2014 SRF Summer Scholar Profile: Shruti Singh

My name is Shruti Singh, and I am a rising senior at the University of Texas at Austin. I am majoring in Human Biology with a concentration in immunity and pathogenesis. I was introduced to research through a program called the Freshman Research Initiative, which allows college freshman at the University of Texas at Austin to perform independent research. I started working in Dr. Andrew Ellington’s Aptamer lab, designing aptamers for specific targets. Aptamers are nucleic acids (RNA or DNA) that can bind to proteins, small molecules, or any other target, with applications in therapeutics, diagnostics, and drug delivery.

The target for aptamer selection in my first research project was a protein called transferrin, which is involved in the transport of ferric iron to rapidly proliferating cells of the body. Tumor cells have been shown to express more transferrin receptors than normal cells, making transferrin a great candidate for drug development in cancer research. I planned to select an aptamer against transferrin and use it for cytotoxic drug delivery to cancer cells. After multiple trials, I observed no significant binding between the target and the nucleic acid. Nevertheless, the results from this project will be used to optimize the selection of future aptamers targeted against transferrin.

In my next project, I helped develop an educational kit that can be utilized in high school classrooms to demonstrate important concepts in evolutionary theory. This kit uses biochemistry and molecular biology techniques, including a fluorescence assay for visualization, to allow students to observe the evolution of a catalytically active ribozyme ligase. A ribozyme is an RNA molecule with catalytic activity similar to proteins, and a ribozyme ligase is a ribozyme that can attach itself to another RNA molecule. In this kit, evolutionary pressure is applied to a T500 ribozyme ligase population to select for a catalytically faster ribozyme . For this project, I tested new iterations of the kit and troubleshot problems that I encountered, so the kit could be distributed to classrooms where it could be used as a tool for teaching evolution.

After watching a family member undergo the ordeal of a kidney transplant, I understand the importance of and the necessity for new therapeutic ideas in the field of organ function renewal. That made me very interested in the field of regenerative medicine. As a SENS Summer Scholar, I hope to both learn and contribute to organ regeneration and renewal research.

Regeneration of a part of Pig Thymus ex vivo using Mouse Thymic Epithelial and Bone Marrow Cells

This summer, I am working on a thymus regeneration project in Dr. John Jackson’s lab at the Wake Forest Institute for Regenerative Medicine. The thymus is a specialized organ in the immune system, and it is involved in the maturation of T-cells. T-cells recognize and attack foreign substances, called antigens, thus protecting the body from developing infections. The thymus consists of two lobes, which can further be divided into two regions – a cortex and a medulla. In the cortex, positive selection of a T-cell’s major histocompatibility complex (MHC) takes place. MHCs are cell surface proteins that present antigen to T-cells. A negative selection of T-cells occurs in the medulla, where the T-cells that recognize self-antigens are destroyed, so the T-cells won’t attack the body’s own cells and tissues.

In old age, the thymus starts to lose its functional abilities, rendering the immune system ineffective. One approach to restore the immune system in aged individuals is the regeneration of the thymus. Thymic tissue regeneration and T-cell maturation also have application in the treatment of autoimmune diseases, immunodeficiencies, and transplant rejection. During the summer, I will work on one part of this larger project.

Figure 1. An example of decellularized thymus section1.

The H&E staining of a section of decellularized pig thymus scaffold reveals that the structure is clear of cellular and nuclear material.

I plan to decellularize a small piece of pig thymus (Figure 1), which entails getting rid of all the cells in the thymus, leaving behind the extracellular structure called a scaffold. After decellularizing the thymus, I will reseed the thymus scaffold with thymus epithelial cells and bone marrow cells from mice, providing a 3-D environment to the cells that resembles their natural environment in the body. I will then analyze the proliferation of these cells in the scaffold and look for the production of mature T-cells. The success of this project will be an important step forward towards the overarching goal of whole thymus regeneration.

Future Plans:

I plan to return to the Ellington lab in the fall of 2014 and continue my work on the high school evolution demonstration kit. I hope to finish troubleshooting the problems being encountered and successfully replicate the experiment multiple times, so that the kit can be ready for distribution. I plan to graduate from the University of Texas in December 2014, after which I hope to attend medical school. I also hope to continue performing medically relevant research in medical school.

References:

(1) Ryu, S.W., Mondal, A.K., Kim, N., Bullock, D., Lee, S.J., Atala, A., Yoo, J.J., Jackson, J.D. “Characterization of thymus scaffolds for engineering thymus tissue.” Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC.

2014 SRF Summer Scholar Profile: Ethan Bassin

My name is Ethan Bassin, and I am a rising senior majoring in Bioengineering at the University of Pennsylvania. I first discovered the field of regenerative medicine in high school when I read an article in Scientific American about growing artificial organs. I was instantly captivated. Even though I already have some research experience, what is exciting about Wake Forest Institute for Regenerative Medicine (WFIRM) is their focus on clinical translation. WFIRM is an international leader in translating scientific discovery into clinical therapies and is working to grow tissues and organs and develop healing cell therapies for more than 30 different areas of the body.

