REVIEW URRENT C OPINION
New strategies in kidney regeneration and tissue engineering Joseph S. Uzarski a,b, Yun Xia c, Juan C.I. Belmonte c, and Jason A. Wertheim a,b,d,e,f
Purpose of review The severe shortage of suitable donor kidneys limits organ transplantation to a small fraction of patients suffering from end-stage renal failure. Engineering autologous kidney grafts on-demand would potentially alleviate this shortage, thereby reducing healthcare costs, improving quality of life, and increasing longevity for patients suffering from renal failure. Recent findings Over the past 2 years, several studies have demonstrated that structurally intact extracellular matrix (ECM) scaffolds can be derived from human or animal kidneys through decellularization, a process in which detergent or enzyme solutions are perfused through the renal vasculature to remove the native cells. The future clinical paradigm would be to repopulate these decellularized kidney matrices with patient-derived renal stem cells to regenerate a functional kidney graft. Recent research aiming toward this goal has focused on the optimization of decellularization protocols, design of bioreactor systems to seed cells into appropriate compartments of the renal ECM to nurture their growth to restore kidney function, and differentiation of pluripotent stem cells (PSCs) into renal progenitor lineages. Summary New research efforts utilizing bio-mimetic perfusion bioreactor systems to repopulate decellularized kidney scaffolds, coupled with the differentiation of PSCs into renal progenitor cell populations, indicate substantial progress toward the ultimate goal of building a functional kidney graft on-demand. Keywords decellularization, extracellular matrix, induced pluripotent stem cells, kidney, tissue engineering
INTRODUCTION The scope of this review is to highlight new research advances that are of importance to the field of whole-organ, kidney tissue engineering, with particular emphasis on studies utilizing renal extracellular matrix (ECM) scaffolds derived from kidney decellularization. In this review, we provide an overview of the recent and early progress made toward the ultimate goal of developing functional kidney grafts in vitro on-demand.
RATIONALE FOR DEVELOPMENT OF ENGINEERED KIDNEYS The global prevalence of chronic kidney disease (CKD) is progressing at an alarming rate, and has correlated with the dramatic increase in the prevalence of obesity over the past 30 years, leading to type II diabetes, metabolic syndrome, and renal failure [1]. Despite the rise in the prevalence of
CKD, current renal replacement therapeutic options remain limited to either peritoneal-dialysis, hemodialysis or kidney transplantation. Kidney transplantation is the optimal treatment for patients suffering from end-stage renal disease because it improves long-term survival [2], leads to better quality of life [3], and is cost-effective compared with a Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, bDepartment of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, cGene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, d Institute for BioNanotechnology in Medicine, Northwestern University, Chicago Illinois, eChemistry of Life Processes Institute and fDepartment of Biomedical Engineering, Northwestern University, Evanston, Illinois, USA
Correspondence to Jason A. Wertheim, MD, PhD, 676 N. St. Clair St., Suite 1900, Chicago, IL 60611, USA. Tel: +1 312 695 0257; fax: +1 312 503 3366; e-mail: [email protected] Curr Opin Nephrol Hypertens 2014, 23:399–405 DOI:10.1097/01.mnh.0000447019.66970.ea
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Renal pathophysiology
KEY POINTS Perfusion decellularization is a reliable technique that has been used to produce acellular, bioactive renal ECM scaffolds from a variety of kidney sources, including rat, rhesus monkey, pig, and human. The specific structural arrangement and complex composition of the renal ECM, which vary among different compartments of the kidney (e.g., vascular, tubular) are significant in directing cellular proliferation, differentiation, morphological arrangement, metabolism, and function. Recently, hPSC differentiation into kidney-specific lineages (including ureteric bud and metanephric mesenchyme) has been independently achieved by several research groups, suggesting that differentiation protocols to generate mature kidney cell types (e.g., proximal tubule epithelial) may be on the horizon.
impossible, task with the current level of technology. The particular structural features of the kidney ECM (e.g., glomerular basement membrane) are critical for appropriate tissue-specific function, and extracellular signaling cues (e.g., integrin-binding ligands of the basement membrane, bound growth factors) are important for driving renal cell growth. A promising alternative approach to create scaffolds for whole kidney engineering would be decellularization of xenogeneic or allogeneic donor kidneys. Detergents, enzymes, or other cell-lysing solutions are perfused antegrade through the renal vasculature to remove the antigenic parenchyma from the entire renal matrix. This technique has been used to produce acellular whole-kidney scaffolds from rodent [6,7 ,8 ,9 ], pig [7 ,10 ,11 ], and human [7 ,12 ] kidneys. A number of kidney decellularization protocols have been reported, most of which rely on the use of detergents, such as the nonionic Triton X-100 [6,7 ,13 ] or the anionic sodium dodecyl sulfate (SDS) [6,7 ,8 ,9 ,11 –14 ], to solubilize and wash out cellular components (see Fig. 1). Other techniques to aid this process include the use of alternating freeze–thaw cycles [8 ,15 ], osmotic shock [6,7 ,8 ,9 ,10 ,11 –14 ,15 ,16], and deoxyribonuclease to degrade nuclear material [6,10 ,13 ]. Regardless of the protocol used, the resulting renal ECM must be rigorously evaluated to confirm sufficient cell removal (e.g., through DNA quantification, histological loss of nuclei or MHC antigens), retention of basement membrane proteins and growth factors (e.g., through immunohistochemical staining, ELISA, proteomic analysis), and preservation of the native ECM microstructure (e.g., through scanning electron microscopy, histological staining) [17]. Further, the capacity of the remaining scaffold to support cellular adhesion, growth, and stem cell differentiation must be evaluated in vitro to confirm the bioactivity of the renal ECM. &&
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long-term dialysis [4]. Unfortunately, despite the rising number of patients on the waiting list for kidney transplantation, the number of available donor kidneys from both cadavers and living donors has stagnated, and the inability to match supply with the growing demand essentially precludes extension of this therapy to all patients in need. To address this discrepancy, the concept of building a functional kidney graft on-demand using patient-specific stem cells has emerged, and in the past 2 years, substantial progress toward this longterm goal has been made. Theoretically, this approach would extend the option of kidney transplantation to more patients suffering from renal failure, potentially minimize or eliminate the need for lifelong immunosuppressive therapy, and reduce the lengthy waiting times during which many patients either die or are removed from the list because of the progression of pathophysiological conditions such as coronary artery disease during prolonged hemo-dialysis [5].
