Karthikeyan Kandasamy

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.

The performance of primary human renal cells in hollow fiber bioreactors for bioartificial kidneys

Bioartificial kidneys (BAKs) containing human primary renal proximal tubule cells (HPTCs) have been applied in clinical trials. The results were encouraging, but also showed that more research is required. Animal cells or cell lines are not suitable for clinical applications, but have been mainly used in studies on BAK development as large numbers of such cells could be easily obtained. It is difficult to predict HPTC performance based on data obtained with other cell types. To enable more extensive studies on HPTCs, we have developed a bioreactor containing single hollow fiber membranes that requires relatively small amounts of cells. Special hollow fiber membranes with the skin layer on the outer surface and consisting of polyethersulfone/polyvinylpyrrolidone were developed. The results suggested that such hollow fiber membranes were more suitable for the bioreactor unit of BAKs than membranes with an inner skin layer. An HPTC-compatible double coating was applied to the insides of the hollow fiber membranes, which sustained the formation of functional epithelia under bioreactor conditions. Nevertheless, the state of differentiation of the primary human cells remained a critical issue and should be further addressed. The bioreactor system described here will facilitate further studies on the relevant human cell type.

Identification of nephrotoxic compounds with embryonic stem-cell-derived human renal proximal tubular-like cells.

The kidney is a major target for drug-induced toxicity, and the renal proximal tubule is frequently affected. Nephrotoxicity is typically detected only late during drug development, and the nephrotoxic potential of newly approved drugs is often underestimated. A central problem is the lack of preclinical models with high predictivity. Validated in vitro models for the prediction of nephrotoxicity are not available. Major problems are related to the identification of appropriate cell models and end points. As drug-induced kidney injury is associated with inflammatory reactions, we explored the expression of inflammatory markers as end point for renal in vitro models. In parallel, we developed a new cell model. Here, we combined these approaches and developed an in vitro model with embryonic stem-cell-derived human renal proximal tubular-like cells that uses the expression of interleukin (IL)-6 and IL-8 as end points. The predictivity of the model was evaluated with 41 well-characterized compounds. The results revealed that the model predicts proximal tubular toxicity in humans with high accuracy. In contrast, the predictivity was low when well-established standard in vitro assays were used. Together, the results show that high predictivity can be obtained with in vitro models employing pluripotent stem cell-derived human renal proximal tubular-like cells.

An in vitro method for the prediction of renal proximal tubular toxicity in humans

The kidney is one of the major target organs for drug-induced toxicity. The renal proximal tubule is frequently affected due to its roles in drug transport and in concentrating the glomerular filtrate. Drug-induced kidney injury is associated with increased morbidity and mortality of patients. During drug development, nephrotoxicity is typically detected only late, which leads to high costs for the pharmaceutical industry. A central problem is the lack of pre-clinical models with high predictability. Regulatory accepted or validated in vitro models for the prediction of nephrotoxicity are not available. We developed a novel in vitro model for the prediction of renal proximal tubular toxicity in humans. It employs human primary renal proximal tubular cells and the expression levels of interleukin (IL)-6 and IL-8 were used as the endpoint. The model was evaluated with 41 well-characterized drugs and chemicals. The median values of the major performance metrics (balanced accuracy, sensitivity, specificity, positive predictive value, negative predictive value and area under the curve of the receiver operating characteristic curve) ranged between 0.76 and 0.85. This revealed that the predictability of the model is high and it would be expected that in ∼76%–85% of the cases where compounds were predicted as positives or negatives the predictions would be correct. Altogether, the data suggest that the model would allow the prediction of drug-induced proximal tubular toxicity at early pre-clinical stages during drug development. Future work will aim at further validating this model and adapting it to recently developed renal proximal tubular-like cells derived from human pluripotent stem cells.

Prediction of drug-induced nephrotoxicity and injury mechanisms with human induced pluripotent stem cell-derived cells and machine learning methods

The renal proximal tubule is a main target for drug-induced toxicity. The prediction of proximal tubular toxicity during drug development remains difficult. Any in vitro methods based on induced pluripotent stem cell-derived renal cells had not been developed, so far. Here, we developed a rapid 1-step protocol for the differentiation of human induced pluripotent stem cells (hiPSC) into proximal tubular-like cells. These proximal tubular-like cells had a purity of >90% after 8 days of differentiation and could be directly applied for compound screening. The nephrotoxicity prediction performance of the cells was determined by evaluating their responses to 30 compounds. The results were automatically determined using a machine learning algorithm called random forest. In this way, proximal tubular toxicity in humans could be predicted with 99.8% training accuracy and 87.0% test accuracy. Further, we studied the underlying mechanisms of injury and drug-induced cellular pathways in these hiPSC-derived renal cells, and the results were in agreement with human and animal data. Our methods will enable the development of personalized or disease-specific hiPSC-based renal in vitro models for compound screening and nephrotoxicity prediction.