Last summer, I worked in Dr. Jason Burdick’s lab in the Department of Bioengineering at the University of Pennsylvania. The title of my project was “Enzymatic Degradation and Cell Patterning of Fibrous Hyaluronic Acid Scaffolds.” Tissue engineering is an alternative to organ transplantation that uses a patient’s own adult stem cells. The stem cells are incorporated into a polymer scaffold, which mimics extracellular matrix (ECM) by providing structural support and regulating cellular processes. Currently, insufficient control over scaffold properties limits clinical translation.

The goals of this study were 1) to develop scaffolds that are composed of nano fibers and degrade enzymatically in an attempt to better replicate natural ECM and 2) to create patterned cell adhesion within nanofibrous scaffolds. Fibrous scaffolds of synthetically modified hyaluronic acid (HA), a natural polymer, were made by electrospinning to produce fibers with sub-micron diameters. The scaffolds were made enzymatically degradable by cross-linking with peptide via a light (ultraviolet) initiated reaction. A peptide sequence was used that is cleaved by matrix metalloproteinase (MMP), an enzyme naturally found in the ECM. In order to create patterned cell adhesion, scaffolds were hydrated in a solution containing RGD, a naturally derived amino sequence responsible for cell adhesion, and re-exposed to ultraviolet light to attach the adhesion sequences to the scaffolds. By using a patterned photo-mask, only certain regions of the scaffold were photo-patterned to include RGD.

This work represents the first time that enzymatic degradation has been engineered into an electrospun fibrous hydrogel scaffold. Enzymatic degradation had previously been incorporated into traditional hydrogel scaffolds but these scaffolds were unable to replicate the topographical cues that fibers provide cells for orientation and differentiation. The control of topographical cues through fibrous enzymatic degradation and spatial location through cell patterning could ultimately be used to form cell structures, such as blood vessels, or organize multiple cell types on the same scaffold, two major challenges to the field of tissue engineering.

This summer, as a SENS Research Foundation (SRF) Summer Scholar at WFIRM, I am working on a whole kidney engineering project under the mentorship of Dr. In Kap Ko, Dr. James Yoo, and Dr. Anthony Atala. SRF is a non-profit organization that sponsors work like mine that aim to repair molecular and cellular damage associated with aging in the hopes of curing age-related disease. Engineering a whole kidney construct to address the lack of transplantable kidneys for renal failure is one such project.

Enhanced Endothelial Cell Attachment Via Antibody Conjugation: Toward Kidney Implantation Using Autologous Cell Sources

Recent progress in whole organ engineering techniques based on decellularization of organs and recellularization of the resulting collagen-based matrix suggests that this method could eventually be used in transplantation. The first step of this technique is the “decellularization” of native whole organs. Most decellularization methods have been based on perfusion of the organ through existing vascular systems with detergents and other agents. These perfusion-based decellularization protocols allow efficient removal of all native cells and residual DNA, while maintaining the structural integrity of the organ’s extracellular matrix. Next, the resulting acellular matrix needs to be re-populated with functional organ-specific cell populations (“re-cellularization”). Available cell sources for targeting specific organs can be seeded onto the decellularized matrix and be allowed to organize into functional components of the organ. Ideally, engineered whole organs should contain an intact 3-dimensional cellular architecture as well as a functional vasculature that is composed of suitable cell types for organ transplantation. Particularly, acellular kidney vasculature needs an endothelial cell lining to avoid blood clotting and thrombus formation when transplanted into a living recipient. To maintain blood circulation and induce renal functions, re-endothelialization of an acellular scaffold is critical.

Figure 1. Whole Organ Engineering Process.

Re-endothelialization of acellular porcine kidney scaffolds via antibody conjugation and characterization using morphological and functional analysis.

The WFIRM team has developed a combination cell seeding system for efficient and functional re-endothelialization of the entire vasculature of an acellular renal scaffold. In their previous study, the team developed a surface modification method to reinforce endothelial cell attachment onto renal vasculature via CD31 antibody conjugation. CD31 antibody binds to an antigen found on endothelial cells. The effectiveness of antibody-conjugation-mediated re-endothelialization was evaluated through an in vitro cell detachment test using a microfluidic flow chamber system and an in vivo implantation test. Encouraged by their promising results using an endothelial cell line, the WFIRM team has recently attempted to re-endothelialize the kidney scaffolds using autologous cell sources for long-term porcine kidney implantation. This approach could potentially be applied to a translational clinical trial.

For my project, I plan to isolate and characterize primary endothelial cells from pigs to determine if the conjugation of CD31 antibody on vasculatures of kidney scaffolds will enhance primary endothelial cell attachment. Primary endothelial attachment will be evaluated by measuring the level of cell detachment in flow chamber systems using either a glass slide or a vascular specimen. For the glass slide, video recording will be taken under various flow conditions and cells will be counted using Image J software. For the vascular specimen, nuclear staining and fluorescent imaging will be performed after the completion of flow conditions and cells will be counted using Image J software.

Future Plans:

My career aspiration is to earn a PhD in biomedical engineering and then work as a senior scientist at a biomedical or regenerative medicine company. The aspects of research that I most enjoy are selecting project ideas and troubleshooting, interpreting the results, and trying to figure out what went wrong if they did not turn out as expected. As a senior scientist, I would be able to select research topics and direct research without directly conducting the experiments. At this point, I want to work in industry instead of academia because industry tends to have applied research. I think I will find applied research even more rewarding than basic research because I would get to see my efforts come to fruition with a product that could benefit the public.

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