Useful properties of undifferentiated mesenchymal stromal cells and adipose tissue as the source in liver-regenerative therapy studied in an animal model of severe acute fulminant hepatitis.

BACKGROUND AIMS: End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. METHODS: AT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbecco's modified Eagle's medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbecco's modified Eagle's medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription-polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. RESULTS: All MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. CONCLUSIONS: AT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.

Zinc protects human peripheral blood lymphocytes from Cr(III)(phenanthroline)3-induced apoptosis.

We have studied the effect of Cr(III)(phen)3 [(tris(1,10-phenanthroline) chromium(III) chloride)] on lymphocytes in order to find out if metallothioneins (MTs) are produced in the process. We also investigated whether zinc pretreatment is able to protect cells from apoptosis reported to occur for this compound. Our results indicate that MT synthesis is induced by Cr(III)(phen)3, and it has been identified as the MT-3 isoform through RT-PCR which has not been reported earlier. By zinc pretreatment, this apoptosis is reversed as inferred from cytotoxicity studies, Annexin-V/PI staining, ethidium bromide/acridine orange staining and DNA fragmentation pattern and ultrastructural investigations using TEM and SEM. The zinc pretreatment reduces the amount of ROS produced by Cr(III)(phen)3. The MT-1a and 1b synthesized by zinc (also evidenced through RT-PCR experiments) is possibly able to scavenge ROS which is one of the early signaling molecules that lead to apoptosis. Zinc pretreatment also reverses the changes in downstream signaling events such as mitochondrial membrane potential, ATP levels and the activation of caspase-3. This is the first report on the induction of MT-3 in lymphocytes due to a metal stress or any other stimuli. Even though MT-3 is synthesized here, apoptosis still occurs due to ROS production on Cr(III)(phen)3 exposure when the cells have not been primed with zinc.

Non-enzymatic phosphorylation of bovine serum albumin by Cr(V) complexes: role in Cr(VI)-induced phosphorylation and toxicity.

Evidence for the non-enzymatic phosphorylation of bovine serum albumin (BSA) by sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[CrVO(ehba)2], 1, sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[CrVO(hmba)2], 2 and potassium dichromate, K2Cr2O7, 3 in the presence of labeled adenosine-5'-triphosphate (ATP) under conditions of physiological pH is presented. Aggregation and extent of phosphorylation of BSA mediated by 1, 2 or 3 seems to increase with the concentration and time of incubation of the reaction mixture containing all the reactants. The [gamma-32P] label in ATP is incorporated into aggregates of BSA in the in vitro reaction of the protein with ATP in the presence of 1, 2 or 3. Phosphorylation of BSA by ATP in the absence of 1, 2 or 3 is negligible. Addition of EDTA reverses aggregation of protein and liberates partially the incorporated phosphate label. The stoichiometry of phosphorylation is found to be the highest and is equal to 12.25 mol PO4(3-)/mol BSA in the presence of 500 microM of 1, which decreases to 10.56 mol PO4(3-)/mol BSA after EDTA treatment. Resistance to the removal of phosphate label by EDTA increases with increase in time of incubation. Dialysis of phosphorylated BSA reverses the incorporated [gamma-(32)P] label only partially, indicating the formation of covalent links of phosphate groups to BSA. Evidence for the site of phosphorylation in the reaction mediated by 1, 2 or 3 being hydroxyl side groups of tyrosine and serine/threonine residues has been gained. Based on the results, a possibility that 1, 2 and 3 mimic the function of tyrosine and serine/threonine kinases has been invoked.