Browsing by Subject "Cell differentiation"

Now showing 1 - 10 of 35
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    Bidirectional Regulatory Cross-Talk between Cell Context and Genomic Aberrations Shapes Breast Tumorigenesis
    (American Association for Cancer Research, 2021) Kumar, Brijesh; Bhat-Nakshatri, Poornima; Maguire, Calli; Jacobsen, Max; Temm, Constance J.; Sandusky, George; Nakshatri, Harikrishna; Surgery, School of Medicine
    Breast cancers are classified into five intrinsic subtypes and 10 integrative clusters based on gene expression patterns and genomic aberrations, respectively. Although the cell-of-origin, adaptive plasticity, and genomic aberrations shape dynamic transcriptomic landscape during cancer progression, how interplay between these three core elements governs obligatory steps for a productive cancer progression is unknown. Here, we used genetic ancestry-mapped immortalized breast epithelial cell lines generated from breast biopsies of healthy women that share gene expression profiles of luminal A, normal-like, and basal-like intrinsic subtypes of breast cancers and breast cancer relevant oncogenes to develop breast cancer progression model. Using flow cytometry, mammosphere growth, signaling pathway, DNA damage response, and in vivo tumorigenicity assays, we provide evidence that establishes cell context-dependent effects of oncogenes in conferring plasticity, self-renewal/differentiation, intratumor heterogeneity, and metastatic properties. In contrast, oncogenic aberrations, independent of cell context, shaped response to DNA damage-inducing agents. Collectively, this study reveals how the same set of genomic aberration can have distinct effects on tumor characteristics based on cell-of-origin of tumor and highlights the need to utilize multiple "normal" epithelial cell types to decipher oncogenic properties of a gene of interest. In addition, by creating multiple isogenic cell lines ranging from primary cells to metastatic variants, we provide resources to elucidate cell-intrinsic properties and cell-oncogene interactions at various stages of cancer progression. IMPLICATIONS: Our findings demonstrate that how an interplay between the normal cell type that encountered genomic aberrations and type of genomic aberration influences heterogeneity, self-renewal/differentiation, and tumor properties including propensity for metastasis.
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    CD45 Phosphatase Inhibits STAT3 Transcription Factor Activity in Myeloid Cells and Promotes Tumor-Associated Macrophage Differentiation
    (Elsevier, 2016-02-16) Kumar, Vinit; Cheng, Pingyan; Condamine, Thomas; Mony, Sridevi; Languino, Lucia R.; McCaffrey, Judith C.; Hockstein, Neil; Guarino, Michael; Masters, Gregory; Penman, Emily; Denstman, Fred; Xu, Xiaowei; Altieri, Dario C.; Du, Hong; Yan, Cong; Gabrilovich, Dmitry I.; Department of Pathology and Laboratory Medicine, IU School of Medicine
    Recruitment of monocytic myeloid-derived suppressor cells (MDSCs) and differentiation of tumor-associated macrophages (TAMs) are the major factors contributing to tumor progression and metastasis. We demonstrated that differentiation of TAMs in tumor site from monocytic precursors was controlled by downregulation of the activity of the transcription factor STAT3. Decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. Upregulation of CD45 tyrosine phosphatase activity in MDSCs exposed to hypoxia in tumor site was responsible for downregulation of STAT3. This effect was mediated by the disruption of CD45 protein dimerization regulated by sialic acid. Thus, STAT3 has a unique function in the tumor environment in controlling the differentiation of MDSC into TAM, and its regulatory pathway could be a potential target for therapy.
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    Derivation of endothelial colony forming cells from human cord blood and embryonic stem cells
    (2013) Meador, J. Luke; Kapur, Ruben; Yoder, Mervin C.; Corson, Timothy W.; Meyer, Jason S.
    Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.
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    The development and in vivo function of T helper 9 cells
    (SpringerNature, 2015-05) Kaplan, Mark H.; Hufford, Matthew M.; Olson, Matthew R.; Department of Pediatrics, IU School of Medicine
    The specialized cytokine secretion profiles of T helper (TH) cells are the basis for a focused and efficient immune response. On the 20th anniversary of the first descriptions of cytokine signals that act to differentiate interleukin-9 (IL-9)-secreting T cells, this review focuses on the extracellular signals and transcription factors that promote the development of what we now term TH9 cells, which are characterized by the production of this cytokine. We summarize our current understanding of the contribution of TH9 cells to both effective immunity and immunopathological disease and propose that TH9 cells could be targeted for the treatment of allergic and autoimmune disease.
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    The Direct Reprogramming of Somatic Cells: Establishment of a Novel System for Photoreceptor Derivation
    (2013-08-22) Steward, Melissa Mary; Meyer, Jason S.; Dai, Guoli; Randall, Stephen Karl, 1953-; Atkinson, Simon
    Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.
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    E2F1 Suppresses Oxidative Metabolism and Endothelial Differentiation of Bone Marrow Progenitor Cells
    (American Heart Association, 2018-03-02) Xu, Shiyue; Tao, Jun; Yang, Liu; Zhang, Eric; Boriboun, Chan; Zhou, Junlan; Sun, Tianjiao; Cheng, Min; Huang, Kai; Shi, Jiawei; Dong, Nian-Guo; Liu, Qinghua; Zhao, Ting C.; Qiu, Hongyu; Harris, Robert A.; Chandel, Navdeep S.; Losordo, Douglas W.; Qin, Gangjian; Biochemistry and Molecular Biology, School of Medicine
    RATIONALE: The majority of current cardiovascular cell therapy trials use bone marrow progenitor cells (BM PCs) and achieve only modest efficacy; the limited potential of these cells to differentiate into endothelial-lineage cells is one of the major barriers to the success of this promising therapy. We have previously reported that the E2F transcription factor 1 (E2F1) is a repressor of revascularization after ischemic injury. OBJECTIVE: We sought to define the role of E2F1 in the regulation of BM PC function. METHODS AND RESULTS: Ablation of E2F1 (E2F1 deficient) in mouse BM PCs increases oxidative metabolism and reduces lactate production, resulting in enhanced endothelial differentiation. The metabolic switch in E2F1-deficient BM PCs is mediated by a reduction in the expression of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase kinase 2; overexpression of pyruvate dehydrogenase kinase 4 reverses the enhancement of oxidative metabolism and endothelial differentiation. Deletion of E2F1 in the BM increases the amount of PC-derived endothelial cells in the ischemic myocardium, enhances vascular growth, reduces infarct size, and improves cardiac function after myocardial infarction. CONCLUSION: Our results suggest a novel mechanism by which E2F1 mediates the metabolic control of BM PC differentiation, and strategies that inhibit E2F1 or enhance oxidative metabolism in BM PCs may improve the effectiveness of cell therapy.
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    Effect of Shear Stress on RhoA Activities and Cytoskeletal Organization in Chondrocytes
    (2013-09-05) Wan, Qiaoqiao; Na, Sungsoo; Li, Jiliang; Yokota, Hiroki
    Mechanical force environment is a major factor that influences cellular homeostasis and remodeling. The prevailing wisdom in this field demonstrated that a threshold of mechanical forces or deformation was required to affect cell signaling. However, by using a fluorescence resonance energy transfer (FRET)-based approach, we found that C28/I2 chondrocytes exhibited an increase in RhoA activities in response to high shear stress (10 or 20 dyn/cm2), while they showed a decrease in their RhoA activities to intermediate shear stress at 5 dyn/cm2. No changes were observed under low shear stress (2 dyn/ cm2). The observed two-level switch of RhoA activities was closely linked to the shear stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. Collectively, these results herein suggest that intensities of shear stress are critical in differential activation and inhibition of RhoA activities in chondrocytes.
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    Endothelial Colony Forming Cells (ECFCs): Identification, Specification and Modulation in Cardiovascular Diseases
    (2009-12) Huang, Lan; Pescovitz, Mark D.; Quilliam, Lawrence A.; Ingram, David A., Jr.; Pescovitz, Mark D.
    A hierarchy of endothelial colony forming cells (ECFCs) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. High proliferative potential ECFCs (HPP-ECFCs) display properties (robust proliferative potential in vitro and vessel-forming ability in vivo) consistent with stem/progenitor cells for the endothelial lineage. Corneal endothelial cells (CECs) are different from circulating and resident vascular endothelial cells (ECs). Whereas systemic vascular endothelium slowly proliferates throughout life, CECs fail to proliferate in situ and merely expand in size to accommodate areas of CEC loss due to injury or senescence. However, we have identified an entire hierarchy of ECFC resident in bovine CECs. Thus, this study provides a new conceptual framework for defining corneal endothelial progenitor cell potential. The identification of persistent corneal HPP-ECFCs in adult subjects might contribute to regenerative medicine in corneal transplantation. While human cord blood derived ECFCs are able to form vessels in vivo, it is unknown whether they are committed to an arterial or venous fate. We have demonstrated that human cord blood derived ECFCs heterogeneously express gene transcripts normally restricted to arterial or venous endothelium. They can be induced to display an arterial gene expression pattern after vascular endothelial growth factor 165 (VEGF165) or Notch ligand Dll1 (Delta1ext-IgG) stimulation in vitro. However, the in vitro Dll1 primed ECFCs fail to display significant skewing toward arterial EC phenotype and function in vivo upon implantation, suggesting that in vitro priming is not sufficient for in vivo specification. Future studies will determine whether ECFCs are amenable to specification in vivo by altering the properties of the implantation microenvironment. There is emerging evidence suggesting that the concentration of circulating ECFCs is closely related to the adverse progression of cardiovascular disorders. In a pig model of acute myocardial ischemia (AMI), we have demonstrated that AMI rapidly mobilizes ECFCs into the circulation, with a significant shift toward HPP-ECFCs. The exact role of the mobilized HPP-ECFCs in homing and participation in repair of the ischemic tissue remains unknown. In summary, these studies contribute to an improved understanding of ECFCs and suggest several possible therapeutic applications of ECFCs.
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    ERG functionally overlaps with other Ets proteins in promoting TH9 cell expression of Il9 during allergic lung inflammation
    (American Association of Immunologists, 2023) Kharwadkar, Rakshin; Ulrich, Benjamin J.; Chu, Michelle; Koh, Byunghee; Hufford, Matthew M.; Fu, Yongyao; Birdsey, Graeme M.; Porse, Bo T.; Randi, Anna M.; Kaplan, Mark H.; Biochemistry and Molecular Biology, School of Medicine
    CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.
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    Exploring dysfunctional barrier phenotypes associated with glaucoma using a human pluripotent stem cell-based model of the neurovascular unit
    (Springer Nature, 2024-11-14) Lavekar, Sailee S.; Hughes, Jason M.; Gomes, Cátia; Huang, Kang-Chieh; Harkin, Jade; Canfield, Scott G.; Meyer, Jason S.; Biology, School of Science
    Glaucoma is a neurodegenerative disease that results in the degeneration of retinal ganglion cells (RGCs) and subsequent loss of vision. While RGCs are the primary cell type affected in glaucoma, neighboring cell types selectively modulate RGCs to maintain overall homeostasis. Among these neighboring cell types, astrocytes, microvascular endothelial cells (MVECs), and pericytes coordinate with neurons to form the neurovascular unit that provides a physical barrier to limit the passage of toxic materials from the blood into neural tissue. Previous studies have demonstrated that these barrier properties may be compromised in the progression of glaucoma, yet mechanisms by which this happens have remained incompletely understood. Thus, the goals of this study were to adapt a human pluripotent stem cell (hPSC)-based model of the neurovascular unit to the study of barrier integrity relevant to glaucoma. To achieve this, hPSCs were differentiated into the cell types that contribute to this barrier, including RGCs, astrocytes, and MVECs, then assembled into an established Transwell®-insert model. The ability of these cell types to contribute to an in vitro barrier model was tested for their ability to recapitulate characteristic barrier properties. Results revealed that barrier properties of MVECs were enhanced when cultured in the presence of RGCs and astrocytes compared to MVECs cultured alone. Conversely, the versatility of this system to model aspects of barrier dysfunction relevant to glaucoma was tested using an hPSC line with a glaucoma-specific Optineurin (E50K) mutation as well as a paired isogenic control, where MVECs then exhibited reduced barrier integrity. To identify factors that could result in barrier dysfunction, results revealed an increased expression of TGFβ2 in glaucoma-associated OPTN(E50K) astrocytes, indicating a potential role for TGFβ2 in disease manifestation. To test this hypothesis, we explored the ability to modulate exogenous TGFβ2 in both isogenic control and OPTN(E50K) experimental conditions. Collectively, the results of this study indicated that the repurposing of this in vitro barrier model for glaucoma reliably mimicked some aspects of barrier dysfunction, and may serve as a platform for drug discovery, as well as a powerful in vitro model to test the consequences of barrier dysfunction upon RGCs in glaucoma.