Measurement of quantum back action in the audio band at room temperature.

Quantum mechanics places a fundamental limit on the precision of continuous measurements. The Heisenberg uncertainty principle dictates that as the precision of a measurement of an observable (for example, position) increases, back action creates increased uncertainty in the conjugate variable (for example, momentum). In interferometric gravitational-wave detectors, higher laser powers reduce the position uncertainty created by shot noise (the photon-counting error caused by the quantum nature of the laser) but necessarily do so at the expense of back action in the form of quantum radiation pressure noise (QRPN)1. Once at design sensitivity, the gravitational-wave detectors Advanced LIGO2, VIRGO3 and KAGRA4 will be limited by QRPN at frequencies between 10 hertz and 100 hertz. There exist several proposals to improve the sensitivity of gravitational-wave detectors by mitigating QRPN5-10, but until now no platform has allowed for experimental tests of these ideas. Here we present a broadband measurement of QRPN at room temperature at frequencies relevant to gravitational-wave detectors. The noise spectrum obtained shows effects due to QRPN between about 2 kilohertz and 100 kilohertz, and the measured magnitude of QRPN agrees with our model. We now have a testbed for studying techniques with which to mitigate quantum back action, such as variational readout and squeezed light injection7, with the aim of improving the sensitivity of future gravitational-wave detectors.

Continuously tunable modulation scheme for homodyne detection and state tomography.

Homodyne detection is a common self-referenced technique to extract optical quadratures. Due to ubiquitous fluctuations, experiments measuring optical quadratures require homodyne angle control. Current homodyne angle locking techniques only provide high quality error signals in a span significantly smaller than π radians, the span required for full state tomography, leading to inevitable discontinuities during full tomography. Here, we present and demonstrate a locking technique using a universally tunable modulator which produces high quality error signals at an arbitrary homodyne angle. Our work enables continuous full-state tomography and paves the way to backaction evasion protocols based on a time-varying homodyne angle.

Laser power stabilization via radiation pressure.

This Letter reports the experimental realization of a novel, to the best of our knowledge, active power stabilization scheme in which laser power fluctuations are sensed via the radiation pressure driven motion they induce on a movable mirror. The mirror position and its fluctuations were determined by means of a weak auxiliary laser beam and a Michelson interferometer, which formed the in-loop sensor of the power stabilization feedback control system. This sensing technique exploits a nondemolition measurement, which can result in higher sensitivity for power fluctuations than direct, and hence destructive, detection. Here we used this new scheme in a proof-of-concept experiment to demonstrate power stabilization in the frequency range from 1 Hz to 10 kHz, limited at low frequencies by the thermal noise of the movable mirror at room temperature.

Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration.

PURPOSE: Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA), but lacked detailed analysis of retinal structure and function in the treated area. DESIGN: Phase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov identifier, NCT01469832). PARTICIPANTS: Twelve participants with advanced Stargardt disease (STGD1), the most common cause of macular degeneration in children and young adults. METHODS: Subretinal transplantation of up to 200 000 hESC-derived RPE cells with systemic immunosuppressive therapy for 13 weeks. MAIN OUTCOME MEASURES: The primary end points were the safety and tolerability of hESC-derived RPE cell administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure and function using microperimetry and spectral-domain OCT. RESULTS: Focal areas of subretinal hyperpigmentation developed in all participants in a dose-dependent manner in the recipient retina and persisted after withdrawal of systemic immunosuppression. We found no evidence of uncontrolled proliferation or inflammatory responses. Borderline improvements in best-corrected VA in 4 participants either were unsustained or were matched by a similar improvement in the untreated contralateral eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In one instance at the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Participant-reported quality of life using the 25-item National Eye Institute Visual Function Questionnaire indicated no significant change. CONCLUSIONS: Subretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of detailed analysis of spatial correlation of retinal structure and function in determining with appropriate sensitivity the impact of cell transplantation and suggest that intervention in early stage of disease should be approached with caution. Given the slow rate of progressive degeneration at this advanced stage of disease, any protection against further deterioration may be evident only after a more extended period of observation.

Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies.

BACKGROUND: Since they were first derived more than three decades ago, embryonic stem cells have been proposed as a source of replacement cells in regenerative medicine, but their plasticity and unlimited capacity for self-renewal raises concerns about their safety, including tumour formation ability, potential immune rejection, and the risk of differentiating into unwanted cell types. We report the medium-term to long-term safety of cells derived from human embryonic stem cells (hESC) transplanted into patients. METHODS: In the USA, two prospective phase 1/2 studies were done to assess the primary endpoints safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium in nine patients with Stargardt's macular dystrophy (age >18 years) and nine with atrophic age-related macular degeneration (age >55 years). Three dose cohorts (50,000, 100,000, and 150,000 cells) were treated for each eye disorder. Transplanted patients were followed up for a median of 22 months by use of serial systemic, ophthalmic, and imaging examinations. The studies are registered with ClinicalTrials.gov, numbers NCT01345006 (Stargardt's macular dystrophy) and NCT01344993 (age-related macular degeneration). FINDINGS: There was no evidence of adverse proliferation, rejection, or serious ocular or systemic safety issues related to the transplanted tissue. Adverse events were associated with vitreoretinal surgery and immunosuppression. 13 (72%) of 18 patients had patches of increasing subretinal pigmentation consistent with transplanted retinal pigment epithelium. Best-corrected visual acuity, monitored as part of the safety protocol, improved in ten eyes, improved or remained the same in seven eyes, and decreased by more than ten letters in one eye, whereas the untreated fellow eyes did not show similar improvements in visual acuity. Vision-related quality-of-life measures increased for general and peripheral vision, and near and distance activities, improving by 16-25 points 3-12 months after transplantation in patients with atrophic age-related macular degeneration and 8-20 points in patients with Stargardt's macular dystrophy. INTERPRETATION: The results of this study provide the first evidence of the medium-term to long-term safety, graft survival, and possible biological activity of pluripotent stem cell progeny in individuals with any disease. Our results suggest that hESC-derived cells could provide a potentially safe new source of cells for the treatment of various unmet medical disorders requiring tissue repair or replacement. FUNDING: Advanced Cell Technology.

Platelet bioreactor-on-a-chip.

Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition,micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs.

First Measurements of High Frequency Cross-Spectra from a Pair of Large Michelson Interferometers.

Measurements are reported of the cross-correlation of spectra of differential position signals from the Fermilab Holometer, a pair of colocated 39 m long, high power Michelson interferometers with flat broadband frequency response in the MHz range. The instrument obtains sensitivity to high frequency correlated signals far exceeding any previous measurement in a broad frequency band extending beyond the 3.8 MHz inverse light-crossing time of the apparatus. The dominant but uncorrelated shot noise is averaged down over 2×10^{8} independent spectral measurements with 381 Hz frequency resolution to obtain 2.1×10^{-20}m/sqrt[Hz] sensitivity to stationary signals. For signal bandwidths Δf>11 kHz, the sensitivity to strain h or shear power spectral density of classical or exotic origin surpasses a milestone PSD_{δh}

Transcription elongation factor Tcea3 regulates the pluripotent differentiation potential of mouse embryonic stem cells via the Lefty1-Nodal-Smad2 pathway.

Self-renewal and pluripotency are hallmark properties of pluripotent stem cells, including embryonic stem cells (ESCs) and iPS cells. Previous studies revealed the ESC-specific core transcription circuitry and showed that these core factors (e.g., Oct3/4, Sox2, and Nanog) regulate not only self-renewal but also pluripotent differentiation. However, it remains elusive how these two cell states are regulated and balanced during in vitro replication and differentiation. Here, we report that the transcription elongation factor Tcea3 is highly enriched in mouse ESCs (mESCs) and plays important roles in regulating the differentiation. Strikingly, altering Tcea3 expression in mESCs did not affect self-renewal under nondifferentiating condition; however, upon exposure to differentiating cues, its overexpression impaired in vitro differentiation capacity, and its knockdown biased differentiation toward mesodermal and endodermal fates. Furthermore, we identified Lefty1 as a downstream target of Tcea3 and showed that the Tcea3-Lefty1-Nodal-Smad2 pathway is an innate program critically regulating cell fate choices between self-replication and differentiation commitment. Together, we propose that Tcea3 critically regulates pluripotent differentiation of mESCs as a molecular rheostat of Nodal-Smad2/3 signaling.

