Differentiation of human induced pluripotent stem cells towards notochordal-like cells: the role of tissue source

INTRODUCTION: Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and they are considered an exciting target for cell-based therapy. However, NCs are scarcely available as they are lost early in life, and attempts at in vivoexpansion have failed because NCs lose their specific phenotype. The production of Notochordal-like cells (NLCs) from human induced pluripotent stem cells (iPSCs) is a viable alternative. However, current attempts have been challenged by the low differentiation efficiency into the NC lineage. Therefore, the aim of this study was to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD progenitor cells (TIE2+-cells) to improve hiPSC differentiation towards mature, matrix-producing NLCs. METHODS: hiPSCs were generated from TIE2⁺ cells of three adult donors. As a comparison, donormatched minimally invasive peripheral blood mononuclear (PBM) cell-derived iPSCs were used. Firstly, the iPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)¹. Thereafter, the cells were further driven towards the NClineage by transfection with synthetic NOTO mRNA¹ and further matured using a 3D pellet culture in discogenic medium containing 10ng/mL TGF-β1. Read-out parameters included cell morphology, gene and protein expression and matrix deposition. RESULTS: Both TIE2⁺ and PBM cell-derived hiPSC showed successful differentiation towards mesendodermal progenitor cells following Wnt activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt-target genes (LEF1, NODAL) and mesendodermal markers (TBXT, FOXA2, TBX6) was observed compared to mTESR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. At day 3, we confirmed a 9-fold increase in NOTO mRNA levels after transfection in all donor lines. At day 28, the appearance of vacuolated NLCs was observed in both TIE2⁺ and PBM cell-derived pellet cultures confirming successful commitment towards the NC-lineage. Interestingly, while DMMB-assay detected GAG deposition in both lines, a significant increase in GAG content was seen in the TIE2⁺ cell-derived pellets. DISCUSSION & CONCLUSIONS: Tissue-specific TIE2⁺ cell-derived iPSCs may allow for an improved iPSNLC differentiation efficiency, indicated by the increased potency for deposition of GAG-rich matrix. Detailed analysis of the phenotypic markers and matrix deposited at the end of the 28 day maturation is ongoing to further document the phenotype of these iPS-NLCs. Delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the differences in their differentiation capacity. REFERENCES: ¹Colombier et al., 202

Dietary Cholesterol-Induced Post-Testicular Infertility

This work shows that an overload of dietary cholesterol causes complete infertility in dyslipidemic male mice (the Liver X Receptor-deficient mouse model). Infertility resulted from post-testicular defects affecting the fertilizing potential of spermatozoa. Spermatozoa of cholesterol-fed lxr−/− animals were found to be dramatically less viable and motile, and highly susceptible to undergo a premature acrosome reaction. We also provide evidence, that this lipid-induced infertility is associated with the accelerated appearance of a highly regionalized epididymal phenotype in segments 1 and 2 of the caput epididymidis that was otherwise only observed in aged LXR-deficient males. The epididymal epithelial phenotype is characterized by peritubular accumulation of cholesteryl ester lipid droplets in smooth muscle cells lining the epididymal duct, leading to their transdifferentiation into foam cells that eventually migrate through the duct wall, a situation that resembles the inflammatory atherosclerotic process. These findings establish the high level of susceptibility of epididymal sperm maturation to dietary cholesterol overload and could partly explain reproductive failures encountered by young dyslipidemic men as well as ageing males wishing to reproduce

