The Importance of in vivo Like Astroglial-Neuron Co-Cultures

The Power of Neuromics’-Aruna Biomedical’s hAstroPro™-NeuroNet™ Co-Cultures. We are pleased to be the first to market with our in vivo like co-cultures. Here are key points of what make these cultures unique:

  • Pure, Potent and Proven

  • Proof That These areTrue Co-Cultures

  •  Co-Cultures vs Neuron Only Cultures

What have we learned? The mix of Astroglia vs Neurons can have a dramatic impact on your data end points. This can lead to wrong conclusion being drawn from your tox and compound/small molecule neurodegenerative disease assays. We stand ready to serve you and your team. Questions? Don not hesitate to call 612-801-1007 or e-mail me Pete Shuster, CEO and Owner, Neuromics

HDAC2 and Anxiety in Alcoholism

The Impact of HDAC2 Gene Expression on Anxiety

Our i-Fect Transfection Kit continues to be a potent tool for testing the impact of altered gene expression on behavior. see: SACHIN MOONAT. The Role of Amygdaloid Chromatin and Synaptic Remodeling in Anxiety and Alcoholism. THESIS Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience in the Graduate College of the University of Illinois at Chicago, 2014.

The author hypothesized that increased HDAC2 would have a positive impact on anxiety in alchohol preferring (P) rats. Specifically, HDAC2-induced histone modifications in the amygdala may play a role in the regulation of synaptic plasticity that may underlie the behavioral phenotypes of P rats. Furthermore, it could be possible that exogenous manipulation of HDAC2 levels in the amygdala may have an effect on anxiety-like behaviors and alcohol preference in P

Figure 1. Chromatin remodeling via histone acetylation and DNA methylation regulates gene transcription associated with changes in synaptic plasticity. During gene transcriptional processes, the chromatin structure associated with DNA to be transcribed is in a relaxed chromatin conformation due to hyperacetylation of histone proteins and hypomethylation of DNA, which allows access to transcriptional machinery. This relaxed chromatin structure results in increased gene transcription, which in neurons may cause increased expression of synaptically active proteins that result in the positive modulation of synaptic plasticity, such as increased dendritic spine density (DSD). DNA methyltransferase (DNMT) methylates DNA at CpG islands, leading to hypermethylated DNA and recruiting of methyl-CpG binding domain protein (MBD) complexes which block binding of transcriptional machinery. The MBD complex can in turn recruit histone deactylases (HDAC) which remove acetyl groups from histone proteins resulting in chromatin condensation thereby decreasing gene transcription. HDACs and histone acetyltransferases (HAT) control the histone acetylation profile, such that HDACs remove acetyl groups and HATs add acetyl groups to histone proteins. In this manner, increased HDAC expression results in hypoacetylation of histones leading to a condensed chromatin structure. Chromatin condensation resulting from HDAC-induced histone deacetylation or DNMT-induced DNA methylation causes reduced gene transcription. In neuronal cells, the reduction in gene transcription may be associated with decreased expression of synaptically active proteins and negative modulation of synaptic plasticity, such as reduced DSD. Treatment with DNMT inhibitors or HDAC inhibitors may block these enzymatic processes and return chromatin to a relaxed state, resulting in increased gene transcription and synaptic plasticity (Moonat and Pandey, 2012).

Methods: P rats that had been previously cannulated for delivery of solutions directly into the CeA were infused with either HDAC2 siRNA, control siRNA or vehicle. The siRNAs were dissolved in iFect solution (Neuromics, Edina, MN), a cationic lipid-based transfection solution, such that the final concentration of the solution was 2 µg/µL. The sequence of the HDAC2 siRNA was as follows: 5’-CAAGUUUCUACGAUCAACATT-3’; 5’-UAUUGAUCGUAGAAACUUGAT-3’. Some of the HDAC2 siRNA (Qiagen, Valencia,
CA) had been modified to include a 5’ Alexa Fluor-488 fluorescent probe in order to
determine the transfection efficiency and cellular localization of transfection. The control
siRNA used was the AllStars Negative Control siRNA (Qiagen), which shows no
homology to any known mammalian gene. To prepare the vehicle, RNase-free water was
dissolved in the iFect solution in place of any siRNA. The solutions (0.5 µL) were
infused bilaterally into the CeA of P rats using an automatic infusion pump which
resulted in a dose of 1 µg of siRNA per side. The automatic pump was attached to a
microdialysis probe which seated in the guide cannula and extended 3 mm past the tip of the cannula into the CeA.

For the experiments which looked at the anxiolytic effect of HDAC2 siRNA
infusion, P rats were infused with either HDAC2 siRNA, control siRNA or vehicle at the
end of the light cycle. 16 hours after the infusion, the rats were tested for anxiety-like behaviors. Immediately following behavioral testing, rats were anaesthetized and brains
were collected for further analysis.
For the voluntary drinking experiment, P rats were infused with either HDAC2
siRNA or vehicle when the bottles were changed following the third day of 9% ethanol
exposure. The rats continued to be monitored for the intake of 9% ethanol for 7 days
following the infusion. After the final day of voluntary drinking, the rats were
anaesthetized for collection of brains and blood to confirm the cannula position and the
blood alcohol levels, respectively.

Figure. The effects of HDAC2 siRNA Infusion into the CeA of P rats on voluntary ethanol consumption as measured by the two-bottle free choice paradigm. Monitoring the voluntary ethanol consumption of alcohol-preferring (P) rats via the two bottle free choice paradigm following infusion of vehicle or histone deacetylase isoform 2 (HDAC2) siRNA into the central amygdala (CeA) demonstrates that high HDAC2 levels may mediate the high alcohol drinking behaviors of P rats. P rats were given access to water and 7% ethanol followed by water and 9% ethanol. On the sixth day of ethanol access P rats received infusion of vehicle or HDAC2 siRNA and consumption of water and 9% ethanol were monitored for sevnfusion. Total fluid intake did not significantly differ between the groups. Values are represented as the mean ± SEM of the ethanol consumption (g / kg / day) and total fluid intake (mL) plotted daily for n=6 rats per treatment group. *Significantly different between the groups.
This data suggest reduction of HDAC2 levels in the CeA leads to reduced DSD associated with a reduction in anxiety-like behaviors and alcohol preference in P rats and could prove to have therapeutic value.

The Dance Between Immune System and Stem Cell Health

We named it the  immunoLinkTM 
We have been testing a growing number of Clients with our Quantibody Arrays. Many of of these clients have Autoimmune Disorder Diseases. These range from Rheumatoid Arthritis to Multiple Sclerosis.

These arrays are designed to precisely measure factors or markers(proteins) that are dysregulated by these diseases. We measure the levels of these biomarkers in our Clients’ Blood serum. The arrays have also been used to measure the levels of markers in plasma and cell culture supernatants.

Based on results, we are finding links between immune system and stem cell health. We call this the immunoLink. The link shows that when immune/inflammatory response markers are elevated, markers related to stem cell health are depleted.

Here we see the immune/inflammatory response markers IL-6, MCP-1 and TNF-alpha are elevated in our Clients with autoimmunity (A) vs Healthy Controls (HC). We also see lower levels of G-CSF and GM-CSF in these Clients.

G-CSF and GM-CSF are know to play a role in increasing circulating stem cells. GM-CSF is also know to be secreted by Mesenchymal Stem Cells (hMSCs) AND GM-CSF has anti-apoptotic functions on neurons, and is neuroprotective in animal stroke models while G-CSF has a prominent effect on the differentiation of adult neural stem cells (see: BMC Neuroscience 2007, doi:10.1186/1471-2202-8-88).

To us, the immunoLink means achieving a balance between immune system and stem cell health.

We provide immune system balancing and stem cell activating therapies for our Autoimmune Disease Clients and Children with Autism. We first do baseline and follow on testing (each 6 months) to determine how well the therapies are working. You can now benefit from one of our first Stem Cell Activating Product named Stem-Kine.

Dr. Joe Smarda, an applied immunologist, is user and promoter of Stem-Kine and oh, by the way. He is a European Porsche Cup Champion and pilots our Stem-Kine/Neuromics sponsored race car.

