Matching Items (3)
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- All Subjects: human pluripotent stem cells
- Creators: Wang, Xiao
- Creators: Muller, Ryan
Description
Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Neurodegenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis are marked by the loss of different types of neurons and glial cells in the central nervous system (CNS). Human Pluripotent Stem Cell (hPSC)-derived Neural Progenitor Cells (hNPCs) have the ability to self-renew indefinitely and to differentiate into various cell types of the CNS. HNPCs can be used in cell based therapies and have the potential to reverse or arrest neurodegeneration and to replace lost neurons and glial cells. However, the lack of completely defined, scalable systems to culture these cells, limits their therapeutic and clinical applications. In a previous study, a completely defined, robust, synthetic peptide- a Vitronectin Derived Peptide (VDP) that supports the long term expansion and differentiation of various embryonic and induced pluripotent stem cell (hESC/hIPSC) derived hNPC lines on two dimensional (2D) tissue culture plates was identified. In this study, the culture of hNPCs was scaled up using VDP coated microcarriers (MC). VDP MC were able to support the long term expansion of hESC and hiPSC derived hNPCs over multiple passages and supported higher fold changes in cell densities, compared to VDP coated 2D surfaces. VDP MC also showed the ability to support the neuronal differentiation of hNPCs, and produced mature neurons expressing several neuronal, neurotransmitter and cortical markers. Additionally, alzheimer’s disease (AD) relevant phenotypes were studied in patient hIPSC derived hNPCs cultured on laminin MC to assess if the MC culture system could be used for disease modelling and drug screening. Finally, a microcarrier based bioreactor system was developed for the large scale expansion of hNPCs, exhibiting more than a five-fold change in cell density and supporting more than 100 million hNPCs in culture. Thus, the development of a xeno-free, scalable system allows hNPC culture under standard and reproducible conditions in quantities required for therapeutic and clinical applications.
ContributorsRajaram Srinivasan, Gayathri (Author) / Brafman, David (Thesis advisor) / Wang, Xiao (Committee member) / Haynes, Karmella (Committee member) / Arizona State University (Publisher)
Created2017
Description
The advent of CRISPR/Cas9 revolutionized the field of genetic engineering and gave rise to the development of new gene editing tools including prime editing. Prime editing is a versatile gene editing method that mediates precise insertions and deletions and can perform all 12 types of point mutations. In turn, prime editing represents great promise in the design of new gene therapies and disease models where editing was previously not possible using current gene editing techniques. Despite advancements in genome modification technologies, parallel enrichment strategies of edited cells remain lagging behind in development. To this end, this project aimed to enhance prime editing using transient reporter for editing enrichment (TREE) technology to develop a method for the rapid generation of clonal isogenic cell lines for disease modeling. TREE uses an engineered BFP variant that upon a C-to-T conversion will convert to GFP after target modification. Using flow cytometry, this BFP-to-GFP conversion assay enables the isolation of edited cell populations via a fluorescent reporter of editing. Prime induced nucleotide engineering using a transient reporter for editing enrichment (PINE-TREE), pairs prime editing with TREE technology to efficiently enrich for prime edited cells. This investigation revealed PINE-TREE as an efficient editing and enrichment method compared to a conventional reporter of transfection (RoT) enrichment strategy. Here, PINE-TREE exhibited a significant increase in editing efficiencies of single nucleotide conversions, small insertions, and small deletions in multiple human cell types. Additionally, PINE-TREE demonstrated improved clonal cell editing efficiency in human induced pluripotent stem cells (hiPSCs). Most notably, PINE-TREE efficiently generated clonal isogenic hiPSCs harboring a mutation in the APOE gene for in vitro modeling of Alzheimer’s Disease. Collectively, results gathered from this study exhibited PINE-TREE as a valuable new tool in genetic engineering to accelerate the generation of clonal isogenic cell lines for applications in developmental biology, disease modeling, and drug screening.
ContributorsKostes, William Warner (Author) / Brafman, David (Thesis advisor) / Jacobs, Bertram (Committee member) / Lapinaite, Audrone (Committee member) / Tian, Xiaojun (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2022