Study of spinal cord blood flow after spinal cord injury with intact dura mater

Après un traumatisme de la moelle épinière (TM), l’ischémieest un facteur d’aggravation des lésions. Cette ischémie peut être aggravée par l’augmentation depression du liquide cérébro-spinal (LCS) par le biais d’un effet tamponnade. Or chez l’homme,après un TM avec préservation de l’intégrité de la dure-mère, la pression de LCS augmentesignificativement. On suppose donc que le maintien d’une pression de LCS à des valeursphysiologique pourrait être une méthode de limitation de l’ischémie post-traumatique et doncd’amélioration du pronostic fonctionnel. Afin de pouvoir réaliser une étude expérimentale de cesphénomènes, nous avons consacré la première partie expérimentale de cette thèse à la mise au pointd’un modèle de TM à dure-mère intacte chez le rat permettant la mesure simultanée de la pressionde LCS et de la perfusion médullaire. Nous avons confirmé expérimentalement que la pression deLCS augmente après TM. Dans la seconde partie expérimentale, nous avons mis au point unetechnique expérimentale de quantification spatiale et temporelle de la perfusion médullaire grâce àl’échographie de contraste. Cette technique permettait aussi un suivi en temps réel de l’évolution dusaignement intra-parenchymateux induit par le TM. Dans la troisième partie expérimentale, nousavons utilisé notre modèle couplé avec l’échographie de contraste et le laser Doppler pour évaluerles effets de la noradrénaline injectée à la phase aigüe d’un TM sur la perfusion médullaire et lesaignement intra-parenchymateux. Nous avons montré que la noradrénaline augmentait trèslégèrement le flux sanguin superficiel mais pas le flux sanguin profond et qu’elle augmentait lataille du saignement.After spinal cord injury (SCI), ischaemia aggravates lesions.Increase in cerebrospinal fluid (CSF) pressure can worsens ischaemia through a tamponnade effect.In humans, it has been shown that after SCI with intact dura mater, CSF pressure significantlyincreases. Therefore, preserving CSF pressure within a physiological range may limit post-traumaischaemia and improve neurological outcome. In order to experimentally study these phenomenon,we have dedicated the first part of that work to create a model of SCI in rats preserving dura’sintegrity and allowing simultaneous measurement of spinal cord blood flow (SCBF) and CSFpressure. We have confirmed that CSF pressure increases after SCI with intact dura. In the secondexperimental part, we have developed a technique allowing to perform spatial and temporalmeasurement of SCBF thanks to contrast enhanced ultrasonography (CEU). Moreover, thistechnique allows real-time measurement of the size of the parenchymal hemorrhage. In the thirdexperimental part, we have used our experimental model in association with CEU and LaserDoppler to assess the effects of early injection of norepinephrine on SCBF and parenchymalhemorrhage. We found that norepinephrine induces a slight increase in superficial SCBF while itdoesn’t modify deep SCBF and significantly increases the size of parenchymal hemorrhage

Quand le langage émerge... (Partie 1)

Objectif : première partie d'un ensemble de deux articles, elle vise à mettre en évidence les processus et les caractéristiques de l'émergence et de l'acquisition du langage dans le développement normal de l'enfant et celui à risque autistique. Méthode : revue critique de la littérature clinique et linguistique. Résultats/discussion : après avoir discuté des expériences interactionnelles (attention conjointe, imitation, constitution de formats...) et des différents éléments du langage chez l'enfant (prosodie, lexique, morphosyntaxe, pragmatique), nous observerons des particularités de ces derniers dans le développement de l'enfant à risque autistique

Quand le langage émerge... (Partie 2)

Objectif : illustrer l'intérêt prospectif du bilan initial et l'utilité du travail orthophonique en très petit groupe auprès d'enfants présentant un trouble envahissant du développement. Méthode : présentation de deux cas cliniques. Résultats/discussion : l'évolution différentielle du retard de communication est discutée à l'aide des observations faites lors des entretiens psychologiques, de l'échelle du CHAT, de l'épreuve du Brunet-Lézine, de l'évaluation orthophonique et des observations cliniques faites en séance d'orthophonie

Hinged elbow fixator: An extracorporeal technique to position thehinge based on an original guidewire device