Our goal is to bring the many markers we test to healthier levels. As stem cell transplants become more common, moderating levels of immune/inflammatory response in patients could improve outcomes. If you would like to learn more, you can contact me at or 612-801-1007.

Stem Cell Activators

Vitro Biopharma Receives Research Award for Innovative Research!

Vitro Biopharma Receives Frost & Sullivan Technology Innovation Leadership Award: 2014 Best Practices for Stem Cell Tools & Technology in North America

Golden, CO / ACCESSWIRE / July 9, 2014 / Vitro Diagnostics, Inc. (VODG), dba Vitro Biopharma, is pleased to announce receipt of a prestigious award from Frost & Sullivan. The award is based on independent analysis of competing companies’ commitment to innovation, commercial success, application diversity and fulfillment of unmet needs. Vitro Biopharma out-ranked competing firms in all areas evaluated.

Cecilia Van Cauwenberghe, industry analyst for Life Science/Biotech with Frost & Sullivan, noted, “The activation of endogenous stem cells to differentiate into specific cell types appears as an alternative to mitigate the significant remaining regulatory obstacles to adult stem cell transplantation in the United States. Vitro Biopharma is aligning its scheduled stages of clinical trials to test mobilization of endogenous stem cells in the treatment of traumatic brain injury and autism spectrum disorders (ASD), in which pre-clinical research strongly suggests the activation of certain biochemical pathways to increase proliferation, migration, and differentiation performance. Vitro Biopharma’s approach does not require stem cell transplantation, while providing a non-controversial, cost- and time-effective alternative to the current methodologies of competitors.” The full Award Statement is posted on our website.

Dr. Jim Musick, Vitro Biopharma’s president and CEO, said, “We are honored to receive this award from Frost & Sullivan, a premier organization dedicated to corporate growth and development, as well as business expansion. While embryonic stem cell research presents numerous ethical problems and has long been the subject of considerable debate, adult stem cells provide the benefits of embryonic stem cells without the problematic issues. A hallmark of embryonic stem cells is pluripotency, a capacity to develop into any cell in the body. While once thought to be exclusive to embryonic stem cells, it is now clear that adult stem cells may be converted to the functional equivalent of embryonic stem cells through methods that manipulate gene expression. These relatively straight-forward methods were developed and validated in several labs, including Vitro Biopharma. We are now also gaining understanding of the cellular signaling processes that activate adult stem cells, including MSCs, neural and muscle stem cells that reside within our bodies. This opens the possibility to elicit stem cell therapy without transplantation. There are also potential enhancements in mental and physical performance and anti-biological aging effects that have been demonstrated in animals. We look forward to expanding the power of stem cell activation to advancing medical treatments.”

Pete Shuster, a director of Vitro Biopharma and the CEO and founder of Neuromics, said, “Endogenous stem cell activation catalyzes the body’s natural healing processes. Proving this has been an integral part of our internally funded research. The ability to activate specific stem cell expansion, migration and differentiation pathways holds great promise for sufferers of traumatic brain injury and autism.”

“We are also encouraged by initial results treating autoimmune disease with natural stem cell activators. As previously reported, this initiative has generated revenue growth for Vitro Biopharma. Participating in TBI- and Autism-related trials would significantly accelerate this growth.”

“I consider this award a validation of our strategy and anticipate more good news to come as we continue to execute and improve this strategy.”

Treating Autism and TBI

I am excited about this important development in the Neuromics and Vitro Biopharma partnership: We are in the planning stages of clinical trials to test mobilization of endogenous stem cells in the treatment of traumatic brain injury and Autism Spectrum Disorders. This is based on substantial pre-clinical research suggesting that activation of certain cellular pathways in combination with epigenetic modulation of select gene expression yields increased proliferation, migration and differentiation of adult stem cells including neural stem cells and MSCs.
See Press Release:

Dr. Valerie Hu-Autism Mother and Researcher

Unraveling complexities in search of potential treatments
I first became aware of Valerie’s Research when she called to explore how our eSC derived Human Neurons could be of value in her research. When I asked, “How do you plan on using the cells?” she gave me an overview of her fascinating research. She went on to tell me about the role her autistic son, Matthew 26, has played in her quest. This resonated with me because my Godson, Stefan 23 is autistic (see: purpose of this backstory is to give an overview of why her research is proving a key piece of the puzzle in understanding the biology of Autism. More importantly, given the lack of research funding, I am hopeful it opens the door to new sources like Microyza. These would enable those most impacted to have a direct way to participate.

