HSU CIRM Scholars Program
 
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Current HSU-CIRM Scholars

HSU-CIRM Scholars participate in a 12-month, research-intensive internship at the Stanford University Center for Human Embryonic Stem Cell Research and Education, the University of California, San Francisco (UCSF) Institute for Regenerative Medicine, or the University of California, Davis (UCSD) Institute for Regenerative Cures. Scholars receive a monthly stipend of $2,500 in addition to a $5,000 scholarship to be put towards their Humboldt State enrollment fees.

To see a list of past HSU-CIRM scholars, click here.



The 2015-2016 HSU-CIRM Scholars List

 

HSU-CIRM Host Labs

Carsten Charlesworth - "Genome Editing for the Treatment of X-linked SCID "
Matthew Porteus, M.D., Ph.D., Stanford University

X linked severe combined immunodeficiency (X-SCID), is a recessive disease caused by mutations to the gene IL2Rγ and is characterized by a non-functional immune system. We have designed a method for functionally correcting patient hematopoietic stem cells, at the IL2Rγ locus, by creating a double strand break at the first exon of IL2Rγ and delivering a repair donor using an AAV6 vector. The repair donor contains a functional cDNA of IL2Rγ and a selectable marker, truncated nerve growth factor receptor (tNGFR). Optimizing our genome editing strategy in K562 cells a myelogenous cell line. We have found we are able to target upwards of 40% of K562 cells with our donor and enrich for cells expressing tNGFR four fold. Using our genome editing strategy in hematopoietic stem and progenitor cells (HSPC’s) we have found we can target a population of CD34+ cells at the IL2RΓ locus with our donor and enrich for targeted cells 90 fold. We have shown that we can target 8% of CD34+ cells at the IL2RΓ locus with a donor containing only a reporter gene however our rates of targeting with our clinical donor for the treatment of X-SCID are below 2%.  

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Zelenia Contreras - “shRNA Knockdown of METTL3 in Human Mesenchymal Stem Cells (hMSCS) to Prolong Expansion and Regenerative Properties"
Jan Nolta, Ph.D., UC Davis Institute for Regenerative Cures

Mesenchymal stem cells (MSCs) are adult stem cells that have significant clinical implications due to their multipotent ability to differentiate into the mesodermal lineage, their involvement in tissue repair, and secretion of cytokines that promote healing. However, it is known that MSCs populate a small fraction of cells in the bone marrow, therefore necessitating in-vitro expansion and pooling different MSCs donor banks for treatment. Unfortunately, challenges arise with expansion of these cells due to the proliferation rate of MSCs decreasing with extended passaging along with the loss of regenerative properties. To overcome these issues, researchers pool multiple donor MSCs however, there is considerable donor-to-donor variability in MSC population in ability to respond to environmental cues, differentiate, and proliferate. The variability of MSCs may decrease the efficacy of these cells in clinical trials. In order to address these issues, this study aims to knockdown (KD) METTL3, a methyltransferase known to be involved in post-transcriptional RNA modification and a regulator of core self-renewal maintenance genes to elucidate the potential of KD METTL3 in MSCs in overcoming in-vitro expansion limits and in reducing the need to pool donor cells.

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Sean de la O - "Generating three dimensional culture of lung epithelial: Modeling disease and treatment "
Calvin Kuo, M.D., Ph.D., Stanford University

Understanding human physiology and disease requires model systems that faithfully capture homo and heterotypic cellular interactions. To study human cells in the laboratory, the most optimal system thus far has been the culture of primary cells in a 3-dimensional format, where cellular division and differentiation occurs with spontaneous organization into ultrastructure mimicking that of parent organs. This organotypic culture methodology has been well described for the gastrointestinal tract, but limited studies have been performed on the respiratory system. My research focus has been to optimize the differentiation of respiratory epithelium from induced Pluripotent Stem Cells (iPSCs) in addition to developing methods for genetic manipulation via genome editing with CRISPR/Cas9. Taken together, my objective will be to develop a human genetic model system for respiratory physiology and disease.

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Haley DuBois - "Injection of Naked DNA as A Potential Gene Therapy Approach for Muscular Dystrophy"
Michele Calos, Ph.D., Stanford University

Muscular dystrophies are a group of degenerative diseases that arise from genetic mutations that cause a loss of function in the corresponding gene products. Of particular interest is Limb Girdle Muscular Dystrophy Type 2B (LGMD2B), which is caused by mutations in the Dysferlin gene (DYSF). Here, we describe a potential gene therapy approach to deliver the healthy, full-length DYSF coding sequence in a safe and long-lasting manner to muscle fibers. We have injected Dysferlin-negative mice with naked DNA designed to knock-in DYSF using φC31 integrase. φC31 is a serine recombinase that mediates site-specific integration by recombining endogenous pseudo attP sites within the mammalian genome with exogenous attB sites that are introduced on a donor therapeutic plasmid. After one delivery with the hydrodynamic limb vein injection method, an average of 20% of muscle fibers expressed DYSF. We will also be testing whether this approach can transfect or stimulate endogenous muscle stem cells. Currently, we are applying this therapy to Dysferlin-null mice. We will also be translating this approach to other mouse models of muscular dystrophy, including Duchene Muscular Dystrophy and Limb Girdle Muscular Dystrophy Type 2D, with an ultimate goal of moving these therapies to the clinic.

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Aja Harding - "Endothelial Progenitor Cells Derived from Pluripotent Stem Cells for Treatment of Critical Limb Ischemia"
Ping Zhou, Ph.D., UC Davis Institute for Regenerative Cures

Pluripotent stem cells provide a promising source to derive endothelial progenitor cells (EPs) for the treatment of vascular disease, ischemia, and stroke. We have developed a robust protocol to differentiate human iPSCs into nearly pure EP population (94-97% CD31+ and 78-83% VE-Cadherin+). These EPs also expressed other endothelial markers, vWF and NOS3. Compared to HUVECs, these EPs had higher level of artery marker, HEY1, and lower level of venous marker, Coup-TFII. They were able to uptake Dil-acetylated low density lipoprotein and formed tubes on matrigel in vitro and in vivo in a matrigel. The iPSC-EPs injected into a hind limb ischemia mouse model formed patent blood vessels as revealed by adjacent localization of human specific CD31 cells and FITC-dextran that labeled blood flow two months after cell transplantation. No teratomas were detected in these mice. Our data suggest that EPs derived from iPSCs have great potential to treat ischemia.  

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Madeline Hoon - "Engineering of Mesenchymal Stem Cell Derived Exosomes with miR-132 to Increase Angiogenesis "
Jan Nolta, Ph.D., UC Davis Institute for Regenerative Cures

Peripheral arterial disease is an ischemic tissue related disorder characterized by a lack of adequate blood flow to the extremities. This occurs when the artery is blocked, typically as a result of atherosclerosis. Mesenchymal stem cells (MSCs) have been shown to be a possible therapeutic for ischemic diseases through their ability to secrete pro-angiogenic signals. Recently, it was shown that exosomes isolated from these MSC are key components in the delivery of pro-angiogenic factors. MSC exosomes have been shown to increase angiogenesis of endothelial cells in vitro as well as in vivo using mice models of peripheral arterial disease. In this study, bone marrow MSC derived exosomes were engineered to have an increased concentration of the pro-angiogenic microRNA, miR-132. Human umbilical vein endothelial cells were treated with these miR-132 exosomes in vitro. Through fluorescence microscopy we confirmed that the endothelial cells were able to uptake the exosomal contents of the engineered exosomes. The cells that up took the exosomal contents from either the miR-132 engineered exosomes or wild type exosomes displayed increased cell proliferation as well as increased angiogenic capabilities. The results of this study highlight the importance of exosomes in how mesenchymal stem cells confer a proangiogenic effect.

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Manal Hosawi - " The Role of VEGF in the Neural Stem and Progenitor cells in the Hippocampus"
Tony Wyss-Coray, Ph.D., Stanford University

Neurogenesis is the process of generating new neurons from undifferentiated neural stem and progenitor cells (NSPCs). Currently, the functional role of the NSPCs in the subgranular zone (SGZ) is largely attributed to the new neurons that NSPCs create. However, Recent study showed the hippocampal progenitors secrete large quantities of the essential growth factor VEGF in vitro and vivo. These findings arise the possibility that NSPCs could be an unexpected source of VEGF in the brain. Interestingly, after a seizure, the VEGF levels highly increase. However, researchers still debate whether these changes improve or hinder recovery after a seizure. Our study showed that NSPC-VEGF knockdown followed by the induction of seizures resulted in increasing the brain injury markers in hippocampus. This result suggested the importance of NSPC-VEGF expression in the recovery from seizure-related damage.

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Michael Lopez - "Correcting Treacher Collins Syndrome Using Patient Derived Neural Crest Stem Cells "
Michael Longaker, M.D., M.B.A., Stanford University

Neural Crest Stem Cells (NCSC) are a multipotent population of stem cells that arise from the neural plate border and possess the ability to migrate ventrally throughout the developing embryo. NCSCs retain multipotency prior to migration, during, and after migration, and are capable of differentiating into a variety of cells including cartilage, bone, melanocytes, autonomic neurons, and Schwann cells among others. During development cranial NCSCs migrate into the first and second pharyngeal arch and differentiate into the bones and cartilage that make up the craniofacial structure. Treacher Collins syndrome (TCS) is a rare congenital disorder that has been associated with aberrant expression of NCSCs. Individuals with the syndrome commonly display hypoplasia of the craniofacial structure usually the maxilla, zygoma, and mandible. In severe cases patients may be born without zygomatic arches. In this study we seek to determine whether TCS pathogenesis is due to NCSCs’ inability to differentiate into bone and cartilage. We investigate the NCSCs’ ability to differentiate using TCS patient derived induced pluripotent stem cells (iPSC). The iPSCs are directed toward a NCSC lineage using defined factors and subsequently differentiated into bone and cartilage. This technology may provide an avenue to generate cartilage and bone in vitro from patient derived NCSCs that can then be transplanted back into the patient to help restore craniofacial structure while circumventing graft versus host disease.

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Michael Olvera - " A Genome-wide CRISPRi Screen for Discovering Novel Factors of iPSC Growth & Survival"
Bruce Conklin, M.D., Gladstone Institute of Cardiovascular Disease

In order to study disease in physiologically relevant cell models, it is important to develop technologies that enable efficient control of gene expression. Using the newly developed CRISPR interference (CRISPRi) technology, we aim to interrogate genes involved in iPSC growth and maintenance at the whole-genome level. CRISPRi utilizes a deactivated Cas9 enzyme with a fused KRABs domain (dCas9-KRAB) for gene silencing. We have generated a genome-wide kinase and phosphatase library of 13,000 single guide RNAs designed to target 2600 genes. Using inducible CRISPRi iPSC, we have used our library to dissect genes paramount to the maintenance of pluripotency. Several highly confident gene hits were identified in our screen. Our highest ranking gene hits include genes validated in pluripotent cells, such as ATR, CHEK1. Several genes of unknown relevance have also been identified, with some of them being novel hits with little literature on their role in growth and survival. Overall this projects will improve our understanding of cellular pluripotency and differentiation.

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