2016 SRF Summer Scholar Profile: Dayne Martinez

My name is Dayne Martinez. I am a rising senior at Grand Valley State University where I am working to earn a degree in Biochemistry. For several years now, I have been excited by the prospect of using strategies from regenerative medicine to develop novel therapies for neurological problems. Getting involved in SRF’s research has given me an opportunity to pursue that passion, and it will give me important experience that will help me later in my career.

Last summer, I worked in the lab of Dr. Michael Uhler at the University of Michigan as an intern for the Summer at Michigan for Undergraduate Research Training Program (UM-SMART). My project was a part of a larger effort to discover chemical compounds and transcription factors that can be used direct human induced pluripotent stem cells to change into specific subtypes of neurons. Being able to generate a specific subtype of neuron has numerous potential applications, such as the development of disease models and drug screening platforms.

Two of the methods that were used to analyze the cells over the course of the study were live cell imaging and FACS (fluorescence-activated cell sorting). To facilitate those analyses, I designed and constructed vectors to insert a fluorescent reporter gene downstream of neuron subtype-specific genes through CRISPR/Cas9-mediated homologous recombination. In particular, I designed vectors to target the genes coding for either VGLUT1 or VGAT, which are vesicular transporter proteins expressed uniquely in glutamatergic and GABAergic neurons respectively. I used PCR to generate homology arms for both genes. These homology arms are regions that facilitate recombination that could be used for sequence-specific insertions. Over several rounds of sub-cloning, I constructed complete targeting vectors that contained the fluorescent reporter gene flanked by the homology arms. I also designed sgRNA that will be used to guide the Cas9 endonuclease. Ideally, Cas9 will generate a double-stranded break at the genome loci specified by the sequence of the sgRNA. This is followed by homology-directed repair, which results in the reporter gene being inserted at the cut site [1].

For the last two years, I have worked in the lab of Dr. Merritt Taylor at Grand Valley State University. My project has revolved around the study of Nato3, which is a transcription factor expressed in the floor plate region of the neural tube during development. Nato3 has been shown to be important for promoting the neurogenic activity of the mesencephalic floor plate region through the repression of HesI [2]. This function is of interest to us because that particular area of the floor plate region gives rise to an important population of dopamine-producing neurons. These neurons, located in the substantia nigra pars compacta, are the primary target of degeneration in Parkinson’s disease (PD). When these neurons die, proper dopamine input to another brain region, the striatum, is disturbed. This disruption leads to the multiple motor and cognitive symptoms associated with PD.

Previous work in our lab has shown that overexpression of Nato3 is sufficient to promote the generation of dopaminergic neurons in the developing chick model. My work has focused on modifying Nato3 to try to increase its capacity for generating dopaminergic neurons from pluripotent progenitors. The ultimate goal of this work is to determine if modified Nato3 can be a useful genetic tool for generating dopaminergic neurons for applications such as disease modelling and cell replacement therapy for PD.

Quality Control for Autologous Cell Therapy for Parkinson’s Disease

As described above, the loss of dopaminergic input to the striatum is the cause of the motor and cognitive symptoms of PD. Current therapies rely upon the administration of L-DOPA, the direct precursor of dopamine, to alleviate these symptoms. Unfortunately, the effectiveness of L-DOPA therapy decreases as the disease progresses. Administration of L-DOPA over an extended period of time can lead to the development of a dyskinesia, which is a condition characterized by difficulty performing voluntary movements. The shortcomings of L-DOPA therapy leave room for the development of better treatments. One promising approach is cell replacement therapy. This therapeutic approach entails transplanting new dopaminergic neurons into the striatum of patients with PD with the goal of replacing the innervations that were lost. One source of dopamine neurons for these grafts is induced pluripotent stem cells (iPSCs), which are stem cells generated from somatic cells. These cells, which can be used to make dopamine neurons for such a graft, are excellent candidates for cell replacement therapy because they are readily sourced from the patient’s own skin cells. Additionally, the grafts should not be rejected by the recipient’s immune system because they are derived from the patient’s own cells [3].