Dr. Valerie Hu

Autism speaks and acts in riddles. This is the story of how Valerie is working to find the clues needed to solves these riddles.

Her Research Journey
Valerie has a bachelor’s degree in chemistry from the University of Hawaii (1972) and a PhD, also in chemistry, from Caltech (1978). She conducted postdoctoral research into membrane biochemistry and immunology at UCLA. She is currently a Professor of Biochemistry and Molecular Medicine at George Washington University in Washington, DC.

Her current research has required a leap from membrane biophysics to functional genomics. The intersecting theme is both disciplines involve complex molecular biology techniques and methods.

Functional genomics adds the challenge of analyzing the expression of genes that could play a role in disease or disorder and comparing them with the same genes expressed in normal or healthy phenotypes. Then an even bigger challenge is having the expertise and tools to discover the context of how these dysfunctional genes relate to one another.

In some of her early research, she found 4,000 genes appear to behave differently in a group of severely autistic people as compared with non-autistic controls—a startling number, considering the human genome comprises 20,000 to 25,000 genes (see: Searching for Autism’s Treatable Roots). But what is causing this large set of genes to behave differently from the norm?

By mapping the relationship between these genes and integrating gene expression profiles with DNA methylation data a picture emerged. This led to the discovery of a suspected master gene whose protein expression regulates the expression of many downstream genes known to play a role in Autism. This includes genes responsible for development of the central nervous system and the ongoing regulation of neurotransmitters.

A Master Gene Speaks-RORA
The gene Valerie and her team discovered as a suspect is the nuclear hormone receptor RORA (retinoic acid-related orphan receptor-alpha-see: They found the expression of this gene is reduced in autistic brain. So how can a reduction of one gene’s protein have such a profound impact?

As nuclear hormone receptor, RORA indeed has the capability of impairing the function of downstream genes. In fact, RORA can impact a lot of them. Further, RORA has the potential to be under negative and positive regulation by androgen and estrogen, respectively, suggesting the possibility that RORA may contribute to the male bias of ASD. (see: Note: This is a highly accessed article: > 11,000 people have already accessed this article.

By mapping the relationship between these genes and integrating gene expression profiles with DNA methylation data a picture emerged. This led to the discovery of a suspected master gene whose protein expression regulates the expression of many downstream genes known to play a role in Autism. This includes genes responsible for development of the central nervous system and the ongoing regulation of neurotransmitters.

She continues to learn more about the biology of RORA. Her recent publication (see: validates many of the transcriptional target genes of RORA.

Figure: Possible downstream consequences of deregulation of the six confirmed transcriptional targets of RORA

This shows that RORA sets off a critical mass of events leading to massive and variable disruption of gene expression. These events are ultimately manifested in the spectrum that marks Autism-impaired social and communication skills, repetitive behaviors, learning difficulties and sleeping disorders.

What’s Next?
These are breakthrough discoveries. Much more needs to be done. Some of the questions that need to be answered include: Can RORA be up regulated and how could this be done? Can RORA be dysregulated by hormone-like environmental pollutants leading to increased risk for Autism? What impact will alterations in RORA expression have on downstream genes? What are the best methods to regulate RORA…small molecule agonists? gene therapies? Cell based therapies? and so many more.

This research requires predictable and ongoing funding. Government funding is harder and harder to find. So many are impacted by children and adults with Austism, given this, I believe this research could be an ideal candidate for Crowd Funding.