THE DERIVATION OF ACELLULAR RENAL EXTRACELLULAR MATRIX SCAFFOLDS THROUGH WHOLE-KIDNEY DECELLULARIZATION The kidney is a spatially heterogeneous organ, in terms of both cellular composition as well as the structural arrangement of proteins, glycosaminoglycans, and bound growth factors that collectively comprise the ECM. Fully recapitulating the intricate architecture and complex composition of the kidney de novo through scaffold engineering technologies [e.g., electrospinning, three-dimensional (3D) printing] would be a technically difficult, if not 400
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IMPLANTATION OF DECELLULARIZED KIDNEY MATRICES To date, few cases have been reported in which decellularized whole-kidney ECM scaffolds were transplanted, mainly because of the inherent thrombogenicity of the nonendothelialized vasculature. Orlando et al. [11 ] anastomosed the renal artery and vein of decellularized porcine kidneys to the aorta and vena cava, respectively, of recipient pigs. The authors observed sufficient blood flow through the scaffold without bleeding during 60 min of intraoperative monitoring, but noted extensive thrombosis throughout the nonendothelialized kidney scaffolds 2 weeks after implantation. &
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FIGURE 1. Whole kidney decellularization. Whole renal extracellular matrix scaffolds can be derived from human or animal kidneys by perfusing detergent or enzyme solutions through the renal vasculature. Shown in this figure is a representative macroscopic view of a Sprague-Dawley rat kidney undergoing a detergent-based decellularization protocol. As cellular material is removed from the scaffold, the kidney gradually fades in color. At the end of the decellularization process, the kidney is transparent in appearance, and the vasculature can easily be visualized. Scale bar: 5 mm.
However, the authors noted no sign of immunological rejection despite a nonspecific inflammatory response, suggesting acceptable compatibility of the decellularized allogeneic scaffolds with the host animals. More recently, Song et al. [7 ] implanted Sprague-Dawley rat kidneys that were either native, decellularized, or recellularized with human umbilical vein endothelial cells and rat neonatal kidney cells into immunodeficient rats in order to analyze rudimentary urine production. The authors noted no thrombosis or bleeding over the implantation period, although the specific duration of in-vivo perfusion was not reported. In order to effectively transplant decellularized kidney scaffolds over prolonged time periods, the vasculature must be preendothelialized in vitro to prevent thrombotic occlusion of the vessels upon implantation. &&
THE RENAL EXTRACELLULAR MATRIX: A SUPPORTIVE SCAFFOLD THAT REGULATES KIDNEY DEVELOPMENT AND STEM CELL DIFFERENTIATION The kidney ECM consists of a variety of proteins, glycosaminoglycans, and growth factors organized in elaborate spatial arrangements that constitute the vascular, glomerular, and tubular basement membranes, as well as the renal interstitium [18]. Specific cues from the renal ECM play essential roles in modulating cellular behavior, as shown in several recent studies (see Table 1) [7 ,8 ,9 ,13 ,14 ,15 ]. O’Neill et al. [15 ] recently demonstrated that decellularized ECM isolated from different regions of porcine kidneys (cortex, medulla, and papilla) differentially regulated the growth of papillae-derived, BrdU label-retaining kidney stem cells in a regionally specific manner. The papillary ECM (the purported renal stem cell niche) inhibited proliferation and increased the metabolic activity of renal stem cells grown on this region. Furthermore, kidney &&
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stem cells were more metabolically active on decellularized kidney ECM than on ECM derived from heart or bladder, indicating a degree of organ recognition by renal papillae-derived parenchymal cells. Similarly, Nakayama et al. [14 ] showed that human embryonic stem cells (ESCs) seeded onto decellularized rhesus monkey kidney ECM formed tubules and expressed several kidney tubule gene markers (e.g., Dipeptidase 1 renal and Heparan sulfate 6-O-sulfotransferase 1), whereas these results were not observed when the same cells were cultured on decellularized lung scaffolds. It has also been demonstrated that specific renal ECM protein additives, such as type I collagen and laminin, contributed to ex-vivo nephrogenesis, whereas others, such as type IV collagen, had an inhibitory effect [19 ]. This was accomplished in a novel organ culture system in which murine embryonic kidney rudiments were allowed to develop in the presence of surfaceimmobilized or solubilized ECM protein molecules, allowing the authors to investigate nephrogenesis in the presence of a variety of exogenous ECM signals. Thus, the specific make-up of the ECM localized in various compartments of the kidney is important for embryonic renal development and kidney-specific stem cell growth. Ross et al. [13 ] showed that mouse ESCs within the glomeruli and blood vessels of decellularized rat kidney scaffolds expressed the endothelial cellspecific markers BSLB4 lectin and vascular endothelial growth factor receptor-2 (VEGFR-2). Interestingly, the same research group previously discovered that some ESC aggregates seeded in the renal cortex expressed Ksp-cadherin, a distal tubule marker [6]. This would suggest that pluripotent stem cell (PSC) differentiation is somehow dependent on the specific niche of the ECM to which the cells are adhered. This concept was shown more recently in a study where mouse ESCs seeded into decellularized rat kidney scaffolds through the renal artery progressively lost
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Renal pathophysiology Table 1. Recent kidney recellularization experiments Authors
Species
Decellularization strategy
Cells utilized
Key results
Sprague-Dawley rat
1% SDS: 12 h; 1% Triton X-100: 30 min
Rat neonatal kidney cells; human umbilical vein endothelial cells
Partial restoration of renal filtration and electrolyte reabsorption in recellularized scaffolds
Sprague-Dawley rat
0.66% SDS: 1 h
Primary human osteoblasts; human umbilical vein endothelial cells
Osteoblast differentiation into osteocytes; increased osteocalcin deposition
Bonandrini et al. [9 ]
Sprague-Dawley rat
1% SDS: 17 h
R1 murine pluripotent ESCs
Loss of staining for Oct4 and increase in NCAM, Tie-2, and CD31 expression in glomeruli co-localized cells
Sullivan et al. [10 ]
Yorkshire pig
0.5% SDS: 36 h; DNase: overnight
Primary human cortical renal cells
Superior viability, proliferation on SDS-decellularized ECM compared to Triton X-100
Ross et al. [13 ]
Sprague-Dawley rat
3% Triton X-100, DNase, 4% SDS; not specified
B5/EGFP murine pluripotent ESCs
Positive staining for BsLB4 and VEGFR2 in ESCs colocalized in glomeruli and vessels
Nakayama & et al. [14 ]
Rhesus monkey (transverse kidney sections)
1% SDS: 7–10 days
WA09 human ESCs
Increased expression of kidney tubule gene markers DPEP1 and HS6T1
Yorkshire pig
0.02% trypsin: 2 h; 3% Tween: 2 h; 4% sodium deoxycholate: 2h
Murine kidney stem cells; mouse mesenchymal stem cells
Lowered metabolic activity, increased expression on papillary ECM
Song et al. [7 ] &&
Burgkart et al. [8 ] &
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This table provides an overview of several recent studies in which decellularized renal ECM scaffolds were repopulated in vitro using various cell types, primarily with the aid of perfusion bioreactor systems. Details of the species from which the kidney is derived, primary decellularization reagents utilized, cell types seeded, and key results are listed. ECM, extracellular matrix; SDS, sodium dodecyl sulphate.
expression of Oct4, while acquiring expression of NCAM (a mesoderm-endothelial cell precursor marker) and the endothelial cell markers Tie2 and CD31 [9 ]. These studies clearly demonstrate that specific cues from different regions of the renal ECM differentially regulate stem cell differentiation in a site-specific manner, but the precise mechanism has yet to be determined. &
RECELLULARIZATION OF WHOLE KIDNEY EXTRACELLULAR MATRIX SCAFFOLDS: PROGRESS AND FUTURE CONSIDERATIONS Custom bioreactor systems have been designed to enable the perfusion of decellularized whole kidneys through their native vasculature, facilitating uniform delivery of nutrient-rich culture media to cells seeded throughout the scaffold [6,7 ,8 ,9 ,13 ,20]. For cell seeding, the route of cell delivery into the kidney scaffold (renal artery, renal vein, ureter, or direct injection through the capsule) is critical because of the ECM-mediated cell signaling effects from &&
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different extracellular compartments, as discussed above. For complete restoration of kidney function (e.g., glomerular filtration, secretion/reabsorption, and concentration of urine), an entire nephron consisting of endothelial cells within the vasculature and glomeruli, epithelial cells in the tubules and collecting ducts, and other supportive cells (e.g., renal fibroblasts, podocytes, and mesangial cells) must be repopulated. Whole-kidney ECM seeding has typically been performed by perfusion of cell suspensions through either the renal artery [6,7 ,8 ,9 ,13 ] or ureter [6,7 ]. It has been reported that retrograde injection of cells through the ureter results in an uneven cellular distribution, with cells failing to reach glomeruli [6,7 ]. A novel strategy was recently introduced by Song et al. [7 ] in which neonatal rat kidney cells were injected into decellularized rat kidney scaffolds through the ureter while a negative pressure gradient was applied to the bioreactor chamber; this approach improved the distribution and retention of cells within the collecting system. Increasing the efficiency of cell seeding through the use of novel bioreactors and inventive cell delivery &&
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strategies is essential to scale-up recellularization operations to human-sized (e.g., porcine, human cadaver-derived) kidney scaffolds in which a substantially greater number of cells may be necessary. For the eventual development of clinically transplantable kidney grafts, extensive in-vitro functional tests of recellularized kidney scaffolds will be necessary to confirm appropriate behavior of the seeded cells, particularly for immature stem or progenitor cells. As mentioned above, the Ott lab recently reported rudimentary urine filtration function both in vitro and in vivo after recellularization of decellularized rat kidney scaffolds with rat neonatal kidney cells and human umbilical vein endothelial cells [7 ]. The authors perfused a standardized solution through the renal artery of cadaveric, decellularized, and recellularized kidney scaffolds and comparatively evaluated the venous and ureteral effluents. In decellularized kidneys, the authors noted increased dilute urine production, decreased albumin retention, and decreased glucose and electrolyte reabsorption compared with cadaveric kidneys, results consistent with a more permeable filtration barrier. Three to 5 days after recellularizing these kidneys, however, they found lower urine production, improved albumin retention, and partial restoration of glucose and electrolyte reabsorption, when compared with decellularized kidneys, indicating that the seeded cells were capable of partially restoring renal function. In-vitro assays such as these will be essential in the future for confirming proper function of engineered kidney grafts prior to implantation. &&
Human PSCs (hPSCs), capable of differentiating into multiple cell types, possess tremendous potential for applications in regenerative medicine. There has been great success in generating various cell types of the human body, including endodermal and ectodermal organoids from hPSCs (e.g., optical cup, liver bud, and mini-brain [24–26]). However, limited success has been achieved in establishing lineage-specific protocols to generate kidney-related cells from hPSCs. It is of utmost importance to establish efficient and reliable methodologies to drive hPSCs toward kidney-related lineages, which would not only serve as a transplantable resource but also as an ideal platform for kidney disease modeling and drug screening. There are two different approaches to address this challenge. One is to generate pure and large quantities of a single differentiated cell type, whereas the other is to generate self-organizing nephron-like structures. In the case of proximal tubule epithelia and glomerular podocytes, although there are reports describing methodologies to differentiate hPSCs toward these two lineages, efficiency and functionality have not matched the criteria for potential clinical use [27,28]. The Osafune group in Japan first reported the differentiation of induced PSCs into nephrogenic intermediate mesoderm, but these cells could not form ureteric bud tissue [29 ]. From there, three papers have gone on to demonstrate successful derivation of kidney-related cell lineages with 3D structure from hPSCs. The Izpisua Belmonte laboratory reported a robust 4-day protocol for generating ureteric bud progenitors, which could integrate into chimeric 3D ureteric bud structures [30 ]. Nishinakamura and colleagues [31] delicately analyzed the specification of intermediate mesoderm so as to fine-tune a protocol that could generate metanephric mesenchyme progenitors with the capacity to selfassemble into 3D kidney structures upon spinal cord induction. The Little group utilized a 3-step protocol that successfully generated both metanephric mesenchyme and ureteric bud from hPSCs [32 ]. For the first time, defined kidney cell types can be generated from hPSCs, taking a big step closer to stem cell-based kidney regeneration. However, two major issues remain to be addressed before functional kidney structures can be generated for the purpose of transplantation. Firstly, in vitro-derived kidney structures/organoids fully and solely derived from hPSCs should contain a single collecting duct tree draining into the ureter. Second, the kidney organoid needs to be properly vascularized to complement its filtration function. Presumably, a renal organoid with proper vascularization and one collecting duct tree would be connected to the host circulatory and urinary systems. &&
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DERIVATION OF RENAL CELL LINEAGES FROM PLURIPOTENT STEM CELLS AND CELL-BASED KIDNEY FORMATION The kidney represents one of the most complex systems in terms of spatial organization and lineage specification. Kidney formation entails a reciprocal interaction between two different populations derived from intermediate mesoderm, metanephric mesenchyme and ureteric bud [21]. The metanephric mesenchyme differentiates into epithelial cell types, from podocytes to the segmental sections of the distal ureteric epithelium, whereas the ureteric bud generates the collecting duct system. The very limited selfrepair capacity of the adult human kidney and the ambiguity of the presence of residential adult kidney stem cells (although recent evidence by Ward et al. [22] suggests a population of CD133/1þ cells in the renal papillae are capable of incorporating into developing proximal tubules) highlight the urgent need for a better knowledge of both kidney disease progression and regeneration [23].
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Bearing these two criteria in mind, it is not difficult to find one common problem yet to be addressed in Izpisua Belmonte’s and Nishinakamura’s reports. The maturation of ureteric bud and metanephric mesenchyme progenitors is dependent on exogenous signals, derived from either embryonic murine kidney or embryonic spinal cord. The approach utilized by the Little group generates pan-renal progenitors with selfassembly capacity independent of exogenous induction. However, instead of delicately organized nephron-like structures, different segments of the nephron are randomly dispersed without appropriate connections. In general, cell differentiation protocols are established based on knowledge obtained from developmental studies. The reciprocal induction between ureteric bud and metanephric mesenchyme gives rise to the metanephric kidney. The origin of glomerular endothelial cells was described by inter-species fetal kidney rudiment engraftment, suggesting that these cells are derived from an extrarenal source [33,34]. In order to fully recapitulate kidney development, three different types of progenitors are required: ureteric bud progenitor, metanephric mesenchyme progenitor, and angioblasts. Once all of these progenitors are successfully generated from hPSCs, the next question of whether they could correctly assemble into a functional kidneylike structure remains to be determined. Regardless, recently established protocols to generate ureteric bud and metanephric mesenchyme progenitors from hPSC will prove extremely valuable for applications in renal tissue engineering and regenerative medicine.
