Browsing by Subject "Stem cell biology"

Now showing 1 - 12 of 12
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    Bottom-up Pediatric Sarcoma Modeling Using Genetic Engineering and Induced Pluripotent Stem Cell Technologies
    (2022-12) Becklin, Kelsie
    From the onset, induced pluripotent stem cell (iPSC) technology radically changed how we study human disease and it continues to improve our understanding of disease ontology. Continual advances in cell culture techniques, genome engineering, and the -omics fields have expanded the use of iPSCs into our daily disease modeling toolkit. Using iPSCs to model cancer is not especially new; in fact, the first iPSC-derived from cancer cells was around 2011. But, the dedifferentiation process is largely inhibited in solid tumor cells and these cancer-derived iPSCs only capture a single cancer cell genome, losing all heterogeneity. Sarcomas are rare, heterogenous tumors arising from the mesenchymal lineage. Many sarcomas, such as Osteosarcoma (OSA) and Ewing sarcoma (ES), have had limited therapeutic advancements since the advent of chemotherapy. To improve therapeutic outcomes for these patients it is becoming clear that we need to identify tumor-promoting molecular profiles and gain a better understanding of tumor evolution. To do this, we implemented the use of genetic engineering strategies in karyotypically normal iPSC to generate bottom-up models of OSA and ES. OSA is the most common pediatric cancer of the bone and is characterized by a complex genome, but few bona fide OSA-dependent mutations have been identified. To make our iPSC-derived iOSA model, we installed OSA-associated mutations in TP53 and RB1 using the CRISPR/Cas9 system to generate knockout iPSC. After differentiation into mesenchymal stromal cells (iMSC) and osteoblasts (iOB) we then used retrovirus to overexpress constitutively active cMYCT58A and/or hRASG12V. The mutated iMSC and iOB cells had differential proliferation rates, colony forming ability, and tumor formation potential in immunodeficient mice, as well as evidence of large karyotype level mutations similar to those seen in human OSA genomics. Additionally, tumors from the iOSA model had RNA-seq profiles resembling primary OSA. This model demonstrates that using iPSCs for cancer modeling in genomically complex cancers is possible and can illuminate how these cancers initiate and evolve. In addition to OSA, I used iPSCs to model ES, a translocation driven cancer, with a 9-fold higher incidence rate in children of European (EUR) ancestry compared to African (AFR). Using iPSC-derived from individuals spanning the polymorphic spectrum of ES diagnosis, I initiated expression of the ES driving alteration, a EWSR1-ETS translocation. Specifically, I used lentivirus to express the EWSR1-FLI1 fusion protein (EWS/FLI) in iPSC-derived neural crest cells (iNCC). Cells of increasing AFR ancestry had lower tolerance to EWS/FLI expression, a result in line with the aforementioned differences in incidence of ES seen in EUR and AFR children. To investigate the molecular basis of this observation, we used RNA-seq and CUT&TAG to determine the gene expression and global occupancy differentials across ancestries driven by EWS/FLI. Genetic loci that were both differentially expressed and bound were nominated as our ancestry-linked differential Ewing sarcoma response (ALDER) loci. To this end we have identified 80 ALDER loci containing established and novel EWS/FLI target genes for further analysis. This study demonstrates the feasibility and utility of ancestry-informed iPSC modeling to identify novel and potentially targetable pathways to treat ES. In this work we applied genetic engineering tools in iPSC to generate novel models of the gnomically complex OSA, and the gnomically quiet, translocation driven ES. Collectively, the models described here provide a baseline system to study how OSA and ES initiate and the early stages of cancer development.
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    Characterization and in vivo tracking of transplanted oligodendrocyte progenitor cells in the injured rat spinal cord
    (2014-12) Mahoney, Rebecca Ann
    new method to track cells in vivo utilizes proton (1H) magnetic resonance imaging (MRI) with 19fluorine MRI. Cells can be labeled ex vivo with the 19F reagent prior to transplantation and imaged with MRI to generate a signal detected by a custom made, dual-tone fluorine coil to show transplanted cell "hotspots" in the host tissue. Oligodendrocyte Progenitor Cells (OPCs) are an ideal cell population for cellular therapy in traumatic spinal cord injury due to their ability to remyelinate and support axons after injury. OPCs can be labeled with 19F at a similar efficiency as other cell types, confirmed by fluorescence microscopy and NMR spectroscopy. Potential real-time, in vivo cellular tracking of transplanted OPCs would allow for analysis of cell migration after transplantation, comparison of functional analysis with transplanted cell numbers and location at multiple time points for each animal, and quantification of transplanted cells. Cell characterization of transplanted iPSC derived OPCs in injured rat spinal cords has been optimized, and can be translated to 19F labeled cell transplants in the future. The FDA approved Cell Sense 19F reagent allows for direct translation to use in humans for cell visualization and tracking that is not possible in current clinical trials for cellular therapies.
