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  Pharmaceutical Patents  


Title:  Induction of tubular morphogenesis using pleiotrophin
United States Patent: 
February 5, 2008

Nigam; Sanjay Kumar (Del Mar, CA), Sakurai; Hiroyuki (San Diego, CA), Bush; Kevin T. (San Diego, CA)
The Regents of the University of California (Oakland, CA)
Appl. No.: 
June 27, 2003


Executive MBA in Pharmaceutical Management, U. Colorado


Provided are methods and compositions for constructing stable mammalian embryonic epithelial tissues and organs as well as constructing kidney tissue, and treating renal failure. Disclosed are methods of using an active epithelial growth factor having the capability of effectuating induction of growth and morphognesis is cells.

Description of the Invention

Many epithelial organs such as kidney, lung, and prostate under go branching morphogenesis in the course of development. The kidney is formed by mutual induction between two tissue derived from the intermediate mesoderm, the metanephric mesenchyme (MM), and the ureteric bud (UB). The UB induces the MM to differentiate and form the proximal nephron, while the UB undergoes dichotomous branching and elongation as it invades the MM, ultimately forming the kidney collecting system.

Soluble factors that have been thought to play a role in morphogenetic capacity include hepatocytes growth factor (HGF) and epidermal growth factor (EGF) receptor ligands, which have been shown to induce branching tubular structures in epithelial cells cultured in collagen gels. However, these studies have been largely carried out on adult cell lines. In a cell culture model that employs a UB cell line, derived from embryonic day 11.5 (E 11.5) mouse, neither HGF, EGF receptor ligands, nor many other factors tested (alone or in combination), were able to induce UBs to form branching tubular structures with lumens.

As used herein, the abbreviation UB includes ureteric bud cells obtained from UB tissue, as well as UB tissue fragments, whole UB tissue, and UB cell lines, unless clearly indicated otherwise in the specification. The UB cells may be primary cells obtained from embryonic kidney tissue by various techniques known in the art. Such primary UB cells are not immortalized (e.g., by SV40), but may be transfected and/or transformed to express a desired product, as discussed in more detail herein.

Accordingly, the identification of specific soluble factors (e.g., MM-derived soluble factors) mediating UB branching morphogenesis remains a central question in this field. Hepatocyte growth factor (HGF) has been shown to induce the formation of branching tubular structures with lumens in three-dimensional cultures of epithelial cell lines derived from adult kidneys (i.e., MDCK and mIMCD cells)(Barros et al., 1995; Cantley et al., 1994; Montesano et al., 1991; Santos et al., 1993). However, incubation of three-dimensional cultures of an embryonic cell line derived from the ureteric bud (UB) with HGF had only a slight morphogenetic effect and the formation of branching tubular structures with lumens was not observed (Sakurai et al., 1997a). Furthermore, HGF, alone or in the presence of GDNF, does not induce branching morphogenesis of the isolated UB. This suggests that HGF is not an essential factor for early branching morphogenesis of the embryonic UB, though it may play a facilitory role. This notion is supported by the fact that genetic deletion of hgf or its receptor (c-met) apparently has little if any effect on kidney development (Bladt et al., 1995; Schmidt et al., 1995).

Another group of soluble factors implicated in branching morphogenesis of epithelial cells are the family of epidermal growth factor (EGF) receptor ligands. EGF receptor ligands are capable of inducing the formation of branching tubular structures with lumens in three-dimensional cultures of mIMCD cells, a kidney cell line derived from adult collecting duct cells (Barros et al., 1995; Sakurai et al., 1997b). However, as is the case with HGF, EGF receptor ligands are not capable of inducing the formation of branching tubular structures in three-dimensional cultures of the embryonically-derived UB cells (Sakurai et al., 1997a), nor are they capable of inducing branching morphogenesis of the isolated UB (Qiao et al., 1999a). Deletion of the EGF receptor gene results in cystic dilation of collecting ducts in mice with certain genetic backgrounds, perhaps suggesting a role in final maturation of these structures (Threadgill et al., 1995). However, as with HGF, most experimental evidence indicates that the EGF receptor ligands are not essential for early steps in UB branching morphogenesis.

In fact, among many growth factors hypothesized to play a role in kidney development, no single factor, or combination of factors, has been shown to induce UBs to form branching tubular structures (undergo tubulogenesis). Tubulogenesis is a phenotypic transformation of the cells such that condensed aggregates of tubule cells form about a central lumen wherein said lumen is bordered by cells possessing a polarized epithelial phenotype and tight junctional complexes along the lumenal border. Conditioned medium elaborated by the MM-derived cell line, BSN-CM, consistently induced UBs in three-dimensional culture to form branching tubular structures with clearly distinguishable lumens. Likewise, in the isolated UB culture system (in the presence of GDNF), no growth factor, alone or in combination, could induce the extensive branching morphogenesis observed when the isolated UB was cultured with BSN-CM, as demonstrated by the invention.

An essential role for GDNF in UB development is supported by a number of studies, including gene knockouts. For example, null mutations of gdnf its receptor, c-ret, or its co-receptor, gfra, result in abnormal kidney development, although variable phenotypes have been reported in the gdnf and c-ret knockout mice (Enomoto et al., 1998; Moore et al., 1996; Schuchardt et al., 1996). Moreover, although the proliferative effect of GDNF on UBs has been debated (Pepicelli et al., 1997; Sainio et al., 1997), GDNF has been shown to initiate UB growth (Sainio et al., 1997), and plays a role in branching morphogenesis of the isolated UB. Nevertheless, GDNF is not alone sufficient to induce branching morphogenesis of either the isolated UB or cultured UBs (Salcurai et al., 1997a), again consistent with the view that there are additional factors which are critical to the branching morphogenesis of the UB.

Studies in the developing mammalian lung and Drosophila trachea indicate that members of the FGF family function in branching morphogenesis of epithelial tissues (Hogan, 1999; Metzger and Krasnow, 1999; Zelzer and Shilo, 2000). Furthermore, null mutations of either fgf7 or fgf10 have also been reported to affect kidney development (Ohuchi et al., 2000; Qiao et al., 1999b), although in both cases the kidneys appear to be modestly affected. For example, infgf7-null kidneys, there is a 30% reduction in the number of nephrons, and the kidneys appear to function normally (Qiao et al., 1999b). Moreover, since FGF7 is detected in the developing kidney only after several iterations of UB branching have already occurred, it is likely that other factors are necessary for the early steps of the branching program. In the case of FGF 10, the defect appears similar, although the phenotype has yet to be investigated in detail since the embryos die at birth due to severe lung defects (Ohuchi et al., 2000). Nevertheless, by potentiating the effect of an essential branching morphogen produced by the MM, certain FGFs may play a facilitory role in early UB branching morphogenesis (see below).

