Neurotrophins (NTs) are members of a neuronal growth factor protein family whose action is mediated by the tropomyosin receptor kinase (TRK) receptor family receptors and the p75 NT receptor (p75NTR), a member of the tumor necrosis factor (TNF) receptor family. Although NTs were first discovered in neurons, recent studies have suggested that NTs and their receptors are expressed in various types of stem cells mediating pivotal signaling events in stem cell biology. The concept of stem cell therapy has already attracted much attention as a potential strategy for the treatment of neurodegenerative diseases (NDs). Strikingly, NTs, proNTs, and their receptors are gaining interest as key regulators of stem cells differentiation, survival, self-renewal, plasticity, and migration. In this review, we elaborate the recent progress in understanding of NTs and their action on various stem cells. First, we provide current knowledge of NTs, proNTs, and their receptor isoforms and signaling pathways. Subsequently, we describe recent advances in the understanding of NT activities in various stem cells and their role in NDs, particularly Alzheimer's disease (AD) and Parkinson's disease (PD). Finally, we compile the implications of NTs and stem cells from a clinical perspective and discuss the challenges with regard to transplantation therapy for treatment of AD and PD.

Neurotrophin Signaling and Stem Cells—Implications for Neurodegenerative Diseases and Stem Cell Therapy

Background/Aims: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs) under simulated microgravity within a fast-rotating clinostat (clinorotation) and capture of microarray-based gene signatures. Methods: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. Results: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g) after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs). Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. Conclusion: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The most significant simulated microgravity-affected genes, signal transduction pathways, and biological processes which are relevant for mESCs differentiation have been identified and discussed below.

Simulated Microgravity Modulates Differentiation Processes of Embryonic Stem Cells

Background/Aims: Common systems for the quantification of cellular contraction rely on animal-based models, complex experimental setups or indirect approaches. The herein presented CellDrum technology for testing mechanical tension of cellular monolayers and thin tissue constructs has the potential to scale-up mechanical testing towards medium-throughput analyses. Using hiPS-Cardiac Myocytes (hiPS-CMs) it represents a new perspective of drug testing and brings us closer to personalized drug medication. Methods: In the present study, monolayers of self-beating hiPS-CMs were grown on ultra-thin circular silicone membranes and deflect under the weight of the culture medium. Rhythmic contractions of the hiPS-CMs induced variations of the membrane deflection. The recorded contraction-relaxation-cycles were analyzed with respect to their amplitudes, durations, time integrals and frequencies. Besides unstimulated force and tensile stress, we investigated the effects of agonists and antagonists acting on Ca2+ channels (S-Bay K8644/verapamil) and Na+ channels (veratridine/lidocaine). Results: The measured data and simulations for pharmacologically unstimulated contraction resembled findings in native human heart tissue, while the pharmacological dose-response curves were highly accurate and consistent with reference data. Conclusion: We conclude that the combination of the CellDrum with hiPS-CMs offers a fast, facile and precise system for pharmacological, toxicological studies and offers new preclinical basic research potential.

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Mechano-Pharmacological Characterization of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

Background/Aims: To investigate whether nucleosome assembly protein 1-like 1 (Nap1l1) regulates the proliferation of induced pluripotent stem cells (iPSC) and the potential mechanisms. Methods: Nap1l1-knockdown-iPSC and Nap1l1-overexpression-iPSC were constructed by transfection of lentiviral particles. The proliferation of iPSC was detected by MTT analysis, and cell cycle was analyzed by flow cytometry. Results: Nap1l1 overexpression promoted iPSC proliferation and induced G2/M transition compared to their control iPSC while Nap1l1-knockdown-iPSC dramatically displayed the reduced proliferation and accumulated G2/M phase cells. Further analysis showed that Nap1l1 overexpression in iPSC increased the expression of cyclin B1, downregulated the expression of p21 and p27, while knockdown of Nap1l1 showed the opposite effects. In addition, overexpression of Nap1l1 promoted the phosphorylation of AKT and ERK in iPSC, while knockdown of Nap1l1 inhibited the effects. However, these effects displayed in Nap1l1-overexpression-iPSC were greatly suppressed by the inhibition of AKT or ERK signaling. Conclusions: The results indicate that Nap1l1 promotes the proliferation of iPSC attributable to G2/M transition caused by downregulation of p27 and p21, and upregulation of cyclin B1, the activation of AKT or ERK is involved in the process. The present study has revealed a novel molecular mechanism involved in the proliferation of iPSC.

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Nucleosome Assembly Protein 1-Like 1 (Nap1l1) Regulates the Proliferation of Murine Induced Pluripotent Stem Cells.

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embr...

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity

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, FGLL, 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.

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Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

Morten Albrechtsen

Papers

Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells