BackgroundMesenchymal stem cells (MSC) are pluripotent cells, present in the bone marrow and other tissues that can differentiate into cells of all germ layers and may be involved in tissue maintenance and repair in adult organisms. Because of their plasticity and accessibility these cells are also prime candidates for regenerative medicine. The contribution of stem cell aging to organismal aging is under debate and one theory is that reparative processes deteriorate as a consequence of stem cell aging and/or decrease in number. Age has been linked with changes in osteogenic and adipogenic potential of MSCs.ResultsHere we report on changes in global gene expression of cultured MSCs isolated from the bone marrow of mice at ages 2, 8, and 26-months. Microarray analyses revealed significant changes in the expression of more than 8000 genes with stage-specific changes of multiple differentiation, cell cycle and growth factor genes. Key markers of adipogenesis including lipoprotein lipase, FABP4, and Itm2a displayed age-dependent declines. Expression of the master cell cycle regulators p53 and p21 and growth factors HGF and VEGF also declined significantly at 26 months. These changes were evident despite multiple cell divisions in vitro after bone marrow isolation.ConclusionsThe results suggest that MSCs are subject to molecular genetic changes during aging that are conserved during passage in culture. These changes may affect the physiological functions and the potential of autologous MSCs for stem cell therapy.

Ischemia followed by reperfusion is the primary cause of tissue injury and infarction during heart attack and stroke. The initiating stimulus is believed to involve reactive oxygen species that are produced during reperfusion when electron transport resumes in the mitochondria after suppression by ischemia. Programmed death has been shown to be a significant component of infarction, and evidence indicates that multiple pathways are initiated during both ischemia and reperfusion phases. Major infarction is preceded by severe ischemia that includes hypoxia, intracellular acidosis, glucose depletion, loss of ATP, and elevation of cytoplasmic calcium. The superimposition of a reactive oxygen surge on the latter condition provides the impetus for maximal damage. Compelling evidence implicates mitochondria not only as the source of initiating ROS but also as the focal sensors that translate the redox stress signal into a cellular-death response. Pivotal to this response are the BH3-only proteins that are activated by death signals and regulate mitochondrial communication with executioner proteins in the cytoplasm. The BH3-only proteins do this by controlling the activity of pores and channels in the outer mitochondrial membrane. To date at least six BH3-only proteins have been shown to contribute to ischemia-reperfusion death pathways in heart and/or brain; these include Bnip3, PUMA, Bid, Bad, HGTD-P, and Noxa. Here we review the evidence for these cell-death pathways and discuss their relevance to ischemic disease and infarction.

Bnip3 is a prodeath member of the so-called BH3-only subfamily of Bcl-2 proteins. A major function of this class of proteins is to regulate the permeability state of the outer mitochondrial membrane by forming homoand hetero-oligomers inside the membrane. We reported previously that Bnip3 accumulates in cardiac myocytes during exposure to hypoxia, but coincident acidosis is required to activate the death program. Acidosis increased the rate of intracellular accumulation of Bnip3 and promoted a tighter association with mitochondria. Here we report that acidic pH mediates increased half-lives of Bnip3 dimers and monomers (>3-) as well as that of a faster-migrating fragment (>10-) and confers protection against degradation by protease. Hydrophobic partitioning experiments revealed that Bnip3 monomers and oligomers from hypoxia-acidic cell fractions associated significantly with the detergent layer, whereas protein from hypoxia-neutral myocytes did not. Acidosis promoted homodimerization of Bcl-xL but did not increase its association with detergent. Neutralization of the extracellular medium of cardiac myocyte cultures under hypoxia-acidosis resulted in rapid degradation of accumulated Bnip3 (half life, <2 h), coincident with cessation of the death program. Bnip3 monomers appear to be the active species because substitution of alanine for histidine at position 173 within the transmembrane (TM) domain prevented homodimerization but did not inhibit the death function. These results demonstrate a pH-sensitive shift in the stability and apparent hydrophobicity of Bnip3 monomers that correlates closely with membrane binding and function.

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Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables-1, LATS and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ-line and a possible role in regulating the cell cycle of mesenchymal stem cells.