2015 SRF Summer Scholar Profile: Le Zhang

My name is Le Zhang. I just completed my Bachelor of Science in biochemistry & biotechnology from Michigan State University. Before joining to the SRF Summer Scholars Program, I worked in Dr. Yonghui Zheng’s lab at MSU searching for novel proteins or molecules from our human body that can limit human immunodeficiency virus-1 (HIV-1) infection. These proteins are called restriction factors. One of the potential restriction factors we researched was the T-cell immunoglobulin and mucin domain (TIM) protein family. TIMs are T cell surface receptors which were reported in the past decade to promote envelope viruses (viruses surrounded by a lipid bilayer) binding and entry into the host cells like hepatitis A and Ebola1. However, one of the most recent studies has shown that TIM proteins 1, 3, and 4 strongly inhibit the release of HIV-1 2, but the study did not establish the mechanism by which viral proteins antagonize TIM proteins.

Under the supervision of Dr. Yonghui Zheng and Dr. Xianfeng Zhang, I focused on verifying if TIMs have the novel ability to inhibit HIV-1 and finding possible viral proteins that interact with TIMs. Our hypothesis was that HIV Negative Regulatory Factor (Nef), a viral protein which can down regulate host surface receptors like CD4, has the ability to also interact with TIMs. To test this hypothesis, I created an HIV-1 Nef knockout mutant in a retro-vector, which is a plasmid that encodes all HIV-1 proteins except for Nef and can assemble HIV particles. Three TIM plasmids were also prepared, which overexpressed TIM 1, 3 & 4 proteins, respectively. I co-transfected HIV-1 Nef knockout and TIMs plasmids in 293T cells, which are specially designed cells to overexpress plasmids. I determined the effect of overexpression of TIMs by measuring the amount of virus released by the Nef knockout strain compared to the non-mutant control. This research helped us better understand how the HIV infection mechanism works and how our immune system responds to the infection. It will serve as the foundation for future anti-viral treatment development in the Zheng lab.

I was attracted to stem cell research after learning about their power in tissue renewal, their utility in fighting aging, and other novel functions. I hope to learn more about stem cells during my internship with the SRF Summer Scholars Program.

Evaluating genomic stability of induced pluripotent stem cells for Parkinson’s disease cell therapy by SNP analysis

This summer, I will be conducting my research project in Dr. Jeanne Loring’s laboratory at the Center for Regenerative Medicine in the Scripps Research Institute. Guided by Dr. Michael Boland and Dr. Andres Bratt-Leal, my main goal for the summer is to analyze the genomic stability of induced pluripotent stem cells (iPSCs) and neuronal progenitor cells (NPCs), which are intended for cell replacement therapies for patients with Parkinson’s disease.

Human iPSCs are usually derived from somatic cells, like skin cells. By using biological or chemical methods, they are reprogrammed into stem cells. iPSCs are well known for their ability to self-renew and their power to differentiate into other cell types. These features make them a promising tool in cell transplantation therapy development. In many age-related diseases, irreversible cell death is often a major contributor. For instance, death of dopamine-generating cells, one type of neuron in the midbrain, leads to Parkinson’s disease. Previous research in rodent and primate models of Parkinson’s disease has shown that midbrain dopaminergic (DA) neurons differentiated from human embryotic stem (ES) cells improved forelimb use and movement control in Parkinsonian animals3. Also, ES cell-derived DA neurons showed long-term survival in animals3.

Use of human ES cells raises some morality concerns and can lead to resistance for use in adult treatments. However, use of iPSCs directly from patients addresses this concern and is more convenient and safer as well. Although iPSC research shows great promise, reprogramming has the potential to induce detrimental genomic changes. Expansion and differentiation of iPSCs over time could lead to genomic change and possibly tumorigenesis4. Past experiments from the Loring lab indicate that cell lines propagated over 100 continuous passages, regardless of passage methodology, experience genomic instability, such as genomic deletions and duplications5.

Figure 1. Parkinson's disease transplantation project overview.

Dermal Fibroblast cells are taken from patients. They are reprogrammed into iPSCs, then differentiated into NPCs, and finally, transplanted back into patients. NPCs will later develop into DA neurons inside patients. I will use SNP genotyping and computational programs to analyze cells from each of the steps and help to select untumorigenic cell lines. From Illumina, Inc. (Laurent, et al, 2011)6

The Loring lab has derived dermal fibroblasts from 10 patients with Parkinson’s disease. These fibroblasts have been reprogrammed to iPSCs, which have been differentiated into midbrain-specific NPCs. These cells will later develop into DA neurons after transplantation. The Loring lab is the first lab conducting iPSC transplantation on Parkinson’s disease patients, so it is essential to ensure genomic stability of the cells being transplanted. An important method to determine genomic integrity of patients’ iPSC lines is single nucleotide polymorphism (SNP) genotyping, which can be used to examine millions of single base pair differences at genomic sites specific to humans.

SNP analysis will enable me to determine if the cell populations are suitable for transplantation or whether they have too much genetic change and, hence, potential risk for tumorigenesis. My research this summer will generate and analyze genomic SNP profiles from patient-specific dermal fibroblasts, iPSCs, and neuronal progenitors. SNP patterns from the three cell types will be compared to determine whether genomic instability has occurred from fibroblasts to iPSCs then to neuronal progenitors. Hopefully, with efforts from other scientists and me, the Loring Lab will successfully identify some cell lines that are suitable for transplantation and pass the FDA approval. 

Future Plans:

Stem cell research is new to me. Through my experiences in the Loring lab, I want to learn more and later contribute to this field. I plan to apply to graduate school after the SRF Summer Scholars Program has ended.

References:

1. Stephanie Jemielity, Jinyize J Wang, Ying Kai Chan, Asim A Ahmed, Wenhui Li, Sheena Monahan, Xia Bu, Michael Farzan, Gordon J Freeman, Dale T Umetsu, Rosemarie H Dekruyff, Hyeryun Choe. TIM-family Proteins Promote Infection of Multiple Enveloped Viruses through Virion-associated Phosphatidylserine. PLOS. Pathogens: 2013. e1003232.

2. Minghua Lia, Sherimay D. Ablanb, Chunhui Miaoa, Yi-Min Zhenga, Matthew S. Fullera, Paul D. Rennertc,Wendy Mauryd, Marc C. Johnsona, Eric O. Freedb,Shan-Lu Liua. TIM-family proteins inhibit HIV-1 release. PNAS: 2014, doi: 10.1073/pnas.1404851111

3. Sonja Kriks, Jae-Won Shim, Jinghua Piao, Yosif M. Ganat, Dustin R. Wakeman, Zhong Xie, Luis Carrillo-Reid, Gordon Auyeung, Chris Antonacci, Amanda Buch, Lichuan Yang, M. Flint Beal, D. James Surmeier, Jeffrey H. Kordower, Viviane Tabar & Lorenz Studer. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature. 2011. Vol 480, 547-551.

4. Fox JL FDA scrutinizes human stem cell therapies. Nat Biotechnol. 2008. Vol 26, 598-599.

5. Ibon Garitaonandia, Hadar Amir, Francesca Sesillo Boscolo, Geral K. Wambua, Heather L. Schultheisz, Karen Sabatini, Robert Morey, Shannon Waltz, Yu-Chieh Wang, Ha Tran, Trevor R. Leonardo, Kristopher Nazor ,Ileana Slavin , Candace Lynch , Yingchun Li , Ronald Coleman , Irene Gallego Romero ,Gulsah Altun , David Reynolds , Stephen Dalton , Mana Parast , Jeanne F. Loring & Louise C. Laurent. Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions. PLOS ONE. 2015. DOI:10.1371/journal.pone.0118307.

6. Louise C. Laurent, Igor Ulitsky, Ileana Slavin, Ha Tran, Andrew Schork, Robert Morey, Candace Lynch, Julie V. Harness, Sunray Lee, Maria J. Barrero, Sherman Ku, Marina Martynova, Ruslan Semechkin, Vasiliy Galat, Joel Gottesfeld, Juan Carlos Izpisua Belmonte, Chuck Murry, Hans S. Keirstead, Hyun-Sook Park, Uli Schmidt, Andrew L. Laslett, Franz-Josef Muller, Caroline M. Nievergelt, Ron Shamir, Jeanne F. Loring. Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ES and iPS Cells during Reprogramming and Time in Culture. Cell Stem Cell. 2011. Vol 8. 106-118.

2015 SRF Summer Scholar Profile: Brian Shing

My name is Brian Shing, and I am an undergraduate at the University of California, Berkeley. I am a rising junior and intend to major in Molecular and Cell Biology.

