Developmental approaches to kidney tissue engineering

【摘要】  Recent advances in our understanding of the developmental biology of the kidney, as well as the establishment of novel in vitro model systems, have potential implications for kidney tissue engineering. These advances include delineation of the roles of a number of growth factors in the developmental programs of branching morphogenesis and mesenchymal differentiation, a new understanding of the roles of the extracellular matrix, identification of potential "renal" stem cells, the ex vivo propagation and subsequent recombination of isolated components of the kidney, and successful transplantation of renal primordia into adult hosts. This review will examine these advances in the context of approaches to tissue engineering. Finally, novel approaches that synthesize advances in both cell-based and organ-based approaches are proposed.

【关键词】  kidney development in vitro organ culture

THE PREVALENCE AND INCIDENCE of chronic kidney disease in the United States have reached epidemic numbers. By some measures, over 8.3 million Americans have a glomerular filtration rate of 60 ml/min or worse (i.e., at least stage 3 chronic kidney disease); of these, a significant number will develop end-stage renal disease (ESRD) ( 7, 23 ). Dialysis is the major treatment modality for ESRD, but it has significant limitations in terms of morbidity, mortality, and cost; allogeneic transplantation options, which provide significant mortality benefit and are ultimately less costly, are hampered by an extreme shortage of available organs. In fact, the rate of kidney transplantation per dialysis patient years has declined over the past decade ( 43 ).

Acute renal failure (ARF) is also quite common and affects up to 7% of all hospitalized patients, with a mortality rate that ranges from 20 to 70% depending on the studied population ( 5, 21 ). For a number of reasons, including aggressive care of an older patient population with potentially nephrotoxic treatments and procedures, the mortality rate due to ARF has not changed over the past 20 years despite advances in life support, medical care, and renal replacement therapies ( 40 ).

Within this context of ESRD and ARF, there is an increasing interest in developing novel therapies for kidney disease. The translation of recent advances in our understanding of normal kidney developmental programs, ranging from epithelial branching morphogenesis to mesenchymal differentiation, into new therapies holds great promise, especially when brought to bear on the field of renal tissue engineering.

KIDNEY DEVELOPMENT

Kidney development originates with the invasion of an undifferentiated metanephric mesenchyme (MM) by the ureteric bud (UB), an epithelial outgrowth of the Wolffian duct. Through complex, reciprocal inductive events, the UB undergoes branching morphogenesis and ultimately forms the arborized collecting system. The MM condenses at the tips of the branching UB, differentiates into epithelium (so-called "mesenchymal-to-epithelial transformation" or MET), and forms the glomerulus and tubular nephron. In the past decade or so, significant advances in the understanding of the cellular and molecular underpinnings of kidney development and renal recovery after injury have been made. These advances, recently reviewed ( 26, 28, 39 ) and described below, include the identification of growth factors responsible for the reciprocal induction of mesenchyme and epithelium, the description of local extracellular and pericellular environments that permit branching morphogenesis and MET, and the characterization of intracellular pathways responsible for translating the inductive signals into molecular and cellular action. In general, most tissue engineering strategies attempt to manipulate or harness developmental or regenerative processes ( Table 1 ).

Table 1. Developmental approaches to kidney tissue engineering

TISSUE ENGINEERING

Cell-Based Strategies

Broadly defined, tissue engineering strategies encompass both cell-based therapies and organ-based therapies. Cell-based strategies often manipulate one or several cell types, in various stages of development, into forming the essential components of the required tissue. Within the context of in vitro kidney engineering, one of the earliest descriptions of this strategy involved the isolation and in vitro propagation of cells derived from both the undifferentiated MM and unbranched UB ( 35 ). This cell culture work was based on earlier approaches that demonstrated that when placed atop an extracellular matrix (ECM) gel seeded with mature renal epithelial cells [Madin-Darby canine kidney (MDCK) or murine inner medullary collecting duct (mIMCD3) cells], either the whole embryonic kidney or MM could induce branching tubulogenesis of the seeded epithelial cells in the absence of cell contact ( 4, 36, 38 ). This suggested that a simple cell culture system using cell lines derived from the MM and UB could be devised to create branching tubular structures similar to those that form in the intact embryonic kidney.

