Nicole M. Kane

Derivation of Endothelial Cells From Human Embryonic Stem Cells by Directed Differentiation: Analysis of MicroRNA and Angiogenesis In Vitro and In Vivo

Objective—To develop an embryoid body-free directed differentiation protocol for the rapid generation of functional vascular endothelial cells derived from human embryonic stem cells (hESCs) and to assess the system for microRNA regulation and angiogenesis. Methods and Results—The production of defined cell lineages from hESCs is a critical requirement for evaluating their potential in regenerative medicine. We developed a feeder- and serum-free protocol. Directed endothelial differentiation of hESCs revealed rapid loss of pluripotency markers and progressive induction of mRNA and protein expression of vascular markers (including CD31 and vascular endothelial [VE]-cadherin) and angiogenic growth factors (including vascular endothelial growth factor), increased expression of angiogenesis-associated microRNAs (including miR-126 and miR-210), and induction of endothelial cell morphological features. In vitro, differentiated cells produced nitric oxide, migrated across a wound, and formed tubular structures in both the absence and the presence of 3D matrices (Matrigel). In vivo, we showed that cells that differentiated for 10 days before implantation were efficient at the induction of therapeutic neovascularization and that hESC-derived cells were incorporated into the blood-perfused vasculature of recipient mice. Conclusion—The directed differentiation of hESCs is efficient and effective for the differentiation of functional endothelial cells from hESCs.

Pluripotent stem cell differentiation into vascular cells: A novel technology with promises for vascular re(generation)

Several types of stem and progenitor cells are currently under investigation for their potential to accomplish vascular regeneration. This review focuses on embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We will discuss the technologies allowing for their derivation, culture expansion and maintenance in a pluripotent status. Moreover, both ESCs and iPSCs can be differentiated in endothelial cells (ECs) and mural cell, including vascular smooth muscle cells (VSMCs). Here, we will describe the involvements of growth factors (vascular endothelial growth factors-VEGFs-, platet-derived growth factors-PDGFs-), Wnt and Notch signal pathways, reactive oxygen species (ROS), histone deacetylases (HDACs), and microRNAs (miRNAs) in vascular cell differentiation from pluripotent stem cells. We will additionally describe the therapeutic potential of stem cells for vascular medicine.

Role of microRNAs 99b, 181a, and 181b in the differentiation of human embryonic stem cells to vascular endothelial cells

MicroRNAs (miRNAs) are short noncoding RNAs, which post‐transcriptionally regulate gene expression. miRNAs are transcribed as precursors and matured to active forms by a series of enzymes, including Dicer. miRNAs are important in governing cell differentiation, development, and disease. We have recently developed a feeder‐ and serum‐free protocol for direct derivation of endothelial cells (ECs) from human embryonic stem cells (hESCs) and provided evidence of increases in angiogenesis‐associated miRNAs (miR‐126 and ‐210) during the process. However, the functional role of miRNAs in hESC differentiation to vascular EC remains to be fully interrogated. Here, we show that the reduction of miRNA maturation induced by Dicer knockdown suppressed hES‐EC differentiation. A miRNA microarray was performed to quantify hES‐EC miRNA profiles during defined stages of endothelial differentiation. miR‐99b, ‐181a, and ‐181b were identified as increasing in a time‐ and differentiation‐dependent manner to peak in mature hESC‐ECs and adult ECs. Augmentation of miR‐99b, ‐181a, and ‐181b levels by lentiviral‐mediated transfer potentiated the mRNA and protein expression of EC‐specific markers, Pecam1 and VE Cadherin, increased nitric oxide production, and improved hES‐EC‐induced therapeutic neovascularization in vivo. Conversely, knockdown did not impact endothelial differentiation. Our results suggest that miR‐99b, ‐181a, and ‐181b comprise a component of an endothelial‐miRNA signature and are capable of potentiating EC differentiation from pluripotent hESCs. STEM CELLS 2012; 30:643–654

