The present disclosure provides human renal stem cells. Also described are human renal stem cells isolated from the papillary region of the human kidney and methods of isolating the same. Also described are methods for culturing, characterizing, and differentiating the same, including methods for identifying human renal stem cells that are positive for Nestin and CD133, and methods for allowing the cells to differentiate into neurons.

Description of the Invention

BACKGROUND OF THE INVENTION

Many renal disorders, including acute and chronic kidney disease, common genetic diseases such as autosomal dominant polycystic kidney disease (ADPKD), renal cell carcinoma, glomerulonephritis and other pathological conditions, lead to kidney damage or loss. In the United States, approximately one person in five reaching 65 years of age will undergo organ-replacement during their remaining life span. It is predicted over 2 million patients will suffer from end-stage renal disease by 2010 [2]. Approximately 58,000 patients in the United States and 300 patients in New Mexico are currently on the waiting list for a kidney transplant, with some waiting for several years before an appropriate donor can be found. Substantial fractions of patients (12-17%) on the waiting list are designated as "most-difficult-to-transplant." Despite the advances in kidney transplantation, a significant shortage of donor organs severely limits treatment for these patients and requires many to remain on dialysis for extended periods of time. The quest for alternate organ restoration methods has resulted in rapid progression of new approaches, such as therapeutic cloning and embryonic/adult stem cell therapy (reviewed in 2-5).

The role of embryonic stem cells in the treatment of pathophysiological disorders has recently attracted significant interest and has been the subject of much controversy. The pluripotency of embryonic stem cells presents the possibility of their use for replacement of damaged kidney tissue. First indications show that mouse embryonic stem cells, develop an endoderm-like tissue in culture and in kidney transplants [6]. However, the in vitro generation of mesodermal precursors that give rise to the adult kidney remains unsubstantiated.

Furthermore, the transplantation of ES cells includes potential complications of immune rejection, teratomas and other cancers [4, 7]. The problems associated with the use of embryonic stem cells and the potential benefit of autologous transplantation has spurred an intensive search for adult stem cell populations. It is increasingly clear that stem cells may originate not only from embryonic, but also from adult tissue, including adult brain, bone marrow, skin and gut, where they can be recruited for organ repair after injury [8, 9]. Until recently, adult stem cells in kidney had not been identified [2-4, 10]. Progenitor-like cells involved in recovery from renal injury in an animal model were first identified in 2003 [11], and in 2004 a population of rodent adult kidney stem cells derived from rat and mouse renal papilla was isolated and characterized [1]. Adult rodent renal stem cells were identified on the basis of their low cell division rate. To detect them, a pulse of bromodeoxyuridine (BrdU) was administered to rat and mouse pups, and after 2-months, a small population of BrdU-positive cells was detected in the renal papilla. These cells had a plastic phenotype and, following injection into the renal cortex, they incorporated into the parenchyma. Adult renal stem cells exhibited features characteristic of other stem cells and expressed both mesenchymal and epithelial proteins [1]. However, while rats and mice serve as important animal models for human physiology, one cannot necessarily predict from the results obtained in a rat or mouse model system that the same results will be obtained in a human system. For example, differing results may be obtained between an animal model, such as rat and mouse, and the human system because of differences in the immune systems, physiology, life spans, and/or hematopoetic pathways between humans and rodents.

Another potential source for endogenous replacement of damaged or lost renal tubular epithelia may be surviving tubular epithelial cells themselves. These cells have a capacity to adapt to the loss of neighboring cells through dedifferentiation and proliferation. Both glomerular and tubular epithelial cells can regress to an embryonic mesenchymal phenotype and can either stimulate a regenerative potential of neighboring surviving cells or replace the damaged cells [10, 11]. A functional role of these cells in organ repair is based on their ability to migrate, proliferate and produce growth and trophic factors.

Accordingly, there remain many open questions regarding the characteristics that define adult human renal stem cells, as well as their precise differentiation potential. However, the identification of adult human renal stem cells holds promise for numerous benefits including the development of renal tissue replacement strategies.

DETAILED DESCRIPTION

The present disclosure provides a method for isolating adult human papillary cells that display stem cell markers and exhibit the capacity to differentiate toward different 20 lineages. The present disclosure further provides methods for determining if the renal papilla in humans provides a niche for a subpopulation of cells that serve as adult renal stem cells and that are able to differentiate into fully differentiated tubular epithelia.