A critical barrier for cell replacement therapy for PD is quality control of the grafts used for transplant. For my project, I will be working in the lab of Dr. Jeanne Loring under the supervision of Dr. Andrés Bratt-Leal at The Scripps Research Institute. I will be generating dopamine neurons from patient-derived iPSC lines. I will then analyze the neurons using several different methods in an effort to develop quality control assays that can be used to verify the purity of cells to be used for a cell replacement therapy graft. These methods include immunocytochemistry, qt-PCR, flow cytometry, and microarray analysis. The goal is to obtain a pool of cells with a high purity of dopamine neurons, so these assays will be aimed at screening for other cell types that should not be present in a potential graft.

Figure 1. Example of immunocytochemistry technique to be used for verifying the identity and purity of cells.

Blue=DAPI (nuclear stain), Red=β-Tubulin III (neurons), Green=TH (dopaminergic neurons). Other positive markers for dopaminergic neurons that will be used include NURR1, LMX1A, and FOXA2. Markers to screen for undesirable cell types include OCT4 (pluripotency marker), PAX6 (non-dopaminergic neural precursors), and SERT (serotonin neurons). Cells that have been stained for these markers can be quantified using flow cytometry. These methods will be combined with other quality control measures such as qt-PCR and gene expression data to verify the purity of a potential graft.

Future Plans:

I am enthusiastic about the field of regenerative medicine. In particular, I would like to work on developing better therapies for various neurological conditions. To this end, I plan on earning a PhD in Neuroscience. My career plans are not set in stone, and I am open to working in industry or academia at this point.


1. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc Nature Protocols, 8(11), 2281-2308. doi:10.1038/nprot.2013.143

2. Ono, Y., Nakatani, T., Minaki, Y., & Kumai, M. (2010). The basic helix-loop-helix transcription factor Nato3 controls neurogenic activity in mesencephalic floor plate cells. Development, 137(11), 1897-1906. doi:10.1242/dev.042572

3. Bratt-Leal, A. M., & Loring, J. F. (2016). Stem Cells for Parkinson’s Disease. Translational Neuroscience, 187-201. doi:10.1007/978-1-4899-7654-3_11

2016 SRF Summer Scholar Profile: Alicia Lee

I recently graduated from Rutgers University, New Brunswick with a Bachelor’s Degree in Biomedical Engineering with a concentration in Tissue Engineering. During my time at Rutgers, I worked under the supervision of Dr. David Shreiber to investigate the mechanical and biological effects of acupuncture so that these mechanisms can be understood and potentially be incorporated into other medical treatments. The Shreiber lab has developed a unique model of acupuncture therapy using acupuncture needle stimulation of collagen gels that mimic loose connective body tissue. I helped refine this model so that it yielded more consistent and reproducible results. I also developed a successful protocol for creating and testing cellular gels so that our model more closely mimicked natural tissue and thereby allowed us to better simulate the mechanical and biological effects of acupuncture therapy.

Additionally, last summer, I interned at the Johns Hopkins Institute of Nanobiotechnology, where I fabricated and developed drug-loaded nanofiber-hydrogel composites to inhibit cancer stem cell migration after tumor resection and thereby prevent metastasis. Under the supervision of Dr. Hai-Quan Mao and Dr. Jisuk Choi, I evaluated the biological effects of the composites and worked with the lab’s collaborator to design a protocol for testing the composites in rats. The drug-loaded composites showed promising results when tested in cell culture, but the animal study was inconclusive. Consequently, there are other studies currently underway to further evaluate the therapeutic effect of these composites. My research experiences have motivated me to learn more about and to contribute to innovations in regenerative medicine and cell-based therapies.

