Article

Ex Vivo Whole Embryonic Kidney Culture: A Novel Method for Research in Development, Regeneration and Transplantation

Division of Urology, Childrens Hospital Los Angeles and Saban Research Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90041, USA.
The Journal of urology (Impact Factor: 4.47). 02/2008; 179(1):365-70. DOI: 10.1016/j.juro.2007.08.092
Source: PubMed

ABSTRACT

Whole metanephric organ culture represents a novel investigatory approach with potential application in many aspects of research in kidney regeneration and transplantation. We report the current status of embryonic kidney culture, discussing issues such as the appropriate culture conditions and methods, histological results, values of and limitations to the different techniques used today. To optimize this system in vitro for the benefit of future studies we focused our efforts on evaluating and developing a new durable 3-dimensional organ culture system using a uniquely modified approach.
Metanephric kidneys were microdissected from the embryos of timed pregnant WT C57BL/C6 mice on days 12 to 16 of gestation (embryonic days 12 to 16). Novel perfusion channels were created in the harvested embryonic kidneys before placing them in culture. Embryonic kidneys were placed on a 0.4 microm pore size Transwell membrane, cultured in base medium at a medium gas interphase and incubated at 37C with fully humidified 5% CO2. Histological and immunocytochemical analysis was performed to evaluate for signs of necrosis, and the structural integrity and functionality of organs during culture.
We confirmed histologically that our organ culture system was capable of maintaining normal kidney structures significantly longer (mean 10 days) than previously reported standard protocols. Condensation and aggregation of the metanephric mesenchyma at the tips of the ureteral bud were observed, including the formation of well developed nephrons and glomeruli without evidence of necrosis. Organ maturation occurred in a developmentally appropriate centrifugal pattern and the expression of key regulatory factors was demonstrated.
Our in vitro model replicates closely the in vivo processes involved in normal kidney development. We also present what is to our knowledge the first demonstration of a durable 3-dimensional kidney culture system reported in the literature. This system may represent an uncomplicated method for in vitro kidney culture that we hope will serve as an effective adjunct to research focused on signaling pathways, development and regeneration as applied to the kidney.

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Available from: Stefano Giuliani, Apr 12, 2014
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    • "In contrast, Zhang et al. [20] observed a stimulatory effect of transient high glucose on ureteric branching in vitro. While metanephric organ culture has proven a major research tool in developmental nephrology, cultured kidneys are unnaturally flattened, avascular and grow at a relatively slow rate; factors which affect and distort ureteric branching morphogenesis [22], [23]. Inconsistencies between findings from previous studies may be due to differences in media and length of culture time. "
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    ABSTRACT: There is strong evidence from human and animal models that exposure to maternal hyperglycemia during in utero development can detrimentally affect fetal kidney development. Notwithstanding this knowledge, the precise effects of diabetic pregnancy on the key processes of kidney development are unclear due to a paucity of studies and limitations in previously used methodologies. The purpose of the present study was to elucidate the effects of hyperglycemia on ureteric branching morphogenesis and nephrogenesis using unbiased techniques. Diabetes was induced in pregnant C57Bl/6J mice using multiple doses of streptozotocin (STZ) on embryonic days (E) 6.5-8.5. Branching morphogenesis was quantified ex vivo using Optical Projection Tomography, and nephrons were counted using unbiased stereology. Maternal hyperglycemia was recognised from E12.5. At E14.5, offspring of diabetic mice demonstrated fetal growth restriction and a marked deficit in ureteric tip number (control 283.7±23.3 vs. STZ 153.2±24.6, mean±SEM, p<0.01) and ureteric tree length (control 33.1±2.6 mm vs. STZ 17.6±2.7 mm, p = 0.001) vs. controls. At E18.5, fetal growth restriction was still present in offspring of STZ dams and a deficit in nephron endowment was observed (control 1246.2±64.9 vs. STZ 822.4±74.0, p<0.001). Kidney malformations in the form of duplex ureter and hydroureter were a common observation (26%) in embryos of diabetic pregnancy compared with controls (0%). Maternal insulin treatment from E13.5 normalised maternal glycaemia but did not normalise fetal weight nor prevent the nephron deficit. The detrimental effect of hyperglycemia on ureteric branching morphogenesis and, in turn, nephron endowment in the growth-restricted fetus highlights the importance of glycemic control in early gestation and during the initial stages of renal development.
    Full-text · Article · Mar 2013 · PLoS ONE
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    • "Metanephric kidneys of timed pregnant mice at 11.5 days of gestation were harvested under sterile conditions using a dissecting microscope essentially as described previously (Giuliani et al., 2008). The embryos were placed in a Petri dish containing cold PBS. "
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    ABSTRACT: Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.
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    • "Although this is a disadvantage in that the three-dimensional (3D) organization of the kidney is largely lost, it is an advantage in that it makes imaging much easier. There are alternative culture conditions in which the kidney will grow in a more 3D pattern with less flattening (Giuliani et al. 2008; Rosines et al. 2010), but these have not been used as extensively. Another recently described culture system, in which kidneys are grown directly on glass, in a low volume of medium, allows more extensive growth than in the standard filter cultures (including development of a distinct cortex and medulla), although the kidneys also flatten in this system (Sebinger et al. 2010). "
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    ABSTRACT: INTRODUCTION: Development of the kidney involves interactions between several cell lineages and complex morphogenetic processes, such as branching of the ureteric bud (UB) to form the collecting duct system and condensation and differentiation of the mesenchymal progenitors to form the nephron epithelia. One of the advantages of the mouse kidney as an experimental system is that it can develop in culture, from the stage of initial branching of the UB (E11.5) for up to a week (although it achieves the size and degree of development of only an E13.5-E14.5 kidney in vivo). The availability of fluorescent proteins (FPs) has provided powerful tools for visualizing the morphogenesis of specific renal structures in organ cultures. Two categories of genetically modified mice that express FPs are useful for visualizing different cell lineages and developmental processes in these organ cultures: (1) transgenic mice that express a fluorescent reporter in the pattern of a specific gene; and (2) Cre reporter mice, which turn on an FP in cells with Cre recombinase activity (and their daughter cells), used in conjunction with cell type-specific Cre transgenic mice. Here, we describe some of the currently available Cre and FP transgenic lines that are useful for the study of kidney development.
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