There is general agreement that adult stem cells should exhibit continuous self-renewal and the capacity to give rise to multiple differentiated cell lineages [3]. Consequently, stem cells have the unusual property of being able to undergo asymmetric cell division, wherein one progeny remains a stem cell and the second progeny is a non-stem cell sister or transient amplifying precursor that undergoes a finite number of divisions prior to giving rise to fully mature, differentiated cells. Asymmetric DNA strand segregation is believed to be a key component of asymmetric stem cell division and serves to reduce the potential for mutation introduced by errors during DNA replication [12]. As shown in FIG. 1 (see Original Patent), in this scenario the chromosomes segregate non-randomly such that the stem cell retains the original chromosome set, while the transient amplifying precursor receives the newly replicated DNA. Such immortal DNA strand cosegregation mechanisms have been shown to be operative in intestinal cells and in cultured cell lines overexpressing p53 [9, 13, 14]. Relatively straightforward microscopic and flow cytometry based assays have been developed to assess the asymmetric cell division properties of stem cells based on such non-random DNA strand segregation [14].

For example, one microscopic assay is based on quenching of the fluorescent Hoechst dye in the presence of DNA labeled by bromodeoxyuridine. When cells are dividing symmetrically and labeled with bromodeoxyuridine, followed by Hoechst dye, all cells exhibit uniformly quenched Hoechst fluorescence. In contrast, if the bromodeoxyuridine labeled cells undergo asymmetric division, the brornodeoxyuridine is lost from the stem cell population and these cells are subsequently brightly stained with Hoechst. The profile of bromodeoxyuridine label retention measured by flow cytometry several passages after labeling may also reveal a population of asymmetrically dividing cells. For example, if cells are dividing symmetrically, all progeny will exhibit uniform label dilution. If cells are dividing asymmetrically, a population of unlabeled cells is expected.

Non-differentiated pluripotent cells, participating in kidney regeneration, are expected to undergo major phenotypic transitions, analogous to those occurring during normal kidney development. The major difference being that in regenerating kidney, progenitor cells have to undergo the necessary developmental stages to form new tubules in the context of the adult, fully mature kidney. It is known that tubulogenesis involves the integration of many cellular processes, such as differentiation, polarization, shape change, proteolysis, growth, mitosis, regulated cell death, motility, adhesion, signaling, ion fluxes, cytoskeletal organization and membrane traffic [15]. Moreover, tubules can be formed by cells under different conditions and induced by different factors. To predict the behavior of non-differentiated pluripotent cells in adult kidney, 2D filter cultures and 3D in vitro tubulogenesis models may serve as useful experimental tools. Filter cultures enable optimal cell polarization in vitro and are useful for monitoring the acquisition of such epithelial hallmarks as apical and basolateral plasma membrane polarity and transepithelial resistance due to the presence of tight junctions [15]. At present, three-dimensional (3D) extracellular matrix gel culture (composed of collagen I) is widely used for in-vitro tubulogenesis studies [15]. This model provides an "in vivo-like" culture condition and represents a simplified model for true tissue/organ development.

An important and complicated stage in kidney repair is epithelial redifferentiation, which requires a mesenchyme-to-epithelium transition of progenitor cells, such as embryonic stem cells or dedifferentiated survivor cells. Growth factors, including insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF) and epidermal growth factor (EGF), in combination with matrix derived cues, most likely contribute to de-novo generation of tubular epithelia. The search for actual inducing signals that directly promote conversion of mesenchyme to epithelia is still in process. Wnt proteins (particularly Wnt4) were implicated to be essential for mesenchyme transition during tubulogenesis [16]. In addition, the soluble tubulogenic factors, such as leukemia inhibitory factor (LIF), transforming growth factor beta2 (TGF beta2), fibroblast growth factor 2 FGF2 (alone and in combination), were identified as the most striking inducers of renal cell differentiation [17-19]. The ability of the cells to respond to inductive signals depends on the expression of a number of renal specific transcription factors such as WTI and Pax2, among others [20, 21].

