The SIVB’s In Vitro Animal Cell Sciences Section (IVACS) held a Student and Post-Doctoral Oral Presentation Competition on Monday, June 13, and it was moderated by Dr. Addy Alt-Holland (Tufts University) and Dr. Kolla Kristjansdottir (Midwestern University). The abstracts that had been submitted to be considered for this competition underwent a rigorous review process, and three abstracts were selected for the competition based on their quality and scientific merit. The top three contestants who presented their work in the session delivered highly professional and stimulating presentations. They answered numerous questions from the judges as well as from SIVB members that attended the session. The panel of IVACS experts that served as judges and evaluated the contestants included: Dr. Brad L. Upham (Michigan State University), Dr. Mae Ciancio and Dr. Michael J. Fay (Midwestern University), Dr. Michael Dame (University of Michigan), Dr. Barbara Doonan (New York Medical College), Dr. Eugene Elmore, Dr. John W. Harbell (JHarbell Consulting LLC), Dr. Dawn Applegate (RegeneMed, Inc.), Dr. Kolla Kristjansdottir and Dr. Addy Alt-Holland.

Certificates and monetary awards were presented to the three contestants during the IVACS Business Meeting later that day. We gladly announced that Allison Songstad from the University of Iowa won the 1st place award for her presentation “Development of a Stepwise Protocol for the Generation of Human Induced Pluripotent Stem Cell-derived Choroidal Endothelial Cells.” Elizabeth Hansen from Midwestern University won the 2nd place award with her presentation “Voluntary Exercise Prevents High-fat Diet-induced Obesity: Effect on Intestinal Barrier Integrity.” Darryl Giambalvo who is also from Midwestern University won the 3rd place award for his presentation titled “Extending Neuronal Regeneration with Nano-sized Topography and Various Matrix Coatings.”

The Student and Post-Doctoral Oral Presentation Competition is a unique session designed to enrich public-speaking experience, exposure and achievements of students and Post-Doctoral candidates. It provides an important venue for them to present their research work to an expert audience in a national conference. We encourage all qualified individuals to submit their research abstracts to the upcoming 2017 competition in Raleigh, NC.

Submitted by Addy Alt-Holland and Kolla Kristjansdottir

First Place

Development of a Stepwise Protocol for the Generation of Human Induced Pluripotent Stem Cell-derived Choroidal Endothelial Cells

asongstadAge-related macular degeneration (AMD) involves dysfunction of choroidal endothelial cells (CEC), retinal pigment epithelial cells (RPE), and photoreceptor cells (PRs). Human induced pluripotent stem cell (iPSC)-based strategies to replace PRs and RPE are a major scientific focus. However, the replacement of damaged and lost CECs, which degenerate early in the pathogenesis of AMD, is crucial for these approaches. The purpose of this study was to design a stepwise differentiation protocol to generate human iPSC-CECs. Fibroblasts from a patient with normal ocular history were reprogrammed into iPSCs using the Yamanaka factors. Pluripotency was assessed via TaqMan Scorecard Assay and immunocytochemistry (ICC). RNA sequencing analysis of cultured monkey RF6A CECs and 2-D gel/mass spec analysis of RF6A-conditioned medium identified seven secreted proteins known to be involved in vascular development. Using the Taguchi statistical strategy, media were developed using combinations of the seven proteins to drive CEC differentiation. In a separate strategy, each protein was sequentially eliminated from the media to identify crucial proteins for CEC differentiation. The iPSC-CECs from both methods were characterized via rt-PCR and ICC for EC- and CEC-specific markers. Using each approach, CTGF, TWEAKR, and VEGF-B generated iPSC-CECs that expressed the EC-specific markers CD31, CD34, eNOS, FOXA2, ICAM1, TIE2, and VE-Cadherin, the CEC-restricted markers CA4 and TTR, and formed vascular tube networks morphologically identical to those formed in primary human CEC cultures. Thus, we developed a stepwise differentiation protocol for the derivation of CECs from patient-specific iPSCs that will be useful for cell replacement therapy and interrogating AMD pathophysiology.

Allison Songstad, Wynn Institute for Vision Research, University of Iowa, 375 Newton Rd., 4156 MERF, Iowa City, IA 52242. In Vitro Cellular and Developmental Biology, 52:S30, 2016

Second Place

Voluntary Exercise Prevents High-fat Diet-induced Obesity: Effect on Intestinal Barrier Integrity

hansenBackground: High-fat diet-induced obesity (HF-DIO) has been linked to changes in intestinal barrier function and gut microbial balance.  Voluntary exercise (Ex) can prevent HF-DIO by changing gut microbial balance (Evans, et al, 2014), but its effect on intestinal barrier is unclear. Objective: Given the interaction between the gut microbiota and the intestinal barrier, we hypothesized that Ex prevents HF-DIO by maintaining tight junctional integrity. Methods: Five week old male, C57Bl/6 littermates were divided into four treatment groups: low fat (LF)/ sedentary (Sed; n=6), LF/Ex (n=5), high fat (HF)/Sed (n=5), and HF/Ex (n=6). After 14 weeks, mice were gavaged with 4 kDa FITC-dextran (0.5mg/g body weight). Mice were euthanized 1 hr post-gavage, and serum and colonic scrapings were collected.  Intestinal permeability was measured by the amount of FITC-dextran in the serum. Western blots determined the relative expression of the colonic tight junction protein occludin compared to constitutive heat shock protein 70. Two-way ANOVA determined significance, p < 0.05, between groups. Results: HF diet resulted in significant increases in body weight, which was prevented by Ex. HF-DIO mice demonstrated no significant changes in gut permeability compared to LF/Sed mice. Neither HF diet nor Ex altered gut permeability or the relative levels of occludin in the colons of mice (p > 0.05). Conclusion: Our results indicate that HF diet can induce obesity without changing intestinal barrier function. Exercise prevented HF-DIO via mechanisms other than stabilization of tight junctional integrity.  Ongoing studies are examining tight junctional protein localization and markers of inflammation to confirm that there is no measurable change in gut barrier associated with HF-DIO.

Elizabeth Hansen, Midwestern University, College of Health Sciences- Biomedical Sciences Program, 555 31st St, Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 52:S29, 2016

Third Place

Extending Neuronal Regeneration with Nano-sized Topography and Various Matrix Coatings

giambalvoPeripheral nerve damage is a common result of many injuries and disease states. While the peripheral nervous system regenerates spontaneously, the extent to which it does is often insufficient to fully restore function. Even though previous research has shown that different biophysical and biochemical cues may increase axonal growth, it is often uncontrolled and random in direction. Therefore, the aim of this study is to control the direction of axonal growth using nano- to micron scale grooves as topographical cues, and optimize the cell-surface contact by modifying the components of the extracellular matrix for the most extensive regeneration. Our hypothesis is that axons will use the biophysical signals as a guide and propagate parallel to them, which when combined with the appropriate matrix, will lead to a longer effective axonal length. For the purposes of our study, we used ex vivo explants of mouse dorsal root ganglia (DRGs). Cervical and thoracic DRGs were harvested and cultured on anisotropic grooves. Four groove widths were tested: 200nm, 400nm, 700nm, and 2,000nm. A chemically identical flat surface was used as a control. Imaging and analysis demonstrated that axons align and grow in a linear fashion along the 700 and 2,000nm grooves significantly more than all other conditions. This direct propagation also significantly extended the average radius of growth of the axons on the 700 and 2,000nm plates. Due to these results, the 700 and 2,000nm grooves were selected to further study the synergistic effect of various extracellular matrix components on axonal growth. We can already assess differences in axonal growth behavior among the different matrices such as Matrigel, collagen IV and Puramatrix.  In conclusion, these results may translationally be applied toward novel treatments to directionally and biochemically enhance spontaneous nerve regeneration and help us elucidate general mechanisms responsible for axonal growth.

Darryl Giambalvo, Midwestern University, Department of Biomedical Sciences, 555 31st Street, Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 52:S29-30, 2016