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    Chromatin accessibility and its relationship to pluripotency and the induction of pluripotent stem cells
    (2015-01) Espland, Eric
    Chromatin within stem cells is dynamic and relaxed, allowing transcription and thus lineage specification to occur rapidly. To determine how this property can be used to enhance the generation of induced pluripotent stem cells (iPSC), I examined whether the expression of histone variants or peptidyl-prolyl isomerases (Ppiases) could increase the hyperdynamic, plastic nature of stem cell chromatin and thereby increase the efficiency and speed of reprogramming. I used molecular cloning to generate expression vectors containing the histone variants H3T and H2A.B. I used line-scanning microscopy to measure chromatin dynamics, with initial results suggesting that stem cells are more hyperdynamic in nature than differentiated cells. Although the research on the effect of histone variants and Ppiases on chromatin dynamics and reprogramming was not completed, another study showed that the expression of other histone variants does enhance reprogramming and may also induce an open chromatin structure. If this holds true for the histone variants studied here or Ppiase B (PpiB), this could further enhance the generation of iPSC and make future autologous engraftments of iPSC more feasible.
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    Epigenetic memory and lineage specific differentiation of myoblast derived induced pluripotent stem cells
    (2012-08) Mull, Jesse Lyle
    Induced pluripotent stem (iPS) cells, reprogrammed from somatic cells with defined factors such as Oct4, Sox2, cMyc and Klf4, hold the potential to produce unlimited numbers of autologous cells to treat and model a variety of muscular dystrophies. However, the derivation of myogenic precursors from iPS cells remains elusive, and current differentiation protocols rely on multi-stage fluorescent cell sorting or the use of transgenes. Reprogrammed somatic cells exhibit epigenetic memory in the form of DNA methylation patterns and gene expression profiles characteristic of their tissue of origin. Here we show that myoblast (Mb) derived iPS cells maintain low level expression of myogenic markers, including MyoD, providing evidence that myogenic genes are not fully silenced in MB-iPSCs during the reprogramming process. In addition, Mb-iPS cells display preferential myogenic differentiation in vitro and in vivo compared to fibroblast (Fb) derived iPS cells. Exploiting this epigenetic memory, we establish a simple method for the derivation of myogenic progenitor cells from iPS cells, a critical step towards efficient cell therapy of Duchenne muscular dystrophy (DMD).
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    Generation of induced pluripotent stem cells and mesenchymal stromal cells.
    (2011-12) Shanmugam, Aarathi
    Mesenchymal stromal cells (MSCs) are a population of mesoderm-derived cells that possess the ability to differentiate into bone, cartilage, and adipose tissue. They are important to understanding the developmental process of musculoskeletal tissue, and can be utilized for novel human cell therapies. Previous studies by our group and others have demonstrated development of MSCs from human embryonic stem cells (hESCs). Now, we have identified a population of potential mesenchymal precursor cells from adult bone marrow derived iPSC lines using CD73 as a selection marker. Sorting and culture of the hESC/iPSC-derived CD73-positive cells lead to development of MSCs capable of making bone, cartilage, and adipocytes. However the variations in differentiation methods have been found to strongly influence their mesenchyme induction and their ability to make bone, cartilage and adipose tissue. We compare the spin EB (embryoid body) versus stromal co-culture techniques to arrive at a MSC population in our studies. Additionally, these studies examined novel ways to derive iPSCs that could be used for derivation of MSCs and other cell populations. Induced pluripotent stem cells have previously been generated from human dermal fibroblast cells. However, the requirement for skin biopsies and the need to expand fibroblast cells for several passages in vitro make it a cumbersome source for generating patient-specific stem cells. Reprogramming from human blood cells represents a consistent method of establishing patient-specific iPSCs.
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    Improving the Differentiation of human pluripotent stem cells to beta-cells.
    (2012-04) Dalton, Joseph Patrick
    Human pluripotent stem cells (hPSCs) have the ability to differentiate into any cell type within the body, thus holding the potential for treating the beta-cell loss in type 1 diabetes through a cell replacement therapy. Though effective protocols have been produced for creating beta-cells from hPSCs, the efficiency of this process can be improved to yield more beta-cells. Here, we hypothesized that the addition of the hormones prolactin and human growth hormone to our established differentiation protocol will result in more beta-cells or beta-cell progenitors. This is based on work with isolated pancreatic islets, which showed that these hormones are able to stimulate proliferation of mature beta-cells and increase islet volume. And as much of the work with these hormones has been in rodent islets, we also present data showing that prolactin is able to stimulate cell division and islet growth in human islets. Any influences of the hormones were observed through gene expression analysis and a cell-death assay. This work will inform future work on creating beta-cells from hPSCs, and hopefully move the potential therapy closer to the clinic.
