Micromorphologic changes in human amnion cells during trypsinization.

Previous studies from our laboratory showed that human amnion epithelial cells (AECs) have multiple functions, such as synthesis and release of catecholamines, acetylcholine, neurotrophic factors, activin, and noggin. In this study, we investigated the identity of neural progenitor cells in human amnion mesenchyme cells (AMCs), which lie immediately adjacent to the AECs. Cryostat sections revealed that vimentin expression was detected in the AMCs and CK19 in AECs. Vimentin-positive cells made up 97.5% of total cells tested in cultured AMCs. Interestingly, 3.6% of total AMCs expressed the phenotype CK19+/vimentin+, indicating coexpression of epithelial and mesenchyme cell markers. In culturing with bromodeoxyuridine (BrdU) for 24 hr, 66-82% of cells were found to be BrdU positive, suggesting that they have proliferating potency. By using RT-PCR, AMCs express mRNA of nestin and Musashi1. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. Nestin (87.7% of total cells tested) and Musashi1 (93.1%) were expressed in undifferentiated cells, and their positively stained cells increased in number slightly after induction. Undifferentiated cells were stained by anti-Tuj1 and NF-M, and their positively stained cells increased significantly in number after induction, to 72.8% and 46.0%, respectively. Meanwhile, glial fibrillary acidic protein-positive cells increased from 25.4% to 43.2% after induction. These studies demonstrate that AMCs have phenotypes of neuroglial progenitor cells and can be differentiated into neuroglial phenotypes by optimal differentiation protocol. Eventually, AMC-derived stem cells may be a favorable cell vehicle in regenerative medicine.

Amnion↗

PubMed2004

Reversible PEGylation: a novel technology to release native interferon alpha2 over a prolonged time period.

Many peptide and protein drugs have a short circulatory half-life in vivo. The covalent attachment of polyethylene glycol (PEG) chains (PEGylation) can overcome this deficiency, but pegylated peptides and proteins are often inactive. In this study, we present a novel PEG-IFNalpha2 conjugate, PEG(40)-FMS-IFNalpha2, capable of regenerating native interferon alpha2 (IFNalpha2) at a slow rate under physiological conditions. A 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) containing bifunctional reagent, MAL-FMS-NHS, has been synthesized, enabling the linkage of a 40 kDa PEG-SH to IFNalpha2 through a slowly hydrolyzable bond. By use of a BIAcore binding assay, the in vitro rate of regeneration of native interferon was estimated to have a half-life of 65 h. Following subcutaneous administration to rats and monitoring circulating antiviral activity, active IFNalpha2 levels peaked at 50 h, with substantial levels still being detected 200 h after administration. This value contrasts with a half-life of about 1 h measured for unmodified interferon. The concentration of active IFNalpha2 scaled linearly with the quantity injected. Comparing subcutaneous to intravenous administration of PEG(40)-FMS-IFNalpha2, we found that the long circulatory lifetime of IFNalpha2 was affected both by the slow rate of absorption of the PEGylated protein from the subcutaneous volume and by the slow rate of discharge from the PEG in circulation. A numerical simulation of the results was in good agreement with the results observed in vivo. The pharmacokinetic profile of this novel IFNalpha2 conjugate combines a prolonged maintenance in vivo with the regeneration of active-native IFNalpha2, ensuring ready access to peripheral tissues and thus an overall advantage over currently used formulations.

Amnion↗

PubMed2004

Epidermal growth factor and interleukin-1beta utilize divergent signaling pathways to synergistically upregulate cyclooxygenase-2 gene expression in human amnion-derived WISH cells.

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intrauterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1beta-driven PGE(2) production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1beta might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1beta resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1beta-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFkappaB) regulatory elements alone, but was evident when a fragment (-891/ +9) of the COX-2 gene 5'-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1beta-driven NFkappaB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1beta appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1beta employs the NFkappaB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1beta and EGF does not rely on augmented NFkappaB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter.

Amnion↗