A devastating 6% mortality has been reported for patients who die while waiting for a kidney to become available for transplant.2
Donor shortages might be overcome with the development and refinement of engineered organs. While some engineered tissues using decellularized matrices such as bladder19
and the upper airway4
are under study in human clinical trials, the kidney remains an organ that has proven difficult to regenerate with 28 different cell types and complex metabolic functions.20
Decellularized renal matrices have been reported to support cell attachment,21
but the formation of renal structures with functional capacity has yet to be shown. The goal of these studies was to study engineered renal tissue from decellularized matrices that possess basic renal ECM in a clinically relevant rhesus monkey model, and to determine whether age has an impact on cellular repopulation. While kidney diseases affect all individuals at all ages it is possible that different approaches may be needed depending on the disease and the age group.22
We previously reported effective methods to decellularize kidney scaffolds that are free of MHC Class II antigens.13
In the studies described herein, we initially recellularized fetal, juvenile, and adult kidney scaffolds using kidney explant culture and showed that the younger donor explants layered on scaffolds derived from all of the age groups demonstrated the most extensive cell infiltration and repopulation. Layered fetal explants were frequently found with large numbers of single cells and unorganized cell clusters of an epithelial and mesenchymal phenotype throughout the scaffold. The distance of tubular branching from the explant was measured to assess scaffold repopulation. The initial use of explants as the cell source maximized scaffold to cell contact surface area and provided a diverse mixture of native kidney cell populations. As the kidney matures (fetus to postnatal), the ECM remodels creating a different ECM landscape. Zhang et al.23
described enhanced proliferation and differentiation of tissue-matched cell types with their tissue specific ECM, which underscored the sensitivity of cells to the unique ECM composition with which they were presented. Tottey et al.24
demonstrated differences in mechanical and biological properties, including growth factor concentration and mitogenic cell response, of decellularized small intestinal submucosa-ECM from different aged pigs. Complex changes in the ECM that occur with age are likely responsible for differing degrees of repopulation of scaffolds with less extensive repopulation of adult scaffolds. This suggests that a more defined cytokine-enhanced culture system may be required for more mature age groups. We have previously demonstrated lifespan differences in a variety of stem and progenitor cell populations, including mesenchymal,25,26
and endothelial progenitor28
cells, highlighting the importance of consideration of age in regenerative medicine strategies. Other studies have indicated that transplantation of fetal kidney cells from earlier gestational age kidneys resulted in enhanced structural formation when compared to cells obtained from later gestation kidneys.29,30
The impact of age has been extended in the current study to span fetuses, juveniles, and adults and has demonstrated age-dependent recellularization of kidney sections, suggesting that critical differences in cell and ECM composition are important factors in renal tissue engineering and must be taken into consideration when addressing the age of the recipient.
