Transdifferentiation of fibroblasts to neural stem cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.The developmental process is carefully controlled by transcriptional and epigenetic changes that occur as a zygote transforms into an adult organism. This process can be reversed by the overexpression of transcription factors Oct4, Sox2, Klf4, and c-Myc, which reprogram a differentiated cell!s nucleus to one that is transcriptionally and epigenetically indistinguishable from an embryonic stem (ES) cell. However, it is still unclear if transcription factors can completely convert the nucleus of a differentiated cell into that of a distantly related somatic cell type with complete transcriptional and epigenetic reprogramming maintained in the absence of exogenous factor expression. To test this idea, we generated doxycyline (dox)-inducible vectors encoding neural stem cell-expressed factors. We found that stable, self-maintaining NSC-like cells could be induced under defined growth conditions. These cells were characterized in the absence of exogenous factor induction and were shown to be transcriptionally, epigenetically, and functionally similar to endogenous embryonic cortical NSCs. Additionally, a cellular system was created for reproducible generation of doxindependent iNSCs without additional factor transduction. Our results show that a transcriptionally and epigenetically reprogrammed somatic nucleus can be stabilized in vitro and provides a tool to study the mechanism of somatic cell conversion.by John P. Cassady.Ph.D

Direct Lineage Conversion of Adult Mouse Liver Cells and B Lymphocytes to Neural Stem Cells

SummaryOverexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors

Reprogramming Factor Stoichiometry Influences the Epigenetic State and Biological Properties of Induced Pluripotent Stem Cells

We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated “all-iPSC mice” by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating “all-iPSC mice” was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a “generic” epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.National Science Foundation (U.S.). Graduate Research Fellowship ProgramNational Institutes of Health (U.S.) (Grant 5-RO1-HDO45022)National Institutes of Health (U.S.) (Grant 5-R37-CA084198)National Institutes of Health (U.S.). (Grant 5-RO1-CA087869

First insights into the vertical habitat use of the whitespotted eagle ray Aetobatus narinari revealed by pop‐up satellite archival tags

The whitespotted eagle ray Aetobatus narinari is a tropical to warm‐temperate benthopelagic batoid that ranges widely throughout the western Atlantic Ocean. Despite conservation concerns for the species, its vertical habitat use and diving behaviour remain unknown. Patterns and drivers in the depth distribution of A. narinari were investigated at two separate locations, the western North Atlantic (Islands of Bermuda) and the eastern Gulf of Mexico (Sarasota, Florida, U.S.A.). Between 2010 and 2014, seven pop‐up satellite archival tags were attached to A. narinari using three methods: a through‐tail suture, an external tail‐band and through‐wing attachment. Retention time ranged from 0 to 180 days, with tags attached via the through‐tail method retained longest. Tagged rays spent the majority of time (82.85 ± 12.17% S.D.) within the upper 10 m of the water column and, with one exception, no rays travelled deeper than ~26 m. One Bermuda ray recorded a maximum depth of 50.5 m, suggesting that these animals make excursions off the fore‐reef slope of the Bermuda Platform. Individuals occupied deeper depths (7.42 ± 3.99 m S.D.) during the day versus night (4.90 ± 2.89 m S.D.), which may be explained by foraging and/or predator avoidance. Each individual experienced a significant difference in depth and temperature distributions over the diel cycle. There was evidence that mean hourly depth was best described by location and individual variation using a generalized additive mixed model approach. This is the first study to compare depth distributions of A. narinari from different locations and describe the thermal habitat for this species. Our study highlights the importance of region in describing A. narinari depth use, which may be relevant when developing management plans, whilst demonstrating that diel patterns appear to hold across individuals

An Analysis of the Legal, Social, and Political Issues Raised by Asbestos Litigation

This Special Project examines the most important issues of the asbestos problem and advocates a congressional solution (1) to relieve the courts of the thousands of present and potential asbestos cases, (2) to protect future claimants\u27 rights to adequate compensation, and (3) to provide for equitable participation by all responsible parties, which, in addition to asbestos manufacturers,include the federal government, insurance companies, and the tobacco industry. The first six parts of the Special Project examine the various issues of asbestos litigation: theories of liability in products liability suits against asbestos manufacturers, causation,defenses, statutory limitations on actions, collateral estoppel, and punitive damages. The Special Project then discusses in parts VIII,IX, and X the methods used by asbestos manufacturers to attempt to spread their liability through asserting insurer liability, the exclusive remedy of workers\u27 compensation, and indemnity and contribution from the United States. Finally, the Special Project evaluates and analyzes recent developments in the asbestos litigation area, including proposals for federal legislative compensation programs and business alternatives available to asbestos manufacturers facing enormous asbestos-related liabilities... This Special Project critically has examined the most important issues concerning the asbestos problem. It has considered the complex legal, legislative, and social questions that society must confront in order to resolve this predicament. Only swift action by Congress in the form of a fair and comprehensive compensation scheme for victims of asbestos-related disabilities will initiate a solution to this difficult and pervasive problem

Metastable Pluripotent States in NOD Mouse Derived ES Cells

Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the post-implantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the non-obese diabetic (NOD) mouse strain, which prior to this study was considered “non-permissive” for ES cell derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ES and iPS cells appear “metastable”, as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.National Institutes of Health (U.S.) (Grant RO1-HDO45022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869

The VASIMR[registered trademark] VF-200-1 ISS Experiment as a Laboratory for Astrophysics

The VASIMR[R] Flight Experiment (VF-200-1) will be tested in space aboard the International Space Station (ISS) in about four years. It will consist of two 100 kW parallel plasma engines with opposite magnetic dipoles, resulting in a near zero-torque magnetic system. Electrical energy will come from ISS at low power level, be stored in batteries and used to fire the engine at 200 kW. The VF-200-1 project will provide a unique opportunity on the ISS National Laboratory for astrophysicists and space physicists to study the dynamic evolution of an expanding and reconnecting plasma loop. Here, we review the status of the project and discuss our current plans for computational modeling and in situ observation of a dynamic plasma loop on an experimental platform in low-Earth orbit. The VF-200-1 project is still in the early stages of development and we welcome new collaborators

Genetic engineering of human ES and iPS cells using TALE nucleases

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022)Howard Hughes Medical Institut

Association between neighborhood need and spatial access to food stores and fast food restaurants in neighborhoods of Colonias

Objective To determine the extent to which neighborhood needs (socioeconomic deprivation and vehicle availability) are associated with two criteria of food environment access: 1) distance to the nearest food store and fast food restaurant and 2) coverage (number) of food stores and fast food restaurants within a specified network distance of neighborhood areas of colonias, using ground-truthed methods. Methods Data included locational points for 315 food stores and 204 fast food restaurants, and neighborhood characteristics from the 2000 U.S. Census for the 197 census block group (CBG) study area. Neighborhood deprivation and vehicle availability were calculated for each CBG. Minimum distance was determined by calculating network distance from the population-weighted center of each CBG to the nearest supercenter, supermarket, grocery, convenience store, dollar store, mass merchandiser, and fast food restaurant. Coverage was determined by calculating the number of each type of food store and fast food restaurant within a network distance of 1, 3, and 5 miles of each population-weighted CBG center. Neighborhood need and access were examined using Spearman ranked correlations, spatial autocorrelation, and multivariate regression models that adjusted for population density. Results Overall, neighborhoods had best access to convenience stores, fast food restaurants, and dollar stores. After adjusting for population density, residents in neighborhoods with increased deprivation had to travel a significantly greater distance to the nearest supercenter or supermarket, grocery store, mass merchandiser, dollar store, and pharmacy for food items. The results were quite different for association of need with the number of stores within 1 mile. Deprivation was only associated with fast food restaurants; greater deprivation was associated with fewer fast food restaurants within 1 mile. CBG with greater lack of vehicle availability had slightly better access to more supercenters or supermarkets, grocery stores, or fast food restaurants. Increasing deprivation was associated with decreasing numbers of grocery stores, mass merchandisers, dollar stores, and fast food restaurants within 3 miles. Conclusion It is important to understand not only the distance that people must travel to the nearest store to make a purchase, but also how many shopping opportunities they have in order to compare price, quality, and selection. Future research should examine how spatial access to the food environment influences the utilization of food stores and fast food restaurants, and the strategies used by low-income families to obtain food for the household