Staged in vitro reconstitution and implantation of engineered rat kidney tissue

Abstract

A major hurdle for current xenogenic-based and other approaches aimed at engineering kidney tissues is reproducing the complex three-dimensional structure of the kidney. Here, a stepwise, in vitro method of engineering rat kidney-like tissue capable of being implanted is described. Based on the fact that the stages of kidney development are separable into in vitro modules, an approach was devised that sequentially induces an epithelial tubule (the Wolffian duct) to undergo in vitro budding, followed by branching of a single isolated bud and its recombination with metanephric mesenchyme. Implantation of the recombined tissue results in apparent early vascularization. Thus, in principle, an unbranched epithelial tubular structure (potentially constructed from cultured cells) can be induced to form kidney tissue such that this in vitro engineered tissue is capable of being implanted in host rats and developing glomeruli with evidence of early vascularization. Optimization studies (of growth factor and matrix) indicate multiple suitable combinations and suggest both a most robust and a minimal system. A whole-genome microarray analysis suggested that recombined tissue recapitulated gene expression changes that occur in vivo during later stages of kidney development, and a functional assay demonstrated that the recombined tissue was capable of transport characteristic of the differentiating nephron. The approach includes several points where tissue can be propagated. The data also show how functional, 3D kidney tissue can assemble by means of interactions of independent modules separable in vitro, potentially facilitating systems-level analyses of kidney development.

Fig. 1.

WD budding systems. The whole mesonephros (A and B), the WD with a thin layer of intermediate mesoderm (C and D), and the WD devoid of other cell layers (E and F) can be induced to bud according to the conditions outlined in the text and SI Table 3. (Scale bar: 500 μm.)

Fig. 2.

WD budding to isolated in vitro-formed UB branching. One bud from a budded WD after 4 days in culture (A) can be excised, suspended in a 3D extracellular matrix gel (B), and induced to branch (C). Please also see Table 1.

Fig. 3.

Recombination of branched in vitro-formed UB with MM. (A and B) The branched in vitro-formed UB from Fig. 2C (A) was mechanically separated from the matrix (delineated by the dotted line) and recombined with freshly dissected undifferentiated MM (B). (C–F) Recombined tissues were grown for approximately 4–6 days (C). A ×10 dual fluorescent micrograph of the recombined tissue stained with FITC-labeled D. biflorus (green) and rhodamine-conjugated peanut agglutinin (PNA) lectin (red) shows the mesenchymal-to-epithelial transition occurring around the UB branches (green) (D and E ×4). A higher-magnification (×40) at the fusion (arrow) of the WD-derived (green) and MM-derived (red) epithelial cells demonstrates a contiguous tubule lumen (F).

Fig. 4.

Differentiated tubules of the recombined tissue are functionally capable of organic anion transport. (A) Accumulation of 6-CF (green), a fluorescent organic anion, in the MM-derived tubules (UB-derived tubules stained with TRITC-conjugated D. biflorus, red) of the recombined tissue (branched T-shaped UB, MM) suggests both differentiation and function of mesenchymal tubules. (B) The accumulation is seen only in the cells of non-UB-derived tubules (arrows). (C) The accumulation is probenecid- (a competitive inhibitor of organic anion transport) sensitive, confirming that the accumulation was transporter mediated. (Scale bars: A and C, 500 μm; B, 200 μm.)

Fig. 5.

Recombined tissue 14 days after implantation into a host rat. (A and B) The dashed line separates recombined tissue (above) from host tissue (below); glomeruli are stained with PNA (red). (C and D) The presence of erythrocytes (arrows) in the glomeruli suggests blood flow in the recombined tissue. The cells of the glomerulus express the endothelial marker PECAM-1 (green) and type IV collagen (red) along its basement membrane. DAPI nuclear stain is blue. (Scale bar: 50 μm.) (Magnification: A, ×4; B, ×10; C, ×40; D, ×60.)

Fig. 6.

Proposed in vitro kidney engineering strategy. First, the WD is isolated and induced to bud. Then, each bud can be isolated and induced to undergo branching. The branched in vitro-formed UB is then recombined with MM; after 4–6 days of mutual induction, the recombined tissue resembles a late-stage embryonic kidney. The recombined tissue is then implanted into a host animal where it is vascularized and forms glomeruli. The possibility of using cells to engineer WD and/or MM-like tissue is also indicated.