In vitro and in vivo models define a molecular signature reference for human embryonic notochordal cells.

Understanding the emergence of human notochordal cells (NC) is essential for the development of regenerative approaches. We present a comprehensive investigation into the specification and generation of bona fide NC using a straightforward pluripotent stem cell (PSC)-based system benchmarked with human fetal notochord. By integrating in vitro and in vivo transcriptomic data at single-cell resolution, we establish an extended molecular signature and overcome the limitations associated with studying human notochordal lineage at early developmental stages. We show that TGF-ß inhibition enhances the yield and homogeneity of notochordal lineage commitment in vitro. Furthermore, this study characterizes regulators of cell-fate decision and matrisome enriched in the notochordal niche. Importantly, we identify specific cell-surface markers opening avenues for differentiation refinement, NC purification, and functional studies. Altogether, this study provides a human notochord transcriptomic reference that will serve as a resource for notochord identification in human systems, diseased-tissues modeling, and facilitating future biomedical research.

Recommendations for intervertebral disc notochordal cell investigation: From isolation to characterization.

Background: Lineage-tracing experiments have established that the central region of the mature intervertebral disc, the nucleus pulposus (NP), develops from the embryonic structure called "the notochord". However, changes in the cells derived from the notochord which form the NP (i.e., notochordal cells [NCs]), in terms of their phenotype and functional identity from early developmental stages to skeletal maturation are less understood. These key issues require further investigation to better comprehend the role of NCs in homeostasis and degeneration as well as their potential for regeneration. Progress in utilizing NCs is currently hampered due to poor consistency and lack of consensus methodology for in vitro NC extraction, manipulation, and characterization. Methods: Here, an international group has come together to provide key recommendations and methodologies for NC isolation within key species, numeration, in vitro manipulation and culture, and characterization. Results: Recommeded protocols are provided for isolation and culture of NCs. Experimental testing provided recommended methodology for numeration of NCs. The issues of cryopreservation are demonstrated, and a pannel of immunohistochemical markers are provided to inform NC characterization. Conclusions: Together we hope this article provides a road map for in vitro studies of NCs to support advances in research into NC physiology and their potential in regenerative therapies.

Two-aperture measurements for GEO-feeder adaptive optics pre-compensation optimization.

We present a method to estimate the pre-compensation phase of ground-to-geostationary orbit (GEO) optical links based on downlink phase and log-amplitude measurements from two ground apertures. This method allows us to reduce the point-ahead anisoplanatism that currently limits the telecom performance of GEO-feeder links. It is shown to reduce the anisoplanatic phase variance by 50%, hence improving the statistics of the coupled flux aboard the satellite. It also outperforms the one-aperture estimation method for very severe atmospheric conditions. Besides, only low-resolution amplitude measurements are required on the second aperture to reach the performance of the novel estimator.

Tbit/s line-rate satellite feeder links enabled by coherent modulation and full-adaptive optics.

Free-space optical (FSO) communication technologies constitute a solution to cope with the bandwidth demand of future satellite-ground networks. They may overcome the RF bottleneck and attain data rates in the order of Tbit/s with only a handful of ground stations. Here, we demonstrate single-carrier Tbit/s line-rate transmission over a free-space channel of 53.42 km between the Jungfraujoch mountain top (3700 m) in the Swiss Alps and the Zimmerwald Observatory (895 m) near the city of Bern, achieving net-rates of up to 0.94 Tbit/s. With this scenario a satellite-ground feeder link is mimicked under turbulent conditions. Despite adverse conditions high throughput was achieved by employing a full adaptive optics system to correct the distorted wavefront of the channel and by using polarization-multiplexed high-order complex modulation formats. It was found that adaptive optics does not distort the reception of coherent modulation formats. Also, we introduce constellation modulation - a new four-dimensional BPSK (4D-BPSK) modulation format as a technique to transmit high data rates under lowest SNR. This way we show 53 km FSO transmission of 13.3 Gbit/s and 210 Gbit/s with as little as 4.3 and 7.8 photons per bit, respectively, at a bit-error ratio of 1 ∙ 10-3. The experiments show that advanced coherent modulation coding in combination with full adaptive optical filtering are proper means to make next-generation Tbit/s satellite communications practical.