The key events in the recovery of renal tissue from injury are cell dedifferentiation, proliferation and subsequent redifferentiation back into fully polarized epithelial cells. Morphogenic processes that could occur during kidney reconstruction (mediated by dedifferentiated surviving cells or transplanted embryonic stem cells) would be highly dependent on the expression of proteins, implicated in renal tissue development. Among these, members of the cadherin super-family are likely to play a critical role [10]. Several cadherins, such as E-cadherin, R-cadherin, cadherin-6, cadherin-1 1, N-cadherin and P-cadherin, are expressed during nephrogenesis [22]. In addition, the kidney-specific cadherin 16 has recently been implicated in the differentiation of adult stem-like cells from mouse kidney [23].

Cadherin-mediated adhesion is critical during embryogenesis, morphogenesis and in the normal function of epithelial tissues. Cadherins promote Ca2+-dependent cell-cell adhesion through hemophilic interactions with cadherins on neighboring epithelial cells [24,25]. Via their carboxy-terminal cytoplasmic tail, classical cadherins associate with cell cytoskeletal molecules. For example, E-cadherin, which is highly expressed in epithelia, associates with .beta.-catenin and .alpha.-catenin to promote interaction with the actin cytoskeleton [25-27]. The E-cadherin/.beta.-catenin complex plays a dual role. On the one hand, it stabilizes cellular adhesion through cytoskeletal attachment and on the other hand, the protein complex modulates signal transduction and consequently imparts gene expression and cell cycle control. During the process of mesenchyme-to-epithelium transition in kidney organogenesis, N-cadherin is replaced by E-cadherin [28]. Expression of different cadherins and so-called cadherin switches (when one of the cadherins is replaced by another member of the same cadherin protein family) is temporally and spatially regulated during kidney development. This precisely regulated expression is implicated in such critical stages as initial aggregation of mesenchymal cells, mesenchyme-to-epithelium transition, cell proliferation, migration, adhesion and other complex signaling events leading to the formation of mature nephrons.

In vivo animal studies have been used to complement in vitro differentiation analyses and provide critical information regarding the ability of stem cells to repopulate specific tissues. Bone marrow derived hematopoietic stem cells have been studied most extensively with animal studies being used to demonstrate pluripotency [29]. Thus, cells were shown to differentiate not only into the hematopoietic lineage, but also to give rise to epithelia of the liver, lung and gut. In the case of kidney, there is conflicting evidence as to the potential of circulating hematopoietic stem cells to contribute to renal repair [30, 31]. The most recent study suggests that increased cytokine levels enhance kidney recovery following ischemic injury by reducing inflammation rather than by increasing the incorporation of bone marrow derived cells into regenerated tubules [31]. The quest to identify and characterize kidney-derived stem cells has also relied on animal models. Rodent renal ischemic injury models have been used to provide evidence for a direct role of tubular epithelial cells in tubule regeneration [11], as well as to support the role of rodent papillary cells in kidney regeneration [1]. Stem cell transplantation with or without renal injury has in turn been used to monitor the capacity of the implanted cells to reintegrate within the intact kidney [1, 32]. In one such study, human CD133 positive cells were xenotransplanted to a SCID mouse following induction of renal ischemia by glycerol injection and the incorporation of the human cells into kidney tubules was scored [32]. At present, the nature and identity of the progeny of renal papillary cells in vivo remains an open question. On the whole, establishing the optimal conditions for induction of differentiation, as well as elucidating the requisite protein expression profiles involved in restoration of injured kidneys, is of particular importance for successful implementation of renal stem cell therapy. There are a number of potential therapeutic applications for stem cells in the future. One is the possible introduction of multipotent cells of different origins for replacement of damaged nephrons following renal injury and monitoring the regenerative process. Another is genetic modification of multipotent cells (stable expression of a selected protein) and their implantation into host renal tissues. This approach may offer improved control over indiscriminate direct injections of gene vectors [10]. The correction of genetic defects would be helpful for the treatment of inherited renal diseases, such as ADPKD. Further, cell therapy could be used as more direct approach for effective delivery of therapeutic drugs. Pluripotent cells, such as adult and embryonic stem cells or dedifferentiated kidney cells, have a rare natural capability of regenerating damaged tissue. Cell therapy will be one of the most advanced therapeutic tools in the nearest future. Tissue based therapy will also support presently used methods, such as kidney transplantation, where genetically modified pluripotent cells may contribute to minimizing immunologic rejection of the transplanted kidney.

As stated above, according to one embodiment, the present invention includes methods for the isolation of renal papillary cells and/or renal stem cells. Such cells may be isolated from one or more human kidneys. As used herein, the term "isolated" means that a cell population is removed from its natural environment. As used herein, the term "purified," means that a cell population is essentially free from any other cell type.

