Programming and reprogramming neural cell types using synthetic transcription factors

Production of large numbers of desirable human cell types in the laboratory is one of the major goals of stem cell research. Current experimental approaches have focused on the strategy of recapitulating the events of normal embryogenesis in culture, by treating cells – either tissue stem cells or pluripotent stem cells (iPS/ES cells) – with cocktails of growth factors, matrix proteins or pharmacological agents. This is challenging and often requires weeks or months of elaborate cell culture regimes. An alternative approach is the forced expression of master regulatory transcription factors; this can bypass developmental programs and drive conversion to the target cell type. Each of these strategies is inefficient and unreliable. Recently a new opportunity has arisen to exploit synthetic transcription factors (sTFs) to program and reprogram cell fate. To create such sTFs the CRISPR/Cas9 system is repurposed through tethering of catalytically dead Cas9 to various transcriptional regulatory effector domains (e.g. VP16, KRAB). In this thesis, we have explored sTFs as tools to reset transcriptional regulatory networks in neural stem cells and mouse embryonic fibroblasts. We tested transcriptional activation of key neural lineage target genes (e.g Olig2, Sox10 and Nkx6.2). We designed and validated a series of sTFs that could effectively activity these. We have found that activation of Sox10 by dCas9-VP160 in mouse neural stem cells can increase the amount of arising oligodendrocyte and oligodendrocyte precursors cells during the differentiation. The activity of sTFs strongly depends on cellular context: i.e. a specific sTF might work well in one cell type but not another. Importantly, these biological barriers are not easily overcome by increasing the strength of the sTF – either through levels or types of effector domains used. Our data inspecting single cells suggests that multiplex delivery of sTFs can indeed cooperate by both increasing the number of cells that activated the gene of interest and increasing the level of transcriptional activation in a given cell. To fully exploit these new technologies, we therefore developed a new construction pipeline that allows easy and efficient assembly of multiple sTFs. Using this approach, we were able to successfully activate three different target genes from a single expression plasmid (Olig2, Sox10 and Nkx6.2) in fibroblasts. These sTFs we able to force fibroblast transdifferentiation towards oligodendrocyte lineage. Future studies will explore further how to exploit these sTFs to augment or replace current reprograming strategies

Biomarkers to identify and isolate senescent cells.

Aging is the main risk factor for many degenerative diseases and declining health. Senescent cells are part of the underlying mechanism for time-dependent tissue dysfunction. These cells can negatively affect neighbouring cells through an altered secretory phenotype: the senescence-associated secretory phenotype (SASP). The SASP induces senescence in healthy cells, promotes tumour formation and progression, and contributes to other age-related diseases such as atherosclerosis, immune-senescence and neurodegeneration. Removal of senescent cells was recently demonstrated to delay age-related degeneration and extend lifespan. To better understand cell aging and to reap the benefits of senescent cell removal, it is necessary to have a reliable biomarker to identify these cells. Following an introduction to cellular senescence, we discuss several classes of biomarkers in the context of their utility in identifying and/or removing senescent cells from tissues. Although senescence can be induced by a variety of stimuli, senescent cells share some characteristics that enable their identification both in vitro and in vivo. Nevertheless, it may prove difficult to identify a single biomarker capable of distinguishing senescence in all cell types. Therefore, this will not be a comprehensive review of all senescence biomarkers but rather an outlook on technologies and markers that are most suitable to identify and isolate senescent cells

Biomarkers to identify and isolate senescent cells

This paper was accepted for publication in the journal Ageing Research Reviews and the definitive published version is available at http://dx.doi.org/10.1016/j.arr.2016.05.003.Aging is the main risk factor for many degenerative diseases and declining health. Senescent cells are part of the underlying mechanism for time-dependent tissue dysfunction. These cells can negatively affect neighbouring cells through an altered secretory phenotype: the senescence-associated secretory phenotype (SASP). The SASP induces senescence in healthy cells, promotes tumour formation and progression, and contributes to other age-related diseases such as atherosclerosis, immune-senescence and neurodegeneration. Removal of senescent cells was recently demonstrated to delay age-related degeneration and extend lifespan. To better understand cell aging and to reap the benefits of senescent cell removal, it is necessary to have a reliable biomarker to identify these cells. Following an introduction to cellular senescence, we discuss several classes of biomarkers in the context of their utility in identifying and/or removing senescent cells from tissues. Although senescence can be induced by a variety of stimuli, senescent cells share some characteristics that enable their identification both in vitro and in vivo. Nevertheless, it may prove difficult to identify a single biomarker capable of distinguishing senescence in all cell types. Therefore, this will not be a comprehensive review of all senescence biomarkers but rather an outlook on technologies and markers that are most suitable to identify and isolate senescent cells

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology