Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular ...arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.
Angiogenesis—the develoment of microvasculature—requires, in part, directed endothelial cell motility and responsiveness to external signals. Several of the proteins, which modulate and/or direct ...endothelial cell motility and morphology in angiogenesis are the Rho GTPases (Rho, Rac, and Cdc42) and Pak (a downstream effector of Rac and Cdc42). Previously, overexpression and activation of Rho GTPases and Pak had been implicated in the development of cancer, through their roles in cancer cell transformation, stimulation of proliferation, inhibition of apoptosis, and migration. Yet regardless of the transformed status of cells within a tumor, without a blood supply most tumors cannot grow larger than 1–2
mm. The blood supply in tumors is provided by capillaries formed of endothelial cells in a process called angiogenesis. Consequently, there is enormous interest in the role of the wild type endothelial cells—and the signaling mechanisms required to support angiogenesis and subsequent growth of metastatic and aggressive cancers. Recent work has begun to uncover the roles of the Rho GTPases and Pak in the regulation of normal endothelial cell function. This review will discuss the current literature regarding the roles of Rho and Rac, and the Rac effector—Pak, in endothelial cells, and we will propose new avenues of research for interaction of the AGC kinase—PKG, with the Rho GTPases and Pak in the cell motility and cell morphology of endothelial cells.
Hypoxia, HIF and the Placenta Fryer, Benjamin H.; Simon, M. Celeste
Cell cycle (Georgetown, Tex.),
20/3/1/, Volume:
5, Issue:
5
Journal Article
Peer reviewed
Open access
Early in mammalian development the placenta, a highly vascularized organ, develops to facilitate exchange of oxygen (O2), nutrients and waste between mother and offspring. This process is intricately ...regulated by O2 tension and the hypoxic (low O2) uterine environment. Consequently, the placenta provides an excellent model for understanding the relationship between hypoxia (low O2 tension), organogenesis (organ development) and angiogenesis (blood vessel development). Herein we describe recent research on Hypoxia Inducible Factor (HIF), a heterodimeric transcription factor regulated by hypoxia that is crucial for proper placental development. Complete disruption of HIF signaling through loss of the HIFβ (ARNT) or HIF1α and HIF2α subunits results in improper placental development, characterized by a diminished spongiotrophoblast layer and insufficient chorio/allantoic fusion. Experiments using placental stem cells (TS cells) derived from Hif1α-/- Hif2α-/- (Hifα-/-) and Arnt-/- mice indicate that there is increased expression of the labyrinthine specific transcription factors GCM and TFEB and a deficiency in the spongiotrophoblast transcription factor Mash2. Furthermore Hifα-/- and Arnt-/- TS cellssubjected to differentiating conditions tend to adopt a labyrinthine like syncytial fate, and do not form giant cells or spongiotrophoblasts. These observations demonstrate a crucial role for HIF in the formation of the spongiotrophoblast that is probably regulated by Mash2, and suggest a complex interaction between hypoxia, HIF and Mash2 in the formation of the spongiotrophoblast.
Magnetosomes are biologically-derived magnetic nanoparticles (MNPs) naturally produced by magnetotactic bacteria (MTB). Due to their distinctive characteristics, such as narrow size distribution and ...high biocompatibility, magnetosomes represent an attractive alternative to existing commercially-available chemically-synthesized MNPs. However, to extract magnetosomes from the bacteria, a cell disruption step is required. In this study, a systematic comparison between three disruption techniques (enzymatic treatment, probe sonication and high-pressure homogenization) was carried out to study their effect on the chain length, integrity and aggregation state of magnetosomes isolated from
MSR-1 cells. Experimental results revealed that all three methodologies show high cell disruption yields (>89%). Transmission electron microscopy (TEM), dynamic light scattering (DLS) and, for the first time, nano-flow cytometry (nFCM) were employed to characterize magnetosome preparations after purification. TEM and DLS showed that high-pressure homogenization resulted in optimal conservation of chain integrity, whereas enzymatic treatment caused higher chain cleavage. The data obtained suggest that nFCM is best suited to characterize single membrane-wrapped magnetosomes, which can be particularly useful for applications that require the use of individual magnetosomes. Magnetosomes were also successfully labelled (>90%) with the fluorescent CellMask™ Deep Red membrane stain and analysed by nFCM, demonstrating the promising capacity of this technique as a rapid analytical tool for magnetosome quality assurance. The results of this work contribute to the future development of a robust magnetosome production platform.
Diabetes is a debilitating disease characterized by a chronic inability to normalize blood glucose levels. Transplanting cadaveric pancreata or isolated pancreatic islets can restore glucose ...homeostasis, but organ demand outstrips supply. Consequently, there is significant interest in alternative tissue sources. This review summarizes state-of-the-art efforts to generate scalable, functional β-cells to treat diabetes.
Applying knowledge gleaned from developmental biology, human pluripotent stem cells can be treated stepwise with combinations of small molecules, developmentally relevant growth factors, and morphogens to generate pancreatic progenitor cells (PPCs) in vitro. Transplanted PPCs can then further mature in vivo into functional islet-like tissues containing all of the endocrine hormone cells present in adult islets and can reverse hyperglycemia in a diabetic animal model. Recent publications demonstrate that skin, liver, and other cell lineages may also be reprogrammed to functional β-like cells.
Although generation of fully functional β-cells in vitro has not yet been achieved, possible intermediate approaches to treat diabetes include using PPCs or reprogramming adult cells to β-like cells. A cell therapy with either approach will require isolation from the host immune response. Ongoing efforts are addressing this need through the use of immune-isolation devices to avoid immunosuppressive drugs.
Sulindac causes regression of and prevents recurrence of colonic adenomas in patients with familial adenomatous polyposis. Although cell cycle arrest and apoptosis have been proposed, the mechanism ...of action is poorly understood. In this study, we characterized the growth-inhibitory effects of active metabolites of sulindac in cultured colon adenocarcinoma cells by determining the contribution of apoptosis and cell cycle arrest and the requirement for cyclooxygenase (COX) inhibition and p53 involvement and compared the effects of sulindac metabolites with the chemotherapeutic drug, 5-fluorouracil (5-FU). Time course and dose-response experiments demonstrated that increased apoptosis paralleled the growth-inhibitory effects of the sulfide and sulfone. A relationship among a series of nonsteroidal anti-inflammatory drugs was observed between potency for growth inhibition and ability to induce apoptosis but not potency to inhibit COX. For example, the sulfone was at least 5000-fold less potent than the sulfide for inhibiting COX but only 6.5-fold less potent for inducing apoptosis. Moreover, the prostaglandin analogue, dimethyl-prostaglandin E2, failed to reverse the apoptosis-inducing effects of the sulfide. Sulindac metabolites caused G1 cell cycle arrest in proliferating cells but were comparably effective in nonproliferating cells. In contrast, 5-FU treatment was less effective in nonproliferating cells. Combined treatment with sulindac metabolites and 5-FU did not result in an additive apoptotic response. Treatment of cells with 5-FU increased p53 protein levels, whereas sulindac metabolites did not induce expression. Saos-2 cells, which lack p53, responded to sulindac metabolites but not 5-FU. These results show that apoptosis primarily contributes to growth inhibition by sulindac metabolites. The biochemical pathway does not require COX inhibition or p53 induction and appears to be fundamentally different from the apoptotic response to 5-FU.
Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, ...cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.
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