Bio

Bio


I am interested in preventing age-related diseases. During my Ph.D. I co-invented the first rapid but transient method of extending telomeres in human cells. Our approach uses modified mRNA encoding TERT. I am continuing this work as a post-doc.

Professional Education


  • Doctor of Philosophy, Stanford University, NEURS-PHD (2014)
  • Bachelor of Science, University of Waterloo, Biochemistry (2001)

Stanford Advisors


Publications

All Publications


  • Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice PLOS ONE Raval, A., Behbehani, G. K., Le Xuan Truong Nguyen, L. X., Thomas, D., Kusler, B., Garbuzov, A., Ramunas, J., Holbrook, C., Park, C. Y., Blau, H., Nolan, G. P., Artandi, S. E., Mitchell, B. S. 2015; 10 (7)
  • Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells FASEB JOURNAL Ramunas, J., Yakubov, E., Brady, J. J., Corbel, S. Y., Holbrook, C., Brandt, M., Stein, J., Santiago, J. G., Cooke, J. P., Blau, H. M. 2015; 29 (5): 1930-1939

    Abstract

    Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24-48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 1.5 and 3.4 0.4 population doublings (PD), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 1.1 PD, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 10(12)-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.-Ramunas, J., Yakubov, E., Brady, J. J., Corbel, S. Y., Holbrook, C., Brandt, M., Stein, J., Santiago, J. G., Cooke, J. P., Blau, H. M. Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells.

    View details for DOI 10.1096/fj.14-259531

    View details for Web of Science ID 000354114600027

  • Direct evaluation of myocardial viability and stem cell engraftment demonstrates salvage of the injured myocardium. Circulation research Kim, P. J., Mahmoudi, M., Ge, X., Matsuura, Y., Toma, I., Metzler, S., Kooreman, N. G., Ramunas, J., Holbrook, C., McConnell, M. V., Blau, H., Harnish, P., Rulifson, E., Yang, P. C. 2015; 116 (7): e40-50

    Abstract

    Rationale: The mechanism of functional restoration by stem cell therapy remains poorly under-stood. Novel manganese-enhanced MRI and bioluminescence reporter gene imaging (BLI) were applied to follow myocardial viability and cell engraftment, respectively. Human-placenta-derived amniotic mesenchymal stem cells (AMCs) demonstrate unique immunoregulatory and pre-cardiac properties. In this study, the restorative effects of three AMC-derived sub-populations were exam-ined in a murine myocardial injury model: 1) unselected AMCs (uAMCs), 2) ckit+AMCs (c+AMCs), and 3) AMC-derived iPSCs (MiPSCs). Objective: Determine the differential restorative effects of the AMC-derived sub-populations in the murine myocardial injury model using multi-modality imaging. Methods and Results: SCID mice underwent left anterior descending artery ligation and were divid-ed into 4 treatment arms: 1) normal saline control (n=14), 2) uAMCs (n=10), 3) c+AMCs (n=13), and 4) MiPSCs (n=11). Cardiac MRI assessed myocardial viability and left ventricular (LV) func-tion while BLI assessed stem cell engraftment over a four-week period. Immunohistological label-ing and RT-PCR of the explanted myocardium were performed. The uAMC and c+AMC treated mice demonstrated transient LV functional improvement. However, the MiPSCs exhibited a signifi-cantly greater increase in LV function compared to all the other groups during the entire four-week period. LV functional improvement correlated with increased myocardial viability and sustained stem cell engraftment. The MiPSCs treated animals lacked any evidence of de novo cardiac differ-entiation. Conclusions: The functional restoration seen in MiPSCs was characterized by increased myocardial viability and sustained engraftment without de novo cardiac differentiation, indicating salvage of the injured myocardium.

    View details for DOI 10.1161/CIRCRESAHA.116.304668

    View details for PubMedID 25654979

  • Direct Evaluation of Myocardial Viability and Stem Cell Engraftment Demonstrates Salvage of the Injured Myocardium CIRCULATION RESEARCH Kim, P. J., Mahmoudi, M., Ge, X., Matsuura, Y., Toma, I., Metzler, S., Kooreman, N. G., Ramunas, J., Holbrook, C., McConnell, M. V., Blau, H., Harnish, P., Rulifson, E., Yang, P. C. 2015; 116 (7): E40-?
  • Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice. PloS one Raval, A., Behbehani, G. K., Nguyen, L. X., Thomas, D., Kusler, B., Garbuzov, A., Ramunas, J., Holbrook, C., Park, C. Y., Blau, H., Nolan, G. P., Artandi, S. E., Mitchell, B. S. 2015; 10 (7)

    Abstract

    Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

    View details for DOI 10.1371/journal.pone.0131722

    View details for PubMedID 26133370

  • High-resolution, serial intravital microscopic imaging of nanoparticle delivery and targeting in a small animal tumor model NANO TODAY Smith, B. R., Zavaleta, C., Rosenberg, J., Tong, R., Ramunas, J., Liu, Z., Dai, H., Gambhir, S. S. 2013; 8 (2): 126-137
  • Toward an Electrolytic Micropump Actuator Design with Controlled Cyclic Bubble Growth and Recombination SENSORS, ACTUATORS, AND MICROSYSTEMS (GENERAL) - 219TH ECS MEETING Hsu, L., Ramunas, J., Gonzalez, J., Santiago, J. G., STRICKLAND, D. G. 2011; 35 (30): 3-11

    View details for DOI 10.1149/1.3653918

    View details for Web of Science ID 000300930300001

  • A self-priming, roller-free, miniature, peristaltic pump operable with a single, reciprocating actuator SENSORS AND ACTUATORS A-PHYSICAL Shkolnikov, V., Ramunas, J., Santiago, J. G. 2010; 160 (1-2): 141-146
  • Design and analysis of a long-term live-cell imaging chamber for tracking cellular dynamics within cultured human islets of Langerhans BIOTECHNOLOGY AND BIOENGINEERING Moogk, D., Hanley, S., Ramunas, J., Blaylock, A., Skorepova, J., Rosenberg, L., Jervis, E. 2007; 97 (5): 1138-1147

    Abstract

    A means of expanding islet cell mass is urgently needed to supplement the limited availability of donor islets of Langerhans for transplant. Live cell imaging of human islets in culture has the potential to identify the specific cells and processes involved in islet expansion. A novel imaging chamber was developed to facilitate long-term three-dimensional imaging of human islets during transformation. Islets have been induced to transform into duct-like epithelial cystic structures and revert back to glucose responsive endocrine cells under appropriate conditions (Jamal et al. Cell Death Differ. 2005 12:702-712). Here we aim to further our understanding by characterizing the process at a single cell level over time-essentially constructing a high resolution recorded history of each cell and its progeny during transformation and reversion. The imaging chamber enables high resolution imaging of three-dimensional islets while maintaining the structure of the islet cells and intercellular matrix components. A mathematical model was developed to validate the imaging chamber design by determining the required chamber dimensions to avoid introduction of oxygen and nutrient transport limitations. Human islets were embedded in collagen in the imaging chamber and differential interference contrast time course images were obtained at 3 min intervals. Immunofluorescent imaging confirmed that islet phenotype was maintained for at least 5 days during imaging. Analysis of the time courses confirms our ability to identify and track individual cells over time and to observe cell death and phenotype transformation in isolated human islets.

    View details for DOI 10.1002/bit.21335

    View details for Web of Science ID 000247960300014

    View details for PubMedID 17243145

  • Real-time fluorescence tracking of dynamic transgene variegation in stem cells MOLECULAR THERAPY Ramunas, J., Montgomery, H. J., Kelly, L., Sukonnik, T., Ellis, J., Jervis, E. J. 2007; 15 (4): 810-817

    View details for DOI 10.1038/mt.sj.6300073

    View details for Web of Science ID 000245215400022

    View details for PubMedID 17285140

  • True monolayer cell culture in a confined 3D microenvironment enables lineage informatics. Cytometry. Part A : the journal of the International Society for Analytical Cytology Ramunas, J., Illman, M., Kam, A., Farn, K., Kelly, L., Morshead, C. M., Jervis, E. J. 2006; 69 (12): 1202-1211

    Abstract

    There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo-like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates "lineage informatics," a lineage-centric approach to cytomics.We cultured cells in a confined monolayer using a novel "gap chamber" that produces images with confocal-like qualities using standard DIC microscopy. Lineage and other cytometric data were semiautomatically extracted from image sets of neural stem and progenitor cells and analyzed using lineage informatics.Cells imaged in the chamber every 3 min could be tracked for at least 6 generations allowing for the construction of extensive lineage trees with multiparameter data sets at hundreds of time points for each cell. The lineage informatics approach reveals relationships between lineage, phenotype, and microenvironment. Mass transfer characteristics and 3D geometry make the chamber more in vivo-like than traditional culture systems.The gap chamber allows cells to be cultured, imaged, and tracked in true monolayers permitting detailed informatics analysis of cell lineage, phenotype, and fate determinants. The chamber is biomimetic and straightforward to build and use, and should find many applications in long-term cell imaging.

    View details for PubMedID 17066473

  • High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dykstra, B., Ramunas, J., Kent, D., McCaffrey, L., Szumsky, E., Kelly, L., Farn, K., Blaylock, A., Eaves, C., Jervis, E. 2006; 103 (21): 8185-8190

    Abstract

    To search for new indicators of self-renewing hematopoietic stem cells (HSCs), highly purified populations were isolated from adult mouse marrow, micromanipulated into a specially designed microscopic array, and cultured for 4 days in 300 ng/ml Steel factor, 20 ng/ml IL-11, and 1 ng/ml flt3-ligand. During this period, each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (>1% contribution to lymphoid and myeloid lineages). In a first experiment, 6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity, demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions.

    View details for DOI 10.1073/pnas.0602548103

    View details for Web of Science ID 000237853900045

    View details for PubMedID 16702542

  • Probabilistic model-based cell tracking. International journal of biomedical imaging Kachouie, N. N., Fieguth, P., Ramunas, J., Jervis, E. 2006; 2006: 12186-?

    Abstract

    The study of cell behavior is of crucial importance in drug and disease research. The fields of bioinformatics and biotechnology rely on the collection, processing, and analysis of huge numbers of biocellular images, including cell features such as cell size, shape, and motility. However manual methods of inferring these values are so onerous that automated methods of cell tracking and segmentation are in high demand. In this paper, a novel model-based cell tracker is designed to locate and track individual cells. The proposed cell tracker has been successfully applied to track hematopoietic stem cells (HSCs) based on identified cell locations and probabilistic data association.

    View details for DOI 10.1155/IJBI/2006/12186

    View details for PubMedID 23165016

  • Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro JOURNAL OF CELL BIOLOGY Karpowicz, P., Morshead, C., Kam, A., Jervis, E., Ramuns, J., Cheng, V., van der Kooy, D. 2005; 170 (5): 721-732

    Abstract

    The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

    View details for DOI 10.1083/jcb.200502073

    View details for Web of Science ID 000231510300007

    View details for PubMedID 16115957

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