We describe the isolation of endothelial cells from the center region of the attached embryoid body (EB) by a two-step enzyme treatment. The isolated cells from the center and outgrowth region of the EB were characterized separately. As human embryonic stem (hES) cells differentiated in EB, they lost expression of the undifferentiated marker Oct-4, whereas expression levels of endothelial-specific markers were increased. Using RT-PCR, fluorescence-activated cell sorting (FACS) analysis, and immunofluorescence, we have shown that various endothelial cell markers, including platelet/endothelial cell adhesion molecule (PECAM), von Willebrand factor (vWF), Flk-1, and Tie2, were expressed on the attached EB. Compared with the outgrowth region of EB, the center region had a higher population of cells with these endothelial cell markers. Once isolated by the twostep enzyme treatment, cells from the center region continued to differentiate into endothelial cell lineage while expression level of endothelial cell markers in cells from the outgrowth region decreased in subcultures. This study has demonstrated that the isolation of EB by a two-step enzyme treatment is a useful technique to obtain endothelial cell marker-positive cells with high yield. Furthermore, a similar approach can be taken to identify the location and distribution of specific cell types in EB and thereby allow us to isolate and expand specific cell types.

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.