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Browsing by Author "Lee, Jiyoon"
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Item Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids(Elsevier, 2019-07-09) Tang, Pei-Ciao; Alex, Alpha L.; Nie, Jing; Lee, Jiyoon; Roth, Adam A.; Booth, Kevin T.; Koehler, Karl R.; Hashino, Eri; Nelson, Rick F.; Otolaryngology, IU School of MedicineMutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders.Item Generation of inner ear organoids with functional hair cells from human pluripotent stem cells(Nature Publishing group, 2017-06) Koehler, Karl R.; Nie, Jing; Longworth-Mills, Emma; Liu, Xiao-Ping; Lee, Jiyoon; Holt, Jeffrey R.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of MedicineHuman inner ear tissue derived from pluripotent stem cells could provide a powerful platform for drug discovery or a source of sound- or motion-sensing cells for patients with hearing loss or balance dysfunction. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and Wnt signaling to generate multiple otic vesicle–like structures from a single stem-cell aggregate. Over two months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR/Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system will be useful for elucidating mechanisms of human inner ear development and testing potential inner ear therapies.Item Hair follicle development in mouse pluripotent stem cell-derived skin organoids(Elsevier, 2018-01-02) Lee, Jiyoon; Boescke, Robert; Tang, Pei-Ciao; Hartman, Byron H.; Heller, Stefan; Koehler, Karl R.; Otolaryngology -- Head and Neck Surgery, School of MedicineThe mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures for use in basic research studies or in vitro drug testing. To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in a co-culture with stem cell-derived cells, combined with in vivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This in vitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases., Lee at el. describe a defined in vitro 3D culture system that generates skin organoids from mouse pluripotent stem cells. The skin organoids contain self-organized skin layers and skin appendages including hair follicles, sebaceous glands, and adipocytes.,Item Hair-bearing human skin generated entirely from pluripotent stem cells(Springer Nature, 2020-06) Lee, Jiyoon; Rabbani, Cyrus C.; Gao, Hongyu; Steinhart, Matthew R.; Woodruff, Benjamin M.; Pflum, Zachary E.; Kim, Alexander; Heller, Stefan; Liu, Yunlong; Shipchandler, Taha Z.; Koehler, Karl R.; Otolaryngology -- Head and Neck Surgery, School of MedicineThe skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.Item Mapping oto-pharyngeal development in a human inner ear organoid model(The Company of Biologists, 2023) Steinhart, Matthew R.; van der Valk, Wouter H.; Osorio, Daniel; Serdy, Sara A.; Zhang, Jingyuan; Nist-Lund, Carl; Kim, Jin; Moncada-Reid, Cynthia; Sun, Liang; Lee, Jiyoon; Koehler, Karl R.; Otolaryngology -- Head and Neck Surgery, School of MedicineInner ear development requires the coordination of cell types from distinct epithelial, mesenchymal and neuronal lineages. Although we have learned much from animal models, many details about human inner ear development remain elusive. We recently developed an in vitro model of human inner ear organogenesis using pluripotent stem cells in a 3D culture, fostering the growth of a sensorineural circuit, including hair cells and neurons. Despite previously characterizing some cell types, many remain undefined. This study aimed to chart the in vitro development timeline of the inner ear organoid to understand the mechanisms at play. Using single-cell RNA sequencing at ten stages during the first 36 days of differentiation, we tracked the evolution from pluripotency to various ear cell types after exposure to specific signaling modulators. Our findings showcase gene expression that influences differentiation, identifying a plethora of ectodermal and mesenchymal cell types. We also discern aspects of the organoid model consistent with in vivo development, while highlighting potential discrepancies. Our study establishes the Inner Ear Organoid Developmental Atlas (IODA), offering deeper insights into human biology and improving inner ear tissue differentiation.Item TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells(Informa UK (Taylor & Francis), 2014-11) Cardenas, Horacio; Vieth, Edyta; Lee, Jiyoon; Segar, Mathew; Liu, Yunlong; Nephew, Kenneth P.; Matei, Daniela; Department of Medicine, IU School of MedicineA key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.Item Tissue Transglutaminase Activates Cancer-Associated Fibroblasts and Contributes to Gemcitabine Resistance in Pancreatic Cancer(Elsevier, 2016-11) Lee, Jiyoon; Yakubov, Bakhtiyor; Ivan, Cristina; Jones, David