Patients with mtDNA depletion syndrome 3 (MTDPS3) often die as children from liver failure caused by severe reduction in mtDNA content. The identification of treatments has been impeded by an inability to culture and manipulate MTDPS3 primary hepatocytes. Here we generated DGUOK-deficient hepatocyte-like cells using induced pluripotent stem cells (iPSCs) and used them to identify drugs that could improve mitochondrial ATP production and mitochondrial function. Nicotinamide adenine dinucleotide (NAD) was found to improve mitochondrial function in DGUOK-deficient hepatocyte-like cells by activating the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). NAD treatment also improved ATP production in MTDPS3-null rats and in hepatocyte-like cells that were deficient in ribonucleoside-diphosphate reductase subunit M2B (RRM2B), suggesting that it could be broadly effective. Our studies reveal that DGUOK-deficient iPSC-derived hepatocytes recapitulate the pathophysiology of MTDPS3 in culture and can be used to identify therapeutics for mtDNA depletion syndromes.

Elucidating the molecular basis of cell differentiation will advance our understanding of organ development and disease. We have previously established a protocol that efficiently produces cells with hepatocyte characteristics from human induced pluripotent stem cells. We previously used this cell differentiation model to identify the transcription factor hepatocyte nuclear factor 4 α (HNF4A) as being essential during the transition of the endoderm to a hepatic fate. Here, we sought to define the molecular mechanisms through which HNF4A controls this process. By combining HNF4A chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) analyses at the onset of hepatic progenitor cell formation with transcriptome data collected during early stages of differentiation, we identified genes whose expression is directly dependent upon HNF4A. By examining the dynamic changes that occur at the promoters of these HNF4A targets we reveal that HNF4A is essential for recruitment of RNA polymerase (RNA pol) II to genes that are characteristically expressed as the hepatic progenitors differentiate from the endoderm.

Fibroblast growth factors (FGFs) are required to specify hepatic fate within the definitive endoderm through activation of the FGF receptors (FGFRs). While the signaling pathways involved in hepatic specification are well understood, the mechanisms through which FGFs induce hepatic character within the endoderm are ill defined. Here we report the identification of genes whose expression is directly regulated by FGFR activity during the transition from endoderm to hepatic progenitor cell. The FGFR immediate early genes that were identified include those encoding transcription factors, growth factors, and signaling molecules. One of these immediate early genes encodes naked cuticle homolog 1 (NKD1), which is a repressor of canonical WNT (wingless-type MMTV integration site) signaling. We show that loss of NKD1 suppresses the formation of hepatic progenitor cells from human induced pluripotent stem cells and that this phenotype can be rescued by using a pharmacological antagonist of canonical WNT signaling. We conclude that FGF specifies hepatic fate at least in large part by inducing expression of NKD1 to transiently suppress the canonical WNT pathway.

We have previously shown that the transcription factor HNF4A is required for the formation of hepatic progenitor cells from endoderm that has been derived from human induced pluripotent stem cells (iPSCs). We reasoned that we could uncover regulatory pathways with new roles in hepatocyte differentiation by identifying cellular processes that regulate HNF4A. We therefore performed a screen of 1120 small molecules with well-characterized mechanisms of action to detect those that affect the abundance of HNF4A in iPSC-derived hepatic progenitor cells. This approach uncovered several small molecules that depleted HNF4A. Of those, we chose to focus on an inhibitor of heat shock protein 90 beta (HSP90β). We show that mutation of the gene encoding HSP90β represses hepatocyte differentiation during the formation of hepatocytes from iPSCs. We reveal that HSP90β, although dispensable for expression of HNF4A mRNA, directly interacts with HNF4A protein to regulate its half-life. Our results demonstrate that HSP90β has an unappreciated role in controlling hepatic progenitor cell formation and highlight the efficiency of using small-molecule screens during the differentiation of iPSCs to reveal new molecular mechanisms that control hepatocyte formation.