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale.

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. µs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.

Phase estimation at the point-ahead angle for AO pre-compensated ground to GEO satellite telecoms.

We present a new method to estimate the off-axis adaptive optics pre-compensation phase of a ground to GEO satellite telecom link suffering from point-ahead anisoplanatism. The proposed phase estimator relies on the downlink phase and log-amplitude measurements that are available at the optical ground station. We introduce the analytical tools, extended from the literature, to build the estimator as well as a general modal formalism to express the reciprocal residual phase covariance matrix resulting from any estimation linear with measurements. We use this residual phase covariance matrix to generate independent coupled flux samples thanks to a pseudo-analytical approach and study the gain offered by the proposed estimator on the coupled flux statistics, in various atmospheric conditions. The estimator is shown to reduce the anisoplanatic residual phase variance by at least 35%, and 46% at best, with a greater impact on the lower modes, especially on the tip and tilt residual phase variances. The phase variance reduction brings a gain up to 15 dB on the cumulative density function at probability 10-3. This gain should allow to relax the power constraints on the link budget at the OGS and renews the interest in large aperture diameter (60 cm class telescopes) for GEO Feeder links by reducing the atmospheric turbulence impact on the uplink coupled signal.

Feasibility demonstration of AO pre-compensation for GEO feeder links in a relevant environment.

Optical technologies are extremely competitive candidates to achieve very-high throughput links between ground and GEO satellites; however, their feasibility relies on the ability to mitigate channel impairments due to atmospheric turbulence. For that purpose, Adaptive Optics (AO) has already proved to be highly efficient on the downlink. However, for the uplink, anisoplanatism induced by point-ahead angle (PAA) compromises AO pre-compensation efficiency to an extent that depends on propagation conditions. The ability to properly assess the anisoplanatism impact in a wide variety of conditions is thus critical in designing the optical ground terminals. In this paper, we demonstrate the consistency of experimental coupled flux statistics with results coming from performance and end-to-end models, on an AO pre-compensated 13 km slant path in Tenerife. This validation is demonstrated in a wide variety of turbulence conditions, hence consolidating propagation channel models that are of critical importance for the reliability of future GEO feeder links. We then compare experimental results to theoretical on-sky performance, and discuss to what extent such slant path or horizontal path experiments can be representative of real GEO links.

An immunohistochemical analysis of fibroblasts in giant cell arteritis.

BACKGROUND: Giant cell arteritis (GCA) is a systemic vasculitis of large and medium vessels characterized by an inflammatory arterial infiltrate. GCA begins in the adventitia and leads to vascular remodeling by promoting proliferation of myofibroblasts in the intima. The morphology of the fibroblasts in the adventitia in GCA is unclear. Access to temporal artery biopsies allows morphological studies and evaluation of the microenvironment of the arterial wall. We evaluated the distribution of vascular fibroblasts and of markers of their activation in GCA. METHODS: Formalin-fixed paraffin-embedded tissue sections from 29 patients with GCA and 36 controls were examined. Immunohistochemistry was performed for CD90, vimentin, desmin, alpha-smooth muscle actin (ASMA), prolyl-4-hydroxylase (P4H), and myosin to evaluate the distribution of fibroblasts within the intima, media, and adventitia. RESULTS: Temporal arteries from patients with GCA showed increased levels of CD90, vimentin, and ASMA in the adventitia and intima compared to the controls. Desmin was expressed only in the media in both groups. P4H was expressed similarly in the adventitia and intima in the two groups. Adventitial and intimal CD90+ cells co-expressed P4H, ASMA, and myosin at a high level in GCA. CONCLUSION: The results suggest a role for adventitial fibroblasts in GCA. Inhibiting the differentiation of adventitial fibroblasts to myofibroblasts has therapeutic potential for GCA.

Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients.