The invention demonstrates that UBs undergo branching tubulogenesis in the presence of a conditioned medium elaborated by a cell line derived from the MM also isolated from an E11.5 mouse (BSN cells). This suggests that other soluble factors present in BSN-CM are important for UB morphogenesis. These novel factors that are secreted by the MM are important for the development of the collecting system in artificial systems as well as in vivo.

This MM-derived cell conditioned medium (BSN-CM), when supplemented with GDNF, also induces the isolated rat UB (in the absence of MM) to undergo dichotomous branching reminiscent of that seen in the developing kidney. This indicates that the MM-derived cell line, presumably reflecting the MM itself, secretes soluble factors capable of inducing branching morphogenesis of the UB. This isolated UB culture system can serve as a powerful assay system since it directly assesses the effect of soluble factors on UB morphogenesis.

The invention demonstrates that serial liquid column chromatographic fractionation of BSN-CM contain an active morphogenetic fraction comprising a single polypeptide (capable of inducing branching morphogenesis comparable to whole BSN-CM). This polypeptide was identified as pleiotrophin. Immunoblot analysis of BSN-CM as well as in situ hybridization data of developing kidney (Vanderwinden et al., 1992), demonstrate that the embryonic MM is a source of pleiotrophin. In addition to its ability to induce branching morphogenesis in the isolated UB, pleiotrophin also induced a UB cell line to form branching tubular structures with lumens, and is thus the only soluble factor so far identified with this capability. Based on this in vitro data with the isolated UB as well as the UB cell line, the invention provides methods and compositions for use in vitro and in vivo to induce morphogenesis and tubular formation of tissues (e.g., kidney tissue).

The invention provides a novel factor, and combination of factors capable of inducing UB branching morphogenetic activity. In one aspect, the invention provides an 18 kDa heparin binding protein, pleiotrophin, obtained from the BSN-CM. This factor has not previously been shown to play a role in kidney morphogenesis. Pleiotrophin was originally discovered as a fibroblast proliferative factor (Milner et al, BBRC, 165:1096-1103, 1989) and a neurite outgrowth-promoting factor (Rauvala, EMBO J, 8:2933-41, 1989). Outside the nervous system pleiotrophin is generally detected in those embryonic organs in which mesenchymal-epithelial interactions are thought to play an important role, such as salivary glands, lung, pancreas, and kidney (Mitsiadis et al., Development 121:37-51, 1995; Vanderwinden et al., Anat Ebryol (Berl) 186:387-406, 1992). Although pleiotrophin has been shown to be mitogenic for certain epithelial cells (Li et al., Science 250:1690-4, 1990; Sato et al., Exp Cell Res 246:152-64, 1999), there has been no compelling pleiotrophin during epithelial organogenesis.

The invention demonstrates that purified pleiotrophin induces impressive branching morphogenesis of the isolated UB in vitro. Thus, the invention provides methods of using pleiotrophin and compositions comprising pleiotrophin to induce morphogenesis in the kidney cells in vitro and in vivo. The invention further demonstrates that pleiotrophin is a key metanephric mesenchymally-derived factor that plays a critical role in branching morphogenesis of the UB during kidney development.

Pleiotrophin and another heparin binding growth factor, midkiue, make up a distinct growth factor family. These proteins share about 50% sequence homology (Rauvala, 1989), both are expressed widely during organogenesis (Mitsiadis et al., 1995), and are highly conserved among species (Kurtz et al., 1995). Both have been implicated in neurite outgrowth (Li et al., 1990; Rauvala et al., 1994), a phenomenon that has some similarity to branching morphogenesis (particularly as it occurs in cultured cells), and they exhibit a spatiotemporal expression pattern in other developing organ systems which suggest a role in mesenchymal-epithelial interactions (Mitsiadis et al., 1995). However, other than the finding that pleiotrophin enhances bone formation (Imai et al., 1998) and limb cartilage formation (Dreyfus et al., 1998), little is known about the role of pleiotrophin in organogenesis. However, the invention demonstrates that pleiotrophin freshly purified to apparent homogeneity from either BSN cells or 3T3 cells induced impressive growth and branching morphogenesis of isolated UBs.

A wide range of concentrations of pleiotrophin has been reported to exhibit biological activity (up to 50 .mu.g/ml) in various systems (Imai et al., 1998; Li et al., 1990; Rauvala et al., 1994; Souttou et al., 1997). Pleiotrophin binds to the extracellular matrix, which may explain why concentrations of 200-600 ng/ml were required for morphogenetic activity in the systems employed in Examples below (see also FIGS. 5A and 6 , see Original Patent). In the examples below the UB cell-line and isolated UBs were cultured within basement membrane Matrigel, which could conceivably bind a large fraction of pleiotrophin. In one aspect of the invention, similar artificial matrix systems, e.g., cell-free extracellular matrices (e.g., obtained by decellularizing a desired tissue), or synthesized matrices (e.g., lactic acid, glycolic acid, or combinations thereof) can be used and may similarly be modified to bind pleiotrophin.

To date, several glycoproteins, including brain-specific proteoglycans, the receptor type tyrosine phosphatase beta (Maeda and Noda, 1998; Meng et al., 2000) and syndecan-3 (Raulo et al., 1994) have been postulated to function as receptors for pleiotrophin. The UB has been shown to express syndecan-1 (Vainio et al., 1989), and pleiotrophin is capable of binding to syndecan-1 (Mitsiadis et al., 1995).