My first research experience was as an intern for BNNI-SHARP (Berkeley Nanosciences and Nanoengineering Institute Summer High School Apprenticeship Program) at UC Berkeley. As an intern in Alex Zettl’s lab, I worked under the guidance of Ashley Gibb. My project aimed to use nanotechnology and materials science to improve existing microscopy capabilities for researchers. I synthesized graphene, a monolayer of carbon atoms, to create an enclosure that could be filled with a solution of biomolecules. This enclosure would allow nanometer resolution imaging of biomolecules under hydrated conditions using transmission electron microscopy (TEM), a microscope that transmits electrons through a sample for imaging.

Currently, I am a research member of NanoNerve, Inc. and Song Li’s lab at UC Berkeley. NanoNerve is a biotechnology start-up specializing in neural regeneration that has developed a synthetic graft to aid nerve regeneration. I work primarily under the guidance of Dr. Shyam Patel. I culture pluripotent stem cells and characterize the graft’s efficiency in differentiating the stem cells into neuronal stem cell lineages. I characterize the differentiation by employing histological techniques to stain the cells for imaging. Next semester, I intend to continue this ongoing research endeavor to develop a product that can regenerate nerves in patients with peripheral nerve damage.

Making the world a better place is a worthwhile goal. I am excited to be working at the Wake Forest Institute of Regenerative Medicine (WFIRM) because the use of stem cells in regenerative medicine could hold the key to treating many currently untreatable diseases and conditions.

Development of a Delivery System of Placental Stem Cell-derived Trophic Factors for Treatment of Kidney Diseases

This summer, I will be working in Drs. In Kap Ko, James J. Yoo and Anthony Atala’s lab on a kidney regeneration project. Kidney disease is a leading cause of death in the United States [1]. Three major forms of kidney disease are acute kidney injury (AKI), chronic kidney disease (CKD), and end-stage renal disease (ESRD). AKI and CKD are conditions where the kidney loses its ability to function and filter blood efficiently [2]. AKI can often worsen into CKD, which affects 8-16% of the population globally [3]. Further degradation of renal function can lead to ESRD, a life-threatening condition where the kidney completely fails.

Current medical therapies for renal disease primarily revolve around hemodialysis or kidney transplantation. Both of these treatments have inherent limitations. Dialysis can replace the kidney in filtering metabolic waste from blood, but it is merely a supportive treatment that only manages symptoms and slows disease progression. Dialysis also cannot replace other critical renal functions, such as synthesizing erythropoietin hormone to stimulate red blood cell production [4]. Consequently, treatment of renal disease should promote efficient regeneration of functional renal-specific cells. Cell-based approaches that can replace or restore damaged renal cells may provide an excellent alternative to current treatments.

Figure 1. Kidney Regeneration Project Overview.

Use of conditioned medium secreted from human placental stem cells for kidney regeneration.

Recent advances in stem cell biology and cell culture techniques have facilitated the development of cell therapy for preclinical and clinical translation [5, 6]. Particularly, recent studies have reported that secretome, such as growth factors, derived from therapeutic cells could enhance regeneration of damaged tissues [7]. WFIRM has recently tested the feasibility of using secretome from therapeutic stem cells for the treatment of kidney diseases.

This summer, I will be working with the WFIRM team to explore a novel delivery system that allows for efficient delivery of the secretome secreted from human placental stem cells (hPSCs) and evaluate the secretome’s effect on renal regeneration. This project could lead to novel therapies that could improve the lives of patients suffering from kidney disease throughout the world.

Future Plans:

After I have finished my education, I would like to work in the biotechnology industry. I am also interested in exploring the possibility of conducting biomedical research as a military researcher.

References:

[1] Centers for Disease Control and Prevention (CDC). CDC National Health Report: Leading Causes of Morbidity and Mortality and Associated Behavioral Risk and Protective Factors—United States, 2005-2013. 2014.

[2] National Institute of Diabetes and Digestive and Kidney Diseases. Kidney Disease Statistics for the United States. 2012.

[3] Jha, Vivekanand, et al. Chronic Kidney Disease: Global Dimension and Perspectives. The Lancet 382 (9888) : 260-272 (2013).

[4] National Institute of Diabetes and Digestive and Kidney Diseases. Anemia in Chronic Kidney Disease. 2014.