Embryonic stem cell trials for macular degeneration: a preliminary report.

BACKGROUND: It has been 13 years since the discovery of human embryonic stem cells (hESCs). Our report provides the first description of hESC-derived cells transplanted into human patients. METHODS: We started two prospective clinical studies to establish the safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium (RPE) in patients with Stargardt's macular dystrophy and dry age-related macular degeneration--the leading cause of blindness in the developed world. Preoperative and postoperative ophthalmic examinations included visual acuity, fluorescein angiography, optical coherence tomography, and visual field testing. These studies are registered with ClinicalTrials.gov, numbers NCT01345006 and NCT01344993. FINDINGS: Controlled hESC differentiation resulted in greater than 99% pure RPE. The cells displayed typical RPE behaviour and integrated into the host RPE layer forming mature quiescent monolayers after transplantation in animals. The stage of differentiation substantially affected attachment and survival of the cells in vitro after clinical formulation. Lightly pigmented cells attached and spread in a substantially greater proportion (>90%) than more darkly pigmented cells after culture. After surgery, structural evidence confirmed cells had attached and continued to persist during our study. We did not identify signs of hyperproliferation, abnormal growth, or immune mediated transplant rejection in either patient during the first 4 months. Although there is little agreement between investigators on visual endpoints in patients with low vision, it is encouraging that during the observation period neither patient lost vision. Best corrected visual acuity improved from hand motions to 20/800 (and improved from 0 to 5 letters on the Early Treatment Diabetic Retinopathy Study [ETDRS] visual acuity chart) in the study eye of the patient with Stargardt's macular dystrophy, and vision also seemed to improve in the patient with dry age-related macular degeneration (from 21 ETDRS letters to 28). INTERPRETATION: The hESC-derived RPE cells showed no signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or apparent rejection after 4 months. The future therapeutic goal will be to treat patients earlier in the disease processes, potentially increasing the likelihood of photoreceptor and central visual rescue. FUNDING: Advanced Cell Technology.

Human embryonic stem cell lines derived from single blastomeres.

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos.

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres.

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.

Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence.

Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony-forming capability. After further differentiation into erythroid cells, >1,000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low-density lipoprotein and forming capillary-vascular-like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods.

Human Hemangioblast-Derived Mesenchymal Stem Cells Promote Islet Engraftment in a Minimal Islet Mass Transplantation Model in Mice.

Islet transplantation can restore glycemic control in patients with type 1 diabetes. Using this procedure, the early stages of engraftment are often crucial to long-term islet function, and outcomes are not always successful. Numerous studies have shown that mesenchymal stem cells (MSCs) facilitate islet graft function. However, experimental data can be inconsistent due to variables associated with MSC generation (including donor characteristics and tissue source), thus, demonstrating the need for a well-characterized and uniform cell product before translation to the clinic. Unlike bone marrow- or adipose tissue-derived MSCs, human embryonic stem cell-derived-MSCs (hESC-MSCs) offer an unlimited source of stable and highly-characterized cells that are easily scalable. Here, we studied the effects of human hemangioblast-derived mesenchymal cells (HMCs), (i.e., MSCs differentiated from hESCs using a hemangioblast intermediate), on islet cell transplantation using a minimal islet mass model. The co-transplantation of the HMCs allowed a mass of islets that was insufficient to correct diabetes on its own to restore glycemic control in all recipients. Our in vitro studies help to elucidate the mechanisms including reduction of cytokine stress by which the HMCs support islet graft protection in vivo. Derivation, stability, and scalability of the HMC source may offer unique advantages for clinical applications, including fewer islets needed for successful islet transplantation.

Biologic properties and enucleation of red blood cells from human embryonic stem cells.

Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (10(10)-10(11) cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESC-derived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult beta-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of beta-globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 mum. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale.

Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration.

Assessments of safety and efficacy are crucial before human ESC (hESC) therapies can move into the clinic. Two important early potential hESC applications are the use of retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration and Stargardt disease, an untreatable form of macular dystrophy that leads to early-onset blindness. Here we show long-term functional rescue using hESC-derived RPE in both the RCS rat and Elov14 mouse, which are animal models of retinal degeneration and Stargardt, respectively. Good Manufacturing Practice-compliant hESC-RPE survived subretinal transplantation in RCS rats for prolonged periods (>220 days). The cells sustained visual function and photoreceptor integrity in a dose-dependent fashion without teratoma formation or untoward pathological reactions. Near-normal functional measurements were recorded at >60 days survival in RCS rats. To further address safety concerns, a Good Laboratory Practice-compliant study was carried out in the NIH III immune-deficient mouse model. Long-term data (spanning the life of the animals) showed no gross or microscopic evidence of teratoma/tumor formation after subretinal hESC-RPE transplantation. These results suggest that hESCs could serve as a potentially safe and inexhaustible source of RPE for the efficacious treatment of a range of retinal degenerative diseases.

Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications.

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications.

alpha-Thalassemia-like globin gene expression by primitive erythrocytes derived from human embryonic stem cells.

Under culture conditions that promote hematopoietic differentiation, human embryonic stem cells (huESC) give rise to primitive erythroid cells that closely resemble the nucleated erythrocytes of early-stage human embryos. The globin chain distribution of these cells is similar to that seen during the embryonic and fetal stages of development. Here we show that huESC-derived erythroid cells produce substantial quantities of homotetrameric hemoglobin (Hb) composed exclusively of gamma-globin-containing subunits. The globin synthesis of these erythroid cells was also significantly unbalanced, with a substantial decrease of alpha-like globin chain synthesis in relation to that of their beta-like globins, a pattern characteristically associated with alpha-thalassemia (alpha-thal). This pattern of unbalanced globin synthesis appears to be an inherent feature of human erythroid cells that synthesize predominantly embryonic-stage globins.

Next-generation stem cells - ushering in a new era of cell-based therapies.

Naturally occurring stem cells isolated from humans have been used therapeutically for decades. This has primarily involved the transplantation of primary cells such as haematopoietic and mesenchymal stem cells and, more recently, derivatives of pluripotent stem cells. However, the advent of cell-engineering approaches is ushering in a new generation of stem cell-based therapies, greatly expanding their therapeutic utility. These next-generation stem cells are being used as 'Trojan horses' to improve the delivery of drugs and oncolytic viruses to intractable tumours and are also being engineered with angiogenic, neurotrophic and anti-inflammatory molecules to accelerate the repair of injured or diseased tissues. Moreover, gene therapy and gene editing technologies are being used to create stem cell derivatives with improved functionality, specificity and responsiveness compared with their natural counterparts. Here, we review these engineering approaches and areas in which they will help broaden the utility and clinical applicability of stem cells.

Efficient differentiation of functional hepatocytes from human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) to specific functional cell types can be achieved using methods that mimic in vivo embryonic developmental programs. Current protocols for generating hepatocytes from hESCs are hampered by inefficient differentiation procedures that lead to low yields and large cellular heterogeneity. We report here a robust and highly efficient process for the generation of high-purity (70%) hepatocyte cultures from hESCs that parallels sequential hepatic development in vivo. Highly enriched populations of definitive endoderm were generated from hESCs and then induced to differentiate along the hepatic lineage by the sequential addition of inducing factors implicated in physiological hepatogenesis. The differentiation process was largely uniform, with cell cultures progressively expressing increasing numbers of hepatic lineage markers, including GATA4, HNF4alpha, alpha-fetoprotein, CD26, albumin, alpha-1-antitrypsin, Cyp7A1, and Cyp3A4. The hepatocytes exhibited functional hepatic characteristics, such as glycogen storage, indocyanine green uptake and release, and albumin secretion. In a mouse model of acute liver injury, the hESC-derived definitive endoderm differentiated into hepatocytes and repopulated the damaged liver. The methodology described here represents a significant step toward the efficient generation of hepatocytes for use in regenerative medicine and drug discovery.