Animals receiving whole tissue transplants display a correlation between dopamine cell numbers and rotation bias.

<p>Each data point represents the number of TH+ cells from a single host (y-axis) versus their rotation score (x-axis). Note (A) how there is no correlation between the number of TH+ cells in dissociated cells transplants and their rotation scores (r² = 0.0006), while there is (B) a significant inverse correlation between the number of TH+ cells and ipsilateral rotation in animals receiving transplants of intact pieces of VM tissue (r² = 0.306; p = 0.04).</p

Dissociated cells and piece of VM tissue improve rotation bias in unilaterally 6-OHDA lesioned rats.

<p>Net ipsilateral rotation scores of animals pre-transplantation (Pre), and 4 and 6 weeks after transplantation of dissociated cells from, or an intact piece of, half an E13 VM versus controls (lesion alone). Data are shown as group mean rotations of control (diamonds), cell transplanted (squares), and whole tissue transplanted (triangle) animals, with bars above and below each data point indicating the ± SEM. Though both groups of animals receiving either transplants of dissociated VM cells (squares) or intact pieces of VM tissue (triangles) recovered some rotational symmetry, animals receiving transplants of pieces of VM tissue continued to improve after 4 weeks post-transplantation to nearly zero rotations per minute by 6 weeks post-transplantation.</p

Dissociated cell and whole tissue transplants contain TH+ cells projecting neuritis into the host striatum.

<p>Tyrosine hydroxylase (TH) staining of coronal sections through transplants of dissociated cells (A–C), or intact pieces of tissue (D–F) from one half of an E13 VM. (A and D) Transplants (arrowheads) were placed centrally in the 6-OHDA denervated striatum of lesion rats. (B and E) Numerous TH+ cell bodies could be seen situated within grafts of either dissociated cells (B) and whole tissue (E), with a halo (arrow heads) of diffuse TH+ cell fibers distributed throughout much of the surrounding host striatum. (C and F) High power images show varied TH+ cell profiles (arrows) throughout both types of grafts, and large, tapering TH+ processes innervating the surrounding host parenchyma (arrow heads). Scale bar for D shown in A, for E shown in B, and for F shown in C.</p

The number of TH+ cells was greater in animals receiving pieces of E13 VM tissue.

<p>After Ambercrombie correction, the total number of TH+ cells in animals receiving dissociated cells from the E13 VM averaged 1290 (+/−159) per transplant. In comparison, grafts of pieces of VM tissue contained significantly greater numbers of TH+ cells 2543 (+/−142; p = 0.003) 6 weeks after transplantation.</p

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

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

Early nicotinamide administration accelerates the conversion of neural progenitor populations to mature cell types.

<p>(A-B) Early nicotinamide treatment (days 0–7) induced a significant decrease in the <i>Sox1</i>GFP⁺ population in comparison to untreated cells. (A,C) Nicotinamide added between days 7 and 14 did not affect the population of <i>Sox1</i>GFP-expressing cells. (D) Histogram showing the proportion of cells with different levels of <i>Sox1</i>GFP expression. Larger numbers of cells displayed weak <i>Sox1</i>GFP expression in cultures that were exposed to 10 mM nicotinamide between days 0 and 7 (red bars), compared with controls. (E-F) Addition of nicotinamide increased the population of GABAergic neurons from mESCs. Nicotinamide (10 mM) added to media generated a significant increase in the percentage of the total GABAergic neuron population at day 14, compared against the control untreated group. Some cells showed intense GABA immunoreactivity (yellow arrows) and some less strong GABA expression (white arrow). Scale bar 50 μm applies to all high magnification images. ***p<0.001, **p<0.01.</p

Nicotinamide enhanced neuronal maturation in GABAergic populations.

<p>(A and E) The proportion of 46C-derived cells displaying “short” primary processes was significantly decreased in cultures exposed to 10 mM nicotinamide between days 0 and 7, concomitant with an obvious increased trend for “longer” neurite processes, compared with controls (F). (B) Total length of neurites was significantly increased in the presence of nicotinamide. (C) There was no difference in the number of primary neurites per neurons between controls and nicotinamide-stimulated conditions. (D) An upward trend in the proportion of mESC-derived cells displaying “strong” levels of GABA expression was demonstrated in cultures exposed to 10 mM nicotinamide, which correlated with a downward trend in “weaker”-expressing GABAergic cells, compared with control conditions. ***p<0.001, **p<0.01, *p<0.05. Micrographs show morphologies of GABA+ neurons differentiated in control conditions (E), or cultures treated with 10 mM nicotinamide (F). Scale bar = 100 μm.</p