ABSTRACT
The use of unrestricted somatic stem cells (USSCs) holds great promise for future clinical applications. Conventionally, mouse embryonic fibroblasts (MEFs) or other animal-based feeder layers are used to support embryonic stem cell (ESC) growth; the use of such feeder cells increases the risk of retroviral and other pathogenic infection in clinical trials. Implementation of a human-based feeder layer, such as hUSSCs that are isolated from human sources, lowers such risks. Isolated cord blood USSCs derived from various donors were used as a novel, supportive feeder layer for growth of C4mES cells (Royan C4 ESCs). Complete cellular characterization using immunocytochemical and flow cytometric methods were performed on murine ESCs (mESCs) and hUSSCs. mESCs cultured on hUSSCs showed similar cellular morphology and presented the same cell markers of undifferentiated mESC as would have been observed in mESCs grown on MEFs. Our data revealed these cells had negative expression of Stat3, Sox2, and Fgf4 genes while showing positive expression for Pou5f1, Nanog, Rex1, Brachyury, Lif, Lifr, Tert, B2m, and Bmp4 genes. Moreover, mESCs cultured on hUSSCs exhibited proven differentiation potential to germ cell layers showing normal karyotype. The major advantage of hUSSCs is their ability to be continuously cultured for at least 50 passages. We have also found that hUSSCs have the potential to provide ESC support from the early moments of isolation. Further study of hUSSC as a novel human feeder layer may lead to their incorporation into clinical methods, making them a vital part of the application of human ESCs in clinical cell therapy.
Subject(s)
Coculture Techniques/methods , Embryonic Stem Cells/physiology , Feeder Cells/physiology , Animals , Biomarkers/metabolism , Cell Differentiation/physiology , Embryoid Bodies/cytology , Embryonic Stem Cells/cytology , Feeder Cells/cytology , Fetal Blood/cytology , Flow Cytometry , Genetic Markers/genetics , Humans , Immunophenotyping , Karyotype , Mice , Mice, SCID , Teratoma/chemistryABSTRACT
PURPOSE: To study the presence of the herpes simplex virus type 1 (HSV-1) glycoprotein D gene in tear films of Iranian patients with herpetic keratitis. METHODS: Twenty-five tear film and eye swab specimens from 25 herpetic keratitis patients and 10 specimens from 10 healthy volunteers were collected in the Farabi Eye Hospital, Tehran, Iran. HSV-1 DNA was detected by using the nested polymerase chain reaction (nPCR) method. Viral isolation was done using conventional viral techniques. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was used for confirmation of positive cytopathic effect cell culture. The results of a diagnosis by an ophthalmologist team were compared with those of nPCR. RESULTS: HSV-1 DNA was identified in tear films of 88% (23/25) of suspected herpetic keratitis patients. All healthy controls (100%) had negative PCR results. HSV-1 was isolated in cell culture and confirmed by ELISA in 12% (3/25) of herpetic keratitis patients who had epithelial keratitis. The kappa value showed a high level of agreement between ophthalmologist team diagnosis and the PCR results (kappa = 0.86, P < 0.0001). CONCLUSIONS: nPCR is a sensitive, rapid, and powerful tool for detection of HSV-1 DNA in tear films of ocular herpetic keratitis patients and can serve as a supplemental method for diagnosis of herpetic keratitis infection.
