Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients.

Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.

Levels of the oxidative stress marker γ-glutamyltranspeptidase at different stages of nonalcoholic fatty liver disease.

OBJECTIVES: This study investigated oxidative stress in the liver, by determining hepatic expression and serum levels of γ-glutamyltranspeptidase (GGT) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in different stages of nonalcoholic fatty liver disease (NAFLD), and assessed whether GGT can differentiate between the various stages of NAFLD. METHODS: Expression of GGT and 8-OHdG was examined in biopsy specimens by immunohistochemistry, and serum GGT and 8-OHdG levels were measured by enzyme-linked immuno sorbent assays in patients with simple fatty liver (n = 10), nonalcoholic steatohepatitis (NASH; n = 10) and, as a control, in alcoholic liver disease (ALD; n = 10). RESULTS: Hepatic tissue expression of GGT and 8-OHdG was seen in ALD, NASH and fatty liver patients. The percentage of hepatocytes positive for 8-OHdG expression and serum 8-OHdG levels was significantly higher in patients with NASH than simple fatty liver. Serum GGT levels were increased in all cases with ALD, NASH and fatty liver, and correlated significantly with serum levels of 8-OHdG in ALD and NASH, but not in simple fatty liver. CONCLUSIONS: Levels of GGT in fatty liver patients may compensate for mild oxidative stress by repressing 8-OHdG levels and preventing progression to NASH; however further oxidative stress leads to increased levels of 8-OHdG and the development of NASH.

Evaluation of phosphate removal from water by immobilized phosphate-binding protein PstS.

The phosphate (P(i))-binding protein PstS is a member of a family of periplasmic proteins that act as high-affinity receptors for active transport systems in bacteria. PstS protein purified from Pseudomonas aeruginosa was immobilized to N-hydroxysuccinimide-activated Sepharose, packed into a plastic column (5 x 70 mm), and examined for its potential ability to remove P(i) from water. The PstS-Sepharose column completely removed P(i) from 32P-labeled pond water containing about 0.5 microM P(i) (0.015 mg P per liter). More than 90% of 32P-P(i) that was retained in the column could be eluted by washing with low-pH water.