ABSTRACT
Transplantation of ex vivo expanded autologous limbal stem cells into the diseased eye of patients with limbal stem cell deficiency (LSCD) has been in practice worldwide. However, isolation of limbal tissue from the normal eye of the patient with unilateral LSCD still remains a major concern for the donor. More importantly, autologous cell transplantation is not a viable option for patients with bilateral LSCD. The objective of the current study was to determine the expansion potential of human limbal epithelial stem cells (hLESCs) for their possible use in allo-transplantation. A total of six limbal biopsy samples were cultured and expanded in vitro up to passage level 1 (P-1), at which point the hLESCs were cryopreserved. Semi-quantitative RT-PCR and immunophenotypic analysis revealed that hLESCs obtained before and after cryopreservation retained the expression of major limbal epithelial stem cell markers such as p63, SSEA-4, ABCG2, cytokeratin 19 (CK19), integrin ß1 and vimentin. One notable difference was that while P-0 hLESCs expressed HLA-DR mRNA, no HLA-DR gene expression was observed with the expanded and cryopreserved samples. Human LESCs did not express costimulatory proteins CD80 or B7-DC but expressed significant levels of CD86, B7-H1 and HLA-ABC molecules on the cell surface. Treatment of hLESCs with IFN-γ induced the expression of HLA-DR, indoleamine 2,3-dioxygenase (IDO) and HLA-G on these cells. Cultured hLESCs were unable to stimulate allogeneic T cell proliferation in vitro even in the presence of pro-inflammatory cytokine, IFN-γ. These results indicate that cryopreserved hLESCs are non-immunogenic in nature and express negative immunoregulatory molecules which may be critical for their survival in an allogeneic environment.
Subject(s)
Cryopreservation , Epithelial Cells/immunology , Epithelium, Corneal , Limbus Corneae/cytology , Stem Cells/immunology , Cell Culture Techniques , Dendritic Cells/immunology , Epithelial Cells/drug effects , Flow Cytometry , HLA Antigens/genetics , HLA-DR Antigens/metabolism , HLA-G Antigens , Histocompatibility Antigens Class I/genetics , Humans , Interferon-gamma/pharmacology , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/drug effects , T-Lymphocytes/immunologyABSTRACT
The present study reports the biological activity of 8-methyl-4-(3-diethylamino-propylamino) pyrimido [4';5';4,5] thieno (2,3-b) quinoline (MDPTQ), a quinoline derivative structurally related to ellipticine and suggests a possible mechanism through which the compound induces apoptosis in carcinoma cell lines. Out of the 8 cell lines used in the study as representatives of different types of cancer, MDPTQ was found to be effective only against leukemia cell lines (HL-60 and K-562) whereas it had no effect on normal human bone marrow cells (BMC) which were used as controls. Fall mitochondrial membrane potential and increased reactive oxygen species (ROS) were mainly responsible for inducing apoptosis in the two cell lines. Cell death was demonstrated by increase in caspase 3 activity as well as phosphatidyl serine exposure. Pre-incubation with N-acetylcysteine (NAC) reduced the increased ROS and caspase 3 activity as well as phosphatidyl serine exposure. MDPTQ also caused cell cycle arrest in these cell lines. The above study for the first time reports the mode of action of a quinoline derivative, which could be a possible future candidate for leukemia therapy. However, there are lot of questions that need to be answered in terms of signalling pathways and its effects on animal models.
