Browsing by Subject "iPSC"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    Automation of Stem Cell Protocols
    (2019-12) Johnson, Blake
    Human induced pluripotent stem cells (hiPSCs) have become a vital resource for researchers and industry due to their differentiation capacity, as well as providing access to the cell phenotypes and genotypes from any individual donor. Despite improvements in stem cell technology, maintaining iPS cell lines still requires a significant amount of time and technical skill from cell culture technicians. Such steps include consistent media changes, cell counting and confluence analyses, cell passaging, cryopreservation, and subsequent thawing and plating of those cells. For this research, these processes have been transitioned onto an automated cell culturing platform. It is shown here that the automated cell culturing platform is able to properly execute DMSO-free cryopreservation, thawing, plating, and cell maintenance. This demonstrated ability to perform these functions completely automated without a technician is a technical advancement in pluripotent stem cell culturing and may provide financial benefits within a cell culture laboratory.
  • Thumbnail Image
    Item
    Differentiation of Human Induced Pluripotent Stem Cells to OLIG2 Positive Ventral Neural Tube Progenitors
    (2020-12) Ravichandran Damodaran, Jeyaram
    Established protocols to generate OPCs from pluripotent stem cells still have several critical drawbacks including being cumbersome, time consuming, and incompatible for autologous stem cell therapies, that have impeded the translation of stem cell derived OPCs in cell transplantation therapies. The 3D culture system employed in these protocols introduces many undefined variables modulating growth factor response by cells. These include variability in culture environment among cells in a batch, differences in cell number and cell density batch to batch, and site to site variations, all of which impact the identity, number and purity of the cells being generated. By constraining to a 2D monolayer system and systematically experimenting with the growth factors and inhibitors currently used to induce OPC differentiation, a shorter, adherent protocol for generating iPSC-derived OLIG2+ ventral neural tube progenitors via an intermediate neuromesodermal progenitor has been developed in the Dutton laboratory.
  • Thumbnail Image
    Item
    Differentiation of human induced pluripotent stem cells to oligodendrocyte progenitor cells
    (2013-01) Subramaniam, Sandhya
    The similarity between induced Pluripotent Stem Cells (iPSCs) and Embryonic Stem (ES) cells motivated the use of the Keirstead protocol in the differentiation of iPSCs to Oligodendrocyte Progenitor Cells (OPCs). The overall concept of the protocol was successful in differentiating the iPSCs to OPCs with modification at each stage to better suit the survival of the aggregates made from iPSCs. The OPCs generated from the iPSCs were primarily confirmed using immunostaining for Olig2 and NG2. The OPCs produced using this protocol, were expanded on matrigel in Glial Restrictive Medium (GRM) supplemented with Epidermal Growth Factor (EGF) and passaged for further expansion.
