The influence of nicotinamide on the development of neurons

This document is the Accepted Manuscript version of a published work that appeared in final form in Journal of Neurology, Neurosurgery and Psychiatry. To access the final edited and published work see http://dx.doi.org/10.1136/jnnp-2014-309236.199A 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.

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

Using bioinformatics for the identification of key peptides to engineer dopamine neurons. Towards a therapy for Parkinson’s disease.

Parkinson’s disease is a widespread condition caused by degeneration of dopamine neurons in the midbrain. A number of proteins are known to be important to signalling mechanisms present in the midbrain during natural dopamine neuron development, and may be utilised to better produce dopamine neurons in vitro. Relative expression levels of proteins were obtained from substantia nigra tissue of rats from embryonic days E11 through E14 using isobaric tagging for relative and absolute quantification. This project analysed the dataset obtained, with an emphasis on relative expression levels of proteins across the four-day period. Bioinformatics searching of online databases reduced the dataset from 3325 proteins to a shortlist of five worthy of further investigation. It is hoped that the proteins identified using these techniques will help to refine protocols for the production of dopamine neurons in vitro

Effect of calcitriol addition to culture media on dopamine neurons.

<p>Dose responsive increase in the number of TH immunoreactive neurons was obtained with addition of calcitriol to media. A) primary E12 control cultures and B) cultures with 10 nM calcitriol showing immunostaining with antibodies specific to tyrosine hydroxylase (red) and Tuj1 (green) counterstained with DAPI (blue). Scale bars: 20 µM. C) The percentage of dopamine neurons obtained from cultures with various concentrations of calcitriol added to media. Error bars represent SEM, ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05. The percentage of dopamine neurons obtained from primary E12 ventral mesencephalic cultures increased when calcitriol was added to media up to an optimum concentration of 10 nM. Dopamine neurons were also shown to be immunoreactive for other ventral midbrain DA neuron marker proteins: (D) aromatic acid decarboxylase (AADC); (E) Nurr1; (F) dopamine transporter (DAT); and (G) vesicular monoamine transporter 2 (VMAT2). Scale bars: 10 µM.</p

Calcitriol is neuroprotective and does not increase differentiation.

<p>The increase in dopamine neurons obtained from cultures was shown to be a result of increased neuroprotection afforded through calcitriol addition, rather than changes in differentiation. (A–D) Immunolabeling with a pan-caspase marker (red) and antibodies to tyrosine hydroxylase (green) counterstained with DAPI (blue), for: A) control media, B) 10 nM calcitriol, C) 0.3 U/ml Heparinase III and D) 10 nM calcitriol with 0.3 U/ml heparinase III. Scale bars: 20 µM. E) The number of apoptotic dopamine neurons was significantly increased when heparinase III was added to media. Bars represent the ratio of caspase+ dopamine neurons to caspase+ DAPI+ cells. Error bars represent SEM, ***<i>p</i><0.001, *<i>p</i><0.05. F) The percentage of TH+ neurons that had undergone terminal mitotic division and specification during culture was not significantly different when 10 nM calcitriol was added to cultures. Bars represent percent BrdU+ dopamine neurons, error bars are SEM.</p

Expression on vitamin D receptor protein.

<p>Vitamin D receptor protein was identified in developing ventral midbrain tissue harvested from embryonic rats. A) Five unique peptides were identified and used to identify VDR. B) VDR protein was also identified by a single band in Western blots of whole tissue lysate obtained from E12 VM of rats. C) Immunohistochemistry of E13 sagittal sections taken through the midbrain show overlapping expression of vitamin D receptor (VDR, green) and tyrosine hydroxylase (TH, red). Scale bar: 20 µM. D) Co-expression of VDR and TH was also observed in single cell cultures of E12 VM tissue. Total cells were labeled with DAPI (blue). Scale bar: 50 µM.</p

Effects of surface chemistry interaction on primary neural stem cell neurosphere responses

The characteristics of a material’s surface are extremely important when considering their interactions with biological species. Despite surface chemistry playing a critical role in mediating the responses of cells, there remains no single rule which dictates absolute performance; this is particularly challenging when considering the response of differing cell types to a range of materials. Here, we highlight the functional behavior of neural stem cells presented as neurospheres, with respect to a range of alkane-based self-assembled monolayers presenting different functional groups: OH, CO2H, NH2, phenyl, CH3, SH, and laminin. The influence of chemical cues was examined in terms of neurosphere spreading on each of these defined surfaces (cell adhesion and migration capacity) and neuronal versus glial marker expression. Measurements were made over a time series of 3, 5, and 7 days, showing a dynamic nature to the initial responses observed after seeding. While OH surfaces presented an excellent platform for glial migration, larger proportions of cells expressing neuronal β3-tubulin were found on SH- and laminin-coated surfaces. Axonal elongation was found to be initially similar on all surfaces with neurite lengths having a wider spread predominantly on NH2- and laminin-presenting surfaces. A generalized trend could not be found to correlate cellular responses with surface wettability, lipophilicity (log P), or charge/ionizability (pKa). These results highlight the potential for chemical cues to direct primary neural stem cell responses in contact with the defined materials. New biomaterials which control specific cell culture characteristics in vitro will streamline the up-scale manufacture of cellular therapies, with the enrichment of the required populations resulting from a defined material interaction

Supplementary information for article Effects of Surface Chemistry Interaction on Primary Neural Stem Cell Neurosphere Responses

Supplementary Information files for Effects of Surface Chemistry Interaction on Primary Neural Stem Cell Neurosphere Responses The characteristics of a material’s surface are extremely important when considering their interactions with biological species. Despite surface chemistry playing a critical role in mediating the responses of cells, there remains no single rule which dictates absolute performance; this is particularly challenging when considering the response of differing cell types to a range of materials. Here, we highlight the functional behavior of neural stem cells presented as neurospheres, with respect to a range of alkane-based self-assembled monolayers presenting different functional groups: OH, CO2H, NH2, phenyl, CH3, SH, and laminin. The influence of chemical cues was examined in terms of neurosphere spreading on each of these defined surfaces (cell adhesion and migration capacity) and neuronal versus glial marker expression. Measurements were made over a time series of 3, 5, and 7 days, showing a dynamic nature to the initial responses observed after seeding. While OH surfaces presented an excellent platform for glial migration, larger proportions of cells expressing neuronal β3-tubulin were found on SH- and laminin-coated surfaces. Axonal elongation was found to be initially similar on all surfaces with neurite lengths having a wider spread predominantly on NH2- and laminin-presenting surfaces. A generalized trend could not be found to correlate cellular responses with surface wettability, lipophilicity (log P), or charge/ionizability (pKa). These results highlight the potential for chemical cues to direct primary neural stem cell responses in contact with the defined materials. New biomaterials which control specific cell culture characteristics in vitro will streamline the up-scale manufacture of cellular therapies, with the enrichment of the required populations resulting from a defined material interaction. </p

Schematic diagram of time-course analysis of neural and neuronal differentiation in the presence of nicotinamide.

<p>Undifferentiated cells were cultured as monolayers in N2B27 medium for different durations to investigate whether nicotinamide would influence the conversion of Oct4⁺undifferentiated stem cells (scale bar = 100 μm) to <i>Sox1</i>GFP neural progenitors (scale bar = 50 μm) and thus drive differentiation toward βIII-tubulin⁺ neurons (scale bar = 50 μm). The potential of nicotinamide was assessed using immunocytochemistry at different culture periods from days 2–14.</p