Neuromics’ hNP1 Neural Progenitors, DJ-1 and Stroke

Dr. Cesar V. Borlongan, University of South Florida and a team of researchers have successfully identified DJ-1 as a potential therapeutic target for treating stroke. They used our hNP1TM Human Neural Progenitors to confirm the neuroprotective properties of the DJ-1 protein: Yuji Kaneko, Hideki Shojo, Jack Burns, Meaghan Staples, Naoki Tajiri, Cesar V. Borlongan, DJ-1 ameliorates ischemic cell death in vitro possibly via mitochondrial pathway, Neurobiology of Disease, Available online 21 September 2013, ISSN 0969-9961,

hNP1 Human Neural Progenitors in Culture

hNP1 Human Neural Progenitors in Culture


•DJ-1 translocation was assayed in oxygen–glucose deprived human neural progenitor cells.
•Immunofluorescent microscopy and ELISA were used to measure DJ-1 translocation.
•DJ-1 translocated preferentially into polarized mitochondria.
•DJ-1 translocation is associated with the preservation of functional mitochondria.
•DJ-1 exhibits antioxidative stress effects following ischemic stroke.

I will continue to post updates on Research success with our Cell Based Assay Solutions.

eSC Derived hNP1 Neural Progenitors Astrocytic Differentiation

Protocol for Driving hNP1TM Human Neural Progenitors to Astrocytes

There is a great demand for an easy way to generate human astrocytes in culture. I am pleased to present a protocol for differentiating our hNP1 Cells to Astrocytes. This comes from my friend Dr. Steve Stice and his team at ArunA Biomedical and University of Georgia: Majumder A, Dhara SK, Swetenburg R, Mithani M, Cao K, Medrzycki M, Fan Y, Stice SL. Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors. Stem Cell Res. 2013 Jul;11(1):574-86. doi: 10.1016/j.scr.2013.03.003. Epub 2013 Apr 2.

These enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5days of differentiation, with near complete suppression of neuronal differentiation.

Images: Morphology and gene expression after 15 and 30 days of differentiation of cells with astrocytic treatment. Bright field images of hNP cells differentiated (A) with or (B) without astrocytic treatment. A and B compare morphology of cultured cells in treated vs. untreated differentiation at 15 days. Treated and untreated cells were cryopreserved at d6 and subsequently thawed and cultured for an additional 9 days. Flow cytometry analysis to determine percent of GFAP+ and S100B+ cells at d15 of differentiation. Data is presented as histograms for (C) GFAP and (D) S100B with corresponding immunoreactive cells in insets from a parallel culture. Immunocytochemistry detects expression of (E) GFAP with S100B (inset showing distinct staining for both markers), (F) GFAP with GLAST, and (G) GFAP with ALDH1L1 at d30 of differentiation.

The Protocol:  For astrocytic differentiation of hNP cells, neuronal differentiation media were supplemented with BMP2 (20 ng/mL) and combinations of Aza-C and TSA; Aza-C (500 nM), TSA (100 nM) and BMP2 (20 ng/mL) for 2 days, with one complete media change in between, followed by differentiation media supplemented with BMP2 but not with Aza-C or TSA. Cells were harvested prior to analysis at 5, 15 or 30 days of treatment or for cryopreservation to d6 or d10 of differentiation. For cryopreservation, cells were
dissociated with Accutase™ and frozen in differentiation media containing 10% DMSO. Viability was assessed at 30 days in Aza-C and TSA treated cultures by trypan blue exclusion, and datawas acquired using a Cellometer Auto T4® (Nexcelom Biosciences).

I will keep you updated on new differentiation protocols for our potent, pure and widely used hNP1 Human Neural Progenitors to new phenotypes.

Update-Cell Based Assay Solutions

We continue to expand our cell centric strategy. With the addition of Cancer Associated Fibroblasts (CAFs), we are now positioned to develop highly engineered customer specific co-cultures. These engineered cells will inculde our hMSCs, Osteoblasts, Chondrocytes and Fibroblasts. Our goal is to develop in vivo like cell based assays to lower the costs of discovery and increase your “hit” rate.

New Products
Cancer Associated Fibroblasts
Image: Culture of Adeno-Carcinoma CAFs at Day 2.

We will keep you posted as we add capability to our cell based assay solutions.