To better understand the mechanism of recellularization, intact
renal fractions were seeded onto fetal or juvenile scaffolds. Only when scaffolds were seeded with intact
Fraction 2 were tubular structures found to extensively repopulate the scaffold based on number of tubules and infiltration distance. All other fraction/scaffold combinations yielded significantly fewer or no tubules. These findings suggest that intact
Fraction 2 was responsible for tubular repopulation of the scaffolds most likely by the infiltration of existing tubules rather than the generation of new tubules from single dissociated cells. Several recent studies have focused on decellularized matrices in other organ systems4,9
and utilized dissociated cell populations; however, a different cell-based approach may be needed to re-engineer the kidney. Although dissociated
Fraction 1 and 2 were shown to form kidney structures in a collagen matrix in other studies,31
the dissociated cells were filtered through a strainer with pore sizes that were of a sufficient size to allow small glomeruli or tubules to readily pass through and thereby contribute to the observed glomerular structures. In addition, these fractions were seeded at a much higher density than in the study described herein. It is possible that greater seeding density may be required to increase cell–cell and cell–ECM contact, although there is currently no data to support this assumption. Although a renal tubular stem cell has not been identified, the possibility of an epithelial stem/progenitor cell within the tubules has been suggested by Humphreys et al.32
These investigators showed that there were no cells of nontubular origin present in renal tubules before or after injury, suggesting that tubular repair may be performed by cells of tubular origin and not from cell populations located at other anatomical sites within the kidney. A recent publication by this group has also noted that injured epithelial cells of the proximal tubule repopulate the kidney epithelium by self-duplication of surviving epithelia, in the absence of any specialized progenitor population.33
In the current study, an epithelial tubule population as described by Humphreys et al.32,33
may be responsible for infiltration into the scaffold and could explain why fewer numbers of tubular structures were observed with Fraction 1 repopulation, which would not have contained many tubular cells. Although the kidney has demonstrated some capacity for repair in response to injury, a renal stem cell has yet to be found which underscores the necessity to explore multiple cell types for recellularization.34
These findings also suggest that a mixture of renal cells using intact fractions may be required for more complete tubular infiltration. The mechanical separation process used to obtain glomeruli in Fraction 1 removes Bowman's capsule, which has been shown to include a CD24+ CD133+ renal progenitor population that is present throughout kidney development and contributes to podocytes and tubular epithelium.35,36
Alternative methods that preserve Bowman's capsule may yield enhanced scaffold recellularization by retaining this cell population. Fraction 2 contained a heterogeneous mixture of intact tubular aggregates, including occasional glomeruli with Bowman's capsule, which may have contributed to enhanced repopulation. Further studies will be required to determine the specific contribution of different renal cell fractions to recellularize kidney scaffolds, particularly those from different fetal and postnatal age groups.
Similar to the human fetus, the nephrogenic zone persists in the fetal rhesus monkey kidney until the third trimester. The cells that infiltrated the scaffolds stained positive for Pax2, which we have previously shown are restricted to the nephrogenic zone during the early third trimester and localized to the cortical collecting ducts and inner medullary regions of the renal papilla.17
Although additional analysis would be required, findings by others37
suggest a renal progenitor population may exist in the renal papilla that may be Pax2+. The tubules found within recellularized scaffolds also expressed calbindin, which has been shown in the ureteric bud and in the collecting ducts during the third trimester and in the postnatal kidney. Markers of mesenchyme (vimentin), epithelium (cytokeratin), and endothelium (CD31), as well as SMA (expressed in metanephric mesenchyme and the glomerular tuft), provided a combination of markers used to characterize cells that infiltrated the scaffold and showed tubular branching. Additional studies will be necessary to more extensively explore the cells that repopulate renal ECM and their functional capabilities.
To enhance repopulation of the scaffolds, cytokine-enriched medium was also assessed. The addition of FGF to the culture medium enhanced formation of mature tubules with scaffold repopulation noted consisting of large clusters of vimentin+ cytokeratin+ cells. Although addition of high doses of FGF to cell culture has been reported to inhibit tubulogenesis, FGF is a strong inhibitor of apoptosis of tubular epithelial precursors, capillary precursors, and regulatory cells of the ureteric bud.38,39
Because we used a low dose of FGF (4
ng/mL) the benefits of tubular epithelial cell rescue may have outweighed the inhibitory effects on tubulogenesis, resulting in enhanced repopulation. These results demonstrate the capacity of decellularized kidney sections to maintain tubules and serve as a framework for tubular growth. Moreover, formation of three-dimensional renal structures did not occur when dissociated cells were used. Intact tubules and renal structures provided the best environment in which to form tubules in the decellularized kidney scaffolds in these studies. Further investigations will be required to assess the functional properties of these engineered renal constructs.
Taken together, these data provide initial insights into the in vitro renal tissue engineering of decellularized kidney scaffolds, and address the impact of donor age and cellular approach on recellularization. These findings provide useful methods to explore tissue regeneration strategies for the potential future treatment of kidney disease, and in a variety of age groups.