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Functional extrinsic and intrinsic apoptotic pathways in human fetal mesenchymal stem cells

Presented at the Neonatal Society 2004 Summer Meeting (programme).

Kennea N, Stratou C, Naparus A, Edwards D, Mehmet H

Department of Paediatrics, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London, UK

Background: While stem cells offer significant promise for the correction of neurodegenerative diseases and acute brain injury, a major obstacle is the poor survival of grafted cells, the majority of which die by apoptosis. We have recently identified a population of human fetal mesenchymal stem cells (FMSC) (1) that can be differentiated into neural cells both in vitro and in vivo. If cell replacement therapy with FMSC is to be considered for brain injury, knowledge of the cell death machinery is essential to develop strategies to prevent graft loss. Currently, nothing is known about the mechanisms by which FMSC undergo cell death.

Objective: To determine whether human FMSCs have functional apoptotic machinery in both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways and to investigate whether stem cell survival can be prolonged by inhibition of death signalling.

Design/Methods: We investigated apoptosis in FMSCs subjected to three different death stimuli: serum withdrawal, Fas ligation or treatment with staurosporine (SSP). We studied the components of the classical mitochondrial and death receptor pathways. Apoptosis was defined using morphological and biochemical methods. Cytochrome c localisation was determined by immunofluorescence, and activation of caspases using cleavage specific antibodies. DNA fragmentation was confirmed by TUNEL.

Results: Activation of the mitochondrial pathway by SSP was demonstrated by the release of cytochrome c from mitochondria and subsequent cleavage of caspases 9 and 3 followed by DNA fragmentation indicated by TUNEL labelling of apoptotic nuclei. Serum withdrawal resulted in a similar pattern of molecular events, although the time course was significantly longer. In addition, the PKB/Akt pathway was down-regulated in serum-starved MSC cultures. Caspase 9 inhibitors, and the pan-caspase inhibitor z-VAD.fmk attenuated apoptosis triggered by SSP treatment. Death receptor machinery was present in FMSCs and was demonstrated to be functional by the rapid induction of apoptotic death by Fas ligand. This effect was attenuated by the selective caspase-8 inhibitor IETD.fmk as well pan-caspase inhibitors.

Conclusions: These results demonstrate intact intrinsic and extrinsic apoptotic pathways in FMSC, and thus provide insights into potential mechanisms of human FMSC apoptosis following transplantation. This may be useful in the design of pharmacological agents to prolong the survival of stem cell grafts.

1. Campagnoli,C. et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 98, 2396-2402 (2001).

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