Despite the enthusiasm for bioengineering of functional renal tissues for transplantation, many obstacles remain before the potential of this technology can be realized in a clinical setting. Viable tissue engineering strategies for the kidney require identification of the necessary cell populations, efficient scaffolds, and the 3D culture conditions to develop and support the unique architecture and physiological function of this vital organ. Our studies have previously demonstrated that decellularized sections of rhesus monkey kidneys of all age groups provide a natural extracellular matrix (ECM) with sufficient structural properties with spatial and organizational influences on human embryonic stem cell (hESC) migration and differentiation. To further explore the use of decellularized natural kidney scaffolds for renal tissue engineering, pluripotent hESC were seeded in whole- or on sections of kidney ECM and cell migration and phenotype compared with the established differentiation assays for hESC. Results of qPCR and immunohistochemical analyses demonstrated upregulation of renal lineage markers when hESC were cultured in decellularized scaffolds without cytokine or growth factor stimulation, suggesting a role for the ECM in directing renal lineage differentiation. hESC were also differentiated with growth factors and compared when seeded on renal ECM or a new biologically inert polysaccharide scaffold for further maturation. Renal lineage markers were progressively upregulated over time on both scaffolds and hESC were shown to express signature genes of renal progenitor, proximal tubule, endothelial, and collecting duct populations. These findings suggest that natural scaffolds enhance expression of renal lineage markers particularly when compared to embryoid body culture. The results of these studies show the capabilities of a novel polysaccharide scaffold to aid in defining a protocol for renal progenitor differentiation from hESC, and advance the promise of tissue engineering as a source of functional kidney tissue.
The expanding field of tissue engineering provides hope for the creation of tissue and organs with functional properties and therapeutic potential for nearly every tissue of the human body. Initial engineering strategies have been successful, particularly for tubular structures such as blood vessels [1, 2], urinary bladder , larynx , and trachea . The clinical need for functional tissue replacements is urgent for patients on the organ transplant waiting list and is particularly critical for patients waiting for a kidney; individuals in need of a kidney represent >80% of all patients on the waiting list (http://optn.transplant.hrsa.gov). It is notable that the kidney which is in greatest demand is also one of the most challenging tissues to engineer due to complex architecture, a spectrum of cell phenotypes, multiple functions, and a lack of an established stem/progenitor cell population in adults from which the kidney can be regenerated. Viable tissue engineering strategies for the kidney requires identification of necessary cell populations, suitable scaffolds to provide structural support and spatiotemporal organizational properties, as well as medium/growth factor/culture combinations to sustain growth and physiological function of the engineered tissue.
One promising approach for renal tissue engineering involves the use of natural scaffolds produced by decellularization of donor kidneys. The decellularization process typically produces a three-dimensional (3D) biological scaffold with native extracellular matrix (ECM) molecules in proper geometric locations and intact vascular conduits. Since initial proof-ofconcept studies , decellularized kidney scaffolds have been produced in rats [7–11], nonhuman primates [12–13], pigs [14–18], and from cadaveric human kidneys failing to meet criteria for transplantation [8, 19–21]. Studies have also demonstrated the presence of other biologic agents  such as growth factors, cytokines [10–11, 20], and bioactive peptides that may be advantageous to growth and functional maturation of cells within the construct. Despite the promise of this approach many challenges remain including determination of a suitable source of cells for recellularization, optimization of in vitro culture systems for the developing tissueengineered construct, elucidating the role and utility of the native ECM in directing cell fate, and defining an appropriate maturational endpoint prior to in vivo transplantation . Given the shortage of available donor kidneys, tissue engineering strategies with natural scaffolding materials that are tailored to the need may further facilitate clinical translation.
Our studies have previously demonstrated that decellularized rhesus monkey kidney sections of all age groups (fetal to aged) provide a natural ECM with sufficient structural properties to support migration of cells from kidney explants in an age-dependent manner [12, 13], and the utility of these scaffolds to provide spatial and organizational influences on human embryonic stem cell (hESC) migration and differentiation . To further explore the use of decellularized renal scaffolds for ex vivo studies of development, disease, and as engineered tissue replacements, strategies to improve recellularization were assessed. The ability of decellularized kidney scaffolds to influence cell migration and phenotype was studied with undifferentiated hESC seeded in sections of kidney versus whole kidneys. For studies on the recellularization of whole kidneys the delivery of cells through the renal artery or the ureter were assessed. In addition, to evaluate the role of the scaffold in guiding hESC renal differentiation, cells were seeded on decellularized kidney sections and compared with the cellular phenotype of cells obtained when using an organic, physiologically inert polysaccharide scaffold.