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    Investigation of intravenous administration of non-hematopoietic umbilical cord blood cells as a therapy for stroke
    (2013-05) Juliano, Mario
    Stroke is one of the leading causes of death in the United States. Limitations of currently available stroke treatment include a 3 hour time window for recombinant tissue plasminogen activator. Stem cells have become promising for the treatment of a variety of neurodegenerative diseases, including stroke. Therapies in this category include stimulation of endogenous neurogenesis, as well as the delivery of exogenous stem cells.This thesis focuses on the investigation of the intravenous delivery of a CD34 negative subset of umbilical cord blood stem cells as a therapy for stroke. Methods employed for this investigation include next generation sequencing technologies for gene expression profiling, 19-fluorine tracking of stem cells with MRI, and fluorescence biodistribution tracking.
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    Lineage specific reprogramming to blood using a cocktail of transcription factors
    (2010-12) Shaikh, Seema
    A single fertilized cell has the ability to develop into any cell depending on the various cues it responds to. This ability to differentiate into desired cell types can be made use of in the field of developmental biology for studying early embryonic development and for regenerative medicine. Previous work in the lab showed that mouse ES cells engineered with an inducible construct co-expressing the hematopoietic regulatory factors SCL, LMO2 and GATA2 give very efficient hematopoiesis. In monolayer differentiation where hematopoiesis does not occur because the majority of cells differentiate towards ectoderm, expression of these 3 factors diverted cells towards hematopoietic lineage. My work in the lab addressed two questions: Can we optimize the system to obtain hematopoietic progenitors instead of differentiated blood cells? and How does the SCL complex reprogram cells at the molecular level? I added a cocktail of cytokines to the serum free, growth factor free medium to obtain undifferentiated hematopoietic progenitors. A short pulse of induction was sufficient to obtain large number of CD41+, hemoglobin expressing, round semi adherent cells. This treatment gave rise to progenitors of myeloid, erythroid and megakaryocytic lineages proving the multipotent nature of the blood cells that differentiated to both hematopoietic progenitors and committed erythroid cells. To understand the reprogramming potential of the SCL complex at the transcriptional level I performed two RNA sequencing experiments. The first experiment evaluated the early changes and showed that the SCL complex up-regulated many important hematopoietic genes including SCL, LMO2, GATA2, Lyl1 and Gfi1 within 6 hours, but other genes (globins) required a longer period of induction. The second experiment evaluated cells 3 days after a reprogramming pulse. The data showed that the non-reprogrammed (CD41-) cells expressed hematopoietic genes, but at lower levels compared to the fully reprogrammed cells (CD41+) indicating that the cells were not completely reprogrammed. Surprisingly the CD41- cells had higher expression of endodermal genes indicating that the cell could have reverted endoderm, a nearby lineage during the chase period. The data also showed a decrease in the ectodermal genes in both CD41+ and CD41- cells suggesting that the triple construct may be stably erasing the ectodermal program even in non-reprogrammed cells. These results improved the efficiency of the system and shed light on the mechanism of the SCL-LMO2-GATA2 action in lineage specific reprogramming.
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    Loss of Oct4 expression during the development of murine embryoid bodies
    (2012-04) Sajini, Abdulrahim Abdulrahman M
    We describe the internal organization of murine embryoid bodies (EBs) in terms of the structures and cell types formed as Oct4 expression becomes progressively lost. This is done by making the EBs from iPS cells carrying an inducible and permanent Oct4 reporter (Oct4-MerCreMer;mTmG). When these EBs are treated with tamoxifen, the Oct4 expressing cells switch from a red to a green fluorescence color, and this is maintained thereafter by their progeny. We show that there is no specific pattern in which Oct4 is downregulated, rather it appears to be spatially random. The earliest cells to lose Oct4 expression are internal and stain positive for -fetoprotein (AFP) indicating that they are visceral endoderm. However, GATA4, characteristic of primitive endoderm, was found in Oct4-expressing cells at this stage. This indicates that the first formed visceral endoderm does not arise from primitive endoderm, a difference from normal embryonic development. Contrary to previous reports, our EBs did not form a layer of primitive endoderm, or visceral endoderm, around the outside. Markers of the early body axis, BRACHYURY (T) and FOXA2, behaved somewhat differently from each other. BRA, which marks the early mesoderm, node and notochord, arises in Oct4 expressing cells on days 3-4. FOXA2, which marks the floor plate of the neural tube and definitive endoderm, as well as the node and notochord, arises at the same time but mostly in cells that have already lost Oct4 expression. Although there is usually a concentration of T or FOXA2 cells in one region of the EB, the morphology is not predictable and there are also scattered cells expressing these markers. Several clumps of cardiomyocytes are visible by day 7 of EB development, and we show that the cells forming these clumps lose Oct4 expression between days 3 and 5. Overall, our results indicate that EBs recapitulate normal development quite well in terms of the tempo of events and the appearance of specific markers, but they do not resemble embryos in terms of their morphology and structure, which is contrary to the previous reports.