Modelling rare neurogenetic diseases to develop new therapeutic strategies is highly challenging. The use of human-induced pluripotent stem cells (hiPSCs) is a powerful approach to obtain specialized cells from patients. For hereditary peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) Type II, spinal motor neurons (MNs) are impaired but are very difficult to study. Although several protocols are available to differentiate hiPSCs into neurons, their efficiency is still poor for CMT patients. Thus, our goal was to develop a robust, easy, and reproducible protocol to obtain MNs from CMT patient hiPSCs. The presented protocol generates MNs within 20 days, with a success rate of 80%, using specifically chosen molecules, such as Sonic Hedgehog or retinoic acid. The timing and concentrations of the factors used to induce differentiation are crucial and are given hereby. We then assessed the MNs by optic microscopy, immunocytochemistry (Islet1/2, HB9, Tuj1, and PGP9.5), and electrophysiological recordings. This method of generating MNs from CMT patients in vitro shows promise for the further development of assays to understand the pathological mechanisms of CMT and for drug screening.

NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate.

The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation.

Statistical properties of single-mode fiber coupling of satellite-to-ground laser links partially corrected by adaptive optics.

In the framework of satellite-to-ground laser downlinks, an analytical model describing the variations of the instantaneous coupled flux into a single-mode fiber after correction of the incoming wavefront by partial adaptive optics (AO) is presented. Expressions for the probability density function and the cumulative distribution function as well as for the average fading duration and fading duration distribution of the corrected coupled flux are given. These results are of prime interest for the computation of metrics related to coded transmissions over correlated channels, and they are confronted by end-to-end wave-optics simulations in the case of a geosynchronous satellite (GEO)-to-ground and a low earth orbit satellite (LEO)-to-ground scenario. Eventually, the impact of different AO performances on the aforementioned fading duration distribution is analytically investigated for both scenarios.

SCO-spondin oligopeptide inhibits angiogenesis in glioblastoma.

Angiogenesis plays a critical role in glioblastoma growth and progression. We therefore aimed at evaluating the anti-angiogenic properties of an oligopeptide originating from SCO-spondin (NX) on a model of human glioblastoma. To this end, we studied the impact of NX treatment on human brain endothelial cells (HBMECs) alone or co-cultured with glioblastoma cells (U87-MG) on apoptosis, proliferation, migration and release of angiogenic factors. We further investigated the anti-angiogenic potential of NX on human glioblastoma cells grown on chorio-allantoic membrane (CAM) or in glioblastoma xenografts. The results of our experiments showed that NX treatment impaired the microvascular network and induced a decrease in cell proliferation, vascularization and tumor growth in the CAM model as well as in xenotransplants. Interestingly, our in vitro experiments showed that NX impairs HBMECs migration but also regulates the release of angiogenic factors from U87-MG. These results are confirmed by the profiling of NX-treated U87-MG grown on CAM that highlighted modifications of several genes involved in angiogenesis. In conclusion, NX inhibits tumorigenesis by impairing the ability of glioblastoma cells to induce angiogenesis and by inhibiting endothelial cell migration. This molecule might therefore be an interesting candidate for future cancer therapies.

Isolation of Astrocytes Displaying Myofibroblast Properties and Present in Multiple Sclerosis Lesions.

A wide heterogeneity of lesions can affect the central nervous system (CNS). In all situations where neurons are damaged, including multiple sclerosis (MS), a common reactive astrocytosis is present. Sedimentation field-flow fractionation (SdFFF) was used to sort astrocyte subpopulations. After SdFFF elution, cells, prepared from rat newborn cortex, were cultured and analyzed by immunocytofluorescence for glial fibrillary acidic protein (GFAP) and α-smooth muscle (SM) actin (a specific marker for myofibroblasts) expression. Cell contractile capacity was studied. Samples from patients with MS were also analyzed. Three main fractions (F1, F2, and F3) were isolated and compared with the total eluted population (TP). TP, F1, F2, and F3, contained respectively 74, 96, 12, and 98% of GFAP expressing astrocytes. In F3, astrocytes only expressed GFAP while in F1, astrocytes expressed both GFAP and α-SM actin. In F2 and TP, α-SM actin expression was barely detected. F3-derived cells showed higher contractile capacities compared with F1-derived cells. In one specific case of MS known as Baló's concentric MS, astrocytes expressing both GFAP and α-SM actin were detected. Using SdFFF, a population of astrocytes presenting myofibroblast properties was isolated. This subpopulation of astrocytes was also observed in a MS sample suggesting that it could be involved in lesion formation and remodeling during CNS pathologies.

