Differentiation of pluripotent stem cells into endothelial cells

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

Endothelial stem and progenitor cells (stem cells): (2017 Grover Conference Series)

The capacity of existing blood vessels to give rise to new blood vessels via endothelial cell sprouting is called angiogenesis and is a well-studied biologic process. In contrast, little is known about the mechanisms for endothelial cell replacement or regeneration within established blood vessels. Since clear definitions exist for identifying cells with stem and progenitor cell properties in many tissues and organs of the body, several groups have begun to accumulate evidence that endothelial stem and progenitor cells exist within the endothelial intima of existing blood vessels. This paper will review stem and progenitor cell definitions and highlight several recent papers purporting to have identified resident vascular endothelial stem and progenitor cells

Endothelial Stem and Progenitor Cells for Regenerative Medicine

Purpose of Review Vascular endothelial stem cell (VESC) and progenitor cell are emerging as local resident regulators of vascular endothelial repair and replacement in mammalian subjects. However, widely recognized and accepted standard measures of stem cell function have yet to be published and, thus, we summarize some recent evidence that VESCs demonstrate stem cell properties in the process of endothelial cell (EC) lineage emergence, repair, and regeneration. Recent Findings Some rare resident ECs have been identified that are quiescent and reside within blood vessels but are activated and proliferate in response to injury. Transcriptome analyses of these ECs at a single cell level are providing new insights into VESC identity, including tissue specific EC heterogeneity. Summary Blood vessels and circulating blood contain rare immature ECs that display stem cell potential. Continuous efforts to define their precise location, origin, surface marker, and molecular signatures would enhance current approaches for purification of cells that would enable us to build new vessels for regenerative medicine

Renal endothelial dysfunction in acute kidney ischemia reperfusion injury

Acute kidney injury is associated with alterations in vascular tone that contribute to an overall reduction in GFR. Studies in animal models indicate that ischemia triggers alterations in endothelial function that contribute significantly to the overall degree and severity of a kidney injury. Putative mediators of vasoconstriction that may contribute to the initial loss of renal blood flow and GFR are highlighted. In addition, there is discussion of how intrinsic damage to the endothelium impairs homeostatic responses in vascular tone as well as promotes leukocyte adhesion and exacerbating the reduction in renal blood flow. The timing of potential therapies in animal models as they relate to the evolution of AKI, as well as the limitations of such approaches in the clinical setting are discussed. Finally, we discuss how acute kidney injury induces permanent alterations in renal vascular structure. We posit that the cause of the sustained impairment in kidney capillary density results from impaired endothelial growth responses and suggest that this limitation is a primary contributing feature underlying progression of chronic kidney disease

Anti-angiogenic activity of kidney derived endothelial cells

poster abstractThe identification of novel endogenous mediators of angiogenic/vasculogenic processes may provide for novel therapeutic targets to modulate blood vessel growth in disease states, such as cardiovascular disease or cancer. Studies in our lab have shown that blood vessels in kidney have little endogenous regenerative capacity. Kidney derived microvascular endothelial cells (KEC) were isolated from rat kidney or from transgenic mice bearing the temperature sensitive SV40 mutant (and subsequently grown at non-permissive temperature, 37oC). Both rat and mouse KECs manifested significantly reduced growth rates when compared with several commonly used EC lines (rat pulmonary EC, HUVEC and human cord blood colony forming ECs). In 2D matrigel assays, all commonly used ECs faithfully formed characteristic branching structures; while all KECs failed to form stabile branching structures. Time-course analysis of branching activity demonstrated that KEC initially formed primitive branching nodes within 3 hours of culture, but these structures regressed such that no branched structures were observed between 6-12 hours. Co-culture of KECs with any branching competent EC impaired branching dose dependently. When co-cultured with ECFC, labeled KECs incorporated into primitive ECFC branches within the first 3 hours of plating. However, when compared with ECFC branches, ECFC-KEC mixed branches showed a more rapid regression of the branched structures between 12-24 hrs. Interestingly, conditioned media from KEC did not affect branching of competent ECFC. Taken together, these data indicate that KEC have anti-angiogenic activity that may destabilize ECs during angiogenesis. The anti-angiogenic activity requires cell-cell contact, suggesting the possible presence of an angio-inhibitory molecule on the cell surface of KECs. Current and future studies seek to generate additional KEC lines, and will determine if KEC cell fractions mediate the anti-angiogenic effect. In addition, we will seek to determine if KECs mitigate progression of angiogenic dependent tumor formation in vivo

Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell–Derived Endothelial Cells

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony–forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols

Assessing identity, phenotype, and fate of endothelial progenitor cells

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, and one merely has to type the keyword search terms, endothelial progenitor cell, to recover more than 8984 articles including 1347 review articles in PubMed (as of June 2008). What can we possibly add in the form of another EPC review that will be considered of significant value for the reader? We will attempt to review some of the early article in the field and reflect on how information in those articles was gradually derivatized into perhaps more conflicting rather than unifying concepts. We will also attempt to concisely address some of the important determinants and principles that are now leading to a new understanding of what functionally constitutes an EPC and outline some of the current measures used to identify, enumerate, and quantify these cells. Finally, we give our opinion of the best definition for an EPC based on some comparative analyses performed primarily in human subjects

Endothelial colony-forming cells and pro-angiogenic cells: clarifying definitions and their potential role in mitigating acute kidney injury

Acute kidney injury (AKI) represents a significant clinical concern that is associated with high mortality rates and also represents a significant risk factor for the development of chronic kidney disease (CKD). This article will consider alterations in renal endothelial function in the setting of AKI that may underlie impairment in renal perfusion and how inefficient vascular repair may manifest post-AKI and contribute to the potential transition to CKD. We provide updated terminology for cells previously classified as ‘endothelial progenitor’ that may mediate vascular repair such as pro-angiogenic cells and endothelial colony-forming cells. We consider how endothelial repair may be mediated by these different cell types following vascular injury, particularly in models of AKI. We further summarize the potential ability of these different cells to mitigate the severity of AKI, improve perfusion and maintain vascular structure in pre-clinical studies

STIFFNESS OF 3D COLLAGEN MATRICES REGULATES CDC42 ACTIVITY OF ENDOTHELIAL COLONY FORMING CELLS DURING EARLY VACUOLE

poster abstractRecent preclinical reports have provided evidence that endothelial colony forming cells (ECFCs), a subset of endothelial progenitor cells, significantly improve vessel formation, largely due to their robust vasculogenic potential. While it has been known that the Rho family GTPase Cdc42 is involved in this ECFC-driven vessel formation process, the effect of extracellular matrix (ECM) stiffness on its activity during vessel formation is largely unknown. Using a fluorescence resonance energy transfer (FRET)-based Cdc42 biosen-sor, we examined the spatio-temporal activity of Cdc42 of ECFCs in three-dimensional (3D) collagen matrices with varying stiffness. The result re-vealed that ECFCs exhibited an increase in Cdc42 activity in a soft (150 Pa) matrix, while they were much less responsive in a stiff (1000 Pa) matrix. In both soft and stiff matrices, Cdc42 was highly activated near vacuoles; how-ever, its activity is higher in a soft matrix than that in a stiff matrix. The ob-served Cdc42 activity was closely associated with vacuole area. Soft matri-ces induced higher Cdc42 activity, faster vacuole formation, and larger vac-uole area than stiff matrices. Time courses of Cdc42 activity and vacuole formation data revealed that Cdc42 activity proceeds vacuole formation. Collectively, these results suggest that matrix stiffness is critical in regulat-ing Cdc42 activity in ECFCs and its activation is an important step in early vacuole formation

Ape1 regulates hematopoietic differentiation of embryonic stem cells through its redox functional domain

Ape1 is a molecule with dual functions in DNA repair and redox regulation of transcription factors. In Ape1-deficient mice, embryos do not survive beyond embryonic day 9, indicating that this molecule is required for normal embryo development. Currently, direct evidence of the role of Ape1 in regulating hematopoiesis is lacking. We used the embryonic stem (ES) cell differentiation system and an siRNA approach to knockdown Ape1 gene expression to test the role of Ape1 in hematopoiesis. Hemangioblast development from ES cells was reduced 2- to 3-fold when Ape1 gene expression was knocked down by Ape1-specific siRNA, as was primitive and definitive hematopoiesis. Impaired hematopoiesis was not associated with increased apoptosis in siRNA-treated cells. To begin to explore the mechanism whereby Ape1 regulates hematopoiesis, we found that inhibition of the redox activity of Ape1 with E3330, a specific Ape1 redox inhibitor, but not Ape1 DNA repair activity, which was blocked using the small molecule methoxyamine, affected cytokine-mediated hemangioblast development in vitro. In summary, these data indicate Ape1 is required in normal embryonic hematopoiesis and that the redox function, but not the repair endonuclease activity, of Ape1 is critical in normal embryonic hematopoietic development