CONCLUSION The past year has been particularly exciting for the field of kidney tissue engineering and regenerative medicine, as a number of studies have been published in which decellularized whole-kidney ECM scaffolds were repopulated with stem or adult renal cells and cultured in perfusion bioreactor systems. Of particular significance are those utilizing PSCs, which have collectively provided substantial evidence that natural cues from the ECM tend to guide their differentiation into kidney-specific lineages. Recent reports from the Izpisua Belmonte and Nishinakamura groups have shown the feasibility of stimulating hPSC differentiation into specific kidney progenitor lineages (ureteric bud or metanephric mesenchyme), suggesting that the eventual derivation of mature renal cell populations (e.g., proximal tubule epithelial cells) may be on the horizon. 404
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Acknowledgements We thank the support of the Zell Family Foundation, the Excellence in Academic Medicine Act through the Illinois Department of Healthcare and Family Services, the Robert R. McCormick Foundation, Northwestern Memorial Foundation Dixon Translational Research Grants Initiative, a grant from the National Kidney Foundation of Illinois and the American Society of Transplant Surgeon’s Faculty Development Grant to J.A.W. Y.X. was supported by a CIRM Training Grant. Work in the laboratory of J.C.I.B. was supported by CIRM, the G. Harold and Leila Y. Mathers Charitable Foundation and The Leona M. and Harry B. Helmsley Charitable Trust. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Nguyen DM, El-Serag HB. The epidemiology of obesity. Gastroenterol Clin North Am 2010; 39:1–7. 2. Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med 1999; 341:1725–1730. 3. Dew MA, Switzer GE, Goycoolea JM, et al. Does transplantation produce quality of life benefits? A quantitative analysis of the literature. Transplantation 1997; 64:1261–1273. 4. Hutton J. The economics of immunosuppression in renal transplantation: a review of recent literature. Transplant Proc 1999; 31:1328–1332. 5. Meeus F, Kourilsky O, Guerin AP, et al. Pathophysiology of cardiovascular disease in hemodialysis patients. Kidney Int Suppl 2000; 76:S140–S147. 6. Ross EA, Williams MJ, Hamazaki T, et al. Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds. J Am Soc Nephrol 2009; 20:2338–2347. 7. Song JJ, Guyette JP, Gilpin SE, et al. Regeneration and experimental orthotopic && transplantation of a bioengineered kidney. Nat Med 2013; 19:646–651. This is an analysis of rudimentary urine generated both in vitro and in vivo after transplantation demonstrating that repopulation of decellularized rat kidney scaffolds with human umbilical vein endothelial cells and rat neonatal kidney cells partially restored the filtrative and reabsorptive functions of the kidney. 8. Burgkart R, Tron AC, Prodinger P, et al. Decellularized kidney matrix for & perfused bone engineering. Tissue Eng Part C Methods 2013; doi:10.1089/ ten.tec.2013.0270. [Epub ahead of print] The authors developed an expedited protocol to decellularize rat kidneys in only 5 h, and used primary human osteoblasts to remodel the ECM scaffold toward a bone-like composition over 2 weeks in culture. These results suggest that decellularized kidney ECM may serve as a scaffold for bone tissue formation. 9. Bonandrini B, Figliuzzi M, Papadimou E, et al. Recellularization of well & preserved acellular kidney scaffold using embryonic stem cells. Tissue Eng Part A 2013; doi:10.1089/ten.tea.2013.0269. [Epub ahead of print] ESCs injected into decellularized rat kidneys through the renal artery differentiated toward a kidney and endothelial-specific phenotype, as evidenced by loss of immunofluorescence staining for Oct4 and increased staining for NCAM, Tie-2, and CD31 expression in cells co-localized with glomeruli. 10. Sullivan DC, Mirmalek-Sani SH, Deegan DB, et al. Decellularization methods && of porcine kidneys for whole organ engineering using a high-throughput system. Biomaterials 2012; 33:7756–7764. A high-throughput system was designed for decellularization of porcine kidneys, and several detergent-based protocols were compared using detailed biochemical, histological, and structural anlayses. 0.5% SDS was most efficient at cellular removal and preserved the renal ECM microstructure, whereas 1% Triton X-100 was less effective at decellularization and proved toxic to primary renal cells in 3-day culture. 11. Orlando G, Farney AC, Iskandar SS, et al. Production and implantation of & renal extracellular matrix scaffolds from porcine kidneys as a platform for renal bioengineering investigations. Ann Surg 2012; 256:363–370. The authors showed that the decellularized porcine renal ECM scaffolds implanted into size-matched pigs were biocompatible, as evidenced by no signs of rejection, though a nonspecific inflammatory response and thrombotic occlusion were observed 2 weeks after transplantation.
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Kidney regeneration and tissue engineering Uzarski et al. 12. Orlando G, Booth C, Wang Z, et al. Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies. Biomaterials 2013; 34:5915–5925. In this article, the authors demonstrated that nontransplantable (fibrotic or glomerulosclerotic) human kidneys could be effectively decellularized by perfusion of SDS through both the renal artery and ureter; perfusion through the renal artery alone did not result in decellularization of the tubular compartment. The authors showed that the decellularized kidneys did not express HLA-ABC or HLA-DR antigens and no cell nuclei were detected, but scaffolds retained collagen IV and laminin, and promoted angiogenesis in a chick chorioallantoic membrane assay. 13. Ross EA, Abrahamson DR, St John P, et al. Mouse stem cells seeded into & decellularized rat kidney scaffolds endothelialize and remodel basement membranes. Organogenesis 2012; 8:49–55. Mouse ESCs seeded into the vasculature of decellularized rat kidneys began to differentiate into endothelial cells. Differentiated ESCs stained positive for endothelial-specific lectin and VEGFR-2, and increased laminin staining over culture time indicated cell-mediated remodeling of the basement membrane. 14. Nakayama KH, Lee CC, Batchelder CA, et al. Tissue specificity of decel& lularized rhesus monkey kidney and lung scaffolds. PLoS One 2013; 8:e64134. Proteomic analysis was used to compare the structural and signaling proteins retained after decellularization of rhesus monkey kidney or lung transverse tissue sections. ESC cultured on kidney sections increased expression of kidney-specific tubule marker genes that were not upregulated when ESCs were cultured on lung tissue. 15. O’Neill JD, Freytes DO, Anandappa AJ, et al. The regulation of growth and && metabolism of kidney stem cells with regional specificity using extracellular matrix derived from kidney. Biomaterials 2013; 34:9830–9841. The authors isolated decellularized ECM from different regions of the rat kidney: cortex, medulla, and papilla, and cultured harvested stem cells on ECM sheets, hydrogels, or solutions derived from each region. Of significance, kidney stem cells consistently demonstrated lower proliferation and higher mitochondrial metabolic activity when cultured on ECM sheets derived from renal papillae, when compared with cortical or medullary ECM, demonstrating regionally specific cues in the ECM direct cellular activity. 16. Nakayama KH, Batchelder CA, Lee CI, et al. Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering. Tissue Eng Part A 2010; 16:2207–2216. 17. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials 2011; 32:3233–3243. 18. Miner JH. Renal basement membrane components. Kidney Int 1999; 56:2016–2024. 19. Sebinger DD, Ofenbauer A, Gruber P, et al. ECM modulated early kidney && development in embryonic organ culture. Biomaterials 2013; 34:6670–6682. Using a novel ex-vivo organ culture system, the authors quantified the number of developing ureteric buds and nephrons on developing embryonic murine kidney rudiments, as well as growth area, to show how exogenous ECM components (e.g., laminin, type I collagen) promote kidney development, whereas others (e.g., collagen type IV) have an inhibitory effect. 20. Bijonowski BM, Miller WM, Wertheim JA. Bioreactor design for perfusionbased, highly-vascularized organ regeneration. Curr Opin Chem Eng 2013; 2:32–40. &