TrkB-containing exosomes promote the transfer of glioblastoma aggressiveness to YKL-40-inactivated glioblastoma cells.

The neurotrophin receptors are known to promote growth and proliferation of glioblastoma cells. Their functions in spreading glioblastoma cell aggressiveness to the microenvironment through exosome release from glioblastoma cells are unknown. Considering previous reports demonstrating that YKL-40 expression is associated with undifferentiated glioblastoma cancer stem cells, we used YKL-40-silenced cells to modulate the U87-MG differentiated state and their biological aggressiveness. Herein, we demonstrated a relationship between neurotrophin-receptors and YKL-40 expression in undifferentiated cells. Differential functions of cells and derived-exosomes were evidenced according to neurotrophin receptor content and differentiated cell state by comparison with control pLKO cells. YKL-40 silencing of glioblastoma cells impairs proliferation, neurosphere formation, and their ability to induce endothelial cell (HBMEC) migration. The modulation of differentiated cell state in YKL-40-silenced cells induces a decrease of TrkB, sortilin and p75NTR cellular expressions, associated with a low-aggressiveness phenotype. Interestingly, TrkB expressed in exosomes derived from control cells was undetectable in exosomes from YKL-40 -silenced cells. The transfer of TrkB-containing exosomes in YKL-40-silenced cells contributed to restore cell proliferation and promote endothelial cell activation. Interestingly, in U87 MG xenografted mice, TrkB-depleted exosomes from YKL-40-silenced cells inhibited tumor growth in vivo. These data highlight that TrkB-containing exosomes play a key role in the control of glioblastoma progression and aggressiveness. Furthermore, TrkB expression was detected in exosomes isolated from plasma of glioblastoma patients, suggesting that this receptor may be considered as a new biomarker for glioblastoma diagnosis.

New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation.

Human induced pluripotent stem cells (hiPSc) are a very useful solution to create and observe the behavior of specific and usually inaccessible cells, such as human motor neurons. Obtained from a patient biopsy by reprograming dermal fibroblasts (DF), hiPSc present the same properties as embryonic stem cells and can generate any cell type after several weeks of differentiation. Today, there are numerus protocols which aim to control hiPSC differentiation. The principal challenge is to obtain a sufficiently enriched specific cell population to study disease pathophysiology and to provide a good model for further investigation and drug screening. The differentiation process is very costly and time-consuming, because many specific factors and different culture media must be used. In this study, we used Sedimentation Field Flow Fractionation (SdFFF) to prepare enriched populations derived from hiPSc after only 10 days of culture in a classical medium. Based on phenotypic and proteomic characterization, "hyperlayer" elution resulted in a fraction expressing markers of endothelial progenitors while another fraction expressed markers of neural progenitors. The isolation of subpopulations representing various differentiation lineages is of major interest for the production of specialized, cell-enriched fractions and in the preparation of increasingly complex models for the development of new therapeutic tools.

Neural Stem Cell Properties of an Astrocyte Subpopulation Sorted by Sedimentation Field-Flow Fractionation.

Astrocytes encompass a heterogeneous cell population. Using sedimentation field-flow fractionation (SdFFF) method, different, almost pure, astrocyte subpopulations were isolated. Cells were collected from cortex of newborn rats and sorted by SdFFF to obtain different fractions, which were subjected to protein analysis and characterized by immunocytofluorescence. The behavior of the cells was analyzed in vitro, under culture conditions used for neural stem cells. These culture conditions were also applied to cells derived from an adult cortical tissue after traumatic brain injury (TBI). Finally, the astrocytic neural stem-like cells were transplanted in damaged sciatic nerve. Protein analysis indicated a high expression of glial fibrillary acidic protein (GFAP) and vimentin in fraction F3-derived cells. These cells formed neurospheres when cultured with epidermal growth factor and large colonies in a collagen-containing semi-solid matrix. Neurospheres expressed GFAP and nestin and were able in addition to generate neurons expressing MAP2 and oligodendrocytes expressing Olig2. When transplanted in a damaged nerve, cells of F3-derived neurospheres colonized the damaged area. Finally, after TBI in adult rats, cells able to form neurospheres containing a subpopulation of astrocytes expressing vimentin were obtained. Using the SdFFF method, an astrocyte subpopulation presenting stem cell properties was isolated from a newborn rat cortex and from an injured adult rat cortex. The specific activation of this astrocyte subpopulation may provide a potential therapeutic approach to restore lost neuronal function in injured or diseased brain.