Concise Review: MicroRNAs as Modulators of Stem Cells and Angiogenesis

MicroRNAs (miRs) are highly conserved, short noncoding RNA molecules that negatively regulate messenger RNA (mRNA) stability and/or translational efficiency. Since a given miR can control the expression of many mRNAs, their importance in governing gene expression in specific cell types including vascular cells and their progenitor cells has become increasingly clear. Understanding how the expression of miRs themselves is regulated and how miRs exert their influence on post‐transcriptional gene control provides novel opportunities to dissect gene regulatory networks in clinically relevant cell types. A multitude of miRs have been identified with key roles in vascular development, homeostasis, function, disease, and regeneration. In this review, we will describe the impact of miRs on angiogenesis and their capacity to modulate the behavior of stem and progenitor cells which may be utilitarian for promoting vascular growth in ischemic tissue. Moreover, we summarize these strategies available for modulating miR expression and function and future therapeutic applications. Stem Cells 2014;32:1059–1066

Lentivirus-mediated Reprogramming of Somatic Cells in the Absence of Transgenic Transcription Factors

Retroviral vectors remain the most efficient and widely applied system for induction of pluripotency. However, mutagenic effects have been documented in both laboratory and clinical gene therapy studies, principally as a result of dysregulated host gene expression in the proximity of defined integration sites. Here, we report that cells with characteristics of pluripotent stem cells can be produced from normal human fibroblasts in the absence of reprogramming transcription factors (TFs) during lentiviral (LV) vector-mediated gene transfer. This occurred via induced alterations in host gene and microRNA (miRNA) expression and detrimental changes in karyotype. These findings demonstrate that vector-induced genotoxicity may alone play a role in somatic cell reprogramming derivation and urges caution when using integrating vectors in this setting. Clearer understanding of this process may additionally reveal novel insights into reprogramming pathways.

Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk

We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer.

MicroRNAs regulating cell pluripotency and vascular differentiation

Human embryonic stem cells (hESC) offer broad potential for regenerative medicine owing to their capacity for self renewal, exponential scale up and differentiation into any cell type in the adult body. hESC have been proposed as a potentially unlimited source for the generation of transplantable, healthy, functional vascular cells for repair of ischemic tissues. To optimally harness this potential necessitates precise control over biological processes that govern maintenance, pluripotency and cell differentiation including signalling cascades, gene expression profiles and epigenetic modification. Such control may be elicited by microRNAs, which are powerful negative regulators of gene expression. Here, we review the role for miRNAs in both the maintenance of pluripotency and differentiation of cells to a cardiovascular lineage including endothelial cells, vascular smooth muscle cells and cardiomyocytes and put this into context for regenerative medicine in the cardiovascular system.

Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells

hESCs (human embryonic stem cells) offer great potential for pharmaceutical research and development and, potentially, for therapeutic use. However, improvements in cell culture are urgently required to allow the scalable production of large numbers of cells that maintain pluripotency. Supplementing feeder-free conditions with either EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine] or readily synthesized analogues of this compound maintains hESC pluripotency in the absence of exogenous cytokines. When the hESC lines SA121 or SA461 were maintained in feeder-free conditions with EHNA they displayed no reduction in stem-cell-associated markers such as Nanog, Oct4 (octamer-binding protein 4) and SSEA4 (stage-specific embryonic antigen 4) when compared with cells maintained in full feeder-free conditions that included exogenously added bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but EHNA did not limit efficient spontaneous or directed differentiation following its removal. We conclude that EHNA or related compounds offers a viable alternative to exogenous cytokine addition in maintaining hESC cultures in a pluripotent state and might be a particularly useful replacement for bFGF for large-scale or GMP (good manufacturing practice)-compliant processes.

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

Viral and non-viral gene delivery and its role in pluripotent stem cell engineering

Human cell culture has become an essential component of biomedical research. The pluripotent phenotype, naturally occurring within the inner cell mass of the early embryo, confers the ability to develop into any cell type of interest. The exploitation of this ability in conjunction with modern genetic engineering tools holds great potential for analysing developmental and adult physiology and pathophysiology as well as for promising an exciting future for regenerative medicine. A broad range of gene delivery systems are available to researchers involved in pluripotent stem cell manipulation. Merits of viral and non-viral systems are driven primarily by the efficiency of stem cell gene transfer. Here, we review the use of viruses (primarily retroviruses, adenoviruses and lentiviruses) and recently optimised non-viral systems for stem cell gene transfer focusing on the recent work on human embryonic stem cells and induced pluripotent stem cells.