Kinetic Evaluation of the Hypoxia Radiotracers [18F]FMISO and [18F]FAZA in Dogs with Spontaneous Tumors Using Dynamic PET/CT Imaging.

Purpose: We evaluated the kinetics of the hypoxia PET radiotracers, [18F]fluoromisonidazole ([18F]FMISO) and [18F]fluoroazomycin-arabinoside ([18F]FAZA), for tumor hypoxia detection and to assess the correlation of hypoxic kinetic parameters with static imaging measures in canine spontaneous tumors. Methods: Sixteen dogs with spontaneous tumors underwent a 150-min dynamic PET scan using either [18F]FMISO or [18F]FAZA. The maximum tumor-to-muscle ratio (TMRmax) > 1.4 on the last image frame was used as the standard threshold to determine tumor hypoxia. The tumor time-activity curves were analyzed using irreversible and reversible two-tissue compartment models and graphical methods. TMRmax was compared with radiotracer trapping rate (k 3), influx rate (K i), and distribution volume (V T). Results: Tumor hypoxia was detected in 7/8 tumors in the [18F]FMISO group and 4/8 tumors in the [18F]FAZA group. All hypoxic tumors were detected at > 120 min with [18F]FMISO and at > 60 min with [18F]FAZA. [18F]FAZA showed better fit with the reversible model. TMRmax was strongly correlated with the irreversible parameters (k 3 and K i) for [18F]FMISO at > 90 min and with the reversible parameter (V T) for [18F]FAZA at > 120 min. Conclusions: Our results showed that [18F]FAZA provided a promising alternative radiotracer to [18F]FMISO with detecting the presence of tumor hypoxia at an earlier time (60 min), consistent with its favorable faster kinetics. The strong correlation between TMRmax over the 90-150 min and 120-150 min timeframes with [18F]FMISO and [18F]FAZA, respectively, with kinetic parameters associated with tumor hypoxia for each radiotracer, suggests that a static scan measurement (TMRmax) is a good alternative to quantify tumor hypoxia. Supplementary Information: The online version contains supplementary material available at 10.1007/s13139-022-00780-4.

Double blind trial of a deuterated form of linoleic acid (RT001) in Friedreich ataxia.

OBJECTIVES: Friedreich ataxia is (FRDA) an autosomal recessive neurodegenerative disorder associated with intrinsic oxidative damage, suggesting that decreasing lipid peroxidation (LPO) might ameliorate disease progression. The present study tested the ability of RT001, a deuterated form of linoleic acid (D2-LA), to alter disease severity in patients with FRDA in a double-blind placebo-controlled trial. METHODS: Sixty-five subjects were recruited across six sites and received either placebo or active drug for an 11-month study. Subjects were evaluated at 0, 4, 9, and 11 months, with the primary outcome measure being maximum oxygen consumption (MVO2) during cardiopulmonary exercise testing (CPET). A key secondary outcome measure was a composite statistical test using results from the timed 1-min walk (T1MW), peak workload, and MVO2. RESULTS: Forty-five subjects completed the protocol. RT001 was well tolerated, with no serious adverse events related to drug. Plasma and red blood cell (RBC) membrane levels of D2-LA and its primary metabolite deuterated arachidonic acid (D2-AA) achieved steady-state concentrations by 4 months. No significant changes in MVO2 were observed for RT001 compared to placebo. Similarly, no differences between the groups were found in secondary or exploratory outcome measures. Post hoc evaluations also suggested minimal effects of RT001 at the dosages used in this study. INTERPRETATIONS: The results of this study provide no evidence for a significant benefit of RT001 at the dosages tested in this Friedreich ataxia patient population.

Novel Oxygen Carrier Slows Infarct Growth in Large Vessel Occlusion Dog Model Based on Magnetic Resonance Imaging Analysis.