Laser beam complex amplitude measurement by phase diversity.

The control of the optical quality of a laser beam requires a complex amplitude measurement able to deal with strong modulus variations and potentially highly perturbed wavefronts. The method proposed here consists in an extension of phase diversity to complex amplitude measurements that is effective for highly perturbed beams. Named camelot for Complex Amplitude MEasurement by a Likelihood Optimization Tool, it relies on the acquisition and processing of few images of the beam section taken along the optical path. The complex amplitude of the beam is retrieved from the images by the minimization of a Maximum a Posteriori error metric between the images and a model of the beam propagation. The analytical formalism of the method and its experimental validation are presented. The modulus of the beam is compared to a measurement of the beam profile, the phase of the beam is compared to a conventional phase diversity estimate. The precision of the experimental measurements is investigated by numerical simulations.

Characterization of Endoneurial Fibroblast-like Cells from Human and Rat Peripheral Nerves.

Endoneurial fibroblast-like cells (EFLCs) are one of the cell populations present in the peripheral nervous system. The role and immunophenotypic characteristics of EFLCs are not well known and led us to perform a histological and cytological study of EFLCs in normal human and rat peripheral nerves. We found that all EFLCs express CD34, neural/glial antigen 2 (NG2), and prolyl-4-hydrolase-beta. In addition, half of the EFLCs in normal peripheral nerves express platelet-derived growth factor receptor-ß (PDGFR-ß) and some also express the intermediate filament nestin in vivo (at a lower level than Schwann cells, which express high levels of nestin). Using cell cultures of purified EFLCs, we characterized subpopulations of EFLCs expressing PDGFR-ß alone or PDGFR-ß and nestin. Experimental nerve lesions in rat resulted in an increase in nestin-positive EFLCs, which returned to normal levels after 8 days. This suggests that some EFLCs could have a different proliferative and/or regenerative potential than others, and these EFLCs may play a role in the initial phase of nerve repair. These "activated" EFLCs share some immunophenotypic similarities with pericytes and Interstitial cells of Cajal, which have progenitor cell potentials. This raises the questions as to whether a proportion of EFLCs have a possible role as endoneurial progenitor cells.

In vitro 3D angiogenesis assay in egg white matrix: comparison to Matrigel, compatibility to various species, and suitability for drug testing.

In vitro angiogenesis assays are commonly used to assess pro- or anti-angiogenic drug properties. Extracellular matrix (ECM) substitutes such as Matrigel and collagen gel became very popular in in vitro 3D angiogenesis assays as they enable tubule formation by endothelial cells from culture or aortic rings. However, these assays are usually used with a single cell type, lacking the complex cellular interactions occurring during angiogenesis. Here, we report a novel angiogenesis assay using egg white as ECM substitute. We found that, similar to Matrigel, egg white elicited prevascular network formation by endothelial and/or smooth muscle cell coculture. This matrix was suitable for various cells from human, mouse, and rat origin. It is compatible with aortic ring assay and also enables vascular and tumor cell coculture. Through simple labeling (DAPI, Hoechst 33258), cell location and resulting prevascular network formation can easily be quantified. Cell transfection with green fluorescent protein improved whole cell visualization and 3D structure characterization. Finally, egg-based assay dedicated to angiogenesis studies represents a reliable and cost-effective way to produce and analyze data regarding drug effects on vascular cells.

Experimental demonstration of the full-wave iterative compensation in free space optical communications.

Long-range free space optical communications suffer from atmospheric turbulence effects. To mitigate them, a bidirectional full-wave compensation technique seems promising. We present an experimental implementation and characterization of this concept on a laboratory breadboard. Experimental results confirm former numerical results for similar propagation conditions. The effects of measurement and control errors are analyzed by numerical modeling.