Integration of Embryonic Stem Cells in Metanephric Kidney Organ Culture

Abstract

Many stages of nephrogenesis can be studied using cultured embryonic kidneys, but there is no efficient technique available to readily knockdown or overexpress transgenes for rapid evaluation of resulting phenotypes. Embryonic stem (ES) cells have unlimited developmental potential and can be manipulated at the molecular genetic level by a variety of methods. The aim of this study was to determine if ES cells could respond to developmental signals within the mouse embryonic day 12 to embryonic day 13 (E12 to E13) kidney microenvironment and incorporate into kidney structures. ROSA26 ES cells were shown to express ␤-galactosidase ubiquitously when cultured in the presence of leukemia inhibitory factor to suppress differentiation. When these cells were microinjected into E12 to E13 metanephroi and then placed in transwell organ culture, ES cell-derived, ␤-galactosidase-positive cells were identified in epithelial structures resembling tubules. On rare occasions, individual ES cells were observed in structures resembling glomerular tufts. Electron microscopy showed that the ES cell-derived tubules were surrounded by basement membrane and had apical microvilli and junctional complexes. Marker analysis revealed that a subset of these epithelial tubules bound Lotus tetragonolobus and expressed ␣ 1 Na ؉ /K ؉ ATPase. ES cells were infected before injection with a cytomegalovirus promoter-green fluorescence protein (GFP) adenovirus and GFP expression was found as early as 18 h, persisting for up to 48 h in cultured kidneys. This ES cell technology may achieve the objective of obtaining a versatile cell culture system in which molecular interventions can be used in vitro and consequences of these perturbations on the normal kidney development program in vivo can be studied.

Embryonic Stem Cells Proliferate and Differentiate when Seeded into Kidney Scaffolds

“…29 Also, ES cells have been transplanted in to murine day 12 and 13 embryos amid the metanephric mesenchyme, and these cells have integrated into the metanephric kidney in the organ culture. 30 ECM proteins have been shown to direct ES cell differentiation and are factors affecting cell behavior during organogenesis. 22,[31][32][33][34][35] Furthermore, differentiation toward a target tissue is improved if the ECM protein architecture closely resembles the in vivo ECM of the targeted tissue.…”

Regrow or Repair

“…Yamamoto et al (72) used this approach to provide evidence that murine ES cells had the potential to give rise to mesonephric ducts and UB in teratomas. In contrast, Steenhard et al (73) reported 50% integration of undifferentiated ES cells into the tubules of embryonic kidneys without evidence for teratomas. Kobayashi et al (74) created Wnt4-transformed murine ES cells and showed in vitro that these had the capacity to form aquaporin 2-positive renal tubules.…”

Stem cell options for kidney disease

“…With respect to technical risks, in vivo injection of these pluripotent cells can give rise to teratomas, as demonstrated by Yamamoto et al [57], who reported the generation of teratomas containing metanephric-mesenchyme-related structures after injection of undifferentiated ES cells within the peritoneum of nude mice [57]. In contrast, Steenhard et al [58] reported a 50% integration of undifferentiated ES cells into the tubules of embryonic kidneys without evidence of teratoma formation. The directed differentiation of ES cells to a renal progenitor fate has been performed by priming embryonic bodies (EBs) with retinoic acid, acitivin-A and BMP7 [59].…”