The cells of the present invention may be isolated from a region deep within the inner medullary region of the kidney. In situ in the kidney, the cells of the present invention may reside within the loops of Henle, the tubules found deep within the kidney papilla.

According to another embodiment, the present invention also includes isolated cells populations with a marker staining pattern similar to the marker staining pattern of isolated human renal papillary cells, wherein the cell populations are isolated from a cell source other than the renal papillary region of a human kidney. Such populations may be isolated, for example, from placental cord blood, bone marrow, embryonic stem cell populations, neonatal stem cell populations, and/or somatic stem cell populations. Such markers may include, but are not limited to CD133 and nestin.

According to yet another embodiment, the present invention also includes methods of culturing, co-culturing, maintaining and/or storing isolated human renal papillary cells so that further differentiation does not occur.

The isolated human renal papillary cells described herein may possess the ability to differentiate into specialized cells having one or more structural and/or functional aspects of a physiologic kidney. The present invention also includes methods of culturing, co-culturing maintaining and/or storing isolated human renal papillary cells so that differentiation occurs. For example, hypoxic conditions, ischemic injury, or conditions mimicking ischemic injury may be used to induce isolated human renal papillary cells to differentiate. As another example, the isolated human renal papillary cells may be co-cultured with other types of cells, such as differentiated adult human kidney tubule cells or embryonic neural stem cells. Accordingly, using various methods described herein, isolated human renal papillary cells may be induced to differentiate into different types of cells such as, for example, into tubular epithelial cells or neuronal cells. According to still another embodiment, the present invention also includes such populations of differentiated cells.

The isolated human renal papillary cells described herein possess the ability to differentiate into specialized cells having one or more structural and/or functional aspects of a physiologic kidney. For example, isolated human renal papillary cells may be induced to differentiate into, for example, tubular epithelial cells or may be used to repopulate kidney tubules.

According to another embodiment, the present invention also includes gene expression profiles of isolated human renal papillary cells. Such gene expression profiles may be used in the identification of markers for stem cells. Such gene expression profiles may be used for monitoring, for example, proliferative potential and differentiation status of stem cells. Such gene expression profiles include, but are not limited to, gene expression profiles from undifferentiated cell populations, gene expression profiles from differentiated cell populations, and gene expression profiles from cell populations obtained from individuals. Individuals from which gene expression profiles may be obtained include, but are not limited to, individuals with normal kidney function, individuals suffering from a kidney disorder, individuals suspected of carrying one or more genes for a heritable kidney condition, individuals undergoing treatment for a kidney disorder, and individuals undergoing treatments with nephrotoxic side effects. Gene expression profiles may be obtained by any known methods. For example, gene expression profiles may be obtained using the Human Genome U133 Plus 2.0 Genechip.RTM. Array (Affymetrix, Santa Clara, Calif.).

According to yet another embodiment, the isolated human renal papillary cells disclosed herein may be administered to a subject for the treatment of a kidney disorder. The cells to be administered include undifferentiated cells and/or cells that have been induced to differentiate.

According to some embodiments, the isolated human renal papillary cells disclosed herein may include an exogenous polynucleotide. According to some embodiments, the exogenous polynucleotide may encode a therapeutic agent. According to another embodiment, the exogenous polynucleotide may include an expression vector.

According to a still further embodiment, the isolated human renal papillary cells of the present invention may be used in bioartificial kidneys such as, for example, the bioartificial kidney described in U.S. Pat. No. 6,150,164.

Those of skill in the art will be familiar with a wide variety of materials and methods that are suitable for use to perform the methods described in the present disclosure. For example, suitable materials and methods include, but are not limited to those described in U.S. Pat. Nos. 5,429,938; 6,060,270; 6,150,164; 6,410,320; and 6,458,588; and U.S. Patent Application Serial No. 20020119566 A1.

