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Item 3D mouse embryonic stem cell culture for generating inner ear organoids(Springer Nature, 2014) Koehler, Karl R.; Hashino, Eri; Otolaryngology -- Head and Neck Surgery, School of MedicineThis protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.Item Aberrant Adult Neurogenesis in the Subventricular Zone-Rostral Migratory Stream-Olfactory Bulb System Following Subchronic Manganese Exposure(Oxford University Press, 2016-04) Fu, Sherleen; Jiang, Wendy; Gao, Xiang; Zeng, Andrew; Cholger, Daniel; Cannon, Jason; Chen, Jinhui; Zheng, Wei; Department of Neurological Surgery, School of MedicineAdult neurogenesis occurs in brain subventricular zone (SVZ). Our recent data reveal an elevated proliferation of BrdU(+) cells in SVZ following subchronic manganese (Mn) exposure in rats. This study was designed to distinguish Mn effect on the critical stage of adult neurogenesis, ie, proliferation, migration, survival and differentiation from the SVZ via the rostral migratory stream to the olfactory bulb (OB). Adult rats received a single ip-dose of BrdU at the end of 4-week Mn exposure to label proliferating cells. Immunostaining and cell-counting showed a 48% increase of BrdU(+) cells in Mn-exposed SVZ than in controls (P< .05). These BrdU(+) cells were identified as a mixed population of mainly GFAP(+) type-B neural stem cells, Nestin(+) type-C transit progenitor cells, DCX(+) migratory neuroblasts and Iba1(+) microglial cells. Another group of adult rats received 3 daily ip-injections of BrdU followed by subchronic Mn exposure. By 4-week post BrdU labeling, most of the surviving BrdU(+) cells in the OB were differentiated into NeuN(+) matured neurons. However, survival rates of BrdU/NeuN/DAPI triple-labeled cells in OB were 33% and 64% in Mn-exposed and control animals, respectively (P< .01). Infusion of Cu directly into the lateral ventricle significantly decreased the cell proliferation in the SVZ. Taken together, these results suggest that Mn exposure initially enhances the cell proliferation in adult SVZ. In the OB, however, Mn exposure significantly reduces the surviving adult-born cells and markedly inhibits their differentiation into mature neurons, resulting in an overall decreased adult neurogenesis in the OB.Item Analysis of differentiation capacity of Cfp1 null embyronic stem cells(2014) Bowen, Tamara R.; Skalnik, David Gordon; Marrs, James; Chang, Hua-ChenEpigenetics is defined as “the study of stable, often heritable, changes that influence gene expression that are not mediated by DNA sequence” (Fingerman et al., 2013). Epigenetic marks such as covalent histone modifications and DNA methylation are important for maintaining chromatin structure and epigenetic inheritance. Several proteins have been found to bind and/ or regulate epigenetic marks. One such protein, CXXC finger protein 1 (Cfp1) is an important chromatin regulator that binds to unmethylated CpG islands. It has been found to be essential for mammalian development. Mice lacking Cfp1 exhibit an embryonic- lethal phenotype. However, the function of Cfp1 can be studied using Cfp1 Null mouse ES cells, which are viable. Thus far, Cfp1 has been shown to be important for cell growth, cytosine methylation, histone modifications, subnuclear localization of Set1A histone H3K4 methyltransferase, and cellular differentiation. When Cfp1 Null ES cells are induced to differentiate by removal of Leukemia Inhibitory Factor (LIF), the cells are not able to turn off pluripotency markers such as Oct4 and alkaline phosphatase and fail to express differentiation markers such as Gata4 and Brachyury. In this study, we used established protocols to further examine the differentiation capacity of Cfp1 Null cells. Specifically, we tested the ability of Cfp1 Null ES cells to retain stem cell properties in the absence of LIF, differentiate into cardiomyocytes in the presence of TGF-β2 and differentiate into neuron precursors in the presence of retinoic acid (RA). While the differentiation effects of RA were inconclusive, Null cells were able to start differentiating in the absence of LIF, either as individual cells or EBs, and the presence of TGF-β2 when seeded on gelatin coated tissue culture dishes. However, no difference was seen between cells treated without LIF and those treated with TGF-β2. In both conditions, only a small portion of cells were able to differentiate, while the majority of the cell population retained stem cell characteristics. Cell growth and the differentiation capacity of Cfp1 Null cells were also compromised in comparison to WT cells. Thus, further supporting the need for the correct epigenetic patterns maintained by Cfp1 during cellular differentiation.Item Assessment of electromechanically stimulated bone marrow stem cells seeded acellular cardiac patch in a rat myocardial infarct model(IOPP, 2021) Öztürk, Şükrü; Shahbazi, Reza; Zeybek, Naciye Dilara; Kurum, Barıs; Gultekinoglu, Merve; Aksoy, Eda Ayse; Demircin, Metin; Ulubayram, Kezban; Medicine, School of MedicineIn this study, we evaluated cardiomyogenic differentiation of electromechanically stimulated rat bone marrow-derived stem cells (rt-BMSCs) on an acellular bovine pericardium (aBP) and we looked at the functioning of this engineered patch in a rat myocardial infarct (MI) model. aBP was prepared using a detergent-based decellularization procedure followed by rt-BMSCs seeding, and electrical, mechanical, or electromechanical stimulations (3 millisecond pulses of 5 V cm-1at 1 Hz, 5% stretching) to enhance cardiomyogenic differentiation. Furthermore, the electromechanically stimulated patch was applied to the MI region over 3 weeks. After this period, the retrieved patch and infarct region were evaluated for the presence of calcification, inflammatory reaction (CD68), patch to host tissue cell migration, and structural sarcomere protein expressions. In