Constructing kidney-like tissues from cells based on programs for organ development: toward a method of in vitro tissue engineering of the kidney

Abstract

The plausibility of constructing vascularized three-dimensional (3D) kidney tissue from cells was investigated. The kidney develops from mutual inductive interactions between cells of the ureteric bud (UB), derived from the Wolffian duct (WD), and the metanephric mesenchyme (MM). We found that isolated MMs were capable of inducing branching morphogenesis of the WD (an epithelial tube) in recombination cultures; suggesting that the isolated MM retains inductive capacity for WD-derived epithelial tubule cells other than those from the UB. Hanging drop aggregates of embryonic and adult renal epithelial cells from UB and mouse inner medullary collecting duct cell (IMCD) lines, which are ultimately of WD origin, were capable of inducing MM epithelialization and tubulogenesis with apparent connections (UB cells) and collecting duct-like tubules with lumens (IMCD). This supports the view that the collecting system can be constructed from certain epithelial cells (those ultimately of WD origin) when stimulated by MM. Although the functions of the MM could not be replaced by cultured mesenchymal cells, primary MM cells and one MM-derived cell line (BSN) produced factors that stimulate UB branching morphogenesis, whereas another, rat inducible metanephric mesenchyme (RIMM-18), supported WD budding as a feeder layer. This indicates that some MM functions can be recapitulated by cells. Although engineering of a kidney-like tissue from cultured cells alone remains to be achieved, these results suggest the feasibility of such an approach following the normal developmental progression of the UB and MM. Consistent with this notion, implants of kidney-like tissues constructed in vitro from recombinations of the UB and MM survived for over 5 weeks and achieved an apparently host-derived glomerular vasculature. Lastly, we addressed the issue of optimal macro- and micro-patterning of kidney-like tissue, which might be necessary for function of an organ assembled using a tissue engineering approach. To identify suitable conditions, 3D reconstructions of HoxB7-green fluorescent protein mouse rudiments (E12) cultured on a filter or suspended in a collagen gel (type I or type IV) revealed that type IV collagen 3D culture supports the deepest tissue growth (600 +/- 8 microm) and the largest kidney volume (0.22 +/- 0.02 mm(3)), and enabled the development of an umbrella-shaped collecting system such as occurs in vivo. Taken together with prior work (Rosines et al., 2007; Steer et al., 2002), these results support the plausibility of a developmental strategy for constructing and propagating vascularized 3D kidney-like tissues from recombinations of cultured renal progenitor cells and/or primordial tissue.

FIG. 1.

WD/MM coculture. (A) Schematic of the procedure followed in engineering kidney tissue from the WD and MM. (B) Embryonic day 13 rat kidney; UB is outlined by dashed red line. (C) Kidney that has been separated into isolated UB and isolated MM; light green oval indicates empty space in MM where the UB was removed. (D) Isolated MM from (C), in which a piece of WD has been used to replace the UB; light green oval indicates empty space in MM where the UB was removed, and dashed blue line demarcates the section of WD. (E) After 7 days, the WD/MM coculture grew similar to traditional in vitro kidney culture. (F) After 7 days, the WD/MM coculture grew similar to traditional in vitro kidney culture (green = Dolichos biflorus lectin, UB-derived tissues; red = E-cadherin, UB- and MM-derived polarized epithelial tissues). (G) After 12 days, peanut agglutinin lectin staining (red) revealed differentiation of glomerular podocytes. Scale bars = B–G 300 μm. UB, ureteric bud; WD, Wolffian duct; MM, metanephric mesenchyme. Color images available online at www.liebertonline.com/ten.

FIG. 2.

IMCD hanging drop cell aggregates. (a) An IMCD aggregate from the hanging drop. (b) After 6 days in the Matrigel suspension culture, the IMCD cell aggregate protruded a tubule with a lumen. (c, d) The tubule continued growing to form a structure resembling a T-shaped UB (compare with e) after 8 days of culture. (e) Phase-contrast photomicrograph of a freshly isolated rat E13 T-shaped UB. Scale bars = 200 μm. IMCD, inner medullary collecting duct.

FIG. 3.

Budding of IMCD cell aggregates. (a, b) IMCD cell aggregates from the handing drop method formed buds with lumens in a manner like WD budding after 7 days in the Matrigel suspension culture. (c) An isolated clean WD. (d) Budding of the clean WD after 5 days culture supplemented with a medium with glial-cell-derived neurotrophic factor and fibroblast growth factor1. Scale bar = 200 μm.

FIG. 4.

UB cell aggregate coculture with MM. (a) Hanging drop aggregate of UB cells (outlined in red) surrounded by numerous freshly isolated MMs. (b) Phase-contrast of coculture after 7 days. (c) Confocal fluorescent photomicrograph of coculture tissue after 7 days of growth in culture (green = Dolichos biflorus lectin, UB-derived tissues; red = E-cadherin, UB- and MM-derived polarized epithelial tissues). (d) Higher magnification examination of the recombined tissue showing that the MM-derived tubule is continuous with the green UB cells. (e) UB-cell-derived multicellular extensions. Scale bars = a–c, 400 μm; d and e, 25 μm. Color images available online at www.liebertonline.com/ten.

FIG. 5.

IMCD cell aggregate coculture with MM. (a) After 7 days, the IMCD cells organized into epithelial tubules; however, MM induction did not appear very widespread. (b–d) Cytokeratin staining (green) demonstrates that the IMCD cell aggregate formed tubular structures with lumens (noted by the asterisk). (e) Occasional comma-shaped bodies (evident by PAX-2 staining, red) were induced by the IMCD cell aggregate. Scale bars = a and b 400 μm; d and e, 50 μm. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Three MM-derived cell lines tested for the ability to induce isolated WD budding. (a–c) The BSN, rat inducible metanephric mesenchyme (RIMM)-18, and MM primary cell lines are all MM-derived cell lines that are mostly vimentin positive and cytokeratin negative or low. (d) 3T3 fibroblasts are also vimentin positive, cytokeratin negative cells, but are not MM derived. (e) Conditioned medium (CM) from BSN cells strongly induced isolated UB branching. (g) CM from primary MM cells only slightly induced branching. (f, h) CM from RIMM-18 or 3T3 cells did not induce branching morphogenesis. (i) Plot of tip number versus cell-CM used (analysis of variance, p ≤ 0.00001); *p ≤ 0.05, **p ≤ 0.00005. Scale bars = a–d, 50 μm; e–g, 250 μm. Color images available online at www.liebertonline.com/ten.

FIG. 7.

Three MM derived cell lines were tested for the ability to support isolated WD budding. (a, b) WD cultured on a filter disintegrated. (c, d) WD cultured on a layer of BSN cells appeared to survive, (e, f) but WD cultured on RIMM-18 cells underwent budding at multiple sites along the WD. (g, h) 3T3 only supported WD survival. (i, j) MM primary cells resulted in WD disintegration. (Scale bar = 500 μm).

FIG. 8.

Examination of engineered kidney tissue 37 days after implantation under the renal capsule of a kidney in a nude mouse. (a, b). Hematoxylin and eosin stained section through the host kidney and implanted kidney tissue. (c–i) Confocal fluorescent micrographs showing extent of implant vascularization. (c) Dolichos biflorus (green); Collagen IV (red). (d) Flk-1 (green); Collagen IV (red). (e) Higher magnification examination of d. (f) Specific staining for mouse podocalyxin (red). (g) Specific staining for rat podocalyxin (red). (h) von Willebrand factor (green); mouse podocalyxin (red). (i) Higher magnification examination of h. (Scale bar = a, 400 μm; b, 150 μm; c, d, h, 50 μm; e, i, 10 μm). Color images available online at www.liebertonline.com/ten.

FIG. 9.

Three-dimensional (3D) projection of the branching UB of E12 HoxB7–green fluorescent protein mouse kidneys cultured for 7 days. (a, b) Kidneys in the traditional filter culture grew flat and along the filter. (c–e) Kidneys cultured in type I collagen or type IV collagen grew much thicker and in a more 3D manner (units, μm). (e, f) Type IV collagen supported the deepest tissue growth, but the least distance from the origin of branching. Color images available online at www.liebertonline.com/ten.

FIG. 10.

(A–D) Phase-contrast photomicrographs of whole embryonic kidneys cultured for 7 days directly on the Transwell filter (A) or in 3D culture suspended in either Matrigel (B, 25%), type I collagen (C, 1 mg/mL), or type IV collagen (0.65 mg/mL). (E, F) Fluorescent photomicrographs of whole embryonic kidneys from the HoxB7–green fluorescent protein transgenic mouse grown for 7 days in 3D culture suspended in either type I collagen (E) or type IV collagen (F). Note especially “umbrella-like” branching of UB in F. Color images available online at www.liebertonline.com/ten.

FIG. 11.

Schematic of developmental approaches to in vitro engineering of kidney-like tissues. Note that methods for propagation of WD buds and branched UBs are discussed elsewhere.^, Color images available online at www.liebertonline.com/ten.