Purpose Stem cell-based therapy has the potential to become one approach to regenerate the damaged trabecular meshwork (TM) in glaucoma. Co-culture of induced pluripotent stem cells (iPSCs) with human TM cells has been a successful approach to generate autologous TM resembling cells. However, the differentiated cells generated using this approach are still problematic for clinical usage. This study aimed to develop a clinically applicable strategy for generating TM-like cells from iPSCs. Methods Highly expressed receptors during iPSC differentiation were identified by AutoSOME, Gene Ontology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. The recombinant cytokines that bind to these receptors were used to generate a new differentiation protocol. The resultant TM-like cells were characterized morphologically, immunohistochemically, and transcriptionally. Results We first determined two stages of iPSC differentiation and identified highly expressed receptors associated with the differentiation at each stage. The expression of these receptors was further confirmed by RT-PCR analysis. Exposure to the recombinant cytokines that bind to these receptors, including transforming growth factor beta 1, nerve growth factor beta, erythropoietin, prostaglandin F2 alpha, and epidermal growth factor, can efficiently differentiate iPSCs into TM-like cells, which express TM biomarkers and can form dexamethasone-inducible CLANs. Conclusions We successfully generated a xeno- and feeder-free differentiation protocol with recombinant cytokines to generate the TM progenitor and TM-like cells from human iPSCs. Translational Relevance The new approach minimizes the risks from contamination and also improves the differentiation efficiency and consistency, which are particularly crucial for clinical use of stem cells in glaucoma treatment.
The α7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca 2+-permeable homopentameric ion channel implicated in cognition and neuropsychiatric disorders. Pharmacological enhancement of α7 nAChR function has been suggested for improvement of cognitive deficits. In the present study, we characterized a thiazolyl heterocyclic derivative, 6-(2-chloro-6methylphenyl)-2-((3-fluoro-4-methylphenyl)amino)thiazolo[4,5-d]pyrimidin-7(6H)-one (JWX-A0108), as a novel type I α7 nAChR positive allosteric modulator (PAM), and evaluated its ability to reverse auditory gating and spatial working memory deficits in mice. In Xenopus oocytes expressing human nAChR channels, application of JWX-A0108 selectively enhanced α7 nAChR-mediated inward current in the presence of the agonist ACh (EC 50 value = 4.35 ± 0.12 µM). In hippocampal slices, co-application of ACh and JWX-A0108 (10 µM for each) markedly increased both the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded in pyramidal neurons, but JWX-A0108 did not affect GABA-induced current in oocytes expressing human GABA A receptor α1β3γ2 and α5β3γ2 subtypes. In mice with MK-801-induced deficits in auditory gating, administration of JWX-A0108 (1, 3, and 10 mg/kg, i.p.) dose-dependently attenuates MK-801-induced auditory gating deficits in five prepulse intensities (72, 76, 80, 84, and 88 dB). Furthermore, administration of JWX-A0108 (0.03, 0.1, or 0.3 mg/kg, i.p.) significantly reversed MK-801-induced impaired spatial working memory in mice. Our results demonstrate that JWX-A0108 is a novel type I PAM of α7 nAChR, which may be beneficial for improvement of cognitive deficits commonly found in neuropsychiatric disorders such as schizophrenia and Alzheimer's disease.
Neuroinflammation plays an early and prominent role in the pathology of Alzheimer's disease (AD). Tumor necrosis factor‐a‐induced protein 8‐like 2 (TIPE2) has been identified as a negative regulator of innate and adaptive immunity. However, whether TIPE2 affects cognitive functions in AD‐like mouse models remains unknown. In this study, we compared the gene and protein expressions of TIPE2 and β‐amyloid in the brain between the APP/PS1 mice and the age‐matched wild type (WT) mice at different stages of developments using assays of immunofluorescence, western blot and qPCR. Deposition of Aβ plaques appeared in the hippocampus from the 6‐month‐old APP/PS1 mice, and the hippocampal expression of the TIPE2 mRNA and protein in APP/PS1 mice were significantly higher than that of the WT mice starting from 6 months to 10 months. However, the difference of the TIPE2 expression between the APP/PS1 mice and the WT mice declined in a time‐dependent manner. The spatial learning and memory deficit from 8‐month‐old APP/PS1 mice was observed in the Y‐maze test and fear conditioning task. Interestingly, overexpression of TIPE2 by intra‐hippocampal injection of AAV‐TIPE2 into the 7‐month old APP/PS1 mice resulted in an improvement of learning and memory. Taken together, our findings show that the TIPE2 expression level was negatively correlated with the Alzheimer's disease, suggesting TIPE2 is a potential target for pharmacological intervention and improvement of cognitive deficits.Support or Funding InformationThis work was supported by research grants to Fang ZHANG from the Natural Science Foundation of Shandong Province of China, No. ZR2016 HM 46; Post doctoral application foundation of Qingdao Municipal Bureau of Social Sciences, No. 2016062; Qingdao science and technology bureau, No 18‐6‐1‐75‐nsh; Clinical medicine +X Project of Qingdao University Medical Department; Innovation and Entrepreneurship Training Program of undergraduate student in Qingdao University. No. 201711065152.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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