Dr. Valerie Hu-Autism Mother and Researcher

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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: http://www.trainmanandmom.com/).The 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: http://www.fasebj.org/content/early/2010/04/07/fj.10-154484.full.pdf). 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: http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0017116&representation=PDF). 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: http://www.molecularautism.com/content/4/1/14) 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

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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, http://dx.doi.org/10.1016/j.nbd.2013.09.007.

hNP1 Human Neural Progenitors in Culture

hNP1 Human Neural Progenitors in Culture

Highlights

•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

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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

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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.

Musculoskeletal Disorders-Stem Cell Based Drug Discovery

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A common Neuromics’ theme is harnessing the power of cellsTM. The raw cost of the cells are often the biggest consideration. We encourage our customers to focus on true costs. These include the # of cells (how many times can they be passaged?), culture viability (how long do the cells live) and bioactivity (how closely do cultures mimic in vivo behavior?). I would like to present a presentation and publication confirming our competitive advantage when analyzing true costs.

Setting a higher bar for Neuron-Glial Based Assays!

Dr. Randen Patterson and his team at UC Davis have developed new culturing techniques using our e18 Rat Primary Hippocampal Neurons. They have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. This is good news indeed: Todd GK, Boosalis CA, Burzycki AA, Steinman MQ, Hester LD, et al. (2013) Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons. PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996.

We will continue to raise the bar. Better cultures=lower costs and better outcomes!

hN2 Human Neurons & hNP1 Neural Progenitors in Action

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I have been promoting Dr. Steve Stice and his team. They are the brains behind our  hN2TM Human Neuron and hNP1TM Human Neural Progenitor Discovery Kits. I would like to share 2 recent publication referencing use of these discovery kits. These validate the postings on capabilities. They are the best solutions available for researchers searching for Neuron or Neural Progenitor Based Assays for basic research, toxicology studies or drug discovery.

Xiugong Gao, Hsiuling Lin, Radharaman Ray, Prabhati Ray. Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX. Neurochemical Research. February 2013…Human hN2 neurons were obtained from Neuromics…

Abstract: Organophosphorus (OP) compounds represent an important group of chemical warfare nerve agents that remains a significant and constant military and civilian threat. OP compounds are considered acting primarily via cholinergic pathways by binding irreversibly to acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Many studies over the past years have suggested that other mechanisms of OP toxicity exist, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. Here we performed 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. Functional annotation and pathway analysis of the differentially expressed genes has revealed many genes, networks and canonical pathways that are related to nervous system development and function, or to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In particular, the neuregulin pathway impacted by VX exposure has important implications in many nervous system diseases including schizophrenia. These results provide useful information valuable in developing suitable antidotes for more effective prevention and treatment of, as well as in developing biomarkers for, VX-induced chronic neurotoxicity.

Protocol: Human hN2 neural cellswere obtained from Neuromics (Edina, MN). The hN2 cells were fully differentiated

normal human neural cells derived as adherent cells from human embryonic stem cell (hESC) WA09 line [34] and thus are considered as ‘‘matured’’ neuronal cells. It should
be noted that the universal neural cell marker Tuj (beta tubulin III) indicates that [80 % of the hN2 cells are neural. The other cell types which constitute\20 % of the cell population are mostly astrocytes and microglia, which are common glial cells found in the brain and spinal cord. The inclusion of the small amount of glial cells in the cell population better mimics real life situation in the central nervous system. The hN2 cells were seeded in 12-well plates at *500,000 cells/well in the AB2 Basal Medium complemented with ANS Supplement (cholinesterase free) provided by Neuromics and cultured at 37OC under
humidified 5 % CO2 for 48 h (without changing media) before VX exposure.

Xiufang Guo, Severo Spradling, Maria Stancescu, Stephen Lambert, James J. Hickman. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials, In Press, Corrected Proof,Mar 2013.doi:10.1016/j.biomaterials.2013.02.061 ...hNP1, were obtained from Neuromics (Edina, Minnesota)…

Abstract: Although sensory neurons constitute a critical component for the proper function of the nervous system, the in vitro differentiation of functional sensory neurons from human stem cells has not yet been reported. This study presents the differentiation of sensory neurons (SNs) from a human neural progenitor cell line, hNP1, and their functional maturation in a defined, in vitro culture system without murine cell feeder layers. The SNs were characterized by immunocytochemistry and their functional maturation was evaluated by electrophysiology. Neural crest (NC) precursors, as one of the cellular derivatives in the differentiation culture, were isolated, propagated, and tested for their ability to generate sensory neurons. The hSC-derived SNs, as well as the NC precursors provide valuable tools for developing in vitro functional systems that model sensory neuron-related neural circuits and for designing therapeutic models for related diseases.

Images: Generation of Schwann cells from the differentiated culture. Immunostaining of a day 38 culture with the Schwann cell marker S100 demonstrating a significant number of Schwann cells in the culture. Schwann cells were located either within the neuronal clusters (A) or along the axonal bundles (B). The neuronal clusters and axonal bundles were marked by Peripherin immunostaining. doi.org/10.1016/j.biomaterials.2013.02.061

I will continue to post more proof regarding the capabilties and value of our human neurons & neural progenitors as pubs/data/images becomes available

New Osteoblasts

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In conjuction with our manuacturing partner, Vitro Biopharma” , we are pleased to announce we now have commercially available unlabeled and FITC-labeled Osteoblasts. These are differentiated from our Umbilical Cord Blood derived Human Mesenchymal Stem Cells.

They are designed for:

  • Osteogenesis/Bone Formation Studies
  • Compound/Small Molecule Testing
  • Gene Expression Analysis
 

Images: (A) Human cord-blood MSCs were expanded in low-serum MSC-GroTM to confluence as shown here. (B) were differentiated in osteogenic MSC-GroTM. Early stage osteoblasts are shown here; the arrow shows early formation of mineralized matrix. (C)&(D) Mature osteoblasts stained positive for Alizarin red. Phase contrast image at 200 x, scale bar is 50 mmeters.

I will continue to update you on new Cell Based Assay Solutions.

QC Study of hMSC Derived Chondrocytes

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Check out the latest images and data of Neuromics‘-Vitro Biopharma’s Human Chondrocytes in Culture Days1-5: MSC-Derived Human Chondrocytes in Culture-01-2013.

Phase contrast image (200x) of chondrocyte and associated cells in day five of culture as described (SC00A7 Protocol). Scale bar = 50 m. Chondrocyte is darker multicellular structure overlying attached cells, ~ 70 m in diameter.