R.; Caperell-Grant, Andrea; Fishel, Melissa; Cardenas, Horacio; Matei, Daniela; Department of Otolaryngology--Head & Neck Surgery, School of MedicineResistance to chemotherapy is a hallmark of pancreatic ductal adenocarcinoma (PDA) and has been partly attributed to the dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca(2+)-dependent enzyme, is secreted by PDA cells and cross-links proteins in the tumor microenvironment (TME) through acyl-transfer between glutamine and lysine residues, promoting PDA growth. The objective of the current study was to determine whether secreted TG2 by PDA cells alters the response of pancreatic tumors to gemcitabine. Orthotopic pancreatic xenografts and co-culture of PDA and stromal cells were employed to determine the mechanisms by which TG2 alters tumor-stroma interactions and response to gemcitabine. Analysis of the pancreatic The Cancer Genome Atlas (TCGA) database demonstrated that increased TG2 expression levels correlate with worse overall survival (hazard ratio=1.37). Stable TG2 knockdown in PDA cells led to decreased size of pancreatic xenografts and increased sensitivity to gemcitabine in vivo. However, TG2 downregulation did not increase cytotoxicity of gemcitabine in vitro. Additionally, multivessel density and gemcitabine uptake in pancreatic tumor tissue, as measured by mass spectrometry (MS-HPLC), were not significantly different in tumors expressing TG2 versus tumors in which TG2 was knocked down. Fibroblasts, stimulated by TG2 secreted by PDA cells, secrete laminin A1, which protects cancer cells from gemcitabine-induced cytotoxicity. In all, our results demonstrate that TG2 secreted in the pancreatic TME orchestrates the cross talk between cancer cells and stroma, impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition to increase the antitumor effects of gemcitabine in PDA.Item Tissue Transglutaminase Mediated Tumor-Stroma Interaction Promotes Pancreatic Cancer Progression.(AACR, 2015-10-01) Lee, Jiyoon; Condello, Salvatore; Yakubov, Bakhtiyor; Emerson, Robert; Caperell-Grant, Andrea; Hitomi, Kiyotaka; Xie, Jingwu; Matei, Daniela; Department of Biochemistry and Molecular Biology, IU School of MedicinePurpose: Aggressive pancreatic cancer is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. The objective of the study was to determine the functions of tissue transglutaminase (TG2), a Ca2+-dependent enzyme which crosslinks proteins through transamidation and is abundantly expressed by pancreatic cancer cells in the pancreatic stroma. Experimental Design: Orthotopic pancreatic xenografts and co-culture systems tested the mechanisms by which the enzyme modulates tumor-stroma interactions. Results: We show that TG2 secreted by cancer cells effectively molds the stroma by crosslinking collagen, which in turn activates fibroblasts and stimulates their proliferation. The stiff fibrotic stromal reaction conveys mechanical cues to cancer cells leading to activation of the YAP/TAZ transcription factors, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells led to decreased size of pancreatic xenografts. Conclusions: Taken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the crosstalk between cancer cells and stroma fundamentally impacting tumor growth. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression.Item Tumor-stroma interaction mediated by tissue transglutaminase in pancreatic cancer(2015-08) Lee, Jiyoon; Matei, Daniela Elena; Harrington, Maureen A.; Herbert, Brittney-Shea; Xie, JingwuPancreatic ductal adenocarcinoma (PDA) is a deadly disease due to early metastasis and resistance to chemotherapy. PDA is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme, is abundantly expressed in pancreatic cancer cells and crosslinks proteins through acyl-transfer transamidation between glutamine and lysine residues. The objective of the study was to determine the functions of TG2 in the pancreatic stroma. Orthotopic pancreatic xenografts and co-culture systems tested the mechanisms by which the enzyme modulates tumor-stroma interactions. We showed that TG2 secreted by cancer cells is enzymatically active and renders the stroma denser by crosslinking collagen, which in turn activates fibroblasts and stimulates their proliferation. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are transcription factors involved in mechanotransduction. The TG2-mediated fibrosis-rich, stiff microenvironment conveys mechanical cues to cancer cells leading to activation of YAP and TAZ, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells led to decreased size of pancreatic xenografts and increased sensitivity of xenografts to gemcitabine. Taken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the crosstalk between cancer cells and the stroma, fundamentally impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression.