into functional renal proximal tubular–like cells 
Karthikeyan Narayanan, Karl M. Schumacher, Farah Tasnim, Karthikeyan Kandasamy, Annegret Schumacher, Ming Ni, Shujun Gao, Began Gopalan, Daniele Zink, Jackie Y. Ying 
Kidney International 
Volume 83, Issue 4, Pages (April 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
The impact of different extracellular matrices (ECMs) on human embryonic stem cell (hESC) differentiation. hESCs were cultivated for 20 days on different ECMs as indicated, except for the experiments related to e. (a) Aquaporin 1 (AQP1) (red) and CK18 (green) were detected by immunostaining (4′,6-diamidino-2-phenylindole (DAPI): blue). Bar=200μm. hESCs differentiated on (b) collagen IV and (c) Matrigel were analyzed by flow cytometry. AQP1 was detected by immunofluorescence using a fluorescein isothiocyanate (FITC)-labeled antibody. The percentages of AQP1-positive cells are indicated. (d) hESCs were cultivated on different ECMs (x axis) and the expression of the marker genes indicated was analyzed by real-time PCR (RT–PCR). The bars display the mean±s.d. (n=3) of the expression levels relative to undifferentiated hESCs. (e) hESCs were cultivated on Matrigel for up to 10 days. The expression levels of AQP1 and paired box gene 2 (PAX2) were determined by RT-PCR. The bars (mean±s.d., n=3) display the relative expression levels (as compared with undifferentiated hESCs). GGT, γ-glutamyl transferase.
Kidney International  , DOI: ( /ki )
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3. Figure 2
The impact of bone morphogenetic proteins (BMPs), retinoic acid (RA), and activin-A on human embryonic stem cell (hESC) differentiation. (a) hESCs were differentiated in complete renal epithelial growth medium (REGM) with fetal bovine serum (FBS) and all supplements, as well as at the indicated concentrations of BMP2 (0, 2.5, 5, and 10ng/ml). Cells were analyzed by flow cytometry, and the fractions of aquaporin 1 (AQP1)-expressing cells are displayed. The images below the respective panels show the representative results obtained by immunostaining of the cells (AQP1: red, 4′,6-diamidino-2-phenylindole (DAPI): blue). (b) hESCs were differentiated in complete REGM medium with FBS supplemented with a combination of BMP2 and BMP7. The concentrations of BMP2 and BMP7 added are indicated at the bottom. Cells were analyzed by flow cytometry, and the numbers of AQP1-expressing cells are shown. The highest numbers of AQP1-expressing cells were obtained with a combination of 10ng/ml of BMP2 and 2.5ng/ml of BMP7. (c) hESCs were differentiated in complete REGM medium with FBS supplemented with the indicated combinations of BMP2 (10ng/ml), BMP7 (2.5ng/ml), RA (0.1μmol/l), and activin-A (Act A; 10ng/ml). Cells were analyzed by flow cytometry, and the numbers of AQP1-expressing cells are shown. The highest numbers of AQP1-expressing cells were obtained with a combination of all four factors. FITC, fluorescein isothiocyanate.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Analysis of marker gene expression. Relative expression levels of the marker genes indicated on the x axis were determined by real-time PCR (RT–PCR). The bars show the mean±s.d. (n=3). Relative gene expression levels were determined for the differentiated human embryonic stem cells (hESCs) (white bars) and the human primary renal proximal tubular cells (HPTCs) (gray bars), relative to the undifferentiated hESCs (control; mean expression level set to 1). Significant differences as compared with undifferentiated hESCs (P<0.05) are indicated by asterisks.
Kidney International  , DOI: ( /ki )
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5. Figure 4
Immunoblot. The expression of the marker proteins indicated on the right (loading control: α-tubulin) was analyzed by immunoblotting in differentiated (Diff.) human embryonic stem cells (hESCs) and human primary renal proximal tubular cells (HPTCs) (two lanes for each cell type). The positions of the size marker bands are shown.