[5] Eirin A, Lerman LO. Mesenchymal Stem Cell Treatment for Chronic Renal Failure. Stem Cell Research & Therapy 2014; 5:83.

[6] Rosenberg ME. Cell-Based Therapies in Kidney Disease. Kidney International Supplements 2013; 3:364-67.

[7] Pawitan JA, Prospect of Stem Cell Conditioned Medium in Regenerative Medicine”, Biomed Res Int. 2014; 2014:965849

2015 SRF Summer Scholar Profile: Blake Johnson

My name is Blake Johnson, and I am a rising senior studying Human Physiology at the University of Iowa. I first became interested in the field of regenerative medicine after viewing Dr. Anthony Atala’s TED Talk on his 3-D kidney printing work. The ability of regenerative medicine to be applied to a vast array of cells, tissues, and organs and the possibility of making patients truly well again, as opposed to managing symptoms, is inspiring. WFIRM is an outstanding research institution, and it is an honor to have been selected to spend the summer learning and growing here.

My previous research under Dr. Janice Staber in the Department of Pediatrics at University of Iowa Carver College of Medicine aimed to improve bleeding symptoms in von Willebrand Disease and Hemophilia A patients. von Willebrand Disease (VWD), which results in prolonged bleeding times following injury or spontaneously due to either qualitative or quantitative deficiencies in von Willebrand Factor (VWF), affects 1% to 3% of people in the United States, more than any other genetically acquired bleeding disorder. VWD results in decreased levels of FVIII, an important clotting factor, due to a lack of protection by VWF in the plasma. We proposed gene transfer strategies that could reduce or eliminate bleeding symptoms in VWD patients through increased production of VWF and, thus, increased stabilization of FVIII. We used a DNA transposon called piggyBac (PB) in our studies. PB is capable of integrating large genes into a host’s genome using a cut and paste method, making it ideal for use in VWF gene therapy. The cut and paste method involves the removal of the gene from the PB vector and subsequent insertion of that gene into the genome of the target.

In order to test the utility of gene therapy in VWF deficiency, I generated PB DNA transposons containing VWF complementary DNA (cDNA). Following the delivery of these DNA constructs into mice, I developed an assay to test for VWF levels. Using this assay, I determined animals receiving VWF cDNA showed a significant increase in VWF production compared to VWF deficient mice. Further testing is needed to determine if these animals have improved bleeding symptoms. I also delivered the VWF gene to liver cells in culture and analyzed their ability to produce VWF protein using the same assay. Analysis revealed the liver cells showed evidence of production of VWF. We concluded that the PB transposon has the potential for therapeutic utility in von Willebrand disease. In the fall, I will continue my work on the VWF project. We will be looking to compare the effects of integrating codon-optimized VWF cDNA to wild-type VWF cDNA and assess the influence of VWF gene therapy in animals with Hemophilia A, a FVIII deficiency. The codon-optimized version of the VWF gene may allow for increased production of VWF protein by utilizing the most efficient tRNA for each amino acid present in the protein. VWF gene therapy concurrent with treatment for Hemophilia A may help to decrease bleeding symptoms in these patients by decreasing clearance of FVIII from the blood.

Generation of Functional Thymus Organoids

This summer, I am working under the direction of Dr. John Jackson to generate thymus organoids capable of producing functional T-cells. The thymus serves an important function as the site of T-cell development. Interestingly, as we age, the thymus undergoes involution, or decreases in size, leading to a decrease in naïve T-cells. The ability to generate a functional thymus outside the body would have a number of clinical applications, including rejuvenation of an aging thymus to boost the immune response in older individuals and development of tolerance in organ transplantation.

Figure 1. Thymus Epithelial Cell Characterization.

Cultured thymus epithelial cells immunologically stained for cytokeratin 5, a thymus epithelial cell marker (red). DNA is imaged using DAPI stain (blue). The images demonstrate the maintenance of thymic epithelial cell phenotype in culture.

I will be using two methods to attempt to generate T-cells outside of the body. 1) I will be seeding decellularized pig thymus with thymus epithelial cells and bone marrow progenitor cells. 2) I will be co-culturing thymus epithelial cells and bone marrow progenitor cells in collagen hydrogels to form small thymic organoids. In the thymus, epithelial cells and progenitor cells work closely together in the development and maturation of T-cells. We hypothesize the interaction between these two cell types is necessary for efficient T-cell production outside the body. In both the decellularized scaffold and hydrogel, I will be using histological techniques to determine if the seeded thymus epithelial cells self-organize into the cortical and medullary regions found in the normal thymus. The regions serve important functions including positive and negative selection of T-cells, respectively. The ability to generate a functional thymus ex vivo has a number of clinical applications and would benefit a large number of patients.

