Apoptosis and necrosis are the two major forms of cell death mechanisms. Both forms of cell death are involved in several physiological and pathological conditions and also in the elimination of cancer cells following successful chemotherapy. Large number of cellular and biochemical assays have evolved to determine apoptosis or necrosis for qualitative and quantitative purposes. A closer analysis of the assays and their performance reveal the difficulty in using any of these methods as a confirmatory approach, owing to the secondary induction of necrosis in apoptotic cells. This highlights the essential requirement of an approach with a real-time analysis capability for discriminating the two forms of cell death. This paper describes a sensitive live cell-based method for distinguishing apoptosis and necrosis at single-cell level. The method uses cancer cells stably expressing genetically encoded FRET-based active caspase detection probe and DsRed fluorescent protein targeted to mitochondria. Caspase activation is visualized by loss of FRET upon cleavage of the FRET probe, while retention of mitochondrial fluorescence and loss of FRET probe before its cleavage confirms necrosis. The absence of cleavage as well as the retention of mitochondrial fluorescence indicates live cells. The method described here forms an extremely sensitive tool to visualize and quantify apoptosis and necrosis, which is adaptable for diverse microscopic, flow cytometric techniques and high-throughput imaging platforms with potential application in diverse areas of cell biology and oncology drug screening.

We demonstrate the importance of combining ABCG2 expression with N/C ratio for identification and quantification of human CESCs. This method will have application in evaluating the role of niche cells/factors in the maintenance of stemness.

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever-increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno-diseases to human system. This study was aimed to establish an efficient xeno-feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using "interactive measurements" of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno-feeder free epithelial equivalents towards ocular surface reconstruction.

Corneal pathologies due to limbal stem cell deficiency or trauma is a common occurrence with high incidence in the modern era. Absence of native limbal stem cells in these cases can even result in blindness unless treated. Identifying alternate autologous stem cells with potential to replace limbal stem cells, gain its significance especially in cases of bilateral limbal damage. Here we look at the suitability of proposing bone marrow derived mesenchymal stem cells as an alternate to limbal stem cells. This work had made use of the corneo -limbal cues for trans differentiating these mesodermal cells to corneal epithelial lineage. Mesenchymal stem cells were isolated from rabbit bone marrow and the culture conditions were optimized. Characterized mesenchymal stem cells were transdifferentiated by simulating limbal niche using extracellular matrix and limbal fibroblast conditioned medium. The differentiation to corneal lineage was evaluated by analyzing the expression of mesenchymal (ABCG2, CD29) and corneal epithelial (CK3/12) markers. Cells cultured in the presence of conditioned medium alone and with conditioned medium/ extracellular matrix showed the expression of CK3/12. CK3/12 expressing cells in these cultures were increasing, with a concomitant decrease in stem cell marker expression. Trans differentiating mesenchymal stem cells to corneal epithelial like cells will provide a useful, autologous source of adult stem cells in therapeutic approaches for patients suffering from bilateral limbal stem cell deficiency.

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