conjunction with any sign of calcification, a higher number of BrdU-labelled cells, and a low level of CD68 positive cells were observed in the infarct region under electromechanically stimulated conditions compared with static conditions. More importantly, MHC, SAC, Troponin T, and N-cad positive cells were observed in both infarct region, and retrieved engineered patch after 3 weeks. In a clear alignment with other results, our developed acellular patch promoted the expression of cardiomyogenic differentiation factors under electromechanical stimulation. Our engineered patch showed a successful integration with the host tissue followed by the cell migration to the infarct region.Item Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap(Nature Research, 2018-11-23) Lee, Youngmin A.; Noon, Luke A.; Akat, Kemal M.; Ybanez, Maria D.; Lee, Ting-Fang; Berres, Marie-Luise; Fujiwara, Naoto; Goossens, Nicolas; Chou, Hsin-I; Parvin-Nejad, Fatemeh P.; Khambu, Bilon; Kramer, Elisabeth G.M.; Gordon, Ronald; Pfleger, Cathie; Germain, Doris; John, Gareth R.; Campbell, Kirk N.; Yue, Zhenyu; Yin, Xiao-Ming; Cuervo, Ana Maria; Czaja, Mark J.; Fiel, M. Isabel; Hoshida, Yujin; Friedman, Scott L.; Pathology and Laboratory Medicine, School of MedicineActivation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.Item A conserved enhancer regulates Il9 expression in multiple lineages(Nature Research, 2018-11-15) Koh, Byunghee; Qayum, Amina Abdul; Srivastava, Rajneesh; Fu, Yongyao; Ulrich, Benjamin J.; Janga, Sarath Chandra; Kaplan, Mark H.; Pediatrics, School of MedicineCytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production.Item Development of astrocytes in the vertebrate eye(Wiley Blackwell (John Wiley & Sons), 2014-12) Tao, Chenqi; Zhang, Xin; Department of Medical & Molecular Genetics, IU School of MedicineAstrocytes represent the earliest glial population in the embryonic optic nerve, contributing critically to retinal angiogenesis and formation of brain-retinal-barrier. Despite of many developmental and clinical implications of astrocytes, answers to some of the most fundamental questions of this unique type of glial cells remain elusive. This review provides an overview of the current knowledge about the origination, proliferation, and differentiation of astrocytes, their journey from the optic nerve toward the neuroretina, and their involvement in physiological and pathological development of the visual system.Item Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells(Nature Publishing Group, 2014-11) Prasain, Nutan; Lee, Man Ryul; Vemula, Sasidhar; Meador, Jonathan Luke; Yoshimoto, Momoko; Ferkowicz, Michael J.; Fett, Alexa; Gupta, Manav; Rapp, Brian M.; Saadatzadeh, Mohammad Reza; Ginsberg, Michael; Elemento, Olivier; Lee, Younghee; Voytik-Harbin, Sherry L.; Chung, Hyung Min; Hong, Ki Sung; Reid, Emma; O'Neill, Christina L.; Medina, Reinhold J.; Stitt, Alan W.; Murphy, Michael P.; Rafii, Shahin; Broxmeyer, Hal E.; Yoder, Mervin C.; Department of Pediatrics, IU School of MedicineThe ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.Item Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein(Elsevier, 2015-07-14) Ehrlich, Marc; Hallmann, Anna-Lena; Reinhardt, Peter; Araúzo-Bravo, Marcos J.; Korr, Sabrina; Röpke, Albrecht; Psathaki, Olympia E.; Ehling, Petra; Meuth, Sven G.; Oblak, Adrian L.; Murrell, Jill R.; Ghetti, Bernardino; Zaehres, Holm; Schöler, Hans R.; Sterneckert, Jared; Kuhlmann, Tanja; Hargus, Gunnar; Department of Pathology and Laboratory Medicine, IU School of MedicineFrontotemporal dementia (FTD) is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein TAU. Because of limited availability of neural cells from patients' brains, the underlying mechanisms of neurodegeneration in FTD are poorly understood. Here, we derived induced pluripotent stem cells (iPSCs) from individuals with FTD-associated MAPT mutations and differentiated them into mature neurons. Patient iPSC-derived neurons demonstrated pronounced TAU pathology with increased fragmentation and phospho-TAU immunoreactivity, decreased neurite extension, and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD neurons showed an activation of the unfolded protein response, and a transcriptome analysis demonstrated distinct, disease-associated gene expression profiles. These findings indicate distinct neurodegenerative changes in FTD caused by mutant TAU and highlight the unique opportunity to use neurons differentiated from patient-specific iPSCs to identify potential targets for drug screening purposes and therapeutic intervention.Item Diverse Perspectives: Considerations About Embryonic Stem Cell Research(2006-09-01T18:18:29Z) Indiana University Center for Bioethics, Stem Cell Study GroupSince the initial isolation of human embryonic stem cells in 1998 (Thomson et al. 1998), important developments in research have offered the promise of valuable therapeutic breakthroughs while continuing to raise significant social, ethical, legal and policy challenges. Among the interests of the Indiana University Center for Bioethics (IUCB) is a desire to engage issues of this kind, and in so doing, to provide a resource to the IU community, to Indiana, and to the entire country. The topic of stem cell research was, therefore, an appropriate one for discussion at the Center. In January 2002, the IUCB created a Stem Cell Study Group (SCSG). Our primary goal was to provide a forum for informed public discussion of the issues by making use of the considerable local scientific, legal and ethical expertise. In other words, we wanted primarily to educate ourselves about these issues. Our secondary goal was to identify and describe those points on which agreement could be achieved, as well as those issues on which agreement proved difficult if not impossible. This paper summarizes our efforts to meet both of these goals.
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