Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration.
Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect.
Blood transfusion is a mainstay of modern clinical medicine. However, a number of fundamental problems persist, including insufficiency of supply, the threat of transfusion transmissible infectious disease and the problem of immune incompatibility. It would be extremely valuable, therefore, to develop a potentially limitless, infection free, immune neutral source of erythrocytes for transfusion. Human embryonic stem cells (hESC), have potentially limitless proliferative capacity and the potential to differentiate into the majority of adult cell types including erythrocytes. A number of barriers to the development of clinical cellular therapeutics from hESC have been posited, including HLA incompatibility between donor and recipient, difficulties in defining optimal cell phenotype and function in vitro and the fact that most tissues consist of complex three-dimensional matrices of cells. Many or most of these problems are circumvented in the generation of erythrocytes and group O RhD negative Kell negative blood would be compatible with the majority of recipients. Red cell transfusion is therefore an attractive goal for pluripotent stem cell derived therapeutics. Much progress has been made however, a number of challenges remain including scale up, ensuring clinical effectiveness and product safety.
Omacetaxine is a natural product extract originating from Chinese medicine and finding therapeutic use as a potent myelosuppressive agent in leukemia. When planning in vitro cell biology experiments to assess omacetaxine activity against primary leukemic stem cells, it became apparent that the literature rarely describes the in vitro stability of the molecule, although accessible chromatographic methods have been published. Clearly whole organisms vs their component cells will differ in the way in which they handle xenobiotics, with the latter more dependent on physiochemical parameters such as pH and temperature in the absence of active metabolism or excretion. This could impact on the cells experience of drug in culture. We therefore report here on examination of a modified, high-performance liquid chromatography (HPLC) method with assessment of degradant production from a 72?h solution stability study, clearly demonstrating that omacetaxine is highly stable in representative cell culture conditions (37?°C, neutral pH) and persists for many days in marked contrast to its short-half life in vivo.
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.
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.
The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of lead-like small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N?,N?-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by off-target inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.
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.
The use of donated red blood cells in transfusion is a well-established cellular therapy. However, problems including insufficient supply, transfusion-transmitted infections and the need for immunological matching hamper even in the best services. These issues may be eliminated by using pluripotent stem cells to generate universal donor group O, Rhesus D-negative red blood cells. Human embryonic stem cells can be maintained and expanded indefinitely and can, therefore, produce the very large cell numbers required for this application. Red blood cell production is also an attractive goal for pluripotent stem cell-derived therapeutics because it is a well-characterized single cell suspension, lacking nucleated cells and with a low expression of HLA molecules. Much progress has been made; however, a number of challenges remain including scale-up, clinical effectiveness and product safety.
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.
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder maintained by cancer stem cells. To target this population, we investigated the mechanism of action of BMS-214662, developed as a farnesyl transferase inhibitor (FTI) and unique in inducing apoptosis in these cells. By contrast, a related congener and equally effective FTI, BMS-225975 does not induce apoptosis, indicating a novel mechanism of action. BMS-214662 significantly and selectively induced apoptosis in primitive CD34(+)38(-) CML compared with normal cells. Apoptosis proceeded via the intrinsic pathway: Bax conformational changes, loss of mitochondrial membrane potential, generation of reactive oxygen species, release of cytochrome c, and caspase-9/3 activation were noted. Up-regulation of protein kinase Cbeta (PKCbeta), down-regulation of E2F1, and phosphorylation of cyclin A-associated cyclin-dependent kinase 2 preceded these changes. Cotreatment of CML CD34(+) and CD34(+)38(-) cells with PKC modulators, bryostatin-1, or hispidin markedly decreased these early events and the subsequent apoptosis. None of these events was elicited by BMS-214662 in normal CD34(+) cells or by BMS-225975 in CML CD34(+) cells. These data suggest that BMS-214662 selectively elicits a latent apoptotic pathway in CML stem cells that is initiated by up-regulation of PKCbeta and mediated by Bax activation, providing a molecular framework for development of novel therapeutics.
The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis.
This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph(+)) cells.
To investigate the interaction of imatinib mesylate (IM) with the clinically relevant adenosine triphosphate-binding cassette efflux transporter MDR1 (ABCB1) in cells from patients with chronic myeloid leukemia (CML) and to explore whether inhibition of this transporter would improve IMs efficacy in the elimination of CML CD34(+) cells by increasing cell-associated drug accumulation.
Spinal cord injury (SCI) is a devastating condition with limited capacity for repair. Cell transplantation is a potential strategy to promote SCI repair with cells from the olfactory system being promising candidates. Although transplants of human olfactory mucosa (OM) are already ongoing in clinical trials, the repair potential of this tissue remains unclear. Previously, we identified mesenchymal-like stem cells that reside in the lamina propria (LP-MSCs) of rat and human OM. Little is known about these cells or their interactions with glia such as olfactory ensheathing cells (OECs), which would be co-transplanted with MSCs from the OM, or endogenous CNS glia such as oligodendrocytes. We have characterized, purified, and assessed the repair potential of human LP-MSCs by investigating their effect on glial cell biology with specific emphasis on CNS myelination in vitro. Purified LP-MSCs expressed typical bone marrow MSC (BM-MSC) markers, formed spheres, were clonogenic and differentiated into bone and fat. LP-MSC conditioned medium (CM) promoted oligodendrocyte precursor cell (OPC) and OEC proliferation and induced a highly branched morphology. LP-MSC-CM treatment caused OEC process extension. Both LP and BM-MSCs promoted OPC proliferation and differentiation, but only myelinating cultures treated with CM from LP and not BM-MSCs had a significant increase in myelination. Comparison with fibroblasts and contaminating OM fibroblast like-cells showed the promyelination effect was LP-MSC specific. Thus LP-MSCs harvested from human OM biopsies may be an important candidate for cell transplantation by contributing to the repair of SCI.
Previous work has shown that the protein kinase A (PKA)-regulated phosphodiesterase (PDE) 4D3 binds to A kinase-anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA-PDE4D3-AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals.
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.
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