21. Little MH, McMahon AP. Mammalian kidney development: principles, progress, and projections. Cold Spring Harb Perspect Biol 2012; 4:a008300. 22. Ward HH, Romero E, Welford A, et al. Adult human CD133/1(þ) kidney cells isolated from papilla integrate into developing kidney tubules. Biochim Biophys Acta 2011; 1812:1344–1357. 23. Humphreys BD, Valerius MT, Kobayashi A, et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2008; 2:284–291. 24. Nakano T, Ando S, Takata N, et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 2012; 10:771–785. 25. Lancaster MA, Renner M, Martin CA, et al. Cerebral organoids model human brain development and microcephaly. Nature 2013; 501:373–379. 26. Takebe T, Sekine K, Enomura M, et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013; 499:481– 484. 27. Song B, Smink AM, Jones CV, et al. The directed differentiation of human iPS cells into kidney podocytes. PLoS One 2012; 7:e46453. 28. Narayanan K, Schumacher KM, Tasnim F, et al. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells. Kidney Int 2013; 83:593–603. 29. Mae S, Shono A, Shiota F, et al. Monitoring and robust induction of nephro&& genic intermediate mesoderm from human pluripotent stem cells. Nat Commun 2013; 4:1367. This article was the first to report derivation of primitive kidney tissue from induced PSCs. The authors utilized a specific temporal combination of growth factors to induce positive expression of OSR1, a marker of intermediate mesoderm, in 90% of induced PSCs after 11 days of culture. 30. Xia Y, Nivet E, Sancho-Martinez I, et al. Directed differentiation of human & pluripotent cells to ureteric bud kidney progenitor-like cells. Nat Cell Biol 2013; 15:1507–1515. This was of the first report of the generation of human ureteric bud-like renal progenitor cells from two different pluripotent sources: human ESCs, and humaninduced PSCs derived from fibroblasts. Using particular combinations of growth factors, the authors were able to generate ureteric bud-like structures within 4 days that incorporated into developing nephrons within chimeric dissociated mouse ESC cultures. 31. Taguchi A, Kaku Y, Ohmori T, et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 2014; 14:53–67. 32. Takasato M, Er PX, Becroft M, et al. Directing human embryonic stem cell & differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol 2014; 16:118–126. In this study, the authors utilized a three-step protocol to synchronously generate both metanephric mesenchyme and ureteric bud from human ESCs. ESCs were differentiated into mesendoderm using Wnt signaling, then FGF9 induced generation of intermediate mesoderm, and finally both BMP7 and retinoic acid were used to induce formation of metanephric mesenchyme and ureteric bud. 33. Hyink DP, Abrahamson DR. Origin of the glomerular vasculature in the developing kidney. Semin Nephrol 1995; 15:300–314. 34. Abrahamson DR. Development of kidney glomerular endothelial cells and their role in basement membrane assembly. Organogenesis 2009; 5:275– 287.
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New strategies in kidney regeneration and tissue engineering Joseph S. Uzarski a,b, Yun Xia c, Juan C.I. Belmonte c, and Jason A. Wertheim a,b,d,e,f
Purpose of review The severe shortage of suitable donor kidneys limits organ transplantation to a small fraction of patients suffering from end-stage renal failure. Engineering autologous kidney grafts on-demand would potentially alleviate this shortage, thereby reducing healthcare costs, improving quality of life, and increasing longevity for patients suffering from renal failure. Recent findings Over the past 2 years, several studies have demonstrated that structurally intact extracellular matrix (ECM) scaffolds can be derived from human or animal kidneys through decellularization, a process in which detergent or enzyme solutions are perfused through the renal vasculature to remove the native cells. The future clinical paradigm would be to repopulate these decellularized kidney matrices with patient-derived renal stem cells to regenerate a functional kidney graft. Recent research aiming toward this goal has focused on the optimization of decellularization protocols, design of bioreactor systems to seed cells into appropriate compartments of the renal ECM to nurture their growth to restore kidney function, and differentiation of pluripotent stem cells (PSCs) into renal progenitor lineages. Summary New research efforts utilizing bio-mimetic perfusion bioreactor systems to repopulate decellularized kidney scaffolds, coupled with the differentiation of PSCs into renal progenitor cell populations, indicate substantial progress toward the ultimate goal of building a functional kidney graft on-demand. Keywords decellularization, extracellular matrix, induced pluripotent stem cells, kidney, tissue engineering
INTRODUCTION The scope of this review is to highlight new research advances that are of importance to the field of whole-organ, kidney tissue engineering, with particular emphasis on studies utilizing renal extracellular matrix (ECM) scaffolds derived from kidney decellularization. In this review, we provide an overview of the recent and early progress made toward the ultimate goal of developing functional kidney grafts in vitro on-demand.
RATIONALE FOR DEVELOPMENT OF ENGINEERED KIDNEYS The global prevalence of chronic kidney disease (CKD) is progressing at an alarming rate, and has correlated with the dramatic increase in the prevalence of obesity over the past 30 years, leading to type II diabetes, metabolic syndrome, and renal failure [1]. Despite the rise in the prevalence of
CKD, current renal replacement therapeutic options remain limited to either peritoneal-dialysis, hemodialysis or kidney transplantation. Kidney transplantation is the optimal treatment for patients suffering from end-stage renal disease because it improves long-term survival [2], leads to better quality of life [3], and is cost-effective compared with a Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, bDepartment of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, cGene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, d Institute for BioNanotechnology in Medicine, Northwestern University, Chicago Illinois, eChemistry of Life Processes Institute and fDepartment of Biomedical Engineering, Northwestern University, Evanston, Illinois, USA
Correspondence to Jason A. Wertheim, MD, PhD, 676 N. St. Clair St., Suite 1900, Chicago, IL 60611, USA. Tel: +1 312 695 0257; fax: +1 312 503 3366; e-mail: [email protected] Curr Opin Nephrol Hypertens 2014, 23:399–405 DOI:10.1097/01.mnh.0000447019.66970.ea
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Renal pathophysiology
KEY POINTS Perfusion decellularization is a reliable technique that has been used to produce acellular, bioactive renal ECM scaffolds from a variety of kidney sources, including rat, rhesus monkey, pig, and human. The specific structural arrangement and complex composition of the renal ECM, which vary among different compartments of the kidney (e.g., vascular, tubular) are significant in directing cellular proliferation, differentiation, morphological arrangement, metabolism, and function. Recently, hPSC differentiation into kidney-specific lineages (including ureteric bud and metanephric mesenchyme) has been independently achieved by several research groups, suggesting that differentiation protocols to generate mature kidney cell types (e.g., proximal tubule epithelial) may be on the horizon.