The involvement of proteoglycans in pleiotrophin-mediated branching morphogenesis of the UB is particularly interesting in light of data demonstrating the importance of proteoglycans in UB development (Bullock et al., 1998; Davies et al., 1995; Kispert et al., 1996). In these studies, chemical or genetic depletion of sulfated proteoglycans inhibits branching morphogenesis, and this is accompanied by decreased GDNF expression, and loss of c-ret at the UB tips (Bullock et al., 1998; Kispert et al., 1996). Even at early time points, when c-ret expression is still preserved, addition of exogenous GDNF alone does not completely restore UB branching morphogenesis (Sainio et al., 1997), again suggesting that other molecules are required in this process. One possibility is that depletion of sulfated proteoglycans also affects pleiotrophin-mediated signaling or binding. Accordingly, the invention provides that pleiotrophin functions as a MM-derived morphogen acting upon the UB. Moreover, the results support the idea that UB branching morphogenesis is likely to be regulated by more than a single factor. At least two soluble factors, GDNF and pleiotrophin can be used to induce morphogenetic changes. Other heparin-binding agents including heregulin can also be used in the methods of the invention along with or substituted for pleiotrophin, for example. GDNF may initiate the UB outgrowth (Sainio et al., 1997), and pleiotrophin (or other heparin binding agent such as heregulin) may induce proliferation and/or facilitate branching. In addition, the invention provides a combination of factors such as pleiotrophin GDNF and may include FGF. FGF and related members may play a facilitory role, since FGF1 potentiates the effects of purified pleiotrophin on UB branching morphogenesis, though by itself (with GDNF present) exerts little if any morphogenetic activity.

Inhibitory factors can also play a role in morphogenesis regulation and can include members of the transforming growth factor-beta family (Sakurai and Nigam, 1997). For example, gradients of positive and negative factors, most of which are matrix-bound, may exist in the mesenchyme and stroma. By regulating proliferation, apoptosis and the expression of morphogenetic molecules at branch tips, branch points and stalks, the global and local balance of these stimulatory and inhibitory factors provide crucial determinant of branching patterns during collecting system development. In addition, sulfated proteoglycans should also be present either to maintain expression of these soluble factors or to secure their binding sites. At later stages, other soluble factors such as HGF and/or EGF receptor ligands may play supplementary roles, either during branching (particularly in the later stages) or shaping/maturation of tubular structures.

It should also be noted that the concentration-dependent morphogenetic changes induced by pleiotrophin in the UB (FIG. 5A, see Original Patent), raise the possibility that pleiotrophin represents a "classical morphogen," in the sense of activin in early Xenopus development (Green and Smith, 1990). Such a molecule might be expected to produce different phenotypic changes in the responding tissue depending upon the concentration of the molecule to which it is exposed. In this regard, the basement membrane of the developing UB, to which pleiotrophin is localized, could potentially act as a "reservoir." Release of pleiotrophin from the basement membrane at the UB tips, perhaps through digestion by matrix degrading proteases, can produce a local concentration gradient, resulting in increased growth and proliferation of tips, while lower amounts of pleiotrophin along the length of the stalk would appear to induce elongation of the forming tubule. Such a concentration gradient of pleiotrophin provides a basis for modulating the shape and directionality of the developing UB. In another aspect of the invention, artificial matrices comprising biocompatible material may be used as a support for cell growth. Such matrices may be designed such that concentrations of pleiotrophin may change at desired branch points within the matrix material. In this manner, kidney cells may grow and proliferate through the matrix and only branch at locations where pleiotrophin concentrations are at a level to induce branching morphogenesis.

In another aspect of the invention there is provided methods and composition for treating acute renal failure (ARF). Acute renal failure is a common condition with a poor outcome. Nearly 200,000 patients in the United States develop ARF annually and the mortality rate remains high in spite of improved techniques of renal replacement therapy. At the present time, treatment of ARF is primarily supportive since no specific therapy is available. Recently improved understanding of the cellular and molecular mechanisms of ARF may be translated into new therapeutic approaches.

The most common etiologies of ARF are renal ischemia or the administration of a nephrotoxic drugs. In many cases, severe ARF results from a combination of renal hypoperfusion and nephrotoxicity. Severe ARF is associated with injury to renal tubular cells leading to both cellular dysfunction and ultimately cell death. The recovery from ARF is characterized by de-differentiation of the tubular epithelium, proliferation, and regeneration of the tubular cell. In many respects, these recovery processes recapitulate the processes fundamental to normal embryonic renal development, such as growth factor expression, matrix digestion, and intercellular tight junction modification.

The conditioned medium (BSN-CM), as well as purified subfractions, appear to be among the most powerful tubulogenic and proliferative growth factors known and have not been studied in the context of acute renal failure. There is a very strong correlation between known branching tubular morphogens and enhancement of recovery in experimental acute renal failure.

The invention provides a novel growth factor-based therapy for acute renal failure (ARF). ARF is primarily caused by acute tubular necrosis (ATN). ATN is manifested by renal cell death, and the recovery from ATN is characterized by a recapitulation of early developmental processes, particularly growth factor-induced tubulogenesis similar to that seen when kidney cells are contacted with BSU-CM or a composition comprising pleiotrophin. As discussed herein, the invention provides methods and compositions that utilize key mediators of kidney development in the treatment and prevention of ARF.

This invention provides a method of tubulogenesis and morphogenesis of kidney cells (e.g., UBs) in vivo in a subject comprising administering to the subject a composition comprising pleiotrophin in an amount effective to induce UB morphogenesis. In one embodiment, kidney cells are treated with a pleiotrophin ex vivo, and the kidney cells are then transplanted into a subject with diminished kidney function.

In another aspect of the invention, there is provided pharmaceutical compositions useful for treating tissue related disorders such as renal disorder and disorders associated with morphogenesis and/or tubulogenesis. In this aspect of the invention, the pharmaceutical composition comprises pleiotrophin in an amount that is effective to treat the particular disorder for which it will be administered. Generally, the pharmaceutical composition comprising pleiotrophin will be administered to a subject at a therapeutically effective doses to prevent, treat, or ameliorate a disorder or disease (e.g., a kidney disease). A therapeutically effective dose refers to that amount of pleiotrophin or pleiotrophin and other agents sufficient to result in a viable or measurable decrease in the disease or disorder symptoms.

Toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD.sub.50/ED.sub.50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such a composition lies typically within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in the invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Thus, for example, pleiotrophin and its physiologically acceptable salts and solvates can be formulated for administration by inhalation or insufflation (either through the mouth or the nose), or oral, buccal, parenteral, intravenous, intramuscular, intradermal, intracerebral, or rectal administration.

For oral administration, the pharmaceutical compositions can take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose; fillers, e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate; lubricants, e.g., magnesium stearate, talc, or silica; disintegrants, e.g., potato starch or sodium starch glycolate; or wetting agents, e.g., sodium lauryl sulphate. The tablets can be coated by methods well known in the art.

Liquid preparations for oral administration can take the form of solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, e.g., lecithin or acacia; non-aqueous vehicles, e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to give controlled release of the active compound.

For administration by inhalation (e.g., where the disorder may be associated with the pulmonary system or may be more readily taken up by the body), the compounds are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Pleiotrophin can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds (e.g., pleiotrophin) can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

This invention provides a method of treating a subject suffering from kidney failure, which comprises administering to the subject, an effective amount of a composition comprising pleiotrophin to treat the subject's kidney failure.

This invention provides methods and compositions comprising pleiotrophin to stimulate endogenous kidney progenitors to differentiate and form tubules for the purpose of restoring kidney function in a subject.

In another embodiment, the goal is to create clonal subcolonies of specifically engineered, functional kidneys that are suitable for use in screening of drugs and agents to measure effects on specific kidney functions as well as for use in transplantation. Using the compositions and methods of the invention, it is possible to culture kidney components derived from a single UB in order to develop a kidney tissue in vitro. Normal kidney development consists of the reciprocal interaction between the UB and the metanephric mesenchyme (MM) as described herein. The methods of the invention provide the ability to reduce the amount of tissue that must be sacrificed from cadaver tissue or through invasive biopsy techniques in order to obtain sufficient tissue for in vitro generation of kidney tissue for screening and transplantation. The invention provides methods and compositions whereby a single progenitor cell or tissue is capable of generating multiple kidney tissues in vitro.

Using the methods of the invention and compositions of the invention it is possible to stimulate UB morphogenesis and MM epithelialization. The methods of and compositions provide for kidney development through co-culturing of MM and UBs in culture systems. The kidney development is initiated when the metanephric mesenchyme (MM) induces an epithelial outgrowth of the Wolfian duct, termed the ureteric bud (UB). The MM induces the UB to elongate and branch, and through multiple iterations of this branching program, the UB subsequently develops into the renal collecting system. In turn, the branching UB initiates the reciprocal induction of the MM and stimulates it to epithelialize and to form the tubular nephron. These nephrons then connect with the UB derived collecting system allowing drainage of fluid (e.g., urine) into the bladder in vivo. This process is repeated through successive iterations to achieve the approximately 1 million nephrons present in the adult human kidney.

For many years, this process of reciprocal induction was thought to depend on direct cell contact between the MM and UB. Although mesenchyme cleanly isolated from the UB could be induced to form tubules by nonspecific inducers such as spinal cord, the UB was not able to undergo branching morphogenesis in vitro when isolated from the surrounding mesenchyme.

As described herein, the invention overcomes these difficulties whereby the isolated UB undergoes impressive branching morphogenesis in vitro when exposed to several growth factors pleiotrophin (PTN) alone or in combination with other factors (e.g. GDNF and/or FGFI). Such other factors include glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor-1 (FGF1), and proteins secreted by a mesenchymally derived cell line. In addition, the invention provides methods for regulating processes that govern UB branching morphogenesis, such as the matrix-binding requirements vis-a-vis integrin expression, the dependence of branching morphogenesis on heparin sulfate proteoglycans, and the roles of positive and negative modulators of branching. Other growth factors present in media conditioned by ureteric bud cells that can induce differentiation of isolated mesenchyme cultured in vitro include, for example, leukemia-inhibitory factor (LIF) and FGF2.

These advances have lead to the possibility of recombination of subcultures of each of the components of the kidney--the ureteric bud and the mesenchyme. Using the methods and compositions of the invention the isolated UB and mesenchyme can be recombined in vitro and grown in an autonomous fashion. The resultant kidney is morphologically and architecturally indistinguishable from a "normal" kidney and can be used for transplantation, as a source for the study of kidney function, and as a resource for determining drug-effects upon kidney function. Furthermore, the invention provides methods for partitioning/propagating the kidney or the cultured isolated ureteric bud into smaller fragments and support the in vitro development of these subfractions through several "generations." The methods of the invention further allow for these subfractions to be recombined with fresh mesenchyme to develop additional kidney tissue through the induction of the mesenchyme. Furthermore, these nascent nephrons formed contiguous connections with limbs of the branched UB. Consequently, the invention provide in vitro engineered kidney tissue comprising a population of renal primordia suitable for transplantation and derived from a single progenitor.

The methods provided by the invention utilize a novel, in vitro, approach to renal engineering that provides an ability to create colonies of kidney tissue (in some cases comprising genetically engineered cells) suitable for transplantation. In one aspect of the invention, an embryonic ureteric bud is separated from the surrounding metanephric mesenchyme and each component (e.g., the MM and UB) is cultured in isolation. In one aspect of the invention, the UB and/or the MM is then modified in vitro (as described herein) in a tailored fashion to express a specific polynucleotide (e.g., a heterologous polynucleotide) to obtain a desired function. The components are then recombined to allow the morphogenesis and development of kidney tissue in vitro (e.g., to generate a in vitro engineered kidney). The in vitro engineered kidney can then be used in transplantation, to screen for desired biological function, and/or to screen for agents, which modulate kidney function.

For example embryonic UB are dissected and separated from the surrounding metanephric mesenchyme (MM) (FIG. 13, see Original Patent). The dissected cells are then used to grow an arborized structure from the isolated UB, which were subdivided into smaller fractions and used to induce additional generations of UBs that grow and branch in vitro. The continued growth and branching ins maintained in the closure of MM cells by culturing and subculturing the UBs in the presence of BSU-CM or a culture medium comprising pleiotrophin (e.g. PTN and GDNF+FGFI). The subfraction of UBs were used through three generations. The UB generations were then recombined with freshly isolated metanephric mesenchyme. The cells retained the ability to induce dramatic tubular epithelial differentiation of the mesenchyme (FIGS. 14 and 15, see Original Patent). Furthermore, there appeared to be connections between induced tubules of the mesenchyme and terminal portions of the UB thereby providing a conduit between the tubule and urinary collecting system. The generated kidney opens up the possibility of uniquely tailoring specific components of either the nephron (derived from the mesenchyme) or tie collecting system (derived from the UB) in vitro in a potentially functional and transplantable organ.