  • Thumbnail Image
    Item
    MODELING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY USING PRIMARY AND PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS
    (2021-01) Goloviznina, Natalya
    Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant degenerative muscle disease with no cure or treatment. The genetic cause of FSHD is the reduction of the copy number of subtelomeric D4Z4 repeats at 4q35 encoding Double Homeobox 4 (DUX4) protein, which is a potent transcription factor that is toxic to the cell. Contractions lead to loss of repeat-induced silencing, allowing for transcription of DUX4. A common diagnostic method for FSHD shows demethylation of D4Z4 using gDNA of blood cells, and Kyba lab has shown that D4Z4 is demethylated in FSHD iPS cells, meaning that any cell type in an FSHD patient could be subject to deregulation by DUX4. DUX4 recruits p300 to induce global changes in histone acetylation, massively disrupting gene expression, thus pathology is not obviously exclusive to myogenic cells. Why the disease is muscle-specific and which cells in muscle may be deregulated by DUX4 are unknowns. Here are examined non-myogenic, muscle-associated cells derived from primary and induced pluripotent stem cells (iPSC) sources for evidence of DUX4 and target gene activation. To better visualize DUX4 expressing cells in vitro and in situ, these studies outline a visualization tool which uses signal amplification combined with a newly developed commercial antibody in both mouse and human tissue. Using this method, DUX4 positive cells have been identified in the human biopsy for the first time, providing a critical and novel insight into the cell types involved in FSHD pathogenesis. In a further attempt to model rare, muscle-associated populations in vitro, a novel fibroadipogenic progenitor (FAP) cell isolation method was developed, which is then used to obtain FAPs from patient biopsies. Using this new isolation method, FAPs were isolated from FSHD patients, and analysis of this rare cell population revealed that FSHD FAPs did not express DUX4 and its target genes, and that patient FAPs remained functionally normal if derived from intact muscle. In addition to developing new methods for detecting rare cell populations in patient muscle, these studies use iPSC modeling to examine DUX4 in other rare cell types which are not accessible in standard patient biopsies. Using this in vitro modeling system this study replicated the myogenic and cardiac phenotypes seen in the patient and examined other rare and difficult-to-access cell types like mesenchymal stem/stromal cells (MSCs) and motor neurons to analyze DUX4 involvement in these cell types. Both MSCs and motor neurons showed evidence of DUX4 target gene expression at the terminal cell type. However, unexpectedly, robust DUX4 expression was seen during early neurogenesis with long-term, significant target gene response persisting through the motor neuron progenitor stage before it tapered off in the mature motor neuron. Analysis of early MSC differentiation showed a similar pattern of target gene expression, though the stage of DUX4 expression was not identified. In aggregate, these data demonstrate that transient activation of DUX4 during differentiation is enough to induce a long-lasting effect that lasts into some terminally differentiated cells.
  • Thumbnail Image
    Item
    Transcriptional disruptions and functional correlates in a human induced pluripotent stem cell – derived motor neuron model of Spinocerebellar ataxia type 1
    (2022-08) Sheeler, Carrie
    It is hypothesized that degeneration of the motor neurons (MNs) in the brain stem and spinal cord contributes to premature lethality in spinocerebellar ataxia type 1 (SCA1) by affecting the strength of swallowing and respiratory drive. While we can recreate some aspects of MN pathology in mouse models, loss of MNs has only been seen in SCA1 patient populations. This, in addition to other species differences that exist between rodents and humans, accentuates the need for translational human models of disease which can be used to uncover therapeutically relevant cellular and molecular mechanisms underlying dysfunction in vulnerable cell types. To investigate potential mechanisms underlying SCA1 pathology in human MNs, I developed a human induced pluripotent stem cell (iPSC)-derived MN model of SCA1. This is, to our knowledge, the first cell-type specific iPSC-derived model made to study SCA1. Previous work in mouse models has demonstrated both that ATXN1 regulates cellular transcription (Ingram et al 2016, Rousseaux et al 2018, Friedrich et al 2019) and that its entry into the nuclei of cells is important for the longevity of mouse models of SCA1 (Handler et al 2022). Thus, I predicted that the presence of mutant ATXN1 in hiPSC-derived MNs would drive measurable transcriptomic changes in SCA1 samples compared to unaffected controls. Furthermore, transcriptomic disruptions might indicate functional pathways of interest for future studies into the lethality of SCA1. I performed bulk RNA sequencing on MN-enriched cultures to assess underlying transcriptional changes that may be affecting SCA1 human MNs, and subsequently identified disruptions in key cellular processes including extracellular matrix (ECM) regulation, calcium ion binding, and mitotic cell cycle regulation. Proper regulation of ECM is key to many aspects of neuronal development and function, including extension of neurites, proper placement and presentation of receptors and ion channels, cell-cell communication, synapse formation, and intracellular transport. As such, I predicted that broad changes in ECM regulation might result in measurable changes in some of these capabilities in SCA1 MN enriched cultures. Neurite outgrowth was measured in motor neuron progenitors (pMNs) and immature motor neurons (iMNs). I determined that SCA1 samples do not exhibit remarkable disruptions in neurite outgrowth at either timepoint. Assessment of spontaneous calcium activity exhibited a similar degree of physiological maturity between SCA1 and unaffected control MN enriched cultures. Additionally, measurement of miniature excitatory postsynaptic currents (mEPSCs) demonstrated formation of synapses but no functional differences in SCA1 samples compared to controls. However, glutamate evoked calcium activity demonstrated a reduced amplitude of calcium response in SCA1 cultures. This occurred despite any measurable transcriptomic changes in glutamate receptor expression. This indicates potential underlying disruptions in receptor activity and calcium dynamics in SCA1 MN-enriched cultures and provides a potential avenue of interest for future work investigating disruptions in communication in the SCA1 spinal cord.
