Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

Improvement of cytokine response and survival time by bioartificial kidney therapy in acute uremic pigs with multi-organ dysfunction.

OBJECTIVE: To explore whether bioartificial kidney (BAK) ameliorates cytokine response and biochemical indices, and prolongs the survival time in acute uremic pigs with multiple organ dysfunction syndrome (MODS). METHODS: Hybridized pigs suffering from MODS and acute renal failure (ARF) were treated with BAK (Group A, n=6) or sham-BAK containing no cells (Group B, n=6), or received no treatment (Group C, n=5). Data on blood pressure, hepatic and renal function, IL-10, TNF-α, arterial blood gas, and survival time of all the pigs was recorded. RESULTS: Mean arterial pressure (MAP, mmHg) responded more rapidly and reached higher values in Group A (91.82 ± 5.73) compared with Groups B and C at 24 hours (p<0.01). The peak level of serum IL-10 (pg/mL) in Group A (249.57 ± 43.51) was significantly higher than in Groups B and C (132.06 ± 17.53, 104.25 ± 13.42, p<0.01). Serum TNF-α level (pg/mL) in Group A dropped gradually to 402.91 ± 32.47 at 24 hours, and showed a significant discrepancy compared with those before treatment (537.16 ± 38.45) and Group B (512.94 ± 19.5, p<0.05). There was no difference in plasma endotoxin and serum IL-6 between pre-treatment and post-treatment in Groups A and B. BAK treatment, however, resulted in a significant decline in IL-6/IL-10 ratios. The average survival time (hours) in Group A (113.01 ± 14.32) was significantly longer, prolonged by 35.93% and 63.90% compared to Groups B and C (p<0.01), respectively. CONCLUSIONS: The addition of renal tubule cell therapy to hemofiltration in an acutely uremic animal model with MODS altered systemic cytokine balance, ameliorated MAP, and prolonged survival time.

Advances in cell therapy for renal failure.

Cell therapy is one of the most exciting fields in translational medicine. It stands at the intersection of a variety of rapidly developing scientific disciplines: stem cell biology, immunology, tissue engineering, molecular biology, biomaterials, transplantation biology, regenerative medicine and clinical research. Cell-based therapy may develop into a new therapeutic platform to treat a vast array of clinical disorders. Blood transfusions and bone marrow transplantation are prime examples of the successful application of cell-based therapeutics; but recent advances in cellular and molecular biology have expanded the potential applications of this approach. Although recombinant genetic engineering to produce a variety of therapeutics, such as human erythropoietin and insulin has proven successful, these treatments are unable to completely correct or reverse disease states, because most common disease processes are not due to the deficiency of a single protein but develop due to alterations in the complex interactions of a variety of cell components. In these complex situations, cell-based therapy may be a more successful strategy by providing a dynamic, interactive and individualized therapeutic approach that responds to the pathophysiological condition of the patient. In this regard, cells may provide innovative methods for drug delivery of biologics, immunotherapy, and tissue regenerative or replacement engineering [Nature 392 (1998) 518-524, Nat Biotechnol 20 (2002) 339-343]. The translation of this discipline to medicinal practice has tremendous potential, but in many applications technological issues need to be overcome. Since many cell-based indications are already being evaluated in the clinic, the field appears to be on the threshold of a number of successes. This review will focus on our group's use of human stem/progenitor cells in the treatment of acute and chronic renal failure as extensions to current successful renal substitution processes of hemodialysis and hemofiltration.

Cell therapy of renal failure.

The kidney is unique in that it is the first organ for which long-term ex vivo substitutive therapy has been available. The first hemodialyzer was successfully applied to a human patient with acute renal failure in 1948, and the first successful allograft transplantation was performed with a kidney in 1951. Both treatments are used today. There is ample evidence that the small solute clearance function provided by hemodialysis does not confer the same survival advantage as a functional kidney, both in acute and in chronic renal failure. To mimic the metabolic, endocrine, and immunologic functions of the kidney, our group has successfully engineered a bioartificial device that includes a conventional dialysis filter and a bioreactor containing 10(9) renal proximal tubule cells. We have demonstrated differentiated activity of these cells both in vitro and ex vivo in a large animal model. The bioreactor has been shown to confer a survival advantage in two large animal models of gram-negative sepsis, seemingly due to modulation of inflammatory mediators. This bioartificial kidney has now completed a Phase I clinical trial in acute renal failure.

Tissue engineering of a bioartificial renal tubule.

Development of a bioartificial renal tubule with a confluent monolayer of renal epithelial cells supported on a permeable synthetic surface may be the first step to further optimization of renal substitution therapy currently used with hemodialysis or hemofiltration. Madin-Darby canine kidney cells, a permanent renal epithelial cell line, were seeded into the lumen of single hollow fibers. Functional confluence of the cells was demonstrated by the recovery of intraluminally perfused 14C-inulin that averaged >98.9% in the cell lined units vs <7.4% in the control noncell hollow fibers during identical pressure and flow conditions. The baseline absolute fluid transport rate averaged 1.4+/-0.4 microl/30 min. To test the dependency of fluid flux with oncotic and osmotic pressure differences across the bioartificial tubule, albumin was added to the extracapillary space, followed by the addition of ouabain, an inhibitor of Na+K+ adenosine triphosphatase, the enzyme responsible for active transport across the renal epithelium. Addition of albumin resulted in a significant increase in volume transport to 4.5+/-1.0 microl/30 min. Addition of ouabain inhibited transport back to baseline levels of 2.1+/-0.4 microl/30 min. These results are the first demonstration that renal epithelial cells have been grown successfully as a confluent monolayer along a hollow fiber, and exhibit functional transport capabilities. The next steps in constructing a bioartificial renal tubule successfully are to develop a multi-fiber bioreactor with primary renal proximal tubule cells that maintain not only transport properties but also differentiated metabolic and endocrine functions, including glucose and ammonia production, and the conversion of vitamin D3 to a more active derivative. A renal tubule device may add critical renal functional components not currently substituted for, thereby improving the treatment regimens for patients with acute and chronic renal failure.