German Group Uses Induced Pluripotent Stem Cells to Model Nonalcoholic Fatty Liver Disease

A German research group has used pluripotent stem cells to design a new in vitro model system for investigating nonalcoholic fatty liver disease (NAFLD).  NAFLD, or steatosis, is a liver disease whose prevalence is probably much higher than estimated, and the new cases of it are increasing every year throughout the world.  NAFLD is typically associated with obesity and type-2 diabetes.  An estimated one-third of the general population of Western countries is thought to be affected with NAFLD, with or without symptoms.  It usually results from a high caloric diet in combination with a lack of exercise.  The liver begins to accumulate fat as lipid droplets.  Initially, this is a benign state, but it can develop into nonalcoholic steatohepatitis (also known as NASH), an inflammatory disease of the liver.  Then many patients develop fibrosis, cirrhosis or even liver cancer.  However, in many cases patients die of heart failure before they develop severe liver damage.

A major obstacle that dogged NAFLD research was that biopsies of patients and healthy individuals were required.  Researchers from the Institute for Stem Cell Research and Regenerative Medicine at the University Clinic of Düsseldorf, Germany solved this problem by reprogramming skin cells into induced pluripotent stem cells (iPSCs) that they differentiated into hepatocyte-like cells.

“Although our hepatocyte-like cells are not fully mature, they are already an excellent model system for the analysis of such a complex disease”, said Nina Graffmann, first author of the paper that appeared in the journal Stem Cells and Development.

The researchers recapitulated important steps of the disease in cultured cells.  They demonstrated up-regulation of PLIN2, a protein called perilipin that surrounds lipid droplets. Mice without PLIN2 do not become obese, even when overfed with a high fat diet.  Also the key role of PPARα, a transcription factor involved in controlling glucose and lipid metabolism, was reproduced in the tissue culture system.  “In our system, we can efficiently induce lipid storage in hepatocyte-like cells and manipulate associated proteins or microRNAs by adding various factors into the culture.  Thus, our in vitro model offers the opportunity to analyse drugs which might reduce the stored fat in hepatocytes,” Graffmann said.

Senior author James Adjaye and his colleagues hope to expand their model by deriving iPSCs from NAFLD patients.  They hope to discover differences that might explain the course of NAFLD.

“Using as reference the data and biomarkers obtained from our initial analyses on patient liver biopsies and matching serum samples, we hope to better understand the etiology of NAFLD and the development of NASH at the level of the individual, with the ultimate aim of developing targeted therapy options,” said Adjayer.

This paper can be found at Nina Graffmann et al., “Modeling NAFLD with human pluripotent stem cell derived immature hepatocyte like cells reveals activation of PLIN2 and confirms regulatory functions of PPARα,”Stem Cells and Development, 2016; DOI: 10.1089/scd.2015.0383.

A New Tool for Gene Editing In Stem Cells Can Drive Changes in Cell Fate Without Causing Mutations

Recently, a new tool is now available to control gene expression in order to understand gene function and manipulate cell fate. This new tool is called CRIPSR/Cas9, which is a gene-editing tool that employs a genetic system that naturally occurs in bacteria, who use it as a protection against viruses. CRISPR/Cas9 allows scientists to precisely add, remove or replace specific sequences of DNA. It is the most efficient, inexpensive and easiest gene editing tool available to date.

Several laboratories have tried to use CRISPR/Cas9 to activate genes in cells, but such an effort has not always succeeded. However a research team at Hokkaido University’s Institute of Genetic Medicine has developed a powerful new method that uses CRISPR/Cas9 to do exactly that.

In cells, genes have an expression switch called “promoters.” Genes are switched off, or silenced, when their promoters are methylated, which means that islands of C-G bases have a methyl group (a –CH3 group) attached to the cytosine base. The Hokkaido University team wanted to turn an inactivated gene on. The ingeniously combined a DNA repair mechanism, called MMEJ (microhomology-mediated end-joining), with CRISPR/Cas9 to do this. They excised a methylated promoter using CRISPR/Cas9 and then used MMEJ to insert an unmethylated promoter. Thus, they replaced the off-switch signal with an on-switch signal.

DNA Methylation

The gene that was activated was the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in order to test the efficiency of this technology in cultured cells. Within five days, they found evidence that the genes were robustly expressed. When they activated the OLIG2 gene in cultured human stem cells, the cells differentiated to neurons in seven days with high-efficiency.

Toru Kondo and his colleagues also discovered that their editing tool could be used to activate other silenced promoters. They also found that their system didn’t cause unwanted mutations in other non-target genes in the cells. According to Kondo, this gene editing tool has wide potential to manipulate gene expression, create genetic circuits, or to engineer cell fates.

See Shota Katayama et al., “A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation,” Angewandte Chemie International Edition, 2016; 55(22): 6452 DOI: 10.1002/anie.201601708.