The field of renal transplantation is exploring new frontiers. Use of traditional whole kidney organ transplants from human deceased or living donors has been limited by the insufficient supply of donor kidneys to meet ever-increasing demand, and by the risks of allograft loss to rejection and toxicity of immunosuppressive therapies.
The transplantation of developing fetal kidney tissues or primordia from animal embryos so that they grow and mature in situ in the recipient offers a number of advantages. Developing metanephric tissue is less immunogenic than developed kidney because it contains fewer antigen-presenting cells and expresses less MHC class I and class II antigens that mediate host recognition.1 Unlike embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, developing metanephric kidney cells are already committed to a genetic program of renal development, obviating the need to preprogram cell fate. Finally, use of animal cells avoids ethical barriers to human ES cell use.
Over the last decade, a number of laboratories around the world have tested the feasibility of xenotransplantation of intact fetal kidneys or fragments of organ primordia from animal embryos.2 By embryonic day (E) 10.5 in the mouse, the ureteric bud evaginates from the nephric duct and contacts the metanephric blastema to initiate the process of nephrogenesis.3,4 Thus, tissue harvesting at E11.5 captures the developmental window when outgrowths of ureteric bud are branching and beginning to induce metanephric mesenchymal progenitor cells to differentiate. Evidence suggests that this midembryonic age of tissue harvesting gives superior results over late time points, probably because of greater tissue plasticity and growth potential as well as lower immunogenicity.5 The induced metanephric mesenchyme will ultimately give rise to the glomeruli and the proximal through distal tubular segments of the nephron. In turn, maturation of the ureteric bud will produce the collecting ducts, renal pelvis, and ureter.
The rich and intricate vascularity of the kidney is a prerequisite for glomerular filtration. Several experimental attempts to “grow” new kidney tissue into animal hosts have shown that intact fetal kidneys or fragments become vascularized by the recipient, undergo morphologic maturation, and may exhibit functional properties.6–12 For example, whole metanephroi transplanted into nonimmunosuppressed hosts are successfully vascularized by host arteries, form mature nephrons, and are not rejected.6 Fetal kidneys maintained in culture become vascularized and form normal glomeruli when grafted into the eye chamber of adult host mice.13 Kidney-like tissues have also been obtained by recombination of in vitro propagated ureteric bud with intact metanephric mesenchyme.14,15 Upon in vivo implantation, these tissues undergo early vascularization and develop morphologically normal glomeruli.14
Bioengineering attempts show that suspensions of murine metanephric mesenchymal E11.5 progenitor cells selected for high expression of Sall-1, a key zinc-finger nuclear transcription factor in renal development, are able to generate in vitro renal epithelia containing rudimentary glomeruli.16 Recently, more progress has been made in the field of engineering fetal kidney tissue from a suspension of single cells by developing a method that allows a simple suspension of E11.5 kidney cells to self-organize in vitro into a tissue mass containing immature nephrons and collecting ducts.17,18 An innovation in this system was the introduction of temporary pharmacologic inhibition of Rho-associated kinase to block the dissociation-induced apoptosis that results from loss of survival signals normally provided by cell-cell and cell-matrix contacts, thereby facilitating reintegration of the dissociated embryonic kidneys.17,19
These attempts, however, failed to develop glomeruli to any significant extent. This is because the avascular in vitro environment is not permissive for glomerulogenesis, which is a critical step toward building functional kidney tissue. This impediment places a serious restriction on the maturation potential of tissue obtained from a simple culture or suspension of precursor cells. One of the greatest challenges in tissue engineering has been the unanswered question of whether single-cell suspensions can generate both glomeruli capable of filtering blood and tubules capable of reabsorbing filtered macromolecules.
In this issue of JASN, Xinaris et al. show for the first time that organoids constructed in vitro from suspensions of fully dissociated cells can be integrated into a living recipient and perform nephron-specific functions.20 This work builds on the previously developed method of embryonic kidney dissociation and reaggregation in vitro.17,18 Renal organoids were grown in vitro from single-cell suspensions derived from E11.5 murine kidneys and then implanted beneath the kidney capsule of athymic rat hosts. Unilateral nephrectomy was performed to enhance the potential for growth and development of the implanted tissue. Unilateral nephrectomy in rodents is known to activate a number of mitogenic, morphogenic, and prosurvival genes such as Wilms tumor-1 (WT-1), EGF, and hepatocyte growth factor. To promote endogenous vascular development, the authors adopted a new technique of preconditioning the organoids in culture with vascular endothelial growth factor (VEGF) followed by injection of VEGF locally into the area of implantation and as intravenous injections three times weekly by tail vein.20 This critical maneuver compensates for the paucity of podocytes in order to replete the podocyte-endothelial axis of VEGF stimulation required for glomerular capillary endothelial induction. Immunostains performed at 3 weeks with antisera specific for murine and rat antigens revealed the vast majority of vessels within the transplant to be of donor (mouse) origin, indicating successful endogenous angiogenesis. This method resulted in the formation in vivo of vascularized glomeruli with fully differentiated glomerular capillary walls, including endothelial fenestrae, foot processes, and slit diaphragms. In addition, erythropoietin-producing cells were detectable in the interstitium, indicating potential hormonal competence. Markers of mature nephron segments were appropriately expressed, including WT-1, nephrin, and synaptopodin in podocytes; claudin-1 in parietal epithelium; megalin in proximal tubular epithelial brush border; calbindin in distal tubules; and Tamm-Horsfall protein in thick ascending limbs. In accordance with this high degree of structural maturation, the implanted tissue exhibited specialized physiologic functions, including proximal tubular reabsorption of tracer macromolecules that gained access to the tubular lumen by glomerular filtration.