Although the concept of transplanting fetal organs is not novel, there are several advantages to the approach provided by the invention. First, by culturing the UB and MM in vitro, the invention provides the unique opportunity to modulate each of their functions in a site-specific manner. For example, transfection of the mesenchyme with constructs expressing organic ion transporters would lead to increased capability to handle drugs and toxins, insertion of genes coding for growth factors, such as insulin-like growth factor (IGF), would lead to markedly enhanced in vitro engineered kidney development and improved functionality, insertion of immunomodulatory elements, such as repressors of co-stimulatory molecules, could be used to improved immune tolerance; stimulation of branching in the UB can lead to an increased number of resultant nephrons and improved renal functionality. Thus, there are numerous of ways to design a in vitro engineered kidney with tailored function. Furthermore, by subcloning either kidneys or UBs, the invention provides the potential to develop a large number of kidneys derived from a single progenitor, thus removing concerns surrounding limited supply of transplantable tissue. Third, it is possible to create a chimeric kidneys using the UB as a scaffold and recombining the UB with heterologous mesenchymal cells. These mesenchymal cells could be derived from embryonic stem cells that, when exposed to kidney derived signals from the UB induce differentiation of the renal mesenchymal cells into epithelial tissues. In normal adults, stem cells originating in the bone marrow repopulate portions of the kidney and differentiate into renal cells, and it is likely that embryonic stem cells also posses this ability. If it were possible to create such a chimeric kidney, it would greatly decrease the likelihood of immunologic problems that currently make transplantation difficult.

The approach provided by the methods and compositions of the invention, whereby in vitro engineered kidneys developed and/or are designed to possess specific functions, such as improved immune tolerance or enhanced tubular secretion of substrate, offer original approaches to transplantation and kidney therapy. Furthermore, creating clonal populations of in vitro engineered kidneys creates the potential for development of organ propagation from a single tissue. This approach is potentially applicable to other epithelial tissues such as lung and pancreas.

Large numbers of patients are awaiting renal transplantation. The development of techniques that allow for safe organ transplantation, especially without excessive immunosuppression, would have enormous commercial application. Transplantation of embryonic tissue appears to be well tolerated. The methods provided by the invention allow for the development of colonies of subcloned in vitro engineered kidneys that have been specifically tailored to express certain functions, and are immune-naive. Immune naive means that the cells lack "self" identification as the cells were fetally or stem cell derived and therefore should be immune tolerant.

Methods of transfecting and transforming cells are known in the art. For example, methods of transfecting/transforming kidney cell are known and include the following methods. Tomita et al. (Biochem. and Biophys. Res. Comm. 186:129-134, 1992) report a method for in vivo gene transfer into the rat kidney. They utilize HVJ (Sendai virus) and liposome methodology. In this protocol, plasmid DNA and a nuclear protein are coencapsulated in liposomes and later cointroduced into cells. The reporter gene utilized in these studies was the SV40 large T antigen. The gene transfer can be performed by inserting a cell or culture of kidney tissue with a liposome suspension. Transfection/transformation of the kidney cells can be assay by detecting SV40 large T antigen immunohistochemically. A study by Zhu et al. (Science 261:209-11, 1993), reports the use of a particular cationic liposome DNA mixture to deliver genes with high efficiency into a vast number of endothelial cells in a rat. Moullier et al. (Kidney International 45:1220-1225, 1994) provides a first report of an adenoviral-mediated gene transfer into a kidney.

As used herein, the term "transfect" or "transform" refers to the transfer of genetic material (e.g., DNA or RNA) of interest via a vector into cells of a mammalian organ or tissue (e.g., kidney/renal tissue). The vector will typically be designed to infect mammalian kidney cell. The genetic material of interest encodes a product (e.g., a protein polypeptide, peptide or functional RNA) whose production by kidney cells is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme or (poly)peptide of therapeutic value. Examples of genetic material of interest include but are not limited to DNA encoding cytokines, growth factors and other molecules which function extracellularly such as chimeric toxins, e.g., a growth factor such as interleukin-2 (IL-2) fused to a toxin, e.g., the pseudomonas exotoxin, dominant negative receptors (soluble or transmembrane forms), truncated cell adhesion or cell surface molecules with or without fusions to immunoglobulin domains to increase their half-life (e.g., CTLA4-Ig). For example, cells of an organ or a tissue do not express a gene product encoded by the genetic material prior to transfection or transformation. Alternatively, infection of the cells of an organ or a tissue may result in an increased production of a gene product already expressed by those cells or result in production of a gene product (e.g., an antisense RNA molecule) which decreases production of another, undesirable gene product normally expressed by the cells of that organ or tissue. Generally, the genetic material encodes a gene product, which is the desired gene product to be supplied to the cells of that organ or tissue. Alternatively, the genetic material encodes a gene product, which induces the expression of the desired gene product by the cells of that organ or tissue (e.g., introduced genetic material encodes a transcription factor which induces the transcription of the gene product to be supplied to the subject). Furthermore, the genetic material could simply contain a polynucleotide, e.g., in the form of single stranded DNA to act as an antisense nucleotide. A genetic material infected into a cell of an organ or a tissue via a vector is in a form suitable for expression in the cell of the gene product encoded by that genetic material. Accordingly, the genetic material includes coding and regulatory sequences required for transcription of a gene (or portion thereof) and, when the gene product is a protein or peptide, translation of the gene product encoded by the genetic material. Regulatory sequences which can be included in the genetic material include promoters, enhancers and polyadenylation signals, as well as sequences necessary for transport of an encoded protein or peptide, for example N-terminal signal sequences for transport of proteins or peptides to the surface of the cell or for secretion, or for cell surface expression or secretion preferentially to the luminal or basal side. Enhancers might be ubiquitous or tissue or cell specific or inducible by factors in the local environment, e.g., inflammatory cytokines.

As used herein, the term an "effective amount" refers to a level of expression of a heterologous polynucleotide transfected or transformed into a kidney cell, which brings about at least partially a desired therapeutic or prophylactic effect in an organ or tissue infected by the method of the invention. For example, expression of genetic material of interest can then result in the modification of the cellular activities, e.g., a change in phenotype, in an organ or a tissue that has been infected by the method of the invention. In one embodiment, an effective amount of the expression of a heterologous genetic material of interest results in modulation of cellular activity in a significant number of cells of an organ transfected or transformed with the heterologous polynucleotide. A "significant number" refers to the ability of the vector to infect at least about 0.1% to at least about 15% of the renal endothelial cells or UBs. Typically, at least about 5% to at least about 15% of the cells are transfected/transformed. Most commonly, at least about 10% of the cells are transfected/transformed.

