Table 1.
Antibodies for immunohistochemical analysis.
Fig 1.
Production of decellularized renal scaffolds.
Whole-kidney scaffolds before (A) and after (B) perfusion with 1% SDS. Kidneys from the same donor were compared after whole-kidney perfusion (C) or static section (D) decellularization methods. Histological comparison of decellularized whole kidney (E) with native kidney (F) demonstrated removal of cellular contents with maintenance of native renal architecture. The section decellularization process at 4°C without agitation (G) was greatly improved when sections were placed on a shaker during the decellularization process at room temperature (H). Scale bars = 100 μm.
Fig 2.
Recellularization of kidney scaffolds with hESC.
(A) Schematic of custom-designed, perfusion bioreactor for cell seeding via the renal artery (RA) or ureter (U). (B) Recellularized kidneys after 7 days of culture. Cells seeded via the renal artery (C-D) or ureter (E-F) were observed in medullary vascular or tubular lumens but not in outer cortical tubules or glomeruli. With longer culture periods enhanced recellularization was observed (G-H), particularly in medullary regions. Representative images from a minimum of three independent experiments are shown. Medulla (M), Cortex (Cx), scale bars = 100 μm.
Fig 3.
Renal developmental markers are upregulated by renal ECM.
hESC were differentiated as embryoid bodies (A) or cultured in whole kidneys (B) or sections of kidneys (C) where cells were typically observed in the medulla and medullary rays. (D-F) Renal developmental markers WT1 and PAX2 were upregulated in whole or sections of decellularized kidneys when compared with embryoid body differentiation. (G-I) AQP1, a marker of proximal tubules, was expressed in tubule-like structures in embryoid bodies and kidney sections, but not in whole kidneys. (J-L) Vimentin, a mesenchymal and mesangial marker, was expressed under all culture conditions. Other markers of mature renal cell types including SMA (mesangial and vascular smooth muscle marker) and Calbindin (renal distal tubules) were not expressed. Nuclei were visualized with DAPI (blue); scale bars = 100 μm.
Fig 4.
Early renal lineage genes were upregulated by renal ECM.
Relative expression of mesodermal (BRY), posterior primitive streak (OSR1), intermediate mesodermal (PAX2), and metanephric mesenchymal (WT1) genes in hESC differentiated as embryoid bodies or in renal scaffolds. Mean ± standard error of the mean (SEM); N≥3 replicates.
Fig 5.
Renal-directed differentiation of hESC on renal ECM or PSS.
(A-D). H&E staining of cell-scaffold constructs after 20 days in culture with two growth factor protocols (A or B). (E-H). Tubule-like structures were present and positive for CK but not VIM. (I-L). Regions positive for PAX2 (intermediate mesoderm and induced metanephric mesenchyme) were typically distinct from regions positive for WT1 (induced mesenchyme) under Protocol A. These markers were expressed in a more diffuse pattern throughout the construct under Protocol B. Tubule-like structures in Protocol B with renal ECM were WT1-positive and surrounded by a ring of PAX2-positive cells. (M-P). The proximal tubule marker AQP1 was expressed with greater frequency under Protocol A and on PSS while the Loop of Henle marker UMOD was rarely expressed in any construct. (Q-T). The distal tubule/collecting duct marker ECAD was widely expressed on cells with epithelial morphology in all constructs while the ascending Loop of Henle and collecting duct marker CALB was only expressed in constructs under Protocol A with PSS. (U-X). The proximal tubule marker EMA was expressed on some tubule structures in all constructs with small vessel-like structures positive for the endothelial marker CD31. Nuclei were visualized with DAPI (blue). Representative images shown; N≥3 experiments. Scale bar = 100 μm.
Fig 6.
Expression of early renal lineage markers in differentiating hESC cultured under Protocol A.
Pluripotent hESC were plated in suspension cultures to form embryoid bodies with supplemental growth factors as shown. Gene expression (qPCR) relative to the housekeeping gene EF1α was calculated using undifferentiated day 0 cells as the comparator. After 12 days of suspension culture, embryoid bodies were collected and plated on renal ECM or PSS at the air-medium interface in basal medium (arrows). Both scaffolds were equal or superior to embryoid body culture in upregulating renal lineage genes (N≥3 replicates). With the exception of SIX2, PSS were equal (OSR1) or superior (BRACHY, LIM1, WT1, PAX2) to renal ECM in supporting renal precursor populations.
Fig 7.
Expression of early renal lineage markers in differentiating hESC cultured under Protocol B.
Pluripotent hESC were differentiated in suspension cultures as embryoid bodies with supplemental growth factors as shown. After 5 days, cells were plated on renal ECM or PSS at the air-medium interface in basal medium (arrows). Gene expression (qPCR) relative to the housekeeping gene EF1α was calculated using undifferentiated day 0 cells as the comparator (N≥3 replicates). Both types of scaffolds were superior to embryoid body cultures in directing upregulation of intermediate mesoderm (OSR1, LIM1, PAX2) and the metanephric mesenchyme gene, WT1. Renal ECM was similar to embryoid body culture in expression of BRY and SIX2, while these genes were upregulated on PSS.