As illustrated in Fig. 1, A - C, when exposed to media conditioned by the cultured mesenchyme, the embryonic UB cells, as single-cell suspensions within a biologically relevant ECM, proliferated and migrated. After several days of culture, the multicellular structures branched and formed polarized tubules with internal lumens. The program of UB branching morphogenesis, initiated by exposure to soluble growth factors but intrinsic to the cell, was dependent on an ECM that included Matrigel (a basement membrane extract that contains laminin, type 4 collagen, sulfated proteoglycans, and fibronectin). Optimal branching occurred only with a specific concentration of these ECM components. This ECM can bind heparin-binding growth factors, and heparin-binding growth factors have subsequently been identified within the mesenchyme-conditioned media ( 36 ). These growth factors, including pleiotrophin and transforming growth factor (TGF)- ( 37 ), have been shown to induce or modulate branching morphogenesis when directly applied to the UB cell culture model. Several other heparin-binding growth factors, including members of the FGF family such as FGF1 and FGF2, can also induce some degree of branching ( 35; Steer DL, Sakurai H, and Nigam S, unpublished observations). It appears that the optimal context for branching morphogenesis involves a specific combination of growth factors, rather than one factor alone, as well as an active ECM that can bind and release these growth factors and thus present a specific growth factor "microenvironment" to the individual cells of the UB.

Fig. 1. Models of in vitro branching morphogenesis. A - C : cell-based approach. Cells are isolated from the ureteric bud (UB; A ) and cultured as single-cell suspensions within a suitable matrix ( B ). When exposed to growth factors found in mesenchyme-conditioned media, the UB cells proliferate, migrate, and form complex 3-dimensional polarized structures with internal lumens ( C ). D - F : organ-based approach. The intact UB is isolated from the surrounding mesenchyme and resuspended within a suitable matrix ( A and B ). When exposed to growth factors, the UB undergoes multiple iterations of branching and elongation.

While the functional capacity of these UB cell-derived tubules has not been further characterized, this approach utilizing cells isolated from discrete portions of the renal primordia has relevance to both tissue engineering and reparative medicine. A number of the growth factors that regulate in vitro tubulogenesis in cell culture are also regulated during renal recovery from injury ( 6 ) ( Table 2 ). Individual growth factors, such as IGF-1, hepatocyte growth factor (HGF), and FGF, which are expressed in the developing mesenchyme and induce early tubulelike structures from UB cells and other epithelial cells, have been utilized to enhance renal recovery after acute tubular injury in experimental models. In a human clinical placebo-controlled randomized trial, however, IGF-1 failed to enhance the recovery from ARF ( 13 ). Other growth factors have been shown to be important in branching tubule formation of the isolated UB. Newer concepts include utilization of a cocktail of "tubulogenic" growth factors that more accurately reflects the developmental milieu and that may potentially further enhance recovery ( 22 ). In terms of augmentation of traditional hemodialysis, a "renal assist device," which is composed of a membrane coated with cultured porcine proximal tubule cells, has been developed ( 14, 15 ). These cells support unidirectional transport, and when added in series to a traditional hemofiltration circuit, the resultant bioartificial kidney appears to significantly improve outcomes of acutely uremic dogs in models of endotoxin-mediated sepsis ( 10 ). Human trials are in progress. Finally, some groups have been interested in utilizing tissue engineering techniques to create bioartificial ureters and lower urinary tract structures based on collagen matrices impregnated with host cells ( 2 ).

Table 2. Growth factors involved in renal development and in in vitro engineering

In fact, the source cells used to ultimately engineer kidney tissues may not be derived from the kidney per se. Pluripotent embryonic stem (ES) cells can serve as progenitor cells for a variety of differentiated cell types, and recent work with human ES cells has opened the doors to some potential beneficial therapeutics for diseases as diverse as neurodegenerative disorders ( 46 ) and cardiac disease ( 12 ). For example, mouse ES cells treated with retinoic acid in vitro can be induced to form neurites in vitro ( 20 ). 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 cells form three-dimensional aggregates, termed 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-. Cells derived from ES cells can organize and display a diverse set of functional properties, such as contractility in ES-derived cardiomyocytes ( 45 ) and insulin secretion from ES-derived "islet-like" cells ( 41 ). 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 renal tubule and glomerular mesangium ( 11, 29 ). Other investigators, however, were not able to demonstrate similar plasticity of hematopoietic stem cells ( 44 ). 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 ( 24 ), but the signals that govern the differentiation of these cells have yet to be determined. The coordination of developmental signals required to produce the large number of terminally differentiated cells thought to exist within the developed kidney (26 by some accounts) ( 1 ) may be a limiting factor in using purely cell-based approaches to engineer a complete kidney in vitro.

Therapeutic cloning strategies provide some insight into normal renal development as well as provide concepts in xeno-, allo-, and autotransplantation. One group has recently employed a nuclear transfer technique to generate cloned bovine fetuses from an adult animal ( 17 ). Renal cells were then isolated from their E56 cloned fetus, passaged and expanded in vitro, and seeded onto a collagen-coated polycarbonate membrane that was then subcutaneously implanted into the nuclear donor. The renal cells self-aggregated into "renal units" within the membrane, were subsequently vascularized by the genetically identical host, and ultimately appeared to produce a concentrated urinelike fluid. As may be expected, there was no evidence of acute rejection of these bioartificial renal units.