impossible, task with the current level of technology. The particular structural features of the kidney ECM (e.g., glomerular basement membrane) are critical for appropriate tissue-specific function, and extracellular signaling cues (e.g., integrin-binding ligands of the basement membrane, bound growth factors) are important for driving renal cell growth. A promising alternative approach to create scaffolds for whole kidney engineering would be decellularization of xenogeneic or allogeneic donor kidneys. Detergents, enzymes, or other cell-lysing solutions are perfused antegrade through the renal vasculature to remove the antigenic parenchyma from the entire renal matrix. This technique has been used to produce acellular whole-kidney scaffolds from rodent [6,7 ,8 ,9 ], pig [7 ,10 ,11 ], and human [7 ,12 ] kidneys. A number of kidney decellularization protocols have been reported, most of which rely on the use of detergents, such as the nonionic Triton X-100 [6,7 ,13 ] or the anionic sodium dodecyl sulfate (SDS) [6,7 ,8 ,9 ,11 –14 ], to solubilize and wash out cellular components (see Fig. 1). Other techniques to aid this process include the use of alternating freeze–thaw cycles [8 ,15 ], osmotic shock [6,7 ,8 ,9 ,10 ,11 –14 ,15 ,16], and deoxyribonuclease to degrade nuclear material [6,10 ,13 ]. Regardless of the protocol used, the resulting renal ECM must be rigorously evaluated to confirm sufficient cell removal (e.g., through DNA quantification, histological loss of nuclei or MHC antigens), retention of basement membrane proteins and growth factors (e.g., through immunohistochemical staining, ELISA, proteomic analysis), and preservation of the native ECM microstructure (e.g., through scanning electron microscopy, histological staining) [17]. Further, the capacity of the remaining scaffold to support cellular adhesion, growth, and stem cell differentiation must be evaluated in vitro to confirm the bioactivity of the renal ECM. &&
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long-term dialysis [4]. Unfortunately, despite the rising number of patients on the waiting list for kidney transplantation, the number of available donor kidneys from both cadavers and living donors has stagnated, and the inability to match supply with the growing demand essentially precludes extension of this therapy to all patients in need. To address this discrepancy, the concept of building a functional kidney graft on-demand using patient-specific stem cells has emerged, and in the past 2 years, substantial progress toward this longterm goal has been made. Theoretically, this approach would extend the option of kidney transplantation to more patients suffering from renal failure, potentially minimize or eliminate the need for lifelong immunosuppressive therapy, and reduce the lengthy waiting times during which many patients either die or are removed from the list because of the progression of pathophysiological conditions such as coronary artery disease during prolonged hemo-dialysis [5].
THE DERIVATION OF ACELLULAR RENAL EXTRACELLULAR MATRIX SCAFFOLDS THROUGH WHOLE-KIDNEY DECELLULARIZATION The kidney is a spatially heterogeneous organ, in terms of both cellular composition as well as the structural arrangement of proteins, glycosaminoglycans, and bound growth factors that collectively comprise the ECM. Fully recapitulating the intricate architecture and complex composition of the kidney de novo through scaffold engineering technologies [e.g., electrospinning, three-dimensional (3D) printing] would be a technically difficult, if not 400
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IMPLANTATION OF DECELLULARIZED KIDNEY MATRICES To date, few cases have been reported in which decellularized whole-kidney ECM scaffolds were transplanted, mainly because of the inherent thrombogenicity of the nonendothelialized vasculature. Orlando et al. [11 ] anastomosed the renal artery and vein of decellularized porcine kidneys to the aorta and vena cava, respectively, of recipient pigs. The authors observed sufficient blood flow through the scaffold without bleeding during 60 min of intraoperative monitoring, but noted extensive thrombosis throughout the nonendothelialized kidney scaffolds 2 weeks after implantation. &
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FIGURE 1. Whole kidney decellularization. Whole renal extracellular matrix scaffolds can be derived from human or animal kidneys by perfusing detergent or enzyme solutions through the renal vasculature. Shown in this figure is a representative macroscopic view of a Sprague-Dawley rat kidney undergoing a detergent-based decellularization protocol. As cellular material is removed from the scaffold, the kidney gradually fades in color. At the end of the decellularization process, the kidney is transparent in appearance, and the vasculature can easily be visualized. Scale bar: 5 mm.
However, the authors noted no sign of immunological rejection despite a nonspecific inflammatory response, suggesting acceptable compatibility of the decellularized allogeneic scaffolds with the host animals. More recently, Song et al. [7 ] implanted Sprague-Dawley rat kidneys that were either native, decellularized, or recellularized with human umbilical vein endothelial cells and rat neonatal kidney cells into immunodeficient rats in order to analyze rudimentary urine production. The authors noted no thrombosis or bleeding over the implantation period, although the specific duration of in-vivo perfusion was not reported. In order to effectively transplant decellularized kidney scaffolds over prolonged time periods, the vasculature must be preendothelialized in vitro to prevent thrombotic occlusion of the vessels upon implantation. &&
THE RENAL EXTRACELLULAR MATRIX: A SUPPORTIVE SCAFFOLD THAT REGULATES KIDNEY DEVELOPMENT AND STEM CELL DIFFERENTIATION The kidney ECM consists of a variety of proteins, glycosaminoglycans, and growth factors organized in elaborate spatial arrangements that constitute the vascular, glomerular, and tubular basement membranes, as well as the renal interstitium [18]. Specific cues from the renal ECM play essential roles in modulating cellular behavior, as shown in several recent studies (see Table 1) [7 ,8 ,9 ,13 ,14 ,15 ]. O’Neill et al. [15 ] recently demonstrated that decellularized ECM isolated from different regions of porcine kidneys (cortex, medulla, and papilla) differentially regulated the growth of papillae-derived, BrdU label-retaining kidney stem cells in a regionally specific manner. The papillary ECM (the purported renal stem cell niche) inhibited proliferation and increased the metabolic activity of renal stem cells grown on this region. Furthermore, kidney &&
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stem cells were more metabolically active on decellularized kidney ECM than on ECM derived from heart or bladder, indicating a degree of organ recognition by renal papillae-derived parenchymal cells. Similarly, Nakayama et al. [14 ] showed that human embryonic stem cells (ESCs) seeded onto decellularized rhesus monkey kidney ECM formed tubules and expressed several kidney tubule gene markers (e.g., Dipeptidase 1 renal and Heparan sulfate 6-O-sulfotransferase 1), whereas these results were not observed when the same cells were cultured on decellularized lung scaffolds. It has also been demonstrated that specific renal ECM protein additives, such as type I collagen and laminin, contributed to ex-vivo nephrogenesis, whereas others, such as type IV collagen, had an inhibitory effect [19 ]. This was accomplished in a novel organ culture system in which murine embryonic kidney rudiments were allowed to develop in the presence of surfaceimmobilized or solubilized ECM protein molecules, allowing the authors to investigate nephrogenesis in the presence of a variety of exogenous ECM signals. Thus, the specific make-up of the ECM localized in various compartments of the kidney is important for embryonic renal development and kidney-specific stem cell growth. Ross et al. [13 ] showed that mouse ESCs within the glomeruli and blood vessels of decellularized rat kidney scaffolds expressed the endothelial cellspecific markers BSLB4 lectin and vascular endothelial growth factor receptor-2 (VEGFR-2). Interestingly, the same research group previously discovered that some ESC aggregates seeded in the renal cortex expressed Ksp-cadherin, a distal tubule marker [6]. This would suggest that pluripotent stem cell (PSC) differentiation is somehow dependent on the specific niche of the ECM to which the cells are adhered. This concept was shown more recently in a study where mouse ESCs seeded into decellularized rat kidney scaffolds through the renal artery progressively lost
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Renal pathophysiology Table 1. Recent kidney recellularization experiments Authors
Species
Decellularization strategy
Cells utilized
Key results
Sprague-Dawley rat
1% SDS: 12 h; 1% Triton X-100: 30 min
Rat neonatal kidney cells; human umbilical vein endothelial cells
Partial restoration of renal filtration and electrolyte reabsorption in recellularized scaffolds
Sprague-Dawley rat
0.66% SDS: 1 h
Primary human osteoblasts; human umbilical vein endothelial cells