A vector refers to a polynucleotide molecule capable of transporting another nucleic acid to which it has been linked into cells. Examples of vectors that exist in the art include: plasmids, yeast artificial chromosomes (YACs) and viral vectors. However, the invention is intended to include such other forms of vectors which serve equivalent functions and which become known in the art subsequently hereto.

The efficacy of a particular expression vector system and method of introducing genetic material into a cell can be assessed by standard approaches routinely used in the art. For example, DNA introduced into a cell can be detected by a filter hybridization technique (e.g., Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase polymerase chain reaction (RT-PCR). The gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product, such as an enzymatic assay. If the gene product of interest to be expressed by a cell is not readily assayable, an expression system can first be optimized using a reporter gene linked to the regulatory elements and vector to be used. The reporter gene encodes a gene product, which is easily detectable and, thus, can be used to evaluate the efficacy of the system. Standard reporter genes used in the art include genes encoding .beta.-galactosidase, chloramphenicol acetyl transferase, luciferase and human growth hormone.

The method of the invention can be used to infect kidney cells to obtain designer kidneys (e.g., genetically engineered kidney cells). As used herein, the term "kidney cells" is intended to including UB and MM cell types as well as the other 15 different cell types, e.g., glomerular cells, mesangial cells, interstitial cells, tubular cell, endothelial cells, are intended to be encompassed by the term "kidney cells".

The method of the invention can also be used to infect a kidney tissue generated ex vivo. For example, in a transplant setting, a kidney is engineered by the methods of the invention, the "in vitro engineered kidney" is then perfused (e.g., the collecting ducts) with a vector carrying genetic material of interest.

One potential application of the invention is in renal allograft or xenograft tissue transplantation. In this aspect, kidney tissue generated by the methods and compositions of the invention are transfected/transformed with an agent (e.g., delivered to MM cells and/or UBS) that results in organ tolerance or might help in the post-operative period for decreasing the incidence of early transplant rejection or function (e.g., due to acute tubular necrosis). Either the organ can be made less immunogenic so as to reduce the number of host T cells generated and/or the endothelial cells (e.g., endothelial cells derived from MM) can be altered so as to prevent the adhesion/transmigration of primed immune T-cells or killer effector T-cells (e.g., by use of IL-2-toxin fusion proteins). Moreover, genes transfected/transformed into in vitro engineered kidney tissue, such as nitric oxide synthetase (NOS), prior to transplantation could also serve to protect the organ post transplantation. These strategies make clinical sense since it is well known that early rejection episodes and malfunction lead to a worse long-term graft survival. Therefore, prevention of acute rejection and preservation of function immediately post transplant are of particular importance. Delivery of the genetic material (i.e., the heterologous polynucleotides) for this purpose can be done using methods known in the art including utilizing an adenovirus vector, lipofection, or other techniques known in the art. In addition to the heterologous polynucleotides mentioned above, these vectors can carry additional sequences comprising anti-sense constructs to one or more cell adhesion molecules (involved in lymphocyte homing) or dominant negative constructs to these molecules, or antisense constructs to MHC antigens in the transplant or locally immune suppressive lymphokines such as interleukin-10 (IL-10) or viral IL-10 or chimeric toxins which would preferentially kill T-cells, e.g., IL-2 toxin fusion protein. It is also possible that one could interfere with the recognition part of the immune system by, for example, the local secretion of CTLA4-IgG fusion proteins. This list of candidate polynucleotides is not exhaustive. Those skilled in the art of transplantation know of others. The genes could be delivered with constitutive promoters or with appropriate inducible enhancers.

The source of cells used to ultimately engineer kidney tissues need not be derived from the kidney per se. Pluripotent embryonic stem (ES) cells and pluripotent embryonic germ (EG) cells can serve as progenitor cells for a variety of differentiated cell types and recent work with human ES and EG cells has opened the doors to some potential beneficial therapeutics for diseases as diverse as neurodegenerative disorders and cardiomyopathy. For example, mouse ES cells treated with retinoic acid in vitro can be induced to form neurites in vitro. When transplanted back into rat spinal cord, these neuronal cell precursors survive, differentiate into distinct and functional neuronal cell types, such as astrocytes, myelin-producing oligodendrocytes, and neurons, migrate to areas of injury/repair, and lead to increased functional outcome. When cultivated in vitro, human ES and EG cells form 3-dimensional aggregates called embryoid bodies (EB) that can then differentiate into derivatives of all three primary germ cell layers. Furthermore, these EB can be induced to differentiate into specific but different cellular subsets based on conditioning by certain growth factors, such as FGF and TGF-beta. Cells derived from ES and EG cells can organize and can display a diverse set of functional properties, such as contractility in ES-derived cardiomyocytes and insulin secretion from ES-derived "islet-like" cells. Finally, multipotent adult bone marrow-derived mesenchymal stem cells (MSC) may serve as an adult source of stem cells readily available for engineering of tissues derived from mesenchyme. Within the context of the kidney, cells derived from the bone marrow were found to repopulate or regenerate a variety of renal territories, including the glomerular podocyte and mesangium, interstitium, and renal epithelial tubule. Recent work suggests that there may exist one or more self-renewing "renal stem cells" found within the MM that can differentiate into the myofibroblasts of the renal stroma and/or endothelium.

