Protein expression profiles during osteogenic differentiation of mesenchymal stem cells derived from human umbilical cord blood.

Mesenchymal stem cells (MSCs) can potentially differentiate along multiple lineages and be expanded in vitro, making them highly attractive candidates for cell therapy and tissue engineering applications. This study sought to investigate the critical proteins involved in osteogenic differentiation of mesenchymal stem cells derived from umbilical cord blood (UCB-MSCs). MSCs, which were isolated from three different preparations of human UCB, were osteoinduced, and total proteins were extracted from the cells. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was performed on the day (d) of induction d0, and on d2, d7, and d21 of differentiation. The optical density (OD) of each spot was measured, and spots with a mean OD of three cell lines of MSCs that increased > 30 or decreased < 0.1 relative to a previous time point were selected. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) was used to identify the proteins. Through database searches, the properties and functions of the proteins were investigated and then classified according to the Gene Ontology classification. Among the 308 spots observed in the 2-D gel, 16 proteins with a mean OD ratio > 30, and 20 proteins with a mean OD ratio < 0.1 were identified during the differentiation process. Additionally, the distribution of differentially expressed proteins according to cellular component and molecular function criteria differed depending on whether protein expression increased or decreased during differentiation. The results of this study will comprise an initial proteomic database for UCB-MSCs differentiation.

Production of recombinant human von Willebrand factor in the milk of transgenic pigs.

Von Willebrand factor (vWF), a large multimeric glycoprotein present in blood plasma, is a blood protein of the coagulation system. It is defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and heyde's syndrome. We have developed a line of transgenic swine harboring recombinant human von Willebrand factor (rhvWF) cDNA through microinjection of fertilized one-cell pig zygotes. Expression of rhvWF in the mammary gland and secretion of rhvWF into the milk of the transgenic swine were confirmed by immunohistochemical and western blot analyses, respectively, and rhvWF proteins were detected in milk from all lactating founder females at concentrations that were 28- to 56-folds greater than that in circulating human plasma. The amino acid sequence of rhvWF protein in the transgenic pig milk matched that of vWF produced from human blood plasma. This study provides evidence that production of rhvWF from transgenic pig milk is a potentially valuable technology and can be used as a cost-effective alternative in clinical applications.

Elevation of CRABP-I in the cerebrospinal fluid of patients with Moyamoya disease.

BACKGROUND AND PURPOSE: The etiology of moyamoya disease (MMD) remains obscure. This study was undertaken to identify specific proteins associated with the pathogenesis of MMD. METHODS: We studied cerebrospinal fluid (CSF) from 20 patients with angiographically confirmed MMD (4 boys and 16 girls; age range, 3 to 13 years; mean, 7.5 years) and 4 control patients with cerebral palsy who underwent selective dorsal rhizotomy (2 boys and 2 girls; age range, 5 to 10 years; mean, 7.3 years). CSF proteins were analyzed by 2-dimensional polyacrylamide gel electrophoresis, and protein identification was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presence of specific CSF protein in patients with MMD was confirmed by Western blotting. In addition, cerebral CSF was also tested in 7 patients who had other brain diseases but no MMD (2 boys and 5 girls; age range, 1 to 12 years; mean, 6.9 years). RESULTS: We identified 1 polypeptide spot (Mr of 13 to 15 kDa and isoelectric point of 5 to 5.5) that was differentially expressed in the CSF samples of MMD patients (mean optical density intensity, 0.36+/-0.24; range, 0.05 to 0.92) and control spinal CSF samples (mean, 0.03+/-0.04; range, 0 to 0.08; P=0.002). This polypeptide was identified as cellular retinoic acid-binding protein (CRABP)-I. High levels of expression of CRABP-I in the CSF from 17 MMD children were confirmed by Western blotting. CONCLUSIONS: The analysis of the CSF of MMD patients reveals high CRABP-I expression. The present study suggests that the elevation of CRABP-I in CSF may be a candidate for pathogenesis of MMD.