Hepatocyte transplantation (HT) has become an effective therapy for patients with metabolic inborn errors. We report the clinical outcome of four children with metabolic inborn errors that underwent HT, describing the cell infusion protocol and the metabolic outcome of transplanted patients. Cryopreserved hepatocytes were used as this allows scheduling of treatments. Functional competence (viability, cell attachment, major cytochrome P450 and UDP-glucuronosyltransferase 1A1 activities, and urea synthesis) and microbiological safety of cell batches were assessed prior to clinical use. Four pediatric patients with liver metabolic diseases [ornithine trans carbamylase (OTC) deficiency, Crigler-Najjar (CNI) syndrome, glycogen storage disease Ia (GSD-Ia), and tyrosinemia type I (TYR-I)] underwent HT. Indication for HT was based on severity of disease, deterioration of quality of life, and benefits for the patients, with the ultimate goal to improve their clinical status whenever liver transplantation (LT) was not indicated or to bridge LT. Cells were infused into the portal vein while monitoring portal flow. The protocol included antibiotic prophylaxis and immunosuppressant therapy. After HT, analytical data on the disease were obtained. The OTC-deficient patient showed a sustained decrease in plasma ammonia levels and increased urea production after HT. Further cell infusions could not be administered given a fatal nosocomial fungus sepsis 2 weeks after the last HT. The CNI and GSD-Ia patients improved their clinical status after HT. They displayed reduced serum bilirubin levels (by ca. 50%) and absence of hypoglycaemic episodes, respectively. In both cases, the HT contributed to stabilize their clinical status as LT was not indicated. In the infant with TYR-I, HT stabilized temporarily the biochemical parameters, resulting in the amelioration of his clinical status while diagnosis of the disease was unequivocally confirmed by full gene sequencing. In this patient, HT served as a bridge therapy to LT.

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocytes. Moreover, the evaluation of urea synthesis from ammonia and UDP-glucuronosyltransferase 1A1 activity, a newly developed assay using beta-estradiol as substrate, allows the possibility of customizing cell preparation for receptors with urea cycle disorders or Crigler-Najjar Syndrome type I. Sources of human liver and factors derived from the procurement of the liver sample (warm and cold ischemia) have also been investigated. The results show that grafts with a cold ischemia time exceeding 15 h and steatosis should not be accepted for hepatocyte transplantation. Finally, livers from non-heart-beating donors are apparently a potential suitable source of hepatocytes, which could enlarge the liver donor pool.

Adipose tissue contains a mesenchymal stem cell (MSC) population known as adipose-derived stem cells (ASCs) capable of differentiating into different cell types. Our aim was to induce hepatic transdifferentiation of ASCs by sequential exposure to several combinations of cytokines, growth factors, and hormones. The most efficient hepatogenic protocol includes fibroblastic growth factors (FGF) 2 and 4 and epidermal growth factor (EGF) (step 1), hepatocyte growth factor (HGF), FGF2, FGF4, and nicotinamide (Nic) (step 2), and oncostatin M (OSM), dexamethasone (Dex), and insulin-tranferrin-selenium (step 3). This protocol activated transcription factors [GATA6, Hex, CCAAT/enhancer binding protein α and β (CEBPα and β), peroxisome proliferator-activated receptor-γ, coactivator 1 α (PGC1α), and hepatocyte nuclear factor 4 α (HNF4α)], which promoted a characteristic hepatic phenotype, as assessed by new informative markers for the stepby-step hepatic transdifferentiation of hMSC [early markers: albumin (ALB), α-2-macroglobuline (α2M), complement protein C3 (C3), and selenoprotein P1 (SEPP1); late markers: cytochrome P450 3A4 (CYP3A4), apolipoprotein E (APOE), acyl-CoA synthetase long-chain family member 1 (ACSL1), and angiotensin II receptor, type 1 (AGTR1)]. The loss of adipose adult stem cell phenotype was detected by losing expression of Thy1 and inhibitor of DNA binding 3 (Id3). The reexpression of phosphoenolpyruvate corboxykinase (PEPCK), apolipoprotein C3 (APOCIII), aldolase B (ALDOB), and cytochrome P450 1A2 (CYP1A2) was achieved by transduction with a recombinant adenovirus for HNF4α and finally hepatic functionality was also assessed by analyzing specific biochemical markers. We conclude that ASCs could represent an alternative tool in clinical therapy for liver dysfunction and regenerative medicine.