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Microfluidic Oxygen Control Demonstrates Crosstalk Between Normoxic and Hypoxic Stem Cells
Megan L. Rexius, David T. Eddington, (University of Illinois at Chicago, Department of Bioengineering) and Jalees Rehman (University of Illinois at Chicago, Department of Pharmacology)

Hypoxia regulates adult and embryonic stem cell function as well as vascular differentiation and regeneration. Homogenous hypoxia is known to increase secretion of pro-angiogenic vascular endothelial growth factor (VEGF) expression in human mesenchymal stem cells (MSCs) which can activate angiogenesis in endothelial cells. Current approaches which use homogenous oxygen levels to study hypoxia in cells and tissues do not replicate physiological oxygen gradients formed as a result of oxygen diffusion from the microvasculature. Furthermore, traditional hypoxia chambers do not allow assessment of real-time paracrine crosstalk which naturally occurs in vivo between cells exposed to varying oxygen levels. The objective of this study was to investigate the effect of close proximity interactions between normoxic and hypoxic cells.

We have developed an open-well microfluidic device with precise control of oxygen gradients. Our cell culture platform allows different oxygen levels to be selectively applied to distinct cell types in a co-culture (e.g. one cell type can be hypoxic while the other is normoxic), while permitting paracrine interactions between the distinct cell types via shared cell culture media. We present for the first time hypoxic upregulation of VEGF in MSCs suppressed in the presence of close-proximity normoxic MSCs, when compared to hypoxic MSCs without adjacent normoxic cells. Similarly, in co-cultures of hypoxic MSCs and normoxic microvascular endothelial cells (MVECs), normoxic endothelial cells also exerted a mitigating effect on VEGF expression. These findings suggest that when cells are exposed to an oxygen landscape with varying oxygen levels, neighboring normoxic cells may modulate cellular hypoxia responses in MSCs. However, this modulating effect does not appear to be a global response and does not impact metabolic genes such as the glucose transporter (Glut1).

Our microfluidic cell culture platform overcomes the limitations of current technology in order to study paracrine interactions across an oxygen gradient. Continuation of this research aims to manipulate the outcomes of hypoxic signaling. We also anticipate that the research results will lead to a better understanding of how to control angiogenesis and improve chronic ischemic disorders. (Back to top) 

Multipotent stromal cells in micromass culture for studying mechanisms of antifibrotic drugs
Chan DD, Li J, Gorski DJ, DelaMotte CA, Predescu DN, Plaas AHK (Rush University and Cleveland Clinic)

TGF-β is a major regulator of numerous cellular processes following soft tissue injury, including proliferation of granulation tissue stromal cells, contraction, and regeneration of collagenous matrices. Dysregulation of TGF-β1 responses during healing can lead to fibrosis, hypertrophic scarring, or contractures. Following cartilage damage in the knee, wild-type mice show a robust healing response, whereas mice lacking hyaluronan synthase 1 (Has1KO) develop extensive soft tissue fibrosis, chronic inflammation, and severe osteoarthritis. Treatment of Has1KO mice with the antifibrotic Pirfenidone reduced inflammation and prevented cartilage and bone damage but only minimally decreased the fibrosis. Since our previous studies showed that chronic intra-articular TGF-β1 application resulted in stromal cell proliferation and fibrotic remodeling, we examined the effect Pirfenidone on TGFb1 responses of stromal cultures from wild-type and Has1KO mice. Cells were isolated from abdominal adipose and passaged 3-times. For micromass cultures, cell suspensions (5×107 cells/mL AMEM/10%-FCS/ascorbate) were dispensed as 20-µL drops, with fresh medium added following adherence and aggregation. After 24h, media was replaced and supplemented with TGF-β1, Pirfenidone and/or TGFβRI/II-inhibitor. Media and cell layers were harvested after 24h for QPCR of Col1a1, Col3a1, Has1, and Has2 mRNA levels and Western blotting for type-1 collagen. In wild-type cells, TGF-β1 increased Col3a1 (~3-fold) and Has1&2 (~4-fold) mRNA levels with no significant changes to Col1a1. Pirfenidone attenuated the increase in Col3a1 mRNA, but had no effect on stimulated levels of Has1&2. TGFβRI/II-inhibitor prevented all TGF-β1-induced effects. In Has1KO cells, TGF-β1 increased Col1a1 mRNA (~5-fold), with no effect on Col3a1 or Has2. Moreover, Pirfenidone and TGFβRI/II-inhibitor only minimally decreased TGF-β1 effects in Has1KO stromal cultures, suggesting the activation of alternative profibrotic signaling pathways, to modulate collagen gene expression in the absence of HAS1. This distinction between phenotypes was also seen when cultures were assayed for collagen-1 production using Western blotting. With Pirfenidone, collagen-1 secretion was reduced in wild-type cells but remained unchanged in Has1KO cells. In conclusion, the use of multipotent stromal cells in micromass cultures is a useful tool to study the effect of antifibrotics and additionally discover new genes in the fibrosis pathways and their sensitivity to such drugs. (Back to top)     

Human neurons derived from induced pluripotent stem cells model chemotherapeutic induced peripheral neuropathy
Heather E. Wheeler, Claudia Wing, Shannon M. Delaney, Masaaki Komatsu, and M. Eileen Dolan, (Section of Hematology/Oncology, Department of Medicine, University of Chicago)

Chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting toxicity of many anti-cancer drugs. The mechanisms underlying CIPN have not been precisely determined and few human neuronal models to study CIPN exist. We describe the development and feasibility of using human neurons derived from induced pluripotent stem cells (iPSCs) as a genetically diverse model for CIPN. Upon treatment of human neurons with the neurotoxic chemotherapeutic paclitaxel, vincristine or cisplatin, we identified reproducible decreases in neurite outgrowth phenotypes. Distinct morphological changes in damage among the drugs reflect differences in their mechanisms of action and clinical manifestations of CIPN. We show the potential of the model for gene perturbation functional studies by optimizing conditions for siRNA transfection. Upon reprogramming four unrelated lymphoblastoid cell lines (LCLs) into iPSCs and differentiating them into neurons, we demonstrate that the variance in several neurite outgrowth phenotypes upon treatment with one of the neurotoxic drugs is greater between than within individuals, demonstrating the potential of this model for larger genetic association studies. Genetically diverse cell types derived from iPSCs relevant to tissues of drug toxicity have the potential to greatly impact the field of pharmacogenomics. We present key chemotherapeutic-induced phenotypes in human iPSC-derived neurons that model CIPN. The human neuron model will allow both for functional studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN. (Back to top)

Disease Modeling Using Patient Specific iPSC
Galat Yekaterina, Irina Elcheva, Mariana Perepitchka, Vasiliy Galat

Defining the earliest phenotypic and molecular signs of abnormal development in patients with various mutations and chromosomal abnormalities will not only contribute to the overall understanding of these syndromes, but also facilitate the development of new treatments that could prevent or lower the risk of congenital and sporadic disorders. Our research, as well as others’, has shown that experimental models of differentiation from pluripotent stem cells (PSCs), both embryonic (ESCs) and induced pluripotent stem cells (iPSCs), recapitulate major steps of the development in embryo. Our lab focuses on deriving iPSC from patients with various disorders and utilizing the method of in vitro differentiation to broaden our understanding of such prominent disorders as Down Syndrome, as well as, increase our knowledge of rare neurobehavioral disorders caused by mutations in RAI1 and GRIN2B genes.

Down syndrome (DS) is a complex disease caused by a trisomy of human chromosome 21 (HSA21) that occurs at a rate of 1 in every 750 live births. Children with DS show a spectrum of clinical anomalies implicating the development of all embryonic lineages. Our lab works to understand how HSA21 affects formation of mesodermal lineages, i.e. mesenchymal, endothelial, cardiac, and blood progenitors on molecular and cellular levels. We anticipate that at least some phenotypic malformations manifested in musculoskeletal, hemato-endothelial, and cardiovascular tissues of DS can be modulated at the mesodermal precursor stage of development.

Additionally, successfully generated and characterized a novel patient specific iPSC model for RAI1 and GRIN2B mutations both of which manifest in a complex phenotype, including intellectual disability. Mutation in the RAI1 gene is characterized by multiple congenital defects including intellectual disabilities, sleep disturbances, craniofacial and skeletal disorders. Several recent studies have identified association of mutations in the GRIN2B gene with autism, schizophrenia and epilepsy. This model provides a unique opportunity to induce neuronal differentiation in stem cells from siblings that differ in single dominant mutations critical to normal neuronal differentiation. The model was established using a non-integrating Sendai virus reprogramming system. The generated iPSCs display embryonic stem cell-like morphology, express stem cell markers, and are capable of differentiation to three germ lineages. We further differentiated the iPSC lines to neural progenitors. We believe that by comparing gene expression data from mutant iPSCs, to ”control” iPSCs and their corresponding neuronal derivatives it will be possible to discover mutation related alterations that can then be linked to impaired human neuronal development and provide a basis for discovery of novel therapeutic targets.

