A team of researchers from Freiburg has used direct programming to successfully produce kidney-like cells very similar to natural renal tubular cells in terms of appearance and function. These cells are thus a promising alternative to kidney cells isolated from animals and cells differentiated from embryonic stem cells. The reprogrammed kidney cells can be used, for example, for pharmacological and toxicological tests and investigating the disease mechanisms of genetic renal diseases.
The in vitro generation of organ-specific cell types has become one of several methods routinely used by cell and developmental biologists. It involves using embryonic stem cells or so-called iPS cells (induced pluripotent stem cells). The latter have the potential to evolve into any cell type whatsoever once they have received the appropriate signals for doing so. In vitro cell differentiation mimics the natural, gradual development of cells into precursor cells during embryonic development and is therefore a rather time-consuming process. A powerful process called direct reprogramming makes it possible to directly transform fully differentiated mature cells into many other cell types without them needing to go through an intermediate pluripotent state. This process uses genes that enable skin cells to transform into other cells, to name but one example. The genes are inserted into the genome of the skin cells to be transformed. This can be done using viral vectors. A group of researchers from Freiburg has now achieved with renal cells what has already been possible with neural and myocardial cells for many years. Teams led by Dr. Soeren Lienkamp from Freiburg University Medical Center and Dr. Sebastian Arnold from the University of Freiburg have successfully transformed connective tissue cells (fibroblasts) from humans and mice into kidney-like cells.
The researchers therefore had to identify genes that mediated the transformation of skin cells into induced renal tubular epithelial cells (iRECs). Computer programmes were used to systematically screen for suitable gene candidates. “We first examined transcription factors that were specifically expressed in large quantities in the kidneys. We also studied the expression and function of specific transcription factors during embryonic kidney development in Xenopus and mouse embryos,” says Dr. Michael Kaminski, lead author of the paper and medical doctor at the Freiburg University Medical Center. The researchers identified 13 genes with the sought-after effect, and eventually chose four that made the planned cell transformation possible. The genes that were detected – Emx2, Hnf1b, Hnf4a and Pax8 – code for transcription factors that control gene expression. When the four genes are inserted into the genome of fibroblasts, they induce the fibroblasts to transform into renal tubular epithelial cells.
The similarity of iRECs with their natural counterparts goes beyond just appearance. The two cell types are also similar in how they behave and grow in cultures. “When grown in 3D culture systems, they form spheroids, i.e. polarised three-dimensional spherical structures, can actively take up molecules by endocytosis and integrate into kidney organoids in vitro,” says Kaminski.
When the iRECs are introduced into kidneys that have previously had all their cells removed, they even grow into elongated tubules, which is what renal cells do. The gene expression profiles of iRECs also show many similarities with genuine renal tubular cells. However, they are not totally identical since some fibroblast-typical genes are still active in iRECs.
Due to their similarity with natural kidney cells, iRECs are suitable for application in a wide range of pharmacological and toxicological tests. “We already know that iRECs are very sensitive to nephrotoxic drugs, i.e. drugs that cause acute tubular and other damage in patients undergoing medical treatment,” says Lienkamp. Comprehensive investigations on whether new drugs can be tested with iRECs are still to be carried out. However, iRECs have already been proved suitable as new in vitro models for investigating genetic renal diseases affecting renal tubules.“ In addition, we might also be able to use the reprogrammed cells for investigating the effect of compounds on renal tubular cells,” explains Arnold. It seems plausible that these cells will contribute to delaying animal experiments to a much later stage of the drug discovery process than at present, or at least help reduce the number of experimental animals used.
The researchers do not currently believe that the new cells are suitable for the regenerative treatment of patients with kidney damage. “Reprogrammed cells might be used for the regenerative treatment of patients with kidney damage at some stage in the future. However, at present, there are still too many obstacles and safety aspects that need to be addressed,” says Lienkamp. As it is impossible to use reporter genes for identifying cells that have been reprogrammed in patients, the recovery of the desired cells is a huge obstacle on its own. Moreover, although the continuous growth of iRECs in culture is a major advantage for many in vitro applications, the cells cannot be used for treating patients due to the risk that the reprogrammed cells might develop into tumours. “The greatest strength of iRECs lies in their use as disease models; using them for regenerative purposes is very much in the background, at least at the moment,” says Lienkamp.
Instead of using the cells for regenerative medicine applications, the researchers are currently specifically focused on optimising the reprogramming process with the able aim of being to control the process more precisely. They are also working on the optimisation of test systems in order to improve the characterisation of iRECs. “We are slowly beginning to understand how cells maintain their identity throughout an organism’s development and life,” says Arnold. The developmental biologist hopes to gain a better understanding of how cell identity is established based on new, genome-wide investigations of reprogrammed cells and normal renal tubular cells. “It will be exciting to learn how reprogramming can be achieved without having to go through all pre-stages of kidney development,” Arnold concludes.