hN2 Primary Human Neurons and Toxicity Assays

Neuromics' hN2 Cells Continue to Shine

I previously posted our Human hN2 Neurons being used for studying the mechanisms of  Nerve Agent VX: hN2 Human Neurons for Toxicity Screening. The versatility of these neurons enabled the researchers to perform a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Solid primary cell based assay results start with healthy and well behaving cells.

I am pleased to announce success in exposing the cells to a specific cytotoxicity inducing compound (small molecule): Mark RichardsChee Wee PhoonGwendoline Tze Wei GohEng Khuan SengXu Ming GuoCherine Mei Fong TanWoon-Khiong ChanJoel Mun Kin Lee. A New Class of Pluripotent Stem Cell Cytotoxic Small Molecules. Research Article | published 19 Mar 2014 | PLOS ONE 10.1371/journal.pone.0085039.

Image: Cytotoxic agent used in dose-response curve

Figure: Dose response curves for 3 specialized somatic cell lines (MRC-5, human primary neurons and human neonatal cardiomyocytes) treated with JC011.

This curve illustrates the sensitivity of human neurons to toxic agents. 

We plan on continuing to use these and our hNP1 Human Neural Progenitors in kinetic, "in vivo like" assays. These assays will give quantitative data on both growth and differentiation inducing agents as well as specifics on how the cells behave when exposed to toxic agents. I will be posting results here.

This data should be of interested to neuro-disease/disorders basic and drug discovery researchers. We plan on making the assays available to researchers. We currently also do small molecule testing and gene expression analysis studies a CRO offering. To learn more, I can be reached at pshuster@neuromics.com or 612-801-1007.

Autism, Inflammation and Stem Cell Enhancers

Proving the Therapeutic Value

We have been running Quantibody® Antibody Arrays on blood serum of children diagnosed with Autism Spectrum Disorder (ASD). All reside in areas of heavy industry in Central Europe. All have elevated levels of one or several heavy metals.

These assays are being run as part of our strategy of treating these children with natural stem cell enhancing supplements. Here are the average serum levels of 2 cytokines (IL-6 and TNF-alpha) and 1 related chemokine (CCL3). All of the children had elevated levels vs healthy controls:

Figure: Serum ASD levels vs Healthy Controls (pg/ml)

TNF-alpha and IL-6 promotes the immune/inflammatory response. These two cytokines are guided to sites (including the CNS) of infection or tissue damage by the chemokine CCL-3 and others. In the normal process, the site(s) of immune response are cleaned (the response) of infection and/or damaged tissue and then repaired. The key with autoimmune diseases and disorders, is that that this process becomes a continuous loop; hence, these cytokines and chemokines are elevated. If the loop is broken or down modulated, the the levels of these should decrease.

Mesenchymal stem cells (MSCs) are immunomodulating and anti-inflammatory. We plan on testing candidate substances (all our currently available as natural supplements) on kinetic assays using our umbilical cord blood human mesenchymal stem cells. These will enable us to quantify  cell growth and expansion. We also plan on testing the best candidates on our human neurons to see the effects on cell behavior.

We will then determine safe dosing working with experts in the U.S. and Europe. During treatment, we will again be testing serum to see if the levels of these and other key Cytokines, Chemokines and Growth Factors. This will give proof as to to whether or not the treatments are working. If so, we should see the serum levels of these move toward to those of healthy controls.

I plan on making more data as it becomes available.

Irritable Bowel Syndrome (IBS) and BDNF

Gene Expression Analysis Determines BDNF's Role in IBS

In this study, Researchers used Neuromics'  i-Fect^TM Transfection Kit to deliver BDNF to determine effect on visceral hypersensitivity (VHS): J. H. Winston1 Q. Li1, S. K. Sarna1. Chronic prenatal stress epigenetically modifies spinal cord BDNF expression to induce sex-specific visceral hypersensitivity in offspring. Article first published online: 4 MAR 2014. Neurogastroenterology & Motility. DOI: 10.1111/nmo.12326. The Journal of Pain, Volume 14, Issue 11, November 2013, Pages 1485–1491 http://dx.doi.org/10.1016/j.jpain.2013.07.007

Intrathecal treatment with brain-derived neurotrophic factor (BDNF) antagonists reduced VMR to colorectal distension (CRD) in female chronic prenatal stress (CPS)+HeICS rats. (A) Graph shows that intrathecal administration of BDNF antagonist trkBFc in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA found a significant main effect of treatment, F1,53 = 10.4, p = 0.015; post hoc tests found significant differences at 30, 40, 50, and 60 mmHg, n = 4). (B) Graph shows that intrathecal administration of BDNF siRNA in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA: treatment 9 pressure interaction, F1,77 = 3.49, p = 0.008, tukey post hoc tests found significance at 30 mmHg, p = 0.013 and at 40, 50 50, 80 mmHg, p < 0.001, n = 7 Ctr., n = 6 BDNF siRNA). (C) Western blot shows a significant decrease in spinal cord BDNF protein expression in rats treated with BDNF siRNA (*p < 0.05).

Conclusion: Chronic prenatal stress followed by a second exposure to HeICS in adult offspring exacerbated visceral hypersensitivity (VHS) greater in female offspring that persisted longer than in male offspring. Chronic prenatal stress up-regulated BDNF expression in the lumbar-sacral dorsal horn that correlated with the exacerbation of VHS in female, but not in male offspring by increasing RNA Pol II binding and histone H3 acetylation, and decreasing histone deacetylase 1 association with the core promoter of BDNF in female offspring.