  • Thumbnail Image
    Item
    Using CRISPR to Model MCT1 Deficiency in Pluripotent Stem Cells.
    (2024) Reutzel, Bryan
    Monocarboxylate transporter 1 (MCT1) plays a key role in transporting monocarboxylates such as lactate, pyruvate, and ketone bodies across the neurovascular unit/blood brain barrier (NVU/BBB). Human MCT1 is a highly conserved 500 amino acid protein embedded in the plasma membrane which contains twelve transmembrane segments with its amino and carboxyl termini in the cytoplasm (Figure). It acts as a facilitative carrier that transports a monocarboxylate and H + in equimolar amounts down a concentration gradient. Some carboxylate drugs may enter the brain via MCT1 and because of its role in metabolism, it has become the target for transport inhibitors (α-cyano-4-hydroxycinnamate, MD-1, AZD3965). Despite the important function of MCT1 in moving metabolically relevant substrates, there have been reports of mutations in the SLC16A1 gene, which codes for MCT1, and render the protein product dysfunctional. Patients with these mutations present with metabolic ketoacidosis that sometimes include seizures, vomiting, and mild to moderate developmental delay. There are currently no in vitro models of MCT1 deficiency in human cells. We hypothesized that creating a cell line of human stem cells in which the SLC16A1 gene is altered would be useful for characterizing the role of MCT1 in the metabolism of cells of the NVU. We therefore used CRISPR/Cas9 editing of human induced pluripotent stem cells (iPSCs). We were able to isolate a heterozygous KO cell line, and were further able to use the isolated line to attempt to create a full/homozygous knockout. The characterization of the resulting cell lines of this study will provide insight into how this pathology may affect the NVU, as well as provide a model system to further investigate this genetic disease.
  • Thumbnail Image
    Item
    Utilization of A 3D Culture System of Collagen-Mimic Peptide Gfoger-Based Hydrogel to Model Osteosarcoma from Engineered Ipsc
    (2021-05) Thueson, Hanna
    Modeling the early stages of human osteosarcoma development remains a significant challenge. Most existing human models are derived from patient tumor tissue which is used to establish tumor cell lines or xenograft models in immunodeficient mice. These models are largely derived from late or end stage disease and do not allow the study of the early events of transformation. Further, 2D cell lines are largely homogenous and do not replicate the heterogeneity of primary tumors. Xenografted models more closely replicate the primary tumor but can have low engraftment rates and are logistically challenging to maintain. The immunocompromised nature of xenografted mice limits the potential for immunotherapy studies. The work presented here establishes a 3D culture system to model early-stage osteosarcoma development from engineered human iPSC. When cultured as aggregates in a GFOGER (integrin-specific glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine) based hydrogel known to promote osteoblastic differentiation, osteoblasts engineered with osteosarcoma-associated mutations readily form 3D organoids. Histological analysis supports that 3D culture of iPSC-derived osteoblasts promotes a more tissue-like phenotype with increased mineralization and ECM development within the tissue construct. In addition, preliminary functional studies suggest that 3D culture promotes transformative properties and an osteosarcoma phenotype. This novel approach has potential for future applications in disease modeling, in vivo studies, and drug discovery.