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Freiburg researchers transform skin cells into renal cells

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.

Dr. Soeren Lienkamp, Dr. Sebastian Arnold and Dr. Michael Kaminski (from left to right) have successfully produced renal cells in “the test tube” using direct reprogramming. © Private

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.

Glossary

  • An expression vector is a kind of ferry for genes, which allows the introduction of a specific gene into a host cell (e.g. E. coli, yeast cells). Furthermore, it contains all the necessary regulatory elements for the transformation of that gene into the protein.
  • A gene is a hereditary unit which has effects on the traits and thus on the phenotype of an organism. Part on the DNA which contains genetic information for the synthesis of a protein or functional RNA (e.g. tRNA).
  • In a biological context, insertion often means the introduction of DNA fragments into another DNA molecule.
  • In vitro (lat.) means: in the test tube, i.e., outside of the organism.
  • Screening is a systematic test procedure that is used to identify certain characteristics within an array of samples or persons. In molecular biology screening is used to filter a designated clone out of a gen bank, for example.
  • Transformation is the natural ability of some species of bacteria to take up free DNA from their surroundings through their cell wall. In genetic engineering, transformation denotes a process which is often used to introduce recombinant plasmids in E. coli, for example. This is a modified version of natural transformation.
  • Transcription in a biological context is the process of transcription from DNA into RNA. In this processes, a single-stranded RNA molecule is synthesized on the basis of the double-stranded DNA with the help of an enzyme named RNA-polymerase.
  • A virus is an infectious particle (no cell!) consisting of a protein envelope and a genome (DNA or RNA). To be able to reproduce it depends entirely on the metabolism of living cells of host organisms (e.g., bacteria for phages, liver cells for Hepatitis A-virus).
  • Embryonic stem cells are cells, which were developed from early blastocyst stage of embryos generated by in vitro fertilisation, or from primordial germ cells of five to nine-week old aborted foetuses. It is possible to proliferate embryonic stem cells in virtually unlimited numbers without showing signs of cell ageing. In addition, these cells are pluripotent, i.e. they possess the ability to differentiate into many, possibly all of the approximately 200 different cell types of the body.
  • A neuron is a nerve cell. A nerve cell consists of a body, an axon and dendrites.
  • Pluripotent cells are able to differentiate into all cell types of the organism. Contrary to totipotent cells they cannot generate a new organism.
  • Stem cells are cells from the embryo, fetus or adult that that have the ability to divide for indefinite periods in culture and give rise to specialized cells. In Germany it is illegal to extract the stem cells from embryos.
  • A tumour is a swelling of a tissue caused by abnormal cell growth, which can be benign or malignant. Benign tumours are local swellings, whereas malign tumours may seed off and spread into other tissues, causing secondary growths (metastases).
  • Expression means the biosynthesis of a gene product. Usually, DNA is transcribed into mRNA and subsequently translated into proteins.
  • The toxicity is the poisonousness of a substance.
  • The differentiation of cells describes their specialisation in their function and structure. Different cell types like myocardial, neural or liver cells with totally different appearance and functions develop from undifferentiated stem cells in this way.
  • Pharmacology is the study of interactions between drugs and organisms. There are two methods of evaluation: The pharmacokinetics describes the uptake, distribution, metabolism and excretion of an active substance. The pharmacodynamics describes the effects of a drug in the organism.

Four genes make this transformation possible

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.

Disease models rather than organ replacements

Induced renal tubular epithelial cells (iRECs) that are grown in 3D cultures can form elongated tubules, which is the typical shape of their natural counterparts. The photo shows the borders (green) of cells and the respective cell nuclei (blue). © Jelena Tosic

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.

Gaining a better understanding of cell identity

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.

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