PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Cardio-protective signalling by glyceryl trinitrate and cariporide in a model of donor heart preservation.

BACKGROUND: Storage of donor hearts in cardioplegic solutions supplemented with agents that mimic the ischaemic preconditioning response enhanced their post-reperfusion function. The present study examines the minimisation of cell death and activation of pro-survival signalling directed towards maintenance of mitochondrial homeostasis in hearts arrested and stored in two such agents, glyceryl-trinitrate, a nitric oxide donor and cariporide, (a sodium-hydrogen exchange inhibitor). METHODS: After baseline functional measurement, isolated working rat hearts were arrested and stored for 6h at 4°C in either Celsior(®), Celsior(®) containing 0.1mg/ml glyceryl-trinitrate, 10µM cariporide or both agents. After reperfusion, function was remeasured. Hearts were then processed for immunoblotting or histology. RESULTS: Necrotic and apoptotic markers present in the Celsior(®) group post-reperfusion were abolished by glyceryl-trinitrate, cariporide or both. Increased phosphorylation of ERK and Bcl2, after reperfusion in groups stored in glyceryl-trinitrate, cariporide or both along with increased phospho-STAT3 levels in the glyceryl-trinitrate/cariporide group correlated with functional recovery. Inhibition of STAT3 phosphorylation blocked recovery. No phospho-Akt increase was seen in any treatment. CONCLUSIONS: Activation of signalling pathways that favour mitophagy activation (ERK and Bcl2 phosphorylation) and maintenance of mitochondrial transition pore closure after reperfusion (STAT3 and ERK phosphorylation) were crucial for functional recovery of the donor heart.

Improved poststorage cardiac function by poly (ADP-ribose) polymerase inhibition: role of phosphatidylinositol 3-kinase Akt pathway.

BACKGROUND: Inhibition of poly(ADP-ribose) polymerase 1 (PARP) has been shown to be effective in minimizing cardiac ischemia reperfusion injury. We investigated the cardioprotective effect of the PARP inhibitor, INO-1153, in isolated working rat hearts after 6 hr of hypothermic storage in Celsior. METHODS: Hearts were treated with 1 muM INO-1153 before hypothermic storage, at cardioplegia and storage or after hypothermic storage. Hearts not exposed to INO-1153 served as controls. Another group was pretreated with the phosphatidylinositol 3-kinase inhibitor Wortmannin (0.1 muM) before storage in INO-1153-supplemented Celsior. After baseline measurement of aortic flow, heart rate, coronary flow, and cardiac output were obtained, hearts were arrested and stored in Celsior at 2-3 degrees C for 6 hr. After storage, hearts were reperfused for 15 min before performing work for a further 30 min, at which time poststorage indices of cardiac function were remeasured then heart tissue was stored at -80 degrees C for Western blot analysis. RESULTS: The presence of INO-1153 during prestorage perfusion or during cardioplegia and storage significantly improved poststorage cardiac function. Functional improvements produced by INO-1153 were completely abolished by Wortmnanin pretreatment. Western blots showed a significant increase in phospho-Akt in presence of INO-1153, which was inhibited by Wortmannin. CONCLUSION: Activation of the prosurvival phosphatidylinositol 3-kinase-Akt pathway was involved in the protective action of PARP inhibition in this model of donor heart procurement and hypothermic storage. Importantly for the logistics of clinical organ procurement, maximum protection is observed when the PARP inhibitor is included in the cardioplegic storage solution.