A novel design of bioartificial kidneys with improved cell performance and haemocompatibility.

Treatment with bioartificial kidneys had beneficial effects in animal experiments and improved survival of critically ill patients with acute kidney injury in a Phase II clinical trial. However, a Phase II b clinical trial failed. This and other results suggested various problems with the current design of bioartificial kidneys. We propose a novel design to improve various properties of device, including haemocompatibility and cell performance. An important feature of the novel design is confinement of the blood to the lumina of the hollow fibre membranes. This avoids exposure of the blood to the non‐haemocompatible outer surfaces of hollow fibre membranes, which usually occurs in bioartificial kidneys. We use these outer surfaces as substrate for cell growth. Our results show that commercial hollow fibre membranes can be directly applied in the bioreactor when human primary renal proximal tubular cells are grown in this configuration, and no coatings are required for the formation of robust and functional renal epithelia. Furthermore, we demonstrate that the bioreactor unit produces significant amounts of interleukins. This result helps to understand the immunomodulatory effects of bioartificial kidneys, which have been observed previously. The novel bioartificial kidney design outlined here and the results obtained would be expected to improve the safety and performance of bioartificial kidneys and to contribute to a better understanding of their effects.

Polysulfone Membranes Coated with Polymerized 3,4-Dihydroxy-l-phenylalanine are a Versatile and Cost-Effective Synthetic Substrate for Defined Long-Term Cultures of Human Pluripotent Stem Cells

Clinical and industrial applications of human pluripotent stem cells (hPSC) require large amounts of cells that have been expanded under defined conditions. Labor-intensive techniques and ill-defined or expensive compounds and substrates are not applicable. Here we describe a chemically defined synthetic substrate consisting of polysulfone (PSF) membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine (DOPA). DOPA/PSF is inexpensive and can be easily produced at various shapes and sizes. DOPA/PSF supports long-term self-renewal of undifferentiated human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) under defined conditions. Pluripotency is maintained for at least 10 passages. Adhesion of hPSC to DOPA/PSF is mainly mediated by a specific integrin heterodimer. Proliferation and gene expression patterns on DOPA/PSF and control substrates are comparable. Labor-intensive cultivation methods and use of serum or coating with proteins are not required. Together, these features make DOPA/PSF attractive for applications where large-scale expansion of human pluripotent stem cells under defined conditions is essential.

The use of a library of industrial materials to determine the nature of substrate-dependent performance of primary adherent human cells

We developed a library of industrial materials, which can be applied to any adherent cell type for determining cell-material interactions. Bulk and surface chemistry as well as other material properties were characterized. The library covered broad ranges of various material properties. We applied the library to primary human endothelial and epithelial cells, which play important roles in tissue engineering and biomedical applications. The results revealed that substrate stiffness was the major determinant of cell performance. The ability to grow and differentiate on stiff or more compliant materials was cell type-dependent, but cell performance was consistently best on stiff and smooth materials. These results give new insights into the nature of substrate-dependent performance of primary human cells and are potentially useful for the development of improved biomaterials. The materials of the library can be easily accessed by the scientific community to determine cell-material interactions of any adherent cell type of interest.

Perinuclear positioning of the inactive human cystic fibrosis gene depends on CTCF, A‐type lamins and an active histone deacetylase

The nuclear positioning of mammalian genes often correlates with their functional state. For instance, the human cystic fibrosis transmembrane conductance regulator (CFTR) gene associates with the nuclear periphery in its inactive state, but occupies interior positions when active. It is not understood how nuclear gene positioning is determined. Here, we investigated trichostatin A (TSA)‐induced repositioning of CFTR in order to address molecular mechanisms controlling gene positioning. Treatment with the histone deacetylase (HDAC) inhibitor TSA induced increased histone acetylation and CFTR repositioning towards the interior within 20 min. When CFTR localized in the nuclear interior (either after TSA treatment or when the gene was active) consistent histone H3 hyperacetylation was observed at a CTCF site close to the CFTR promoter. Knockdown experiments revealed that CTCF was essential for perinuclear CFTR positioning and both, CTCF knockdown as well as TSA treatment had similar and CFTR‐specific effects on radial positioning. Furthermore, knockdown experiments revealed that also A‐type lamins were required for the perinuclear positioning of CFTR. Together, the results showed that CTCF, A‐type lamins and an active HDAC were essential for perinuclear positioning of CFTR and these components acted on a CTCF site adjacent to the CFTR promoter. The results are consistent with the idea that CTCF bound close to the CFTR promoter, A‐type lamins and an active HDAC form a complex at the nuclear periphery, which becomes disrupted upon inhibition of the HDAC, leading to the observed release of CFTR. J. Cell. Biochem. 113: 2607–2621, 2012.