International audienceThe application of a hinged elbow external fixator is technically demanding because the hinge axis must coincide exactly with theflexion–extension axis of the elbow. The standard technique involves inserting a 3-mm K-wire freehand into the distal humerus to materialize theflexion–extension axis. We designed a guidewire device for extracorporeal hinge positioning without K-wire insertion. In a cadaver study, wecompared freehand K-wire insertion and our extracorporeal technique.Methods. – In 12 cadaveric elbows, we induced acute elbow instability by sectioning the medial collateral ligament complex and the anterior andposterior capsule. A hinged external fixator was applied to each elbow using both techniques. The outcome measures were procedure duration,number of image-intensifier shots (as a measure of radiation exposure), and passive motion range after fixator implantation.Results. – Compared with the freehand K-wire technique, the extracorporeal technique provided greater range of motion and significantly lowervalues for procedure duration and number of image-intensifier shots. Data dispersion was less marked with the extracorporeal technique, indicatingbetter reproducibility.Conclusion. – The extracorporeal technique based on a guidewire device enabled non-invasive positioning of a hinged elbow external fixator. Thistechnique was faster, less irradiating, and more reproducible than the freehand K-wire technique

Effect of norepinephrine on spinal cord blood flow and parenchymal hemorrhage size in acute-phase experimental spinal cord injury.

International audiencePURPOSE: In the acute phase of spinal cord injury (SCI), ischemia and parenchymal hemorrhage are believed to worsen the primary lesions induced by mechanical trauma. To minimize ischemia, keeping the mean arterial blood pressure above 85 mmHg for at least 1 week is recommended, and norepinephrine is frequently administered to achieve this goal. However, no experimental study has assessed the effect of norepinephrine on spinal cord blood flow (SCBF) and parenchymal hemorrhage size. We have assessed the effect of norepinephrine on SCBF and parenchymal hemorrhage size within the first hour after experimental SCI. METHODS: A total of 38 animals were included in four groups according to whether SCI was induced and norepinephrine injected. SCI was induced at level Th10 by dropping a 10-g weight from a height of 10 cm. Each experiment lasted 60 min. Norepinephrine was started 15 min after the trauma. SCBF was measured in the ischemic penumbra zone surrounding the trauma epicenter using contrast-enhanced ultrasonography. Hemorrhage size was measured repeatedly on parasagittal B-mode ultrasonography slices. RESULTS: SCI was associated with significant decreases in SCBF (P = 0.0002). Norepinephrine infusion did not significantly modify SCBF. Parenchymal hemorrhage size was significantly greater in the animals given norepinephrine (P = 0.0002). CONCLUSION: In the rat, after a severe SCI at the Th10 level, injection of norepinephrine 15 min after SCI does not modify SCBF and increases the size of the parenchymal hemorrhage

Rat model of spinal cord injury preserving dura mater integrity and allowing measurements of cerebrospinal fluid pressure and spinal cord blood flow.

International audiencePURPOSES: Cerebrospinal fluid (CSF) pressure elevation may worsen spinal cord ischaemia after spinal cord injury (SCI). We developed a rat model to investigate relationships between CSF pressure and spinal cord blood flow (SCBF). METHODS: Male Wistar rats had SCI induced at Th10 (n = 7) or a sham operation (n = 10). SCBF was measured using laser-Doppler and CSF pressure via a sacral catheter. Dural integrity was assessed using subdural methylene-blue injection (n = 5) and myelography (n = 5). RESULTS: The SCI group had significantly lower SCBF (p < 0.0001) and higher CSF pressure (p < 0.0001) values compared to the sham-operated group. Sixty minutes after SCI or sham operation, CSF pressure was 8.6 ± 0.4 mmHg in the SCI group versus 5.5 ± 0.5 mmHg in the sham-operated group. No dural tears were found after SCI. CONCLUSION: Our rat model allows SCBF and CSF pressure measurements after induced SCI. After SCI, CSF pressure significantly increases

Projet COPAIRNIC

National audienc

Projet COPAIRNIC

National audienc