Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype.

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

Oxidative Stress, Glutathione Metabolism, and Liver Regeneration Pathways Are Activated in Hereditary Tyrosinemia Type 1 Mice upon Short-Term Nitisinone Discontinuation.

Hereditary tyrosinemia type 1 (HT1) is an inherited condition in which the body is unable to break down the amino acid tyrosine due to mutations in the fumarylacetoacetate hydrolase (FAH) gene, coding for the final enzyme of the tyrosine degradation pathway. As a consequence, HT1 patients accumulate toxic tyrosine derivatives causing severe liver damage. Since its introduction, the drug nitisinone (NTBC) has offered a life-saving treatment that inhibits the upstream enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD), thereby preventing production of downstream toxic metabolites. However, HT1 patients under NTBC therapy remain unable to degrade tyrosine. To control the disease and side-effects of the drug, HT1 patients need to take NTBC as an adjunct to a lifelong tyrosine and phenylalanine restricted diet. As a consequence of this strict therapeutic regime, drug compliance issues can arise with significant influence on patient health. In this study, we investigated the molecular impact of short-term NTBC therapy discontinuation on liver tissue of Fah-deficient mice. We found that after seven days of NTBC withdrawal, molecular pathways related to oxidative stress, glutathione metabolism, and liver regeneration were mostly affected. More specifically, NRF2-mediated oxidative stress response and several toxicological gene classes related to reactive oxygen species metabolism were significantly modulated. We observed that the expression of several key glutathione metabolism related genes including Slc7a11 and Ggt1 was highly increased after short-term NTBC therapy deprivation. This stress response was associated with the transcriptional activation of several markers of liver progenitor cells including Atf3, Cyr61, Ddr1, Epcam, Elovl7, and Glis3, indicating a concreted activation of liver regeneration early after NTBC withdrawal.

Inflammation Alters the Secretome and Immunomodulatory Properties of Human Skin-Derived Precursor Cells.

Human skin-derived precursors (SKP) represent a group of somatic stem/precursor cells that reside in dermal skin throughout life that harbor clinical potential. SKP have a high self-renewal capacity, the ability to differentiate into multiple cell types and low immunogenicity, rendering them key candidates for allogeneic cell-based, off-the-shelf therapy. However, potential clinical application of allogeneic SKP requires that these cells retain their therapeutic properties under all circumstances and, in particular, in the presence of an inflammation state. Therefore, in this study, we investigated the impact of pro-inflammatory stimulation on the secretome and immunosuppressive properties of SKP. We demonstrated that pro-inflammatory stimulation of SKP significantly changes their expression and the secretion profile of chemo/cytokines and growth factors. Most importantly, we observed that pro-inflammatory stimulated SKP were still able to suppress the graft-versus-host response when cotransplanted with human PBMC in severe-combined immune deficient (SCID) mice, albeit to a much lesser extent than unstimulated SKP. Altogether, this study demonstrates that an inflammatory microenvironment has a significant impact on the immunological properties of SKP. These alterations need to be taken into account when developing allogeneic SKP-based therapies.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

BACKGROUND AND AIMS: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.