In summary, iPSC offer an invaluable disease modeling system, as well as, drug screening platform by providing the type of cells that are not attainable directly from patients. (Back to top)

A 3D High-Content/High-Throughput Screening Platform to Enhance Multilineage Stem Cell Differentiation and Improve Human Pancreatic Islet Viability
Amit Paul, Enza Marchese, David Franz, Sumaira Yahya, Kirstie Danielson, Jose Oberholzer, and Michael Cho (Department of Bioengineering, University of Illinois at Chicago and Division of Transplant Surgery, University of Illinois at Chicago)

The cytoskeleton of human mesenchymal stem cells (MSCs) alters dramatically during differentiation. These lineage-dependent changes cause MSCs to exhibit unique cytoskeletal “fingerprints” along the various stages of differentiation. To investigate the relationship between these dynamic cytoskeletal configurations and MSC lineage commitment on a large scale, we created a novel computational algorithm that integrates high-content screening (HCS, detailed analysis of single cells) and high-throughput screening (HTS, rapid/high-volume experimentation). Most notably, the entire HCS/HTS process is performed in an automated environment using a custom-engineered 3D cell/tissue imaging system. While undergoing multilineage differentiation (adipogenesis, osteogenesis, chondrogenesis, myogenesis, neurogenesis, vasculogenesis, and hepatogenesis), MSCs were fluorescently labeled at crucial differentiation time points for cytoskeletal and lineage-specific markers. All samples were spatially-resolved/scanned/imaged via high-resolution (100-200nm) 3D confocal/multiphoton laser scanning microscopy. This HCS/HTS system was integrated with laser capture microdissection to automatically identify and capture differentiating stem cells in a selective manner for gene expression profiling. Additionally, the dynamic lineage-dependent biomechanical changes were correlated using atomic force microscopy. With the abundance of quantitative experimental data, we developed a sophisticated cytoskeletal fingerprinting process that accurately analyzes & predicts the mechanical transformations which are potentially required to initiate the onset of stem cell differentiation into multiple lineages. With the assistance of this advanced HCS/HTS platform, many aspects of MSC differentiation were thoroughly elucidated. Perhaps most significant was a consistent pattern observed in all types of differentiation: the cytoskeleton remodeled significantly before lineage-specific cellular changes occurred. This strongly suggests that cellular mechanical transformations are a precursor to stem cell differentiation. The combinatorial approach of 3D high-resolution imaging and computational modeling provides a robust system to better understand MSC differentiation and ultimately reduce heterogeneity in multipotent/pluripotent stem cell populations. This 3D HCS/HTS imaging system has also been applied to assessing isolated human pancreatic islets from cadaveric donors (large/thick multicellular structures) for their active beta-cell/alpha-cell/delta-cell content. We are utilizing the results of this analysis to engineer superior methods of improving human islet survival/viability for clinical transplantation. (Back to top)

Endothelin ETA Receptor Antagonist Reverses Naloxone-precipitated Opioid Withdrawal in Mice
Shaifali Bhalla, Melissa Tapia, Gwendolyn Pais, and Anil Gulati (Chicago College of Pharmacy and College of Health Sciences, Midwestern University, IL)

Background: The long term use of opioid analgesics for the treatment of pain results in the rapid development of tolerance and dependence leading to severe withdrawal symptoms. Several neurotransmitter mechanisms have been proposed to play a role in the actions of opioid analgesia and withdrawal. We have previously demonstrated that endothelin-A (ETA) receptor antagonists potentiate morphine and oxycodone analgesia in rodents. We also demonstrated that ETA receptor antagonists eliminated tolerance by restoring the antinociceptive responses to both morphine and oxycodone in opioid tolerant animals.
  
Objective: The present study was designed to investigate the involvement of central endothelin mechanisms in opioid withdrawal. The effect of intracerebroventricular administration of ETA receptor antagonist, BQ123, on morphine and oxycodone withdrawal was determined in male Swiss Webster mice.

Methods: Opioid tolerance was induced by twice-daily injections of morphine for three days, and once-daily injections of oxycodone for five days. Withdrawal was precipitated by opioid receptor antagonist, naloxone, on day 4 for morphine studies and day 6 for oxycodone studies. Expression of ETA receptors, ETB receptors, VEGF and NGF was determined using Western Blotting technique.