BACKGROUND: Tissue hypoxia plays a critical role in the events leading to cell death in ischemic stroke. Despite promising results in preclinical and small clinical pilot studies, inhaled oxygen supplementation has not translated to improved outcomes in large clinical trials. Moreover, clinical observations suggest that indiscriminate oxygen supplementation can adversely affect outcome, highlighting the need to develop novel approaches to selectively deliver oxygen to affected regions. This study tested the hypothesis that intravenous delivery of a novel oxygen carrier (Omniox-Ischemic Stroke [OMX-IS]), which selectively releases oxygen into severely ischemic tissue, could delay infarct progression in an established canine thromboembolic large vessel occlusion stroke model that replicates key dynamics of human infarct evolution. METHODS: After endovascular placement of an autologous clot into the middle cerebral artery, animals received OMX-IS treatment or placebo 45 to 60 minutes after stroke onset. Perfusion-weighted magnetic resonance imaging was performed to define infarct progression dynamics to stratify animals into fast versus slow stroke evolvers. Serial diffusion-weighted magnetic resonance imaging was performed for up to 5 hours to quantify infarct evolution. Histology was performed postmortem to confirm final infarct size. RESULTS: In fast evolvers, OMX-IS therapy substantially slowed infarct progression (by ≈1 hour, P<0.0001) and reduced the final normalized infarct volume as compared to controls (0.99 versus 0.88, control versus OMX-IS drug, P<0.0001). Among slow evolvers, OMX-IS treatment delayed infarct progression by approximately 45 minutes; however, this did not reach statistical significance (P=0.09). The final normalized infarct volume also did not show a significant difference (0.93 versus 0.95, OMX-IS drug versus control, P=0.34). Postmortem histologically determined infarct volumes showed excellent concordance with the magnetic resonance imaging defined ischemic lesion volume (bias: 1.33% [95% CI, -15% to 18%). CONCLUSIONS: Intravenous delivery of a novel oxygen carrier is a promising approach to delay infarct progression after ischemic stroke, especially in treating patients with large vessel occlusion stroke who cannot undergo definitive reperfusion therapy within a timely fashion.

Correction: Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

[This corrects the article DOI: 10.1371/journal.pbio.2005924.].

Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

The heart exhibits the highest basal oxygen (O2) consumption per tissue mass of any organ in the body and is uniquely dependent on aerobic metabolism to sustain contractile function. During acute hypoxic states, the body responds with a compensatory increase in cardiac output that further increases myocardial O2 demand, predisposing the heart to ischemic stress and myocardial dysfunction. Here, we test the utility of a novel engineered protein derived from the heme-based nitric oxide (NO)/oxygen (H-NOX) family of bacterial proteins as an O2 delivery biotherapeutic (Omniox-cardiovascular [OMX-CV]) for the hypoxic myocardium. Because of their unique binding characteristics, H-NOX-based variants effectively deliver O2 to hypoxic tissues, but not those at physiologic O2 tension. Additionally, H-NOX-based variants exhibit tunable binding that is specific for O2 with subphysiologic reactivity towards NO, circumventing a significant toxicity exhibited by hemoglobin (Hb)-based O2 carriers (HBOCs). Juvenile lambs were sedated, mechanically ventilated, and instrumented to measure cardiovascular parameters. Biventricular admittance catheters were inserted to perform pressure-volume (PV) analyses. Systemic hypoxia was induced by ventilation with 10% O2. Following 15 minutes of hypoxia, the lambs were treated with OMX-CV (200 mg/kg IV) or vehicle. Acute hypoxia induced significant increases in heart rate (HR), pulmonary blood flow (PBF), and pulmonary vascular resistance (PVR) (p < 0.05). At 1 hour, vehicle-treated lambs exhibited severe hypoxia and a significant decrease in biventricular contractile function. However, in OMX-CV-treated animals, myocardial oxygenation was improved without negatively impacting systemic or PVR, and both right ventricle (RV) and left ventricle (LV) contractile function were maintained at pre-hypoxic baseline levels. These data suggest that OMX-CV is a promising and safe O2 delivery biotherapeutic for the preservation of myocardial contractility in the setting of acute hypoxia.

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active ß-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.

Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles.

We have derived hESC from biopsied blastomeres of cleavage stage embryos under virtually the same conditions we used for the derivation of hESC lines from inner cell mass of blastocyst stage embryos. Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo. To examine whether hESC lines derived from two developmental stages of the embryo differ in gene expression, we have subjected three blastomere-derived hESC lines and two ICM-derived hESC lines grown under identical culture conditions to transcriptome analysis using gene expression arrays. Unlike previously reported comparisons of hESC lines which demonstrated, apart from core hESC-associated pluripotency signature, significant variations in gene expression profiles of different lines, our data show that hESC lines derived and grown under well-controlled defined culture conditions adopt nearly identical gene expression profiles. Moreover, blastomere-derived and ICM-derived hESC exhibited very similar transcriptional profiles independent of the developmental stage of the embryo from which they originated. Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging. These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population.

GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate.

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ∼6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical-basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical-basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation-ßIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell-cell contacts can trigger adoption of a neuronal fate.

Use of human embryonic stem cell-based models for male reproductive toxicity screening.