Osteoblast differentiation into osteocytes; increased osteocalcin deposition
Bonandrini et al. [9 ]
Sprague-Dawley rat
1% SDS: 17 h
R1 murine pluripotent ESCs
Loss of staining for Oct4 and increase in NCAM, Tie-2, and CD31 expression in glomeruli co-localized cells
Sullivan et al. [10 ]
Yorkshire pig
0.5% SDS: 36 h; DNase: overnight
Primary human cortical renal cells
Superior viability, proliferation on SDS-decellularized ECM compared to Triton X-100
Ross et al. [13 ]
Sprague-Dawley rat
3% Triton X-100, DNase, 4% SDS; not specified
B5/EGFP murine pluripotent ESCs
Positive staining for BsLB4 and VEGFR2 in ESCs colocalized in glomeruli and vessels
Nakayama & et al. [14 ]
Rhesus monkey (transverse kidney sections)
1% SDS: 7–10 days
WA09 human ESCs
Increased expression of kidney tubule gene markers DPEP1 and HS6T1
Yorkshire pig
0.02% trypsin: 2 h; 3% Tween: 2 h; 4% sodium deoxycholate: 2h
Murine kidney stem cells; mouse mesenchymal stem cells
Lowered metabolic activity, increased expression on papillary ECM
Song et al. [7 ] &&
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This table provides an overview of several recent studies in which decellularized renal ECM scaffolds were repopulated in vitro using various cell types, primarily with the aid of perfusion bioreactor systems. Details of the species from which the kidney is derived, primary decellularization reagents utilized, cell types seeded, and key results are listed. ECM, extracellular matrix; SDS, sodium dodecyl sulphate.
expression of Oct4, while acquiring expression of NCAM (a mesoderm-endothelial cell precursor marker) and the endothelial cell markers Tie2 and CD31 [9 ]. These studies clearly demonstrate that specific cues from different regions of the renal ECM differentially regulate stem cell differentiation in a site-specific manner, but the precise mechanism has yet to be determined. &
RECELLULARIZATION OF WHOLE KIDNEY EXTRACELLULAR MATRIX SCAFFOLDS: PROGRESS AND FUTURE CONSIDERATIONS Custom bioreactor systems have been designed to enable the perfusion of decellularized whole kidneys through their native vasculature, facilitating uniform delivery of nutrient-rich culture media to cells seeded throughout the scaffold [6,7 ,8 ,9 ,13 ,20]. For cell seeding, the route of cell delivery into the kidney scaffold (renal artery, renal vein, ureter, or direct injection through the capsule) is critical because of the ECM-mediated cell signaling effects from &&
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different extracellular compartments, as discussed above. For complete restoration of kidney function (e.g., glomerular filtration, secretion/reabsorption, and concentration of urine), an entire nephron consisting of endothelial cells within the vasculature and glomeruli, epithelial cells in the tubules and collecting ducts, and other supportive cells (e.g., renal fibroblasts, podocytes, and mesangial cells) must be repopulated. Whole-kidney ECM seeding has typically been performed by perfusion of cell suspensions through either the renal artery [6,7 ,8 ,9 ,13 ] or ureter [6,7 ]. It has been reported that retrograde injection of cells through the ureter results in an uneven cellular distribution, with cells failing to reach glomeruli [6,7 ]. A novel strategy was recently introduced by Song et al. [7 ] in which neonatal rat kidney cells were injected into decellularized rat kidney scaffolds through the ureter while a negative pressure gradient was applied to the bioreactor chamber; this approach improved the distribution and retention of cells within the collecting system. Increasing the efficiency of cell seeding through the use of novel bioreactors and inventive cell delivery &&
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strategies is essential to scale-up recellularization operations to human-sized (e.g., porcine, human cadaver-derived) kidney scaffolds in which a substantially greater number of cells may be necessary. For the eventual development of clinically transplantable kidney grafts, extensive in-vitro functional tests of recellularized kidney scaffolds will be necessary to confirm appropriate behavior of the seeded cells, particularly for immature stem or progenitor cells. As mentioned above, the Ott lab recently reported rudimentary urine filtration function both in vitro and in vivo after recellularization of decellularized rat kidney scaffolds with rat neonatal kidney cells and human umbilical vein endothelial cells [7 ]. The authors perfused a standardized solution through the renal artery of cadaveric, decellularized, and recellularized kidney scaffolds and comparatively evaluated the venous and ureteral effluents. In decellularized kidneys, the authors noted increased dilute urine production, decreased albumin retention, and decreased glucose and electrolyte reabsorption compared with cadaveric kidneys, results consistent with a more permeable filtration barrier. Three to 5 days after recellularizing these kidneys, however, they found lower urine production, improved albumin retention, and partial restoration of glucose and electrolyte reabsorption, when compared with decellularized kidneys, indicating that the seeded cells were capable of partially restoring renal function. In-vitro assays such as these will be essential in the future for confirming proper function of engineered kidney grafts prior to implantation. &&
Human PSCs (hPSCs), capable of differentiating into multiple cell types, possess tremendous potential for applications in regenerative medicine. There has been great success in generating various cell types of the human body, including endodermal and ectodermal organoids from hPSCs (e.g., optical cup, liver bud, and mini-brain [24–26]). However, limited success has been achieved in establishing lineage-specific protocols to generate kidney-related cells from hPSCs. It is of utmost importance to establish efficient and reliable methodologies to drive hPSCs toward kidney-related lineages, which would not only serve as a transplantable resource but also as an ideal platform for kidney disease modeling and drug screening. There are two different approaches to address this challenge. One is to generate pure and large quantities of a single differentiated cell type, whereas the other is to generate self-organizing nephron-like structures. In the case of proximal tubule epithelia and glomerular podocytes, although there are reports describing methodologies to differentiate hPSCs toward these two lineages, efficiency and functionality have not matched the criteria for potential clinical use [27,28]. The Osafune group in Japan first reported the differentiation of induced PSCs into nephrogenic intermediate mesoderm, but these cells could not form ureteric bud tissue [29 ]. From there, three papers have gone on to demonstrate successful derivation of kidney-related cell lineages with 3D structure from hPSCs. The Izpisua Belmonte laboratory reported a robust 4-day protocol for generating ureteric bud progenitors, which could integrate into chimeric 3D ureteric bud structures [30 ]. Nishinakamura and colleagues [31] delicately analyzed the specification of intermediate mesoderm so as to fine-tune a protocol that could generate metanephric mesenchyme progenitors with the capacity to selfassemble into 3D kidney structures upon spinal cord induction. The Little group utilized a 3-step protocol that successfully generated both metanephric mesenchyme and ureteric bud from hPSCs [32 ]. For the first time, defined kidney cell types can be generated from hPSCs, taking a big step closer to stem cell-based kidney regeneration. However, two major issues remain to be addressed before functional kidney structures can be generated for the purpose of transplantation. Firstly, in vitro-derived kidney structures/organoids fully and solely derived from hPSCs should contain a single collecting duct tree draining into the ureter. Second, the kidney organoid needs to be properly vascularized to complement its filtration function. Presumably, a renal organoid with proper vascularization and one collecting duct tree would be connected to the host circulatory and urinary systems. &&
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DERIVATION OF RENAL CELL LINEAGES FROM PLURIPOTENT STEM CELLS AND CELL-BASED KIDNEY FORMATION The kidney represents one of the most complex systems in terms of spatial organization and lineage specification. Kidney formation entails a reciprocal interaction between two different populations derived from intermediate mesoderm, metanephric mesenchyme and ureteric bud [21]. The metanephric mesenchyme differentiates into epithelial cell types, from podocytes to the segmental sections of the distal ureteric epithelium, whereas the ureteric bud generates the collecting duct system. The very limited selfrepair capacity of the adult human kidney and the ambiguity of the presence of residential adult kidney stem cells (although recent evidence by Ward et al. [22] suggests a population of CD133/1þ cells in the renal papillae are capable of incorporating into developing proximal tubules) highlight the urgent need for a better knowledge of both kidney disease progression and regeneration [23].
