RESEARCH

Projects I’ve worked on are listed chronologically below. Related publications, patents, or resources are outlined for each project, along with links to other media, such as interviews or videos. ‘Show Abstract’ expands a technical summary.

 
Lung Cancer & Elevation: Evidence for Oxygen as an Inhaled Carcinogen – Independent Research – {2013–15}
Show Abstract

The atmospheric concentration of oxygen, a driver of free radical damage and tumorigenesis, decreases sharply with rising elevation. To understand whether ambient oxygen concentrations play a role in human carcinogenesis, we characterized age-adjusted cancer incidence (compiled by the National Cancer Institute from 2005–2009) across counties of the elevation-varying Western United States and compared trends displayed by respiratory cancer (lung) and non-respiratory cancers (breast, colorectal, and prostate). To adjust for important demographic and cancer-risk factors, 8–12 covariates were considered for each cancer. We produced sensible regression models that captured known risks. Models demonstrated that elevation strongly, negatively associates with lung cancer incidence (p < 10−16), but not with incidence of non-respiratory cancers. For every 1000 meter rise in elevation, lung cancer incidence decreased by 7.23 [99% CI: 5.18–9.29] cases per 100,000 individuals, equivalent to 12.7% of the mean incidence, 56.8. As a predictor of lung cancer incidence, elevation was second only to smoking prevalence in terms of significance and effect size. Furthermore, no evidence of uncontrolled confounding or ecological fallacy was detected: the lung cancer association was robust to varying regression models, county stratification, and population subgrouping; additionally seven environmental correlates of elevation, such as exposure to sunlight and fine particulate matter, could not capture the association. Overall, our findings suggest the presence of an inhaled carcinogen inherently and inversely tied to elevation, offering epidemiological support for oxygen-driven tumorigenesis. Finally, highlighting the need to consider elevation in studies of lung cancer, we demonstrated that previously reported inverse lung cancer associations with radon and UVB became insignificant after accounting for elevation.

PublicationSimeonov KPHimmelstein DS. (2015Lung cancer incidence decreases with elevation: evidence for oxygen as an inhaled carcinogen PeerJ 2:e705. | pdf | preprint | peer reviews | code | data

Press: Author interview | SciShow video summary | EurekAlert! | more stuff…

Awards: Top 10 Cancer Biology Papers PeerJ | Feature article Jan. 2015 | Abramson Cancer Center Basic Research Prize

 
 

mRNA Reprogramming of Human Fibroblasts to Hepatocyte-Like Cells
Investigative Toxicology Group – {2011–14} – Genentech
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Direct lineage conversion or reprogramming by overexpression of defined transcription factors is a promising new method of deriving useful but rare cell types from readily available ones. While the method presents numerous advantages over induced pluripotent stem (iPS) cell approaches, a focus on murine conversions and a reliance on retroviral vectors limit potential human applications. Here we address these concerns by demonstrating direct conversion of human fibroblasts to hepatocyte-like cells via repeated transfection with synthetic modified mRNAs. Conversion was achieved with as little as three transcription factor mRNAs encoding HNF1A plus any two of the factors, FOXA1, FOXA3, or HNF4A in the presence of an optimized hepatic growth medium. We show that the absolute necessity of exogenous HNF1A mRNA delivery is explained both by the factor’s inability to be activated by any other factors screened and its simultaneous ability to strongly induce expression of other master hepatic transcription factors. Further analysis of factor interaction showed that a series of robust cross-activations exist between factors that induce a hepatocyte-like state. Transcriptome and small RNA sequencing during conversion toward hepatocyte-like cells revealed global preferential activation of liver genes and miRNAs over those associated with other endodermal tissues, as well as downregulation of fibroblast-associated genes. Induced hepatocyte-like cells also exhibited hepatic morphology and protein expression. Our data provide insight into the process by which direct hepatic reprogramming occurs in human cells. More importantly, by demonstrating that it is possible to achieve direct reprogramming without the use of retroviral gene delivery, our results supply a crucial step toward realizing the potential of direct reprogramming in regenerative medicine.

PublicationSimeonov KP, Uppal H (2014) Direct Reprogramming of Human Fibroblasts to Hepatocyte-like Cells by Synthetic Modified mRNAs. PLoS ONE 9(6): e100134. | pdf

Patents: Simeonov KP, Uppal H. Methods and Compositions for Producing Induced Hepatocytes. | pdf

EP 2892999 A1, 15-July-2015 | EPO
CN 104781393 A, 15-July-2015 | SIPO
US 20140087416 A1, 27-March-2014 | USPTO
CA 2883714 A1, 13-March-2014 | CIPO
WO 2014039768 A1, 13-March-2014 | WIPO

Protocols: DNA Template Construction | Modified In Vitro Transcription


Identifying Differentiation Hierarchies by Telomere Length Analysis

Clarke Lab – {2009–11} – Stanford Inst. for Stem Cell Biology & Regenerative Med.
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Identification of adult stem cells is a vital but difficult aspect of stem cell research and regenerative medicine. Due to natural telomerase expression, stem cells are likely to have telomeres markedly longer than their more differentiated counterparts. Unfortunately at this point no method for quick, precise, accurate, and small-scale telomere length measurement exists to add a genetic component to the current arsenal of gene and protein expression analysis used to identify stem cells. To show that we could accurately measure telomere length in adult tissues, qPCR telomere length analysis was applied to human bone marrow and breast tissue demonstrating that in these well-characterized systems, the more immature cells had longer telomeres. Using as a little as 1000 cells sorted by FACS, we used qPCR analysis in the relatively uncharacterized colonic epithelium to show that stem and immature colon cells display longer telomeres than the total colonic epithelium. Applying the telomere length assay to colon cancer showed that tumorigenic colon cancer cells have longer telomeres than the non-tumorigenic bulk of the tumor, as predicted by cancer stem cell theory. Finally, we corroborated our results for normal human colon in mice, by miniaturizing the assay and detecting a difference in telomere length of immature and mature normal mouse colonocytes at an unprecedented single-cell level.

Honors Thesis: Simeonov KP (2011) Establishing a Hierarchy of Differentiation in Normal and Malignant Colonic Epithelium using Telomere Length. Stanford Thesis Symposium. Stanford, CA. | pdf

Funding: Stanford Bio-X Fellowship 2010


Nanoscale Surface Morphology of Al2O3 after High Temperature Annealing

Lederman Lab – {2006–09} – West Virginia University Dept. of Physics
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The surface structure of a-plane (1120) Al2O3 single crystals with a inclined direction was studied as a function of annealing temperature using atomic force microscopy. The samples had a large increase in step height and terrace width with increasing annealing temperature. For the samples annealed at 1500 °C and 1600 °C the step edge structure became significantly faceted. The facets consisted of (0001) and (1012) planes as a result of these two planes being the ones with the lowest surface energies in Al2O3. In addition, the surface morphology displayed the formation of ridges 0.3 nm high and ∼20 nm wide at 1600 °C which originate from a tendency of the surface to form a (4 × 1) reconstruction with the sharp step edges acting as reconstruction nucleation sites.

Publication: Simeonov KP, Lederman D (2009) Surface Structure of (11-20) Al2O3 Single Crystals After High Temperature Annealing. Surface Science 603(1): 232-236. | pdf

Funding: WV Nano Fellowships 2007, 2008


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