Optimizing the Formation of Renal Tubules in 3D Cell Culture

This summer, I will be working with Dr. Anthony Atala, Dr. James Yoo, Dr. In Kap Ko, and Jennifer Huling at the Wake Forest Institute for Regenerative Medicine towards creating an implantable tissue construct that restores function to damaged or diseased kidney tissue. Chronic Kidney Disease (CKD), which is the gradual loss of kidney function, is an increasingly prevalent condition that often develops into End-Stage Renal Disease (ESRD), an extremely debilitating condition which results in multiple organ failures. Currently, CKD and ESRD patients are treated with either dialysis or kidney implants, but these methods have severe limitations. Although dialysis filters toxins from patients’ blood, it cannot replicate several important functions of the kidney and is not a curative treatment. Furthermore, the average life expectancy of dialysis patients is 5-10 years [1]. Kidney transplantation is currently the only curative treatment, but the critical shortage of available donor kidneys has resulted in waiting times of over 3 years, and graft recipients often experience acute rejection and graft failure [2]. Therefore, there is a need for a comprehensive, biocompatible, and widely available treatment that restores kidney function.

One solution is the creation of tissue-engineered constructs that restore function to damaged or diseased tissue. These constructs can either be partial constructs (which augment the function of existing tissue) or complete constructs (which can completely replace existing tissue). One major challenge in engineering 3D tissue replacements is vascularization, or the incorporation of a blood supply. Pre-vascularization of constructs is critical for cell survival and tissue function. The Kidney Group at WFIRM has done extensive work in developing pre-vascularized partial kidney constructs that have a high degree of vascularization, which can improve the viability of cells in these constructs. Another major challenge is ensuring that the kidney cells in the 3D renal constructs are able to grow, proliferate, and perform their desired functions. Kidney cells in 3D culture tend to self-assemble into renal tubule segments, which are the functional components in normal kidneys (See Figure 1). My project will be “Optimizing the Formation of Renal Tubules in 3D Cell Culture.” I will be seeding renal cells into collagen gels and experimenting with different renal cell concentrations, collagen concentrations, and growth factors to determine which parameters will promote the quickest and the most efficient formation of renal tubules. I plan to incorporate these parameters with the Kidney Group’s pre-vascularized constructs to create optimized partial renal constructs.

Figure 1. Renal tubule self-assembly after 10 days in 3D collagen gel culture at 100x and 400x magnification.

Sections were stained with H&E, staining cell nuclei dark blue and the collagen gel pink. Tubule structures are visible throughout the entire collagen gel and can be identified by the hollow, gel-free lumen formed inside the cells (arrows).

Future Plans:

After my internship at WFIRM, I hope to work in Singapore before returning to the US to pursue a graduate degree. I plan to eventually pursue either a PhD or an MD/PhD degree, and I seek to combine my interests in regenerative medicine and marine biology. I am especially interested in the work that scientists have done in isolating cytotoxic factors from shark immune cells to treat human cancer and in investigating the unusually rapid and wound-free healing process in sharks to see if it can be applied towards humans.


1. Ko, In Kap, Mahran Abolbashari, Jennifer Huling, Cheil Kim, Sayed-Hadi Mirmalek-Sani, Mahmoudreza Moradi, Giuseppe Orlando, John D. Jackson, Tamer Aboushwareb, Shay Soker, James J. Yoo, and Anthony Atala. “Enhanced Re-Endothelialization of Acellular Kidney Scaffolds for Whole Organ Engineering via Antibody Conjugation of Vasculatures.” Technology 2.3 (2014): n. pag. 1 Sept. 2014. Web. 18 Apr. 2016

2. Song, Jeremy J., Jacques P. Guyette, Sarah E. Gilpin, Gabriel Gonzalez, Joseph P. Vacanti, and Harold C. Ott. “Regeneration and Experimental Orthotopic Transplantation of a Bioengineered Kidney.” Nature Medicine (2013): n. pag. Nature Medicine. Nature Publishing Group, 14 Apr. 2014. Web. 29 Apr. 2016.

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