A limitation of this study was that the organoids were only viable for 3–4 weeks and began to involute thereafter.20 Thus, a challenge will be the maintenance of greater longevity, perhaps by use of antirejection therapy and exploration of other potential sites for implantation that might enhance growth and function. For example, xenotransplantation of the developing metanephros into the omentum and para-aortic area in the rat has been shown to promote excellent vascularization and good cortical-medullary differentiation and hormonal function, including production of renin and erythropoietin.8 Transplantation under the renal capsule, as performed in this study, may be more spatially restrictive and inhibit the integration of individual nephrons with the distant ureteric bud-derived medullary collecting ducts and renal pelvis, thereby posing a barrier to excretory function. Indeed, a major challenge in constructing fully efficient kidney tissue from cell suspensions is the need for proper tissue patterning through successive generations of ureteric bud branching and linkage of the nascent nephrons to a single draining collecting system. Single-cell dissociations formed from embryonic kidney also generally have a limited potential for reconstitution of a viable kidney because the ureteric bud formed upon reaggregation exhibits less growth and branching capacity.19 Although this is partially alleviated by temporary pharmacologic inhibition of Rho kinase activity in vitro to reduce dissociation-induced apoptosis of the ureteric bud17,19 and treatment with VEGF in vivo,20 the organoids form fewer nephrons than in a whole transplanted kidney.
Despite these limitations, the system described in this study introduces a novel direction for generating donor tissue and offers the methodologic basis for a number of exciting investigative approaches. First, it provides a nephrogenic environment suitable to validate the renal differentiation potential of kidney stem cells established from various sources, such as iPS or ES cells. It is an outstanding model system in which to dissect the mechanisms and properties of self-organization at both early and advanced developmental stages. Because the system dissociates embryonic kidney down to the single-cell level, it provides a valuable window of experimental accessibility before the in vitro reaggregation step.19 At this point, it is possible to introduce agent or transfection-based interventions to study cell autonomy or to simulate genetic diseases. By permitting the aggregation of genotypically different cells derived from single gene transfection, it supports the production of chimeras to test effects of a particular mutation on cell fate. For example, a fraction of the admixed cells could be modified to express a mutated gene, such as Pax-2 or WT-1 required for renal development, and the fate of these cells can then be studied in mature developmental stages in vivo. The in vitro applicability of these techniques has already been tested.17,21 Moreover, disease-related genes can be introduced in the organoid to study mechanisms of complex diseases and possibly to perform preliminary pharmacologic screening of therapeutic agents, without the need for time-consuming and expensive studies using whole animals. This might apply, for instance, to a model using a mutated podocyte gene that produces a form of FSGS or a major defect in glomerular basement membrane collagen as in Alport syndrome. The single-cell stage also allows the use of genetic engineering approaches to humanize cells, possibly by introducing immunomodulatory genes or by switching off others. For example, silencing of the gene for α-1,3-galactosyl transferase22 in porcine reforming tissues could diminish the potential for rejection via naturally occurring anti-gal antibodies and facilitate xenotransplantation of swine organoids.
Important future perspectives are opened by this system. It can be exploited to generate human renal tissue by constructing chimeric kidneys that combine animal progenitor cells and human stem cells,21,23,24 followed by the selective elimination of animal cells after transplantation. Another attractive approach would be to apply this technology to large animals, such as pigs, whose nephron structure and size closely approximate human nephrons. These new methodological advances, previously unfeasible in mammalian kidneys, represent a considerable step toward the practical goal of engineering renal tissues suitable for transplantation and provide inherent technical flexibility for designing investigations into the molecular and cellular mechanisms of kidney development.
Disclosure
None.
Published online ahead of print. Publication date available at www.jasn.org.
See related article, “In Vivo Maturation of Functional Renal Organoids Formed from Embryonic Cell Suspensions,” on pages 1857–1868.
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