Organ-Based Strategies

Several laboratories are investigating embryonic organ-based approaches to kidney engineering and transplantation. As opposed to purely cell-based strategies, these strategies take advantage of the fact that the multicellular embryonic metanephros has already begun the coordinated process of differentiation and is, in essence, programmed to develop into a kidney. These approaches extend some of the concepts developed in cell-based kidney engineering strategies, and many of the growth factors and ECM elements are similar. Whole embryonic kidney organ culture as an experimental model for renal development has been utilized for many years. For example, this model system has yielded insight into the developmental roles that pericellular sulfated proteoglycans play in mediating epithelial-mesenchymal interactions (Steer DL, Shah MM, Bush KT, Stuart RO, Sampogna RV, Esko JD, Meyer TN, Schwesinger C, and Nigam S, unpublished observations). The individual contribution of the MM or UB to renal development, however, is difficult to determine in this whole kidney culture model. Recently, culture of isolated intact UB and MM has helped tease out these influences. Whole isolated intact UB (i.e., composed of hundreds of cells and cleanly separated from surrounding MM) can be induced to undergo branching morphogenesis in vitro in a manner similar to the three-dimensional cell culture model (i.e., single-cell suspension within an ECM) ( 31 ) ( Fig. 1, D - F ). Interestingly, in these organ-based experiments, the matrix requirements and several of the growth factor requirements are similar to the previously detailed cell-based experiments. After suspension within a Matrigel/collagen gel and when exposed to a mixture of mesenchyme-cultured media augmented with glial cell line-derived neurotrophic factor, the unbranched isolated UB rapidly forms a polarized, extensively branched structure with an internal lumen. Again, major changes in the matrix composition did not support this branching morphogenesis of the UB. Pleiotrophin, which induces branching of UB-derived cells, also induces impressive branching morphogenesis of the whole UB ( 36 ). Recently, other factors have been found to modulate the branching effect of MM cell-derived conditioned medium on the UB ( 30 ). These modulatory effects are 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-, 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 ( 16 ). Recent work to determine factors that promote mesenchymal differentiation has taken a similar approach. Growth factors, such as leukemia-inhibitory factor, have been isolated from UB-conditioned media and induce mesenchymal-to-epithelial transformation of cultured mesenchyme ( 3 ). Other factors, such as FGF2, appear to promote the survival but not the differentiation of mesenchyme ( 25 ). These findings indicate that a variety of growth factors act in a coordinated fashion to organize the branching and elongation of the UB and the survival and differentiation of the MM.

The branching cultured UB retains the ability to induce freshly isolated mesenchyme when recombined in vitro ( 42 ). As depicted in Fig. 2, by the careful removal the surrounding Matrigel-based ECM from the cultured isolated rat UB and placement of freshly harvested 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 significant finding has implications for in vitro kidney engineering. It appears possible to independently culture UB and MM, modify their phenotypes in vitro, and then recombine them. For example, it may be possible to develop "designer" 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, etc. As described below, we have recently demonstrated that within these recombined "neokidneys," 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 ( 42 ).

Fig. 2. Recombination of isolated UBs and mesenchyme. Cultured UBs ( A ) can be recombined with isolated mesenchyme ( B ). The recombined structures display invasion of the mesenchyme by extensions of the UB, and the mesenchyme undergoes mesenchymeto-epithelium transformation ( C ). Adapted from Ref. 42.

It has now been shown that it is possible to propagate the isolated rat UB in vitro through several generations ( 42 ). The isolated UB was cultured in vitro and induced to undergo branching morphogenesis in the presence of BSN-CM, FGF1, and glial cell line-derived neurotrophic factor. As exemplified in Fig. 3, A - C, after 8 days of in vitro culture, the UB was subdivided into approximate thirds and each subdivision was resuspended within a suitable Matrigel/collagen gel. This second-generation bud was further subdivided after another 8 days of culture, and the third-generation bud was cultured for 8 days. Each progenitor bud thus yielded at least nine subdivided buds. These subsequent clonal generations of cultured UB retain the ability of the progenitor bud to induce mesenchyme on in vitro recombination. Importantly, these buds also retained the capacity to form contiguous conduits with the mesenchyme-derived tubules that they induced. This strategy would provide the ability to develop and propagate a clonal, expanded, and long-lived colony of UBs, derived from a single progenitor bud, that retains the inductive properties of the progenitor bud. Using similar techniques with the MM, it may be possible to develop colonies of mesenchyme derived from a single progenitor mesenchyme that can then be recombined with a propagated UB.