Materials and Methods
Tissue Collection No animal subjects were involved in the study. A biorepository of previously obtained rhesus monkey kidney sections or whole kidneys were used for these studies; specimens were obtained through the tissue procurement program (www.cnprc.ucdavis.edu/our-services) (N = 12 included in the study).
Whole kidneys or sections of kidneys were decellularized using the following protocols. Whole kidneys were perfused for decellularization as follows: (1) 100 USP/ml Heparin (Sagent Pharmaceuticals, Schaumburg, IL) in phosphate buffered saline (PBS; Life Technologies, Grand Island, NY) at 1 ml/minute for 15 minutes; (2) 1% sodium dodecyl sulfate (SDS, Life Technologies) in distilled water at 5 to 20 ml/hour (based on kidney size, e.g., ~5–10 g kidney with flow rate of 5 ml/hour for 3–4 days with a total perfusion volume of ~350–500 ml) until the tissue was transparent and the decellularization solution draining from the organ was colorless; and (3) 1x antibiotic-antimycotic (Life Technologies) in PBS wash at 1 to 20 ml/hour for 72–96 hours. Transverse kidney sections were also obtained (2–3 mm thickness) and rinsed briefly in PBS and decellularized in 1% SDS for 5–8 days at 4°C or on a continuous shaker for 48–72 hours at room temperature until translucent. SDS solution was changed daily until decellularization was completed. For decellularization at 4°C the sections and SDS solutions were brought to room temperature prior to SDS replacement. Sections of kidneys were washed at room temperature on a rotator as follows: (1) 1x antibiotic-antimycotic in PBS for 24 hours (repeated 3 times); (2) 70% ethanol for 24 hours; and (3) 1x antibiotic-antimycotic in PBS for 24 hours. Both the decellularized sections and whole kidneys were stored at 4°C in PBS with 1x antibiotic-antimycotic solution until recellularization. Prior to use, an 8 mm diameter biopsy punch (Fisher Scientific) was used for the decellularized kidney sections to ensure consistency in scaffold dimensions. Polysaccharide discs (PSS; GroCell-3DTM, Molecular Matrix, Inc., Davis, CA) with 8 mm diameter and 2–3 mm thickness were utilized as an organic, inert 3D polysaccharide scaffold matrix for comparison.
All hESC studies were approved by the UC Davis Stem Cell Research Oversight Committee. Medium and reagents were purchased from Life Technologies and growth factor supplements from R&D Systems unless otherwise noted. The federally approved hESC line WA09 (H9, WiCell Research Institute) was maintained on irradiated mouse embryonic fibroblasts according to established protocols in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% Knockout Serum Replacer (Life Technologies), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-Mercaptoethanol, and 4 ng/ml fibroblast growth factor 2 (FGF2) .
Decellularized whole kidneys were repopulated with hESC in a closed-system custom bioreactor designed to perfuse oxygenated medium at a constant flow rate through the renal artery and/or the ureter. The scaffold was conditioned prior to cell seeding by perfusion with endothelial growth medium with supplements (EGM2, Lonza, Allendale, NJ) for at least 24 hours. hESC were prepared for seeding with the use of collagenase to remove colonies from the feeder-cell monolayer. After centrifugation to remove collagenase, hESC were dissociated to a single-cell suspension with StemPro Accutase cell dissociation reagent (Life Technologies) for 2–3 minutes. Once single cells were observed, the dissociation process was neutralized with the addition of medium. Following centrifugation, cells (20x106 ) were resuspended in 1 ml of medium, loaded into a syringe, and infused into the scaffold via a 3-way bioreactor access port at 0.1 ml/minute flow rate. After seeding, perfusion of the construct was halted for 2 hours to allow time for cell attachment. Bioreactor culture (37°C, 5% CO2) and perfusion of the construct (flow rate 0.1 ml/minute) was then maintained for up to 7 days to assess cell distribution. For studies on recellularization via the ureter, tubing was used to connect the cannulated ureter to a port located outside the bioreactor and through which cells were manually infused.
Kidney sections were preconditioned for seeding by soaking in EGM2 for 24 hours then placed in a 12-well plate and gently blotted with a sterile cotton-tipped swab to remove excess medium. hESC were prepared as described above in a single-cell suspension and were resuspended in 10 μl of medium and gently pipetted onto the section-scaffold surface. Seeded scaffolds were returned to the incubator for 2 hours to allow time for cell attachment after which additional medium was added to the edges of the wells in order to ensure that the surface of the scaffold was maintained at the air-medium interface. Culture medium was changed every 3 days and kidney sections were cultured for up to 3 weeks.