Future Plans:

My career goal is to complete an MD and become a surgeon. I hope to continue my work in research and, in particular, regenerative medicine. I am inspired by the advances I have seen so far in the field of regenerative medicine, and I believe at some point during my career regenerative medicine technologies will be used regularly. I also hope to work in the health policy sector.

2015 SRF Summer Scholar Profile: Amanda Paraluppi Bueno

My name is Amanda Paraluppi Bueno and I am pursuing my Bachelor’s degree in Biomedical Science at the Centro Universitario Heminio Ometto, Araras-SP (Brazil). I came as an exchange student to study at University of Idaho, Moscow-ID, for one year through the Science Without Borders Program.

I conducted research during my junior and senior year (Fall 2013 – Summer 2014) under Dr. Flávia C. M. C. Alves at Centro Universitário Hermínio Ometto, Araras, São Paulo (Brazil). The project was selected to be funded by the National Council of Scientific and Technological Development (CNPq) in Brazil. In this project, I incorporated growth factors into scaffolds to improve the healing process of chronic wounds in the skin that are common in some diseases like leprosy. This gave me the passion for research and helped me to decide to focus my studies on regenerative medicine.

This summer, I have the opportunity to do what I love: research in the field of regenerative medicine. I am very excited to work for SENS Research Foundation because I will have the chance to learn and contribute to research centered around the diseases of aging at the Wake Forest Institute for Regenerative Medicine (WFIRM), which is an extraordinary place for this field.

Human Mesenchymal Stromal Cells and Endothelial Progenitor Cells as Allogenic Treatment for Inflammatory Bowel Disease in mice

This summer, my Principal Investigator is Dr. Graça Almeida-Porada and my mentors are Saloomeh Mokhtari and Steven Greenberg. Our goal is to develop novel cell-based therapies that could provide a curative treatment for Inflammatory Bowel Disease (IBD). IBD includes a group of chronic inflammatory illnesses of the gastro-intestinal tract, the most common forms of which are Crohn’s disease and ulcerative colitis. IBD is a significant and rapidly growing health care burden that affects millions of people around the world. In the US alone, these costs total approximately $6.3 billion per year. There are several therapeutic approaches to induce remission and/or to prevent relapse, however the side effects, toxicity, and lack of response to the existing drugs highlight the need to develop a cure for this devastating disease. The cause of IDB is unknown, but data suggests that it is caused by the dysregulation of the immune system and alteration of the intestinal microflora balance. Mesenchymal stromal cells (MSC) have been shown to improve IBD in a small percentage of patients. When infused into patients, MSCs migrate (or home) directly to inflammatory sites in the body, modulate the inflammatory response, and stimulate the resident stem cells.

Figure 1. Progression of Inflammatory Bowel Disease (IBD)

By transplanting MSC and EPC, I hope to increase in the number of regulatory cells to modulate the immune system of the disease (phase III). From Neurath (2014).

The Almeida-Porada lab has already shown that increasing the expression of immunomodulatory molecules on MSC leads to better immunosuppression and improvement of IBD in a murine model. Other cells that could help in the treatment of the gut inflammation are endothelial progenitor cells (EPC). These cells are known to increase the vascularization in ischemic tissues. Therefore, EPC could help normalize vascularization in the intestinal submucosa of IBD patients . Hence, I plan to treat IBD in mice using MSC and EPC as cell therapy to promote the modulation of the immune system and increase the vascularization in the intestine.

Future Plans:

After I finish the summer program, I will go back to Brazil to complete my Bachelor’s degree in December 2015. This summer research certainly will help me to pursue a PhD degree and make a contribution as a researcher to combat the diseases of aging. In addition, I would like to instill the same passion for research in other students that my experiences instilled in me. I plan to achieve this goal by teaching students biomedical procedures and advising them how best to achieve a future career in research.

References:

Neurath M.F. Cytokines in inflammatory bowel disease. Nature Reviews: Immunology. May 2014, vol. 14, 329-342.

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