Results: Pretreatment with BQ123 reversed the hypothermia and loss of body weight in mice undergoing morphine and oxycodone withdrawal. BQ123 also significantly reduced the number of wet shakes, rearing and jumping behavior during withdrawal. Western blotting studies indicated no changes in the expression of VEGF, ETA receptors, and ETB receptors following administration of vehicle or BQ123 in the brain. Although statistically insignificant, we observed a slight tendency toward decrease in ETA receptor expression in BQ123-treated animals in control and withdrawal groups. NGF expression was not affected in morphine withdrawal but significantly decreased in the brain during oxycodone withdrawal. Expression of NGF was not altered by BQ123 pretreatment.

Conclusion: These studies are the first to demonstrate that ETA receptor antagonists not only eliminate antinociceptive tolerance but also reverse withdrawal symptoms of morphine and oxycodone. These findings support the hypothesis that ETA receptor antagonists in combination with opioid analgesics provide adequate analgesic response without the addiction potential and withdrawal symptoms of opioids. (Back to top)

   

The Marine Pharmacology and Pharmaceuticals Pipeline in 2014
A.M.S. Mayer, Dom Dop Van Nguyen and K.B.Glaser (Department of Pharmacology, CCOM, Chicago College of Pharmacy, Midwestern University, IL and AbbVie Inc.)

The status of the marine pharmacology and pharmaceutical pipelines was assessed in early 2014. There were five FDA-approved marine-derived drugs in the US market, namely cytarabine for cancer (Cytosar-U®, Depocyt®, FDA-approved 1969), ziconotide for pain (Prialt®, FDA-approved 2004), omega-3-acid ethyl esters for hypertriglyceridemia (Lovaza®, FDA-approved 2004), eribulin mesylate for cancer (Halaven®, FDA-approved 2010), and brentuximab vedotin for cancer (Adcertis®, FDA-approved 2011), while vidarabine as an antiviral (Vira-A®, FDA-approved 1976) was no longer available, and trabectedin for cancer (Yondelis®, FDA-orphan drug approval 2005) being EU-registered. In early 2014, the clinical marine pharmaceutical pipeline (Reviewed in Mayer et al. TIPS 31:255-265, 2010) consisted of 10 marine-derived compounds in clinical development. Included in the clinical marine pharmaceutical pipeline were three new monoclonal antibodies conjugated to monomethyl auristatin E, a synthetic analog of the marine compound dolastatin, which were in either Phase I, Phase II or Phase III clinical trials. Of note is that there are currently at least 14 other auristatin-containing antibody drug conjugates in clinical development. Updated information on the clinical marine pharmaceutical pipeline is available at http://marinepharmacology.midwestern.edu/clinPipeline.htm. Furthermore, the global preclinical marine pharmacology pipeline reported multiple marine chemicals with novel mechanisms of action (Reviewed in Mayer et al. MARINE DRUGS 11:2510-2573, 2013). Thus in early 2014, both the marine pharmacology preclinical pipeline as well as the marine clinical pharmaceutical pipeline remained very active. Supported by Midwestern University. (Back to top)

Effects of Metformin on Bethanechol-Induced Contractions of the Lower Esophageal Sphincter of the Rat
Jacob D. Peuler, Laura E. Phelps and Patrick W. Murphy (CCOM and Midwestern University)

Metformin is widely used in the treatment of diabetes. We have shown 1) that at millimolar concentrations it can markedly inhibit contractions of the isolated rat bladder as induced by the muscarinic receptor agonist bethanechol and 2) that this effect can be antagonized by the potassium (K) channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4AP) but not barium or glyburide (The FASEB Journal 2012 ; 26 :1049.2). Thus, metformin may be opening some but not other subpopulations of K channels known to exist in smooth muscle cell membranes of the bladder wall. We tested for these same effects in the lower esophageal sphincter of the rat because 1) by contracting this tissue bethanechol is used to treat gastroesophageal reflux disease (which is common in diabetic patients) and 2) metformin is known to reach millimolar concentrations in gastrointestinal tissues (including the esophagus) after standard oral dosing. Our results were the same as we observed previously in rat bladder. At 2-3 minutes after its administration, 5 millimolar metformin inhibited bethanechol’s contraction of the isolated esophageal sphincter by 61±3% in the absence of K channel blockers (control). In the presence of TEA and 4AP, it only inhibited that contraction by 18±4% and 28±5%, respectively (p<0.05 versus control). But in the presence of glyburide and barium, its inhibition of that contraction was no different from control (64±4% and 60±3%, respectively). Results were similar at 20-30 minutes after administration of metformin. Thus, metformin’s ability to inhibit muscarinic receptor-mediated smooth muscle contractions may be related in general to an ability to open both TEA-sensitive and 4AP-sensitive K channels in all smooth muscle cell membranes.