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    Oct4 lineage tracing of spermatogonial stem cells in the adult mouse testes
    (2013-12) Haider, Kerri Tana
    Spermatogenesis in mammals occurs in the testes to produce mature sperm for reproductive purposes. Spermatogonia are derived from primordial germ cells. Undifferentiated spermatogonia are thought to arise from a small population of cells arranged around the basal membrane of the seminiferous tubule, called spermatogonial stem cells (SSCs). SSCs divide either symmetrically or asymmetrically to maintain the stem cell pool and give rise to daughter cells. The identity of the spermatogenic stem cell remains unclear. Numerous researchers have attempted to isolate the SSC population but in most cases they also labeled progeny of the SSCs, so a definitive population of SSCs has yet to be isolated. Oct4 is a protein that is expressed in pluripotent cells and has been described as being expressed in undifferentiated SSCs. In adult male Oct4:CreER mTmG transgenic mice GFP expression indicating Oct4 driven tamoxifen inducible CreER recombination is observed only in the testes. Here we describe using this transgenic mouse to identify and lineage trace the progeny of Oct4:CreER expressing cells in the adult mouse testes using multiple tamoxifen pulse and chase experiments. In 4-day chase experiments single GFP expressing cells along the edge of the basal lamina are observed. Longer 10-day chase experiments show aligned spermatogonia expressing GFP in chains of 2, 4 and 8 cells linearly placed on the basal lamina of the tubule. Fallowing extended chase periods after tamoxifen administration GFP expressing cells are seen in all stages of spermatogenesis. Our results are consistent with the cells labeled immediately after tamoxifen addition being the spermatogenic stem cell.
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    The role of decellularized matrix in directing differentiation of pancreatic progenitor cells in pancreatic endocrine cell fate.
    (2011-11) Boyce, Emma Jean
    The direct differentiation of induced pluripotent stem cells derived from somatic cells and pancreatic progenitor cells could generate functional β-cells that secrete insulin, and are also glucose responsive. However, the cellular signals and interactions between the pancreatic epithelium and its surrounding mesenchyme that govern pancreatic specification and differentiation of endoderm into pancreatic progenitor cells and eventually mature beta cells are not fully understood. In this study, I examined various conditions that would direct the induced pluripotent stem cells (iPSCs) derived from a pdx1: GFP mouse, or normal pancreatic progenitor cells, to predominately β cell fate. It involves the “guided” differentiation of iPSCs to a pancreatic lineage and also the use of a decellularized matrix to induce differentiation into. A decellularized matrix may help mature β-cells since decellularization has been shown to remove all the organ’s cells while preserving the composition and biological activity of the extracellular matrix1. Thus, decelluarization has several advantages: it removes cells to avoid any immune response post-implantation and maintains the native environment and membrane components that provide cellular growth and maturation of β-cells. The results showed that the guided differentiation of iPSCs by activin A induced definitive endodermal and pancreatic progenitor cells. Moreover, the decellularized matrix increased exocrine and endocrine gene expression as compared to gelatin and fibronectin, and assisted survival and maturation of all pancreatic cell types. Hence, this novel approach would be useful to produce insulin-expressing β-cells that are also glucose responsive and generate surrogate cells for diabetes therapy.
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    Side population cells possess a stem cell nature: cytoprotection, proliferation, and role of functional recovery in the murine heart.
    (2011-02) Maher, Travis Joseph
    Stem and progenitor cell populations occupy a specialized niche and are consequently exposed to hypoxic and oxidative stresses. We have previously established that the multidrug resistance protein Abcg2 is the molecular determinant of the side population (SP) progenitor cell population. There is an increase in Abcg2 expression, as well as increase in SP cell number, following injury. Transcriptome analysis of the SP cells isolated from the injured adult murine heart reveals enhanced expression of cytoprotective transcripts. We have previously demonstrated that hypoxia-inducible factor 2α (HIF-2α) binds an evolutionary conserved HIF-2 response element (HRE) in the murine Abcg2 promoter and activates Abcg2 expression. Overexpression of Abcg2 results in an increased ability to consume hydrogen peroxide. Importantly, overexpression of Abcg2 also conferred a cell survival benefit following exposure to hydrogen peroxide. In this study we have demonstrated that cytoprotection is dependent upon Abcg2 functioning as an active transporter. In-vitro analysis revealed SP cells proliferate in a delayed-onset manner, and such proliferation was affected by exposure to oxidative stress. Cytoprotective ‘priming’ also served to increase SP cell long-term viability. These results indicate that Abcg2 has a critical role in cytoprotection and Abcg2-expressing cells have important potential as progenitors in the adult.