• Accelerated and efficient neuronal differentiation of Sox1GFP mouse embryonic stem cells in vitro using nicotinamide

      Griffin, Sile; Pickard, Mark R.; Fricker, Rosemary; Keele University, United Kingdom (NECTAR (Network of European CNS Transplantation and Restoration) 24th Annual Meeting, 27/28th November 2014, Galway, Ireland, 2014)
      A major challenge for advancement of clinical neuronal replacement therapies is the production of high yields of purified neuronal populations of appropriate phenotype with control over proliferation to prevent tumorigenesis. We previously reported that treatment of mouse embryonic stem cell (mESC;46CSox1GFP reporter cell line) monolayer cultures with the vitamin B3 metabolite nicotinamide at the early onset of development not only increased the efficiency of neuronal generation by two-fold but also enriched the ratio of purified neurons to non-neuronal cells in culture. This study aimed to investigate if nicotinamide enhances neural induction in this model and whether it also promotes the production/differentiation of specific neuronal subtypes. To address these aims, monolayer mESC cultures were treated with nicotinamide (10 mM) for different durations and immunocytochemistry/fluorescence microscopy was performed to assess the expression of stem cell, neural progenitor (NP) and neuronal subtype markers. Morphometric analyses were also performed to assess the extent of differentiation. Nicotinamide treatment significantly decreased Oct4+ pluripotent cells and concomitantly increased GFP+ cells at day 4, suggesting enhanced neural lineage commitment. By day 14, nicotinamide treatment (from day 0-7) reduced both Oct4+ and GFP expression concomitant with enhanced expression of neuron-specific β-tubulin, indicative of accelerated neuronal differentiation. Nicotinamide selectively enhanced the production of catecholaminergic, serotonergic and GABAergic neurons and, moreover, enhanced various aspects of neuronal morphology and maturation. Collectively, these data demonstrate a direct effect of nicotinamide at the initial stages of embryonic stem cell differentiation which could be critical for rapidly andefficiently promoting neural commitment to highly enriched neuronal lineages. The strong clinical potential of nicotinamide could successfully be applied to future neural cell-based therapies including Parkinson’s and Huntington’s disease, both to eradicate proliferating cells and for a more enhanced and specific differentiation
    • The influence of nicotinamide on the development of neurons

      Griffin, Sile; Pickard, Mark R.; Hawkins, Clive P.; Williams, Adrian C.; Chari, Divya M.; Fricker, Rosemary; Orme, Rowan P.; Keele University, University Hospital of North Staffordshire NHS Trust, University of Birmingham, United Kingdom (2014-09-09)
      A major challenge in translating the promise of stem cell therapies to treat a myriad of neurodegenerative disorders is to rapidly and efficiently direct pluripotent stem cells to generate differentiated neurons. The application of active vitamin metabolites known to function in embryonic development and maintenance in the adult brain such as retinoic acid (vitamin A), ascorbic acid (vitamin C) and calcitriol (vitamin D3) have proven effective in current in-vitro differentiation protocols. Therefore, in this study we investigated whether the biologically active vitamin B3 metabolite, nicotinamide could enhance the differentiation of mouse embryonic stem cells, cultured as monolayers, into mature neurons at either early or late stages of development. Interestingly, nicotinamide elicited a dose-responsive increase in the percentage of neurons when added at an early developmental stage to the cells undergoing differentiation (days 0–7). Nicotinamide (10 mM) increased the proportion of β-III tubulin positive neurons by two fold and concomitantly decreased the total number of cells in culture, measured by quantification of 4′, 6-diamidino-2-phenylindole positive cells. This effect could result from induction of cell-cycle exit and/or selective cell death in non-neural populations. Higher levels of nicotinamide (20 mM) induced cytoxicity and cell death. This study supports previous evidence that vitamins and their metabolites can efficiently direct stem cells into neurons. Current work is focusing on the effect of nicotinamide on the process of neural induction and whether nicotinamide influences the generation of particular neuronal subtypes implicated in neurodegenerative diseases, specifically focusing on midbrain dopamine neurons; towards a therapy for Parkinson's disease.
    • Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations.

      Griffin, Sile M.; Pickard, Mark R.; Orme, Rowan P.; Hawkins, Clive P.; Williams, Adrian C.; Fricker, Rosemary; Keele University; University of Chester; University Hospital of North Staffordshire; University of Birmingham (Public Library of Science, 2017-08-17)
      Vitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington's disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons. METHODS: Nicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods. RESULTS: Results presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons. CONCLUSIONS: Our results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons.