Browsing by Subject "Endothelial colony forming cells"

Now showing 1 - 4 of 4
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    Differentiation of pluripotent stem cells into endothelial cells
    (Wolters Kluwer, 2015-05) Yoder, Mervin C.; Department of Pediatrics, IU School of Medicine
    PURPOSE OF REVIEW: Methods to isolate endothelial cells from murine and human pluripotent stem cells continue to evolve and increasingly diverse endothelial cell populations have been generated. This review provides an update of key articles published within the past year that report on some of those advances. RECENT FINDINGS: Cooperative interactions among microRNA (miRNA), transcription factors and some downstream interacting proteins have been reported to enhance endothelial specification from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Endothelial cell differentiation can also be modulated by various growth factor additions, Notch pathway activation or inhibition, and modulation of the microenvironment of the differentiating ESC and iPSC. Functionality of the derived endothelium has been demonstrated by a variety of in-vitro and in-vivo assays. Finally, two recent reports have identified endothelial progenitor populations with robust proliferative potential. SUMMARY: Progress in differentiating endothelial cells from ESC and iPSC has been made. The recent report of formation of endothelial colony forming cells from human ESC and iPSC provides a protocol that can generate clinically relevant numbers of cells for human cell therapy.
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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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    Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro
    (JoVE, 2018-01-31) Varberg, Kaela M.; Winfree, Seth; Dunn, Kenneth W.; Haneline, Laura S.; Cellular and Integrative Physiology, School of Medicine
    Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.
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    Mesenchyme homeobox 2 regulation of fetal endothelial progenitor cell function
    (2017-06-19) Gohn, Cassandra Rebekah; Haneline, Laura S.; Elmendorf, Jeffrey S.; Herring, B. Paul; Tune, Johnathan D.
    In the United States, 10% of pregnancies are complicated by diabetes mellitus (DM). Intrauterine DM exposure can have long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that fetal endothelial colony forming cells (ECFCs) from DM pregnancies have decreased vessel-forming ability and increased senescence. However, the molecular mechanisms responsible for this dysfunction remain largely unknown. The objective of this thesis was to understand how Mesenchyme Homeobox 2 (MEOX2) interacts with pathways that regulate cell cycle progression and migration, and how this interaction results in impaired vasculogenesis in DM exposed ECFCs. We tested the hypothesis that upregulated MEOX2 in DM-exposed ECFCs decreases network formation through impairments in senescence, cell cycle progression, migration, and adhesion. MEOX2 is a transcription factor which inhibits angiogenesis by upregulating cyclin dependent kinase inhibitors. Here, data show that nuclear MEOX2 is increased in DM-exposed ECFCs. Lentiviral-mediated overexpression of MEOX2 in control ECFCs increased network formation, altered cell cycle progression, increased senescence, and enhanced migration. In contrast, MEOX2-knockdown in DM-exposed ECFCs decreased network formation and migration, while cell cycle progression and senescence were unchanged. Adhesion and integrin expression defects were evaluated as mechanisms by which MEOX2 altered ECFC migration. While MEOX2-overexpression did not alter adhesion or cell surface integrin levels in control cells, MEOX2 overexpression in DM-exposed ECFCs resulted in an increase in α6 integrin surface expression. Similarly, MEOX2-knockdown in DM-exposed ECFCs did not alter adhesion, though did reduce α6 integrin surface expression and total cellular α6 mRNA and protein levels. Together, these data suggest that alterations in cell cycle progression and senescence are not responsible for the disrupted vasculogenesis of DM-exposed ECFCs. Importantly, these data suggest that altered migration may be a key mechanism involved and that altered cell surface levels of the α6 integrin may modify migratory capacity. Moreover, these data suggest that the α6 integrin may be a previously unrecognized transcriptional target of MEOX2. Ultimately, while initially believed to be maladaptive, these data suggest that increased nuclear MEOX2 in DM-exposed ECFCs may serve a protective role, enabling vessel formation despite exposure to a DM intrauterine environment.