Kidney International  , DOI: ( /ki )
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6. Figure 5
Immunofluorescence analysis of marker protein expression and localization. The upper four panels show epifluorescence images of differentiated human embryonic stem cells (hESCs) and human primary renal proximal tubular cells (HPTCs) after coimmunostaining with antibodies against aquaporin 3 (AQP3) (green) and URO-10 (red). Cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI): blue. Arrowheads point to cells expressing both proteins. The lower four panels show cells after immunostaining with an antibody against CD13 (green). The left-hand and right-hand panels show apical and basal focal planes of the same field of either differentiated hESCs or HPTCs, respectively. Bar=20μm.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Cell morphology and formation of tubule-like structures in vitro. (a, b) Differentiated human embryonic stem cells (hESCs) formed confluent epithelia. Zonula occludens (ZO)-1 (red) was enriched at the lateral cell borders (4′,6-diamidino-2-phenylindole (DAPI): blue). Panel b shows an enlargement of the area that was boxed in (a). Bar=200μm (a, b). (c, d) Scanning electron microscopy (SEM) images of differentiated hESCs. Cells were polarized and displayed microvilli on the apical surface. Bars=3μm (c) and 4μm (d). (e) Overview of the network of cords and tubule-like structures on Matrigel (DAPI: blue). Bar=500μm. (f) Cords consisting of rows of single cells (arrowheads) and multicellular tubule-like structures (arrows) on Matrigel. Structures marked with arrows are shown enlarged in the insets (DAPI: blue). Bar=200μm.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Functional assays with differentiated human embryonic stem cells (hESCs) in static cultures. Functional assays were performed with differentiated hESCs (open bars or symbols), human primary renal proximal tubular cells (HPTCs) (positive control, black bars or symbols), or undifferentiated hESCs (negative control, gray bars in panel (a)). (a) Response to parathyroid hormone (PTH). Cells were kept in normal cell culture medium (-PTH) or in medium supplemented with 100nmol/l of PTH (+PTH). The cyclic adenosine monophosphate (cAMP) concentration was measured (pmol) and normalized to the cellular protein content (mg). The bars show the mean±s.d. (n=3). (b) γ-glutamyl transferase (GGT) activity. The concentration of p-nitroaniline (pNA) produced by the cells was measured and normalized to their protein content (mg). The bars show the mean±s.d. (n=3). (c) Ammonia production. Cells were kept in medium adjusted to the pH values indicated on the x axis. The amount of ammonia produced by the cells (μg) was measured and normalized to their protein content (mg). The bars show the mean±s.d. (n=6, two independent experiments). (d) Water transport assay. Fluorescence images of calcein-AM-loaded cells were captured at the time points indicated on the x axis. The cells were transferred to a hypotonic solution at time point 0. Fluorescence intensity was measured, and the mean fluorescence intensity measured at time point 0 was set to 100%. The values represent the mean±s.d. (n=3).
Kidney International  , DOI: ( /ki )
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9. Figure 8
Structures formed by differentiated human embryonic stem cells (hESCs) ex vivo and in vivo. (a, b) Differentiated hESCs were labeled with cell tracker (red) and injected into the cortex of an excised kidney of a newborn mouse. After 3 days of in vitro culture, the kidney was cryosectioned and stained with a CK18 antibody (green). hESCs contributed to CK18-positive simple epithelia (CK18-positive epithelia (green) with hESCs (red) appeared yellow on the merged images), as well as to CK18-negative structures (hESCs in CK18-negative structures appeared red). (b) An enlargement of the area boxed in a, bars=50μm (a, b). (c–f) Formation of tubular structures in vivo. Differentiated hESCs encapsulated in Matrigel were implanted subcutaneously in severe combined immunodeficient (SCID) mice. (c, d) Hematoxylin and eosin-stained cryosections showed that the explants consisted of tubular structures. An enlargement of the area boxed in c is shown in d. Bars=(c) 200μm and (d) 100μm. (e) A cryosection of an explant was immunostained with an anti-AQP1 (aquaporin 1) antibody (green, left-hand panel) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue, middle panel). The right-hand panel shows the merged colors. Bar=100μm. (f) Another cryosection of an explant that was similarly stained as the explant shown in e, bar=100μm. The boxed area of the left panel is shown enlarged in the three right-hand panels, which display the blue and the green fluorescences, as well as the merged colors.
Kidney International  , DOI: ( /ki )
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10. Figure 9
Functional assays with cells cultivated in bioreactors. Bioreactors were seeded with differentiated human embryonic stem cells (hESCs) (white bars), human primary renal proximal tubular cells (HPTCs) (black bars, positive control), or NIH 3T3 cells (gray bars, negative control). (a) Response to parathyroid hormone (PTH). Bioreactors were perfused with medium without PTH or with PTH supplement (+ PTH). The cyclic adenosine monophosphate (cAMP) content of the cells was determined and normalized to their protein content. The bars show the mean±s.d. (n=3). (b) γ-glutamyl transferase (GGT) activity. Bioreactors were perfused with medium containing GGT substrates, and the concentration of p-nitroaniline (pNA) (product of the reaction catalyzed by GGT) was measured in the medium collected at the inlet (medium not exposed to cells) and the outlet (medium exposed to cells) of the bioreactors. The bars indicate the concentrations of pNA (nmol/ml) produced per hour (mean±s.d., n=3). * indicates significant differences (P<0.05).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions