The majority of patients with acute liver failure (ALF) die waiting for orthotopic liver transplantation (OLT). No other treatment modality is shown to improve survival. This study was conducted to assess the safety and feasibility of hepatocyte transplantation (HT) and subsequent engraftment and function of donor cells. Functional and structural integrity of cryopreserved and thawed human hepatocytes were assessed by their morphological characteristics, induction of P-4501A1 transcription, and survival in vivo by xenotransplantation into rats. Five patients with severe ALF underwent intrasplenic (4 patients) and/or intrahepatic (2 patients) HT through angiography under cyclosporine immunosuppression. All patients had grade III to IV encephalopathy and factor V levels less than 0.5 U/mL, were ventilator and dialysis dependent, and were not OLT candidates. Three of the 5 patients who survived 48 hours after HT had substantial improvement in encephalopathy scores, arterial ammonia levels, and prothrombin times. Clinical improvement was paralleled by an increase in aminopyrine and caffeine clearances. All 3 patients lived substantially longer than expected based on clinical experience after HT (12, 28, and 52 days) but eventually died. Postmortem examination showed the presence of transplanted hepatocytes in liver and spleen by light microscopy and fluorescent in situ hybridization (FISH). Cryopreserved and thawed human hepatocytes can be transplanted into recipients with ALF with some acceptable but definite complications. Engraftment of donor hepatocytes was proven by histological examination and FISH by both transjugular biopsy and at autopsy. Improvement in brain edema, encephalopathy grade, and clearance of antipyrine and caffeine suggested function, albeit with a 24- to 72-hour delay posttransplantation.

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements – it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid, guggulsterone, was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinson's disease patients.

Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated b-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.

Until now little is known about the functional integrity of human hepatocytes after hypothermic storage. In order to address this limitation, we evaluated several commercially available hypothermic preservation media for their abilities to protect freshly isolated hepatocytes during prolonged cold storage. Human hepatocytes were isolated from non-transplantable/rejected donor livers and resuspended in ice-cold University of Wisconsin solution (UW), HypoThermosol-Base (HTS-Base), or HypoThermosol-FRS (HTS-FRS) with or without the addition of fetal bovine serum. Cells were stored at 4 degrees C for 24-72 h, and evaluated for hepatocyte viability (trypan blue exclusion, or labeling with fluorochromes), cell attachment, and function. The energy status of hepatocytes was evaluated by measurement of intracellular adenosine 5'-triphosphate. To determine whether the test cells expressed metabolic functions of freshly isolated cells, the activities of major phase I (cytochromes P450, FMO) and phase II (UGT, ST) drug-metabolizing enzymes were examined. Although hepatocytes are shown to be satisfactory after 24 h storage in all of the tested solutions, the cell viability, energy status, and xenobiotic metabolism following cold preservation in HTS-FRS was consistently and, in some cases, markedly higher when compared with other systems. The same metabolites for each of the tested substrates were detected in all groups of cells. Moreover, the use of HTS-FRS eliminates the need for serum in preservation solutions. HTS-FRS represents an improved solution compared to HTS-Base and UW for extending the shipping/storage time of human hepatocytes.