As discussed herein, the invention provide methods and compositions whereby isolated UBs can be co-cultured and stimulated by extrinsic factors to induce branching and kidney development. For example, whole isolated intact UB (cleanly separated from surrounding MM) can be induced to undergo branching morphogenesis in vitro in a manner similar to UB culture. Suspension of the isolated UBs within, or on, a natural or artificial biocompatible substrate (e.g., Matrigel/collagen gel) and when exposed to a mixture of mesenchyme-cultured media augmented with GDNF, results in the isolated unbranched UB rapidly forming a polarized, extensively branched structure with an internal lumen. As described further herein, pleiotrophin, which induced branching of UB-derived cells, also induces impressive branching morphogenesis of the whole ureteric bud. Other factors have been found to modulate the branching effect of BSN-CM or pleiotrophin on the UB. This modulation is typically branch-promoting, elongation promoting, or branch-inhibiting. For example, FGF1 induced the formation of elongated UB branching stalks whereas FGF7 induced amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. TGF-beta, which inhibits branching in several cell-culture model systems, also appears to inhibit the branching of the isolated UB. Endostatin, which is a cleavage product of collagen XVIII normally found in the UB basement membrane, also selectively inhibits branching of the UB. Growth factors, such as LIF, have been isolated from UB conditioned media and induce mesenchymal-to-epithelial transformation of cultured mesenchyme. Other factors, such as FGF2, appear to promote survival but not differentiation of mesenchyme.

The branching isolated ureteric bud retains the ability to induce freshly isolated mesenchyme when recombined in vitro without exogenous growth factors. By carefully removing the surrounding biocompatible matrix from the cultured UB and placing mesenchyme in close proximity, the UB continues to grow and extend branches into the surrounding mesenchyme. Furthermore, the mesenchyme condenses in areas where the UB has extended branches, and then epithelializes in a manner similar to normal kidney development. This has wide-ranging implications for in vitro kidney engineering, including the ability to independently culture ureteric bud and metanephric mesenchyme, modify their phenotypes in vitro, and then recombine them. For example, it may be possible to develop engineer kidneys with properties such as enhanced drug or toxin secretion by in vitro modification of organic anion transporters, improved immune tolerance by suppression of costimulatory molecules, as discussed herein. The invention demonstrates that these recombined "in vitro engineered kidneys," comprised of cultured isolated UB and freshly isolated mesenchyme, form cohesive intact tubular conduits. That is, the nascent tubular nephron, derived from MM, has a tubular lumen in direct connection with the tubular lumen of the collecting system, derived from the UB.

In addition, the culture system and methods of the invention provide the ability to propagate the isolated UB in vitro through several generations. For example, isolated UB are cultured in vitro and induced to undergo branching morphogenesis in the presence of BSN-CM, FGF1, and GDNF. After 8 days, the cultured bud is subdivided into approximate 3rds and resuspended within a suitable biocompatible matrix (matrigel/collagen gel). This 2nd generation bud is further subdivided after another 8 days of culture, and the 3rd generation bud is cultured for 8 days (thus yielding at least 9 subdivided buds from one progenitor bud). These subsequent clonal generations of cultured UB retain the ability of the progenitor bud to induce mesenchyme upon in vitro recombination. The buds also retained the capacity to form cohesive conduits with the mesenchymally-derived tubules that they induced. Thus, the invention provides the ability to develop and propagate a clonal, expanded, and long-lived colony of ureteric buds, derived from a single progenitor bud that retains the properties of the progenitor. Using similar techniques with the MM, it is possible to develop colonies of mesenchyme derived from a single progenitor mesenchyme that can then be recombined with a propagated UB.

Whole embryonic kidneys can be propagated in a similar manner in vitro. After culturing these kidneys for 3 days, it is possible, using the methods of the invention, to subdivide into approximate 3rds the whole cultured kidney and then propagate the subsequent generations in vitro. At least 3 generations, yielding 9 kidneys, were generated from a single progenitor kidney using the methods and compositions of the invention. Thus, the methods and compositions of the invention provide the ability for expansion of syngenic rudiments in vitro prior to transplantation into suitable hosts.

In many tissue-engineering technologies, an extrinsic biocompatible scaffold is required to provide orientation and support to the developing tissue. In one sense, the native polymeric basement membrane (BM) is a bioactive scaffold directing the normal development of the kidney. BM constituents such as endostatin can directly influence branching of the UB, and other components, such as HSPGs, can indirectly regulate growth by binding and releasing growth factors. These processes may be developmentally coordinated by the regulated expression of matrix metalloproteases (e.g. MMP2, MMP9, and TIMP-1). The bioartificial scaffolds used in tissue engineering can be synthetic or biologic and contain or can be coated with ECM constituents, such as collagen or proteoglycans. Exciting new techniques in materials science are emerging that allow these scaffolds to be impregnated with drugs, proteins, or even DNA, and thus may be more biologically relevant. By combining a truly bioactive scaffold with cultured pluripotent cells, such as ES cells, or multipotent cells, such as MSCs or other progenitor cells derived from the mesenchyme, it may be possible to coordinate inductive signals required to derive/engineer an organ such as the kidney. For example, vary the concentration of factors at points where branching is desired, it is possible to design a tissue having a predicted number of branch point.

Biocompatible support materials (biocompatible scaffolds) for culturing kidney cells include any material and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b) allows cells to grow in more than one layer. A number of different materials may be used as a culture support, including, but not limited to, nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE; teflon), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures, cellulose, gelatin, dextran, collagen, decellulularized tissue (both allogenic and xenogeneic), and like.

In one aspect of the invention a ureteric bud is used as a bioactive scaffold, which could then serve as a biologically-relevant orchestrator (conductor) of the complex inductive signals that underlie "normal" renal development. In an organ such as the kidney, where development is dependent on coordinated interactions between epithelium and mesenchyme, utilization of a biologically active epithelial scaffold to induce proper differentiation, maturation and integration of surrounding multipotent cells may provide a unique opportunity to modify specific cellular functions in vitro but yet to retain the complex organizational direction required to develop a mature kidney. This principle is applicable to engineering of other organs, such as lung, liver, pancreas, salivary gland or breast, which are also dependent upon mesenchymal-epithelial interactions within the context of a branching epithelial derivative. In one experiment, the UB, co-cultured with lung mesenchyme, began to express surfactant protein. Accordingly, the UB serves as a scaffold for a number of novel "chimeric" organs. The ability to independently culture and then combine mesenchymally-derived elements with epithelial-derived elements allow for the integration of cellular and organ-based approaches to tissue engineering. This approach would allow one to modify cell-based elements in vitro to possess certain desirable properties but still take advantage of an organ-based approach to tissue engineering.

In another aspect of the invention, the kidney cultures of the invention (UB alone, MM alone, and co-cultures thereof) may be used in vitro to screen a wide variety of compounds, such as cytotoxic compounds, growth/regulatory factors, pharmaceutical agents, and the like to identify agents that modify kidney function and/or cause cytotoxicity and/or kidney cell death or modify kidney proliferative activity. Examples of such agents or compounds include growth factors, peptides, and small organic molecules.

