Introduction: Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are self-renewing multipotent progenitors with the potential to differentiate into multiple lineages of mesoderm, in addition to generating ectodermal and endodermal lineages by crossing the germline barrier. In the present study we have investigated the ability of UCB-MSCs to generate neurons, since we were able to observe varying degrees of neuronal differentiation from a few batches of UCB-MSCs with very simple neuronal induction protocols whereas other batches required extensive exposure to combination of growth factors in a stepwise protocol. Our hypothesis was therefore that the human UCB-MSCs would contain multiple types of progenitors with varying neurogenic potential and that the ratio of the progenitors with high and low neurogenic potentials varies in different batches of UCB. Methods: In total we collected 45 UCB samples, nine of which generated MSCs that were further expanded and characterized using immunofluorescence, fluorescence-activated cell sorting and RT-PCR analysis. The neuronal differentiation potential of the UCB-MSCs was analyzed with exposure to combination of growth factors. Results: We could identify two different populations of progenitors within the UCB-MSCs. One population represented progenitors with innate neurogenic potential that initially express pluripotent stem cell markers such as Oct4, Nanog, Sox2, ABCG2 and neuro-ectodermal marker nestin and are capable of expanding and differentiating into neurons with exposure to simple neuronal induction conditions. The remaining population of cells, typically expressing MSC markers, requires extensive exposure to a combination of growth factors to transdifferentiate into neurons. Interesting to note was that both of these cell populations were positive for CD29 and CD105, indicating their MSC lineage, but showed prominent difference in their neurogenic potential. Conclusion: Our results suggest that the expanded UCB-derived MSCs harbor a small unique population of cells that express pluripotent stem cell markers along with MSC markers and possess an inherent neurogenic potential. These pluripotent progenitors later generate cells expressing neural progenitor markers and are responsible for the instantaneous neuronal differentiation; the ratio of these pluripotent marker expressing cells in a batch determines the innate neurogenic potential.

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.

Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators.

Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.

Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based.