Fig. 3. Propagation of isolated UBs and kidneys. Cultured UBs ( A ) can be subdivided into thirds, and each portion can be cultured independently ( B ). After 8 days of culture, each derivative can be subdivided again and recultured independently for an additional 8 days ( C ). After 3 days of in vitro culture, whole embryonic kidneys can be subdivided and propagated similarly through 2 subsequent generations ( D - F ). Adapted from Ref. 42.

Whole embryonic kidneys can be propagated in a similar manner in vitro ( 42 ). After culturing these kidneys for 3 days, we were able to subdivide the whole cultured kidney into approximate thirds and then propagate the subsequent generations in vitro ( Fig. 3, D - F ). We were again able to extend this to three generations, yielding nine kidney rudiments from a single progenitor kidney. Although functional analysis has not been done, the resultant kidney rudiments appeared morphologically intact and retained the overall architecture of the progenitor metanephros. This technique may allow for the expansion of syngenic rudiments in vitro before transplantation into suitable hosts.

Several groups have recently described transplantation of embryonic metanephroi into adult hosts. In one model ( 32, 33 ), prevascular metanephroi were harvested, cultured ex vivo, and transplanted across the rodent MHC or even xenogeneically, such as pig to rodent ( 34 ). Remarkably, the transplanted metanephroi grew, developed a largely host-derived vasculature, and produced a concentrated filtrate. Finally, these prevascular metanephroi appeared to be immunogenically relatively well tolerated and required little if any adjunctive immunosuppression. The major limitation to therapeutic use was the relatively small amount of end product ultimately created.

Metanephroi harvested from E70 humans and transplanted either intraperitoneally or into the renal subcapsular space also appear to recruit a host-derived vasculature ( 8 ). Furthermore, these metanephroi differentiate, maintain viability for over 60 days, and express a genetic profile similar to "normal" human kidneys. There appears to be a well-defined temporal window that is optimal for transplantation of either pig metanephroi or human metanephroi ( 9 ). This period of metanephric development, which is before vascularization by the recipient of the donor metanephric structure, allows for optimal growth, differentiation, and immunological acceptance in transplantation of both human and porcine embryonic renal precursors.

Scaffolds

In many tissue engineering technologies, an extrinsic biocompatible scaffold is required to provide orientation and support to the developing tissue. In many of the experiments detailed above, the ECM acts as a kind of scaffold. In one sense, the native polymeric basement membrane 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 heparan sulfate proteoglycans, 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) ( 18, 27 ). The bioartificial scaffolds used in tissue engineering can be synthetic or biological and are often 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 simplify the coordination of inductive signals required to engineer an organ such as the kidney.

One novel proposed methodology would be to use the UB as a bioactive scaffold that could then serve as a biologically relevant 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 retain the complex organizational direction required to develop a mature kidney. This principle may be applicable to engineering of other organs, such as the lung, liver, pancreas, salivary gland, or breast, which are also dependent on mesenchyme-epithelium interactions within the context of a branching epithelial derivative. In one interesting experiment, the UB, when cocultured with lung mesenchyme, expresses surfactant protein ( 19 ). It is thus conceivable that the UB could serve as a scaffold for a number of novel "chimeric" organs. The ability to independently culture and then combine mesenchyme-derived elements with epithelium-derived elements may allow for the integration of cell- 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.

CONCLUSION

There are a number of cell-based and organ-based approaches to renal tissue engineering that take advantage of newly discovered developmental programs and experimental strategies. These approaches fundamentally begin with cells (either as individual cells or as structured within an organ) that are induced to develop in a coordinated and regulated fashion by growth factors within a supportive and biologically active matrix. Normal kidney development integrates these various processes, and it is likely that effective tissue engineering strategies will require similar integration. Thus the ultimate goal of therapy will likely dictate which combination of cells, growth factors, and matrix is ultimately employed. For example, renal recovery from acute tubular necrosis may be enhanced by coadministration of isolated cells with tubulogenic combinations of growth factors, whereas generation of transplantable renal tissue may require whole UBs recombined in vitro with isolated mesenchyme in a proper growth factor and ECM milieu. Some kidney tissue engineering strategies are presently being evaluated as therapeutic modalities, but most are undergoing further elaboration at the benchtop and await translation to the bedside.

Address for reprint requests and other correspondence: S. Nigam, Depts. of Medicine, Pediatrics, and Cellular Molecular Medicine, Univ. of California, 9500 Gilman Dr., La Jolla, CA 92093-0693 (E-mail: snigam{at}ucsd.edu