Male reproductive toxicity examines harmful effects of various agents on all aspects and developmental stages of the male reproductive system, including germ cell development and spermatogenesis. In developing a model for reproductive toxicity screening it is important to define the developmental stage that this model is going to recreate in vitro and to identify critical molecular targets of this stage. In this review we focus our discussion on the potential for using embryonic stem cell (ESC)-derived models for male reproductive toxicity screening. The rationale for developing novel toxicity models is that despite significant advances in our biological understanding and clinical treatment of infertility, many unresolved cases still remain. This is likely due to our lack of knowledge about environmental influences on the critical stages of gamete development. Many practical and ethical difficulties are associated with the collection of human tissue samples to explore the unknown causes of infertility. Thus, a readily available in vitro model that mimics human gamete development would be an extremely valuable research tool for establishing novel toxicity assays. ESC exhibit a high degree of similarity with primordial germ cells (PGC) at the level of gene expression and molecular signaling. In addition, recent evidence shows that ESC can be differentiated into PGC and spermatids in culture. Multiple lines of evidence point to the differences between mouse and human ESC (hESC). In light of these data, we present the case that hESC are better suited as in vitro toxicity screening models than their mouse counterparts. We then describe some of the most promising hESC-based systems that are used today to model certain aspects of male gamete development and that have a potential to be used for toxicity screening. We conclude by discussing the potential of these existing models in toxicology studies and the possibilities for their improvement in the future.

A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen.

Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that "senescent" mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-alpha. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

The senescence-associated secretory phenotype: the dark side of tumor suppression.

Cellular senescence is a tumor-suppressive mechanism that permanently arrests cells at risk for malignant transformation. However, accumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescence-associated secretory phenotype (SASP) that turns senescent fibroblasts into proinflammatory cells that have the ability to promote tumor progression.

Effect of karyotype on successful human embryonic stem cell derivation.

The success rate of human embryonic stem cell (hESC) derivation depends on both culture conditions and embryo quality and is routinely determined by morphological criteria. However, high incidence of chromosomal abnormality even in high-grade cleavage embryos from in vitro fertilization (IVF) patients suggests that the morphological grade of supernumerary embryos obtained from IVF clinics may not be a good prediction factor for successful hESC derivation. We show here that from one donor under identical derivation conditions 12 karyotypically abnormal post-bioptic embryos did not yield hESC lines, whereas two out of four normal embryos did. This suggests that the capacity of embryos to give rise to hESC line is likely to be influenced by their genetic status.

Human embryonic stem cells as a model for embryotoxicity screening.

Reproductive toxicity encompasses harmful effects of various agents on all aspects and stages of the reproductive cycle, including infertility and the induction of adverse effects in the embryo/fetus. In developing a model for reproductive toxicity screening, it is important to define the stage of the human reproductive cycle that this specific model is going to recreate in vitro and to identify molecular targets that are critical for this stage of development. In this review, we focus our discussion on modeling pre-implantation embryotoxicity. The rationale for this is that despite advances on both clinical and biological levels, many unresolved infertility cases may be due to our lack of knowledge regarding environmental influences on this short, but critical stage of development. Data from in vitro fertilization practice suggest that the early-dividing embryo is very sensitive to numerous factors present in its microenvironment. In vivo, as the embryo travels down the oviduct, physical or chemical insults can directly damage the embryo and/or prevent implantation, and cause infertility. Multiple lines of evidence point to the differences between mouse and human pre-implantation development and between mouse and human embryonic stem cells (hESCs). In light of these data we present the case that hESCs and their derivatives are better suited as in vitro models for human pre-implantation development than their mouse counterparts. We then describe some of the most promising hESC-based systems that are used today to model certain aspects of development in the human pre-implantation embryo and that have the potential to be used for embryo toxicity screening tests in the near future. Described systems model two major events during differentiation of the human pre-implantation embryo: differentiation of the trophectoderm and segregation of the inner cell mass into epiblast and hypoblast. The first event is replicated in vitro by triggering either direct or indirect (through embryoid body stage) differentiation into trophectoderm. The second event can be modeled using the recently described system of high-throughput generation of embryoid bodies that recapitulate segregation of inner cell mass. We conclude by discussing the potential of these existing models in toxicology studies and the possibilities for their improvement in the future.

Derivation of human embryonic stem cell lines from biopsied blastomeres on human feeders with minimal exposure to xenomaterials.

In a continuous effort to improve the generation of therapeutic grade human embryonic stem cell (hESC) lines, we focused on preserving developmental capacity of the embryos, minimizing the exposure to xenomaterials, increasing derivation efficacy, and reducing the complexity of the derivation procedure. In this study, we describe an improved method for efficient derivation of hESC lines from blastomeres of biopsied embryos. Our protocol substituted feeder cells of mouse origin with human foreskin fibroblasts (HFFs), limited serum exposure of cells to formation of the initial outgrowth, and increased derivation efficacy from 12.5% (one hESC line out of 13 biopsies) to 50% (3 out of 6 biopsies) by using early population doubling (PD) HFFs. In addition, it eliminated a need for embryo-blastomere coculture, thus reducing the complexity of the culture and enabling continued development of the biopsied embryo under optimal conditions. All derived lines maintained normal karyotype and expressed totipotent phenotype including the ability to differentiate into trophectoderm and all three germ layers.