To this end, the cultures (e.g., UB primary cells, UB cell lines, MM cells, whole organ cultures, MM/spinal cord co-cultures, and UB/MM co-cultures) are maintained in vitro and exposed to the compound to be tested. The activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture or by its ability to modify the function of kidney cells (e.g., UB proliferative capacity, branching capacity, MM epithelialization capacity, particular gene expression, cell size, cell morphoogy, protein expression, and the like). This may readily be assessed by vital staining techniques, ELISA assays, immunohistochemistry, PCR, microarray analysis, and the like. The effect of growth/regulatory factors on the kidney cells (e.g., UBs, MMs) may be assessed by analyzing the cellular content of the culture, e.g., by total cell counts, and differential cell counts, including the number of branch points. This may be accomplished using standard cytological and/or histological techniques including the use of immunocytochemical techniques employing antibodies that define type-specific cellular antigens. The effect of various drugs on normal cells cultured in the culture system may be assessed. For example, UB primary cells or cell lines may be cultured in vitro under conditions that stimulate branching morphogenesis/tubulogenesis (e.g., in the presence of BSN-CM, pleiotrophin, or pleiotrophin+other factors). A test compound is then contacted with the culture and the effect the test compound has on branching morphogenesis/tubulogenesis can be compared to a control, wherein a difference is indicative of an compound that increases or decreases branching morphogenesis.

The cytotoxicity to kidney cells (e.g., human UBs and co-cultures of MM and UBs) of pharmaceuticals, anti-neoplastic agents, carcinogens, food additives, and other substances may be tested by utilizing the culture system of the invention.

First, a stable, growing kidney culture comprising UB and/or MM cells is established. Then, the culture is exposed to varying concentrations of a test agent. After incubation with a test agent, the culture is examined by phase microscopy to determine the highest tolerated dose--the concentration of test agent at which the earliest morphological abnormalities appear. Cytotoxicity testing can be performed using a variety of supravital dyes to assess cell viability in the culture system, using techniques well-known to those skilled in the art.

Once a testing range is established, varying concentrations of the test agent can be examined for their effect on viability, growth, and/or morphology of the different cell types constituting the kidney culture by means well known to those skilled in the art.

Similarly, the beneficial effects of drugs may be assessed using the culture system in vitro; for example, growth factors, hormones, drugs which enhance kidney formation, or activity (e.g., branching activity) can be tested. In this case, stable cultures may be exposed to a test agent. After incubation, the cultures may be examined for viability, growth, morphology, cell typing, and the like as an indication of the efficacy of the test substance. Varying concentrations of the drug may be tested to derive a dose-response curve.

The culture systems of the invention may be used as model systems for the study of physiologic or pathologic conditions. For example, in a specific embodiment of the invention, the culture system can be optimized to act in a specific functional manner as described herein by modifying genome of the cells.

The kidney culture system of the invention may also be used to aid in the diagnosis and treatment of malignancies and diseases. For example, a biopsy of a kidney tissue may be taken from a subject suspected of having a malignancy or other disease or disorder of the kidney. The biopsy cells can then be separated (e.g., UBs from MM cells etc.) and cultured in the according to the methods of the invention. UBs from the subject can be co-cultured with normal (e.g., heterologous MM cells) to determine biological function of the UBs compared to UBs derived from a normal kidney. Similarly MM cells from the subject can be cultured with normal UBs to examine MM function and activity. In addition, such cultures obtained from biopsies can be used to screen agent that modify the activity in order to identify a therapeutic regimen to treat the subject. For example, the subject's culture could be used in vitro to screen cytotoxic and/or pharmaceutical compounds in order to identify those that are most efficacious; i.e. those that kill the malignant or diseased cells, yet spare the normal cells. These agents could then be used to therapeutically treat the subject.

Where in vitro engineered kidney tissue is generated according to the methods and compositions of the invention transplantation of the tissue can be performed as follows. Surgery is performed on the recipient subject to expose one or both kidneys. The in vitro engineered kidney tissue is implanted directly into/adjacent to the recipient subject's kidney to result in the formation of chimeric kidney, or into a fold of the omentum where it forms a chimeric kidney that functions independently of the recipient's kidney. The omentum, which is a membranous structure the connects the bowels, is a highly vascularized tissue sufficient for the transplantation of the in vitro engineered kidney. The in vitro engineered kidney can be placed adjacent to any portion of the omentum, however, in one aspect the in vitro engineered kidney is transplanted at or near an omental fold. In another aspect, the in vitro engineered kidney is transplanted at an omental fold located near one of the recipient's kidneys, particularly near the ureter, so that the developing ureter of the metanephros can be readily connected to the recipient's excretory system.

When implanted into the recipient's kidney, an incision, large enough to receive the in vitro engineered kidney tissue is made in the fibrous renal capsule that surrounds the recipient's kidney. The location of the incision can be anywhere in a viable portion of the recipient's kidney, but most conveniently will be at an external border of the kidney that is easily accessible during surgery. The in vitro engineered kidney tissue is placed between the capsule and the cortex of the recipient kidney.

The implanted in vitro engineered kidney tissue is allowed to grow within the recipient under conditions that allow the tissue to vascularize. Suitable conditions may include the use of pre or post-operative procedures to prevent rejection of the implant as well as the administration of factors (e.g., pleotrophin, FGF1, GNDF, and the like) that stimulate tubulogenesis and/or morphogenesis of the in vitro engineered kidney tissue. Immunosuppression techniques (in the absence or combined with genetically engineered techniques) such as cyclosporin A (CSA) to prevent rejection of the donor tissue are known in the art.

Claim 1 of 6 Claims

1. A method of propagating ureteric bud cells in culture, the method comprising: a) culturing ureteric bud cells on a biocompatible matrix in a pleiotrophin-supplemented medium, wherein the medium has not previously been conditioned with cells from metanephric mesenchyme, to induce the ureteric bud cells to undergo branching morphogenesis to generate a population of ureteric bud cells with tubular branches; and b) subculturing said ureteric bud cells with tubular branches on a biocompatible matrix in a pleiotrophin-supplemented medium, wherein the medium has not previously been conditioned with cells from metanephric mesenchyme.

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