Foad J. Rouhani

Human iPS cells have been generated using a diverse range of tissues from a variety of donors using different reprogramming vectors. However, these cell lines are heterogeneous, which presents a limitation for their use in disease modeling and personalized medicine. To explore the basis of this heterogeneity we generated 25 iPS cell lines under normalised conditions from the same set of somatic tissues across a number of donors. RNA-seq data sets from each cell line were compared to identify the majority contributors to transcriptional heterogeneity. We found that genetic differences between individual donors were the major cause of transcriptional variation between lines. In contrast, residual signatures from the somatic cell of origin, so called epigenetic memory, contributed relatively little to transcriptional variation. Thus, underlying genetic background variation is responsible for most heterogeneity between human iPS cell lines. We conclude that epigenetic effects in hIPSCs are minimal, and that hIPSCs are a stable, robust and powerful platform for large-scale studies of the function of genetic differences between individuals. Our data also suggest that future studies using hIPSCs as a model system should focus most effort on collection of large numbers of donors, rather than generating large numbers of lines from the same donor.

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.

, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.