Efficient method for slow cryopreservation of human embryonic stem cells in xeno-free conditions.

An effective, consistent and xeno-free cryopreservation technique is crucial for any human embryonic stem cell (hESC) laboratory with future perspectives for clinical application. This study presents a new slow freezing-rapid thawing method in serum-free conditions that allows the cryopreservation of a large number of colonies without the use of a programmable freezer. To test its efficacy, this method has been compared with two established vitrification methods and applied to three different hESC lines (H9, VAL-3 and VAL-5). The method is based on an increasing concentration of dimethylsulphoxide (1.0, 1.2, 1.5 and 2.0 mol/l) with a slow or a rapid cooling system. Using this method, approximately 60 colonies per cryovial could be cryopreserved, the survival rate ranged between 15 and 68% depending on the cell line used, and the majority of the surviving colonies were grade A. Post-cryopreserved hESC have been cultured for 20 passages, re-cryopreserved and re-thawed with consistent results. After thawing, cells retained the inherent undifferentiated characteristics of hESC and growth rate curve, with a stable karyotype, telomerase activity and teratoma formation when injected into severe combined immunodeficient animals, which was comparable with the fresh lines. This method has been tested for 3 years in two different laboratories.

A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion.

Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.

Disruption of apical-basal polarity of human embryonic stem cells enhances hematoendothelial differentiation.

During murine development, the formation of tight junctions and acquisition of polarity are associated with allocation of the blastomeres on the outer surface of the embryo to the trophoblast lineage, whereas the absence of polarization directs cells to the inner cell mass. Here, we report the results of ultrastructural analyses that suggest a similar link between polarization and cell fate in human embryos. In contrast, the five human embryonic stem cell (hESC) lines displayed apical-basal, epithelial-type polarity with electron-dense tight junctions, apical microvilli, and asymmetric distribution of organelles. Consistent with these findings, molecules that are components of tight junctions or play regulatory roles in polarization localized to the apical regions of the hESCs at sites of cell-cell contact. The tight junctions were functional, as shown by the ability of hESC colonies to exclude the pericellular passage of a biotin compound. Depolarization of hESCs produced multilayered aggregates of rapidly proliferating cells that continued to express transcription factors that are required for pluripotency at the same level as control cells. However, during embryoid body formation, depolarized cells differentiated predominantly along mesenchymal lineage and spontaneously produced hematoendothelial precursors more efficiently than control ESC. Our findings have numerous implications with regard to strategies for deriving, propagating, and differentiating hESC.

Derivation of hESC from intact blastocysts.

This unit describes protocols for culturing human embryos and deriving human embryonic stem cells from the intact blastocyst. Description of the culturing begins with methods for obtaining human blastocysts using pronuclear or cleavage stage embryos left over after in vitro fertilization. Then there is a description of methods that can be used to derive human embryonic stem cell lines from the blastocyst without trophectoderm removal. Also included is a discussion of the critical steps and parameters such as zona pellucida removal, embryo quality assessment, feeder selection, when and how to transfer early embryonic outgrowths. In addition, there are protocols for embryo thawing, seeding of feeder cells, gelatin coating of plates, cleavage and blastocyst stage embryo grading, preparation and storage of reagents and solutions. Finally, there is a discussion of alternative derivation approaches as well as the timeline for the procedures.

Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders.

OBJECTIVE: To derive new human embryonic stem cell (hESC) lines on pathogen-free human placental fibroblast feeders under serum-free conditions. Because the embryo develops in close contact with extraembryonic membranes, we hypothesized that placental mesenchyme might replicate the stem cell niche in situ. DESIGN: We isolated and characterized human placental fibroblast lines from individual donors and tested their ability to support growth of federally registered hESC lines. Moreover, we performed extensive pathogen testing to ensure their suitability as feeders for the derivation of therapy-grade hESCs. RESULT(S): Human placental fibroblasts were comparable or superior to mouse embryo fibroblasts as hESC feeders. We used these qualified placental fibroblasts to derive two new hESC lines in knockout Dulbecco's modified Eagle's medium with serum-free 20% knockout serum replacement. The cells, which had a normal karyotype, were grown for more than 25 passages, expressed markers of stemness including Oct-3/4, Tra 1-60, Tra 1-80, and SSEA-4, exhibited high telomerase activity, and differentiated in vitro and in vivo into cells derived from all three germ layers, confirming their pluripotency. Additionally, newly derived hESCs were adapted to growth on a human placental laminin substrate in a defined medium. CONCLUSION(S): To our knowledge, this is the first report of hESC derivation in the absence of serum on qualified pathogen-free human feeders.