Matching Items (83)
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May I Cut In? Gene Editing Approaches in Human Induced Pluripotent Stem Cells

Description

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies…

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.
ContributorsBrookhouser, Nicholas (Author) / Raman, Sreedevi (Author) / Potts, Chris (Author) / Brafman, David (Author) / Ira A. Fulton Schools of Engineering (Contributor)
Created2017-02-06
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Enhancing CXCR4 expression of hNPCs using Hyaluronic Acid - Laminin Hydrogel: A Potential Treatment for TBI

Description
Traumatic brain injury (TBI) may result in numerous pathologies that cannot currently be mitigated by clinical interventions. Stem cell therapies are widely researched to address TBI-related pathologies with limited success in pre-clinical models due to limitations in transplant survival rates. To address this issue, the use of tissue engineered scaffolds…

Traumatic brain injury (TBI) may result in numerous pathologies that cannot currently be mitigated by clinical interventions. Stem cell therapies are widely researched to address TBI-related pathologies with limited success in pre-clinical models due to limitations in transplant survival rates. To address this issue, the use of tissue engineered scaffolds as a delivery mechanism has been explored to improve survival and engraftment rates. Previous work with hyaluronic acid \u2014 laminin (HA-Lm) gels found high viability and engraftment rates of mouse fetal derived neural progenitor/stem cells (NPSCs) cultured on the gel. Furthermore, NPSCs exposed to the HA-Lm gels exhibit increased expression of CXCR4, a critical surface receptor that promotes cell migration. We hypothesized that culturing hNPCs on the HA-Lm gel would increase CXCR4 expression, and thus enhance their ability to migrate into sites of tissue damage. In order to test this hypothesis, we designed gel scaffolds with mechanical properties that were optimized to match that of the natural extracellular matrix. A live/dead assay showed that hNPCs preferred the gel with this optimized formulation, compared to a stiffer gel that was used in the CXCR4 expression experiment. We found that there may be increased CXCR4 expression of hNPCs plated on the HA-Lm gel after 24 hours, indicating that HA-Lm gels may provide a valuable scaffold to support viability and migration of hNPCs to the injury site. Future studies aimed at verifying increased CXCR4 expression of hNPCs cultured on HA-Lm gels are necessary to determine if HA-Lm gels can provide a beneficial scaffold for stem cell engraftment therapy for treating TBI.
ContributorsHemphill, Kathryn Elizabeth (Author) / Stabenfeldt, Sarah (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Large Scale Biomanufacturing of Human Induced Pluripotent Stem Cell-Derived Neurons

Description
Current culturing methods allow for human neural progenitor cells to be differentiated into neurons for use in diagnostic tools and disease modeling. An issue arises in the relatively low number of cells that can be successfully expanded and differentiated using these current methods, making the progress of research dependent on…

Current culturing methods allow for human neural progenitor cells to be differentiated into neurons for use in diagnostic tools and disease modeling. An issue arises in the relatively low number of cells that can be successfully expanded and differentiated using these current methods, making the progress of research dependent on these cultures as a large number of cells are needed to conduct relevant assays. This project focuses on the expansion and differentiation of human neural progenitor cells cultured on microcarriers and within a rotating bioreactor system as a way to increase the total number of cells generated. Additionally, cryopreservation and the characteristics of these neurons post thaw is being investigated to create a way for long term storage, as well as, a method for standardizing cell lines between multiple experiments at different time points. The experiments covered in this study are aimed to compare the characteristics of differentiated human neurons, both demented and non-demented cell lines between pre-cryopreservation, freshly differentiated neurons and post-cryopreservation neurons. The assays conducted include immunofluorescence, calcium imaging, quantitative polymerase chain reaction, flow cytometry and ELISA data looking at Alzheimer’s disease traits. With the data collected within this study, the use of bioreactors, in addition to, cryopreservation of human neurons for long term storage can be better implemented into human neural progenitor cell research. Both of these aspects will increase the output of these cultures and potentially remove the bottleneck currently found within human neural disease modeling.
ContributorsHenson, Tanner Jay (Author) / Brafman, David (Thesis director) / Kodibagkar, Vikram (Committee member) / School of Life Sciences (Contributor) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05