In vitro Differentiation of Endothelial Precursor Cells Derived from Umbilical Cord Blood

BACKGROUND AND OBJECTIVES: Ex vivo expansion of endothelial cells is important when applying cell therapy to therapeutic angiogenesis in ischemic tissues. Endothelial precursor cells (EPCs) from the umbilical cord blood are one of adult stem cell. In order to establish the culture system for EPCs, we examined the effects of the media and matrix on the differentiation of a subset of mononuclear cells to endothelial cells, and analyzed their endothelial-lineage phenotype. MATERIALS AND METHODS: Mononuclear cells isolated from human umbilical cord blood were cultured in a chamber slide coated with fibronectin or gelatin in a M199 medium supplemented with 10% fetal bovine serum (FBS) (the normal medium) or with 20% FBS and ECGS (the rich medium). Changes in the morphology and the attainment of DiI-ac-LDL uptake ability were examined during a 7 day period. The attached cells were immunostained for CD31, KDR, and vWF. RESULTS: The fibronectin matrix gave rise to more attached cells than the gelatin matrix (about 1.5 fold). The numbers of attached cells were no different between the normal medium and the rich medium at day 3 and 7, and were about 12% of the seeded mononuclear cells. However, the cell size and the numbers of longer spindle-shaped cells increased with the rich medium. Moreover, there was no increase in cellular population, but a 2-3 fold increase in the cellular size between day 3 and 7. About 20-40% of the attached cells acquired the DiI-ac-LDL uptake ability at day 3, whereas more than 85% of the attached cells could be stained with fluorescent DiI-ac-LDL at day 7 (p<0.001). The attached cells after being cultured for 7 days were stained moderately with the antibodies of CD31, or KDR. However, the cells at day 7 were only weakly immunostained with the vWF antibody, whereas more than 90% of cells were strongly stained at day 14. CONCLUSION: These results suggest that a subset of mononuclear cells derived from cord blood cells can give rise to cells with an endothelial cell-like phenotype, in vitro, at high percentages, which could be applied to in vivo vasculogenesis.

A Case of Charcot-Marie-Tooth 1A Showing Atypical Clinical and Pathological Features

A 7 year-old girl presented with generalized muscle weakness and delayed motor development. She was able to stand up at 15 months and began to walk at 4 years of age. A nerve conduction study showed severe demyelinating neuropa-thy .There was no family history of peripheral neuropathy, and her parents and younger brother were clinically and electrophysiologically normal. A sural nerve biopsy showed moderate loss of myelinated fibers with onion-bulb forma-tions. Many teased nerve fibers revealed typical tomaculous changes. However the molecular genetic study of the patient confirms the duplication of 17p11.2-p22 on a polymerase chain reaction using D17S261 as a primer but not in her parents.