Effects of synthetic neural adhesion molecule mimetic peptides and related proteins on the cardiomyogenic differentiation of mouse embryonic stem cells.

BACKGROUND/AIMS: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. METHODS: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGL(L), hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. RESULTS: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. CONCLUSION: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.

The Neural Cell Adhesion Molecule-Derived (NCAM)-Peptide FG Loop (FGL) Mobilizes Endogenous Neural Stem Cells and Promotes Endogenous Regenerative Capacity after Stroke.

The neural cell adhesion molecule (NCAM)-derived peptide FG loop (FGL) modulates synaptogenesis, neurogenesis, and stem cell proliferation, enhances cognitive capacities, and conveys neuroprotection after stroke. Here we investigated the effect of subcutaneously injected FGL on cellular compartments affected by degeneration and regeneration after stroke due to middle cerebral artery occlusion (MCAO), namely endogenous neural stem cells (NSC), oligodendrocytes, and microglia. In addition to immunohistochemistry, we used non-invasive positron emission tomography (PET) imaging with the tracer [18F]-fluoro-L-thymidine ([18F]FLT) to visualize endogenous NSC in vivo. FGL significantly increased endogenous NSC mobilization in the neurogenic niches as evidenced by in vivo and ex vivo methods, and it induced remyelination. Moreover, FGL affected neuroinflammation. Extending previous in vitro results, our data show that the NCAM mimetic peptide FGL mobilizes endogenous NSC after focal ischemia and enhances regeneration by amplifying remyelination and modulating neuroinflammation via affecting microglia. Results suggest FGL as a promising candidate to promote recovery after stroke.

Screening of bioactive peptides using an embryonic stem cell-based neurodifferentiation assay.

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.

The synthetic NCAM mimetic peptide FGL mobilizes neural stem cells in vitro and in vivo.

The neural cell adhesion molecule (NCAM) plays a role in neurite outgrowth, synaptogenesis, and neuronal differentiation. The NCAM mimetic peptide FG Loop (FGL) promotes neuronal survival in vitro and enhances spatial learning and memory in rats. We here investigated the effects of FGL on neural stem cells (NSC) in vitro and in vivo. In vitro, cell proliferation of primary NSC was assessed after exposure to various concentrations of NCAM or FGL. The differentiation potential of NCAM- or FGL-treated cells was assessed immunocytochemically. To investigate its influence on endogenous NSC in vivo, FGL was injected subcutaneously into adult rats. The effects on NSC mobilization were studied both via non-invasive positron emission tomography (PET) imaging using the tracer [(18)F]-fluoro-L-thymidine ([(18)F]FLT), as well as with immunohistochemistry. Only FGL significantly enhanced NSC proliferation in vitro, with a maximal effect at 10 µg/ml. During differentiation, NCAM promoted neurogenesis, while FGL induced an oligodendroglial phenotype; astrocytic differentiation was neither affected by NCAM or FGL. Those differential effects of NCAM and FGL on differentiation were mediated through different receptors. After FGL-injection in vivo, proliferative activity of NSC in the subventricular zone (SVZ) was increased (compared to placebo-treated animals). Moreover, non-invasive imaging of cell proliferation using [(18)F]FLT-PET supported an FGL-induced mobilization of NSC from both the SVZ and the hippocampus. We conclude that FGL robustly induces NSC mobilization in vitro and in vivo, and supports oligodendroglial differentiation. This capacity renders FGL a promising agent to facilitate remyelinization, which may eventually make FGL a drug candidate for demyelinating neurological disorders.