Browsing by Subject "Stem Cells"

Now showing 1 - 10 of 17
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    Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA
    (Wiley, 2019-02-05) McDaniel, Kelly; Wu, Nan; Zhou, Tianhao; Huang, Li; Sato, Keisaku; Venter, Julie; Ceci, Ludovica; Chen, Demeng; Ramos‐Lorenzo, Sugeily; Invernizzi, Pietro; Bernuzzi, Francesca; Wu, Chaodong; Francis, Heather; Glaser, Shannon; Alpini, Gianfranco; Meng, Fanyin; Medicine, School of Medicine
    Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell–derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)−/− mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2−/− mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2−/− mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.
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    Analysis of differentiation capacity of Cfp1 null embyronic stem cells
    (2014) Bowen, Tamara R.; Skalnik, David Gordon; Marrs, James; Chang, Hua-Chen
    Epigenetics 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.
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    Barriers to mental health service use among hematopoietic SCT survivors
    (Springer, 2010) Mosher, C.E.; Duhamel, K.N.; Rini, C.M.; Li, Y.; Isola, L.; Labay, L.; Rowley, S.; Papadopoulos, E.; Moskowitz, C.; Scigliano, E.; Grosskreutz, C.; Redd, W.H.
    This study examined barriers to mental health service use and the demographic, medical and psychosocial correlates of these barriers among hematopoietic SCT (HSCT) survivors. A sample of 253 HSCT survivors who were 1 to 3 years posttransplant completed measures of demographic, physical, psychological and social characteristics as well as a newly modified measure of barriers to mental health service use. Only 50% of distressed HSCT survivors had received mental health services. An exploratory factor analysis of the barriers to mental health service use scale yielded four factors: scheduling barriers, knowledge barriers, emotional barriers and illness-related barriers. Patients with higher social constraints (perceived problems discussing the illness experience with significant others) reported higher levels of all four types of barriers. General distress and transplant-related posttraumatic stress symptoms were positively associated with emotional, knowledge and illness-related barriers to mental health service use, whereas physical and functional well-being were inversely associated with these barriers. Having more knowledge barriers and more emotional barriers predicted a lower likelihood of receiving mental health services, as did lower levels of education and general distress. Results suggest that a significant number of HSCT survivors may benefit from education about mental health services that is tailored to individual barriers.
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    Cardiac engraftment of genetically-selected parthenogenetic stem cell-derived cardiomyocytes
    (Public Library of Science, 2015) Yang, Tao; Rubart, Michael; Soonpaa, Mark H.; Didié, Michael; Christalla, Peter; Zimmermann, Wolfram-Hubertus; Field, Loren J.; Department of Pediatrics, IU School of Medicine
    Parthenogenetic stem cells (PSCs) are a promising candidate donor for cell therapy applications. Similar to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PSCs exhibit self-renewing capacity and clonogenic proliferation in vitro. PSCs exhibit largely haploidentical genotype, and as such may constitute an attractive population for allogenic applications. In this study, PSCs isolated from transgenic mice carrying a cardiomyocyte-restricted reporter transgene to permit tracking of donor cells were genetically modified to carry a cardiomyocyte-restricted aminoglycoside phosphotransferase expression cassette (MHC-neor/pGK-hygror) to permit the generation of highly enriched cardiomyocyte cultures from spontaneously differentiating PSCs by simple selection with the neomycin analogue G148. Following engraftment into isogenic recipient hearts, the selected cardiomyocytes formed a functional syncytium with the host myocardium as evidenced by the presence of entrained intracellular calcium transients. These cells thus constitute a potential source of therapeutic donor cells.
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    Diverse Perspectives: Considerations About Embryonic Stem Cell Research
    (2006-09-01T18:18:29Z) Indiana University Center for Bioethics, Stem Cell Study Group
    Since 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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    The Effects of a Pyk2 Kinase Inhibitor on the Proliferation and Differentiation of Human Dental Pulp Stem Cells
    (2021) McIntyre, Patrick; Bruzzaniti, Angela; Ehrlich, Ygal; Bringas, Josef; Spolnik, Kenneth
    Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.
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    Epigenetic Regulation of Placenta-Specific 8 Contributes to Altered Function of Endothelial Colony-Forming Cells Exposed to Intrauterine Gestational Diabetes Mellitus
    (American Diabetes Association, 2015-07) Blue, Emily K.; Sheehan, BreAnn M.; Nuss, Zia V.; Boyle, Frances A.; Hocutt, Caleb M.; Gohn, Cassandra R.; Varberg, Kaela M.; McClintick, Jeanette N.; Haneline, Laura S.; Department of Pediatrics, IU School of Medicine
    Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation.
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    Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate
    (American Physiological Society, 2015-06-15) Wang, Liang; Zoetemelk, Marloes; Chitteti, Brahmananda R.; Ratliff, Timothy L.; Myers, Jason D.; Srour, Edward F.; Broxmeyer, Hal; Jerde, Travis J.; Department of Pharmacology and Toxicology, IU School of Medicine
    Prostatic inflammation is a nearly ubiquitous pathological feature observed in specimens from benign prostate hyperplasia and prostate cancer patients. The microenvironment of the inflamed prostate is highly reactive, and epithelial hyperplasia is a hallmark feature of inflamed prostates. How inflammation orchestrates epithelial proliferation as part of its repair and recovery action is not well understood. Here, we report that a novel epithelial progenitor cell population is induced to expand during inflammation. We used sphere culture assays, immunofluorescence, and flow cytometry to show that this population is increased in bacterially induced inflamed mouse prostates relative to naïve control prostates. We confirmed from previous reports that this population exclusively possesses the ability to regrow entire prostatic structures from single cell culture using renal grafts. In addition, putative progenitor cells harvested from inflamed animals have greater aggregation capacity than those isolated from naïve control prostates. Expansion of this critical cell population requires IL-1 signaling, as IL-1 receptor 1-null mice exhibit inflammation similar to wild-type inflamed animals but exhibit significantly reduced progenitor cell proliferation and hyperplasia. These data demonstrate that inflammation promotes hyperplasia in the mouse prostatic epithelium by inducing the expansion of a selected epithelial progenitor cell population in an IL-1 receptor-dependent manner. These findings may have significant impact on our understanding of how inflammation promotes proliferative diseases such as benign prostatic hyperplasia and prostate cancer, both of which depend on expansion of cells that exhibit a progenitor-like nature.
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    HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction Implications for Statin Treatment in HIV Patients
    (American Heart Association, 2019-08-27) Chelvanambi, Sarvesh; Gupta, Samir K.; Chen, Xingjuan; Ellis, Bradley W.; Maier, Bernhard F.; Colbert, Tyler M.; Kuriakose, Jithin; Zorlutuna, Pinar; Jolicoeur, Paul; Obukhov, Alexander G.; Clauss, Matthias; Medicine, School of Medicine
    Rationale Even in antiretroviral therapy (ART) treated patients, HIV continues to play a pathogenic role in cardiovascular diseases. A possible cofactor may be persistence of the early HIV response gene Nef, which we have demonstrated recently to persist in the lungs of HIV+ patients on ART. Previously, we have reported that HIV strains with Nef, but not Nef-deleted HIV strains, cause endothelial proinflammatory activation and apoptosis. Objective To characterize mechanisms through which HIV-Nef leads to the development of cardiovascular diseases using ex vivo tissue culture approaches as well as interventional experiments in transgenic murine models. Methods and Results EV (extracellular vesicles) derived from both peripheral blood mononuclear cells (PBMC) and plasma from HIV+ patient blood samples induced human coronary artery endothelial cells dysfunction. Plasma derived EV from ART+ patients that were HIV-Nef+ induced significantly greater endothelial apoptosis compared to HIV-Nef- plasma EV. Both HIV-Nef expressing T cells and HIV-Nef-induced EV increased transfer of cytosol and Nef protein to endothelial monolayers in a Rac1-dependent manner, consequently leading to endothelial adhesion protein upregulation and apoptosis. HIV-Nef induced Rac1 activation also led to dsDNA breaks in endothelial colony forming cells (ECFC), thereby resulting in ECFC premature senescence and eNOS downregulation. These Rac1 dependent activities were characterized by NOX2-mediated ROS production. Statin treatment equally inhibited Rac1 inhibition in preventing or reversing all HIV-Nef-induction abnormalities assessed. This was likely due to the ability of statins to block Rac1 prenylation as geranylgeranyl transferase inhibitors were effective in inhibiting HIV-Nef-induced ROS formation. Finally, transgenic expression of HIV-Nef in endothelial cells in a murine model impaired endothelium-mediated aortic ring dilation, which was then reversed by 3-week treatment with 5mg/kg atorvastatin. Conclusion These studies establish a mechanism by which HIV-Nef persistence despite ART could contribute to ongoing HIV related vascular dysfunction which may then be ameliorated by statin treatment.