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Bearing these two criteria in mind, it is not difficult to find one common problem yet to be addressed in Izpisua Belmonte’s and Nishinakamura’s reports. The maturation of ureteric bud and metanephric mesenchyme progenitors is dependent on exogenous signals, derived from either embryonic murine kidney or embryonic spinal cord. The approach utilized by the Little group generates pan-renal progenitors with selfassembly capacity independent of exogenous induction. However, instead of delicately organized nephron-like structures, different segments of the nephron are randomly dispersed without appropriate connections. In general, cell differentiation protocols are established based on knowledge obtained from developmental studies. The reciprocal induction between ureteric bud and metanephric mesenchyme gives rise to the metanephric kidney. The origin of glomerular endothelial cells was described by inter-species fetal kidney rudiment engraftment, suggesting that these cells are derived from an extrarenal source [33,34]. In order to fully recapitulate kidney development, three different types of progenitors are required: ureteric bud progenitor, metanephric mesenchyme progenitor, and angioblasts. Once all of these progenitors are successfully generated from hPSCs, the next question of whether they could correctly assemble into a functional kidneylike structure remains to be determined. Regardless, recently established protocols to generate ureteric bud and metanephric mesenchyme progenitors from hPSC will prove extremely valuable for applications in renal tissue engineering and regenerative medicine.
CONCLUSION The past year has been particularly exciting for the field of kidney tissue engineering and regenerative medicine, as a number of studies have been published in which decellularized whole-kidney ECM scaffolds were repopulated with stem or adult renal cells and cultured in perfusion bioreactor systems. Of particular significance are those utilizing PSCs, which have collectively provided substantial evidence that natural cues from the ECM tend to guide their differentiation into kidney-specific lineages. Recent reports from the Izpisua Belmonte and Nishinakamura groups have shown the feasibility of stimulating hPSC differentiation into specific kidney progenitor lineages (ureteric bud or metanephric mesenchyme), suggesting that the eventual derivation of mature renal cell populations (e.g., proximal tubule epithelial cells) may be on the horizon. 404
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Acknowledgements We thank the support of the Zell Family Foundation, the Excellence in Academic Medicine Act through the Illinois Department of Healthcare and Family Services, the Robert R. McCormick Foundation, Northwestern Memorial Foundation Dixon Translational Research Grants Initiative, a grant from the National Kidney Foundation of Illinois and the American Society of Transplant Surgeon’s Faculty Development Grant to J.A.W. Y.X. was supported by a CIRM Training Grant. Work in the laboratory of J.C.I.B. was supported by CIRM, the G. Harold and Leila Y. Mathers Charitable Foundation and The Leona M. and Harry B. Helmsley Charitable Trust. Conflicts of interest There are no conflicts of interest.
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21. Little MH, McMahon AP. Mammalian kidney development: principles, progress, and projections. Cold Spring Harb Perspect Biol 2012; 4:a008300. 22. Ward HH, Romero E, Welford A, et al. Adult human CD133/1(þ) kidney cells isolated from papilla integrate into developing kidney tubules. Biochim Biophys Acta 2011; 1812:1344–1357. 23. Humphreys BD, Valerius MT, Kobayashi A, et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2008; 2:284–291. 24. Nakano T, Ando S, Takata N, et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 2012; 10:771–785. 25. Lancaster MA, Renner M, Martin CA, et al. Cerebral organoids model human brain development and microcephaly. Nature 2013; 501:373–379. 26. Takebe T, Sekine K, Enomura M, et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013; 499:481– 484. 27. Song B, Smink AM, Jones CV, et al. The directed differentiation of human iPS cells into kidney podocytes. PLoS One 2012; 7:e46453. 28. Narayanan K, Schumacher KM, Tasnim F, et al. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells. Kidney Int 2013; 83:593–603. 29. Mae S, Shono A, Shiota F, et al. Monitoring and robust induction of nephro&& genic intermediate mesoderm from human pluripotent stem cells. Nat Commun 2013; 4:1367. This article was the first to report derivation of primitive kidney tissue from induced PSCs. The authors utilized a specific temporal combination of growth factors to induce positive expression of OSR1, a marker of intermediate mesoderm, in 90% of induced PSCs after 11 days of culture. 30. Xia Y, Nivet E, Sancho-Martinez I, et al. Directed differentiation of human & pluripotent cells to ureteric bud kidney progenitor-like cells. Nat Cell Biol 2013; 15:1507–1515. This was of the first report of the generation of human ureteric bud-like renal progenitor cells from two different pluripotent sources: human ESCs, and humaninduced PSCs derived from fibroblasts. Using particular combinations of growth factors, the authors were able to generate ureteric bud-like structures within 4 days that incorporated into developing nephrons within chimeric dissociated mouse ESC cultures. 31. Taguchi A, Kaku Y, Ohmori T, et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 2014; 14:53–67. 32. Takasato M, Er PX, Becroft M, et al. Directing human embryonic stem cell & differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol 2014; 16:118–126. In this study, the authors utilized a three-step protocol to synchronously generate both metanephric mesenchyme and ureteric bud from human ESCs. ESCs were differentiated into mesendoderm using Wnt signaling, then FGF9 induced generation of intermediate mesoderm, and finally both BMP7 and retinoic acid were used to induce formation of metanephric mesenchyme and ureteric bud. 33. Hyink DP, Abrahamson DR. Origin of the glomerular vasculature in the developing kidney. Semin Nephrol 1995; 15:300–314. 34. Abrahamson DR. Development of kidney glomerular endothelial cells and their role in basement membrane assembly. Organogenesis 2009; 5:275– 287.
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