As stated above, according to one embodiment of the present invention, a potential adult stem cell population has been isolated and characterized from human kidney papilla. The cells occupy a niche that is deep within the inner medulla and relatively hypoxic providing protection against oxidative stress. Preliminary ultrastructural analyses show these cells in situ exclusively in the loops of Henle of tubules found deep within the kidney in papilla. The cells are embedded between the tubular epithelia and have a characteristic morphology with large nuclei and a limited cytoplasm that is readily distinguishable from that of tubular epithelial cells. These putative adult human renal stem cells express markers that are characteristic of stem cells, including CD133 and nestin. In the presence of serum or specific growth factors the cells are able to adopt tubular epithelial-like or neuronal-like phenotypes. In vitro studies show the papillary cells readily associate with cortical tubular epithelia, both on filter cultures and in 3D collagen gel cultures, and in serum-free, 2D cultures form structures resembling neurospheres.

Various methods for demonstrating that a subpopulation of human papillary cells constitutes adult renal stem cells that can differentiate into tubular epithelia are described below. Methods for characterizing isolated human papillary cells for their self-renewal potential, ability to give rise to tubular epithelia and their ability to repopulate kidney tubules are also described. At present there is limited information about the origin and identity of adult renal stem cells, making the work highly relevant for the development of tissue engineering and alternate renal tissue repair strategies.

Self-Renewal Potential and Asymmetric DNA Segregation

Two hallmarks ascribed to bona fide stem cells is their ability to undergo indefinite self-renewal and asymmetric DNA strand segregation. To assay self-renewal potential, primary human papillary cells can be maintained in culture under conditions designed to maximize the maintenance of undifferentiated multipotent adult progenitor cells. The cells can be continuously cultured and passaged prior to confluence. By measuring the cell number at each passage and monitoring the number of passages we can establish if renal papillary, CD133 and nestin positive cells can be maintained in culture long-term, for example, logarithmic growth for up to one year. Cells can be screened by flow cytometry for various markers of cell differentiation at the start and at defined intervals. Microscopic and flow cytometry based assays can be used to assess the potential for asymmetric DNA segregation. These studies enable the distinction between isolated human papillary cells that resemble a bona fide stem cell population and cells that are transient amplifying precursor cells.

Pluripotent Phenotype and Ability to Differentiate into Tubular Epithelia

A third hallmark ascribed to stem cells is the ability to give rise to cell populations with various differentiated phenotypes. In vitro cell differentiation assays can be used to characterize the potential of the isolated papillary cells to yield distinct cell populations with a particular emphasis on their ability to differentiate into tubular epithelia. In vitro, 2D filter cultures and 3D extracellular matrix gel systems are widely used to study renal tubule epithelial cell function. Epithelial cells grown on 2D filter cultures form cellular junctions and establish a transepithlial resistance. Epithelial cells, grown in 3D collagen gel cultures, form complex cyst-like or tubule-like epithelial structures. 2D and 3D cell cultures supplemented with select growth factors or tubulogenic factors can be studied as a function of time. To assess differentiation status, cell morphology, expression of differentiation specific markers, transepithelial resistance and tubulogenesis can be analyzed. Once a bona fide renal stem cell population is identified, a renal stem cell gene expression profile can be established relative to fully differentiated tubular epithelia to facilitate future stem cell isolation and characterization. Studies monitoring the differentiation potential of human papillary progenitor cells can provide insights into the possible utility of these cells in renal tubular epithelial tissue regeneration.

Contribution to Tubular Regeneration

A fourth hallmark ascribed to stem cells is in vivo regenerative capacity following renal injury. Such analyses necessitate animal studies. Pilot studies can be performed with xenotransplanted, immunosuppressed rats to evaluate the optimal model for in vivo functional analyses of the differentiation and regenerative potential of the putative human papillary stem cells. The incorporation of labeled human cells into various tubules versus the kidney parenchyma can be quantified, as can the dependence on renal injury for incorporation. An optimal animal model system can thus be defined, laying the foundations for more detailed animal studies.

Various embodiments of the present invention are illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly and as non-limiting. Accordingly, it will be appreciated that while may of the examples included herein describe the use of specific instruments, assay reagents, assay systems, material sources, etc., those of skill in the art will be familiar with many instruments, assay reagents, assay systems, material sources, and the like, which will or could be used for the same purposes. Accordingly, such description is intended to be exemplary in nature and should not be interpreted as limiting the invention to any of the specifically described instruments, assay reagents, assay systems, material sources etc.
 

Claim 1 of 8 Claims

1. Isolated human renal adult stem cells, where the human renal stem cells were isolated from only the papillary region of human kidney, wherein the renal adult stem cells are surface positive for CD133 and intracellular nestin.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.