Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

<p>Abstract</p> <p>Background</p> <p>The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin⁺Sox2⁺neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium).</p> <p>Results</p> <p>Here we report the isolation and long term propagation of another population of Nestin⁺cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges.</p> <p>Conclusion</p> <p>Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.</p

Pratiques anesthésiques pour prélèvement d'organes chez le sujet en mort encéphalique (influence sur le pronostic du greffon rénal)

MONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Isolation and Culture of Precursor Cells from the Adult Human Spinal Cord

International audienceWe demonstrated the presence of neural stem cells and/or progenitor cells in the adult human spinal cord. This chapter provides materials and methods to harvest high-quality samples of thoracolumbar, lumbar, and sacral adult human spinal cord and human dorsal root ganglia isolated from brain-dead patients who had agreed before passing to donate their bodies to science for therapeutic and scientific advances. The methods to culture precursor cells from the adult human spinal cord are also described

Coherent Anti-Stokes Raman Scattering Microscopy: A Label-Free Method to Compare Spinal Cord Myelin in Different Species

International audienceMany histological techniques are used to identify and characterize myelin in the mammalian nervous system. Due to the high content of lipids in myelin sheaths, coherent anti-stokes Raman scattering (CARS) microscopy is a label-free method that allows identifying myelin within tissues. CARS excites the CH 2 vibrational mode at 2845 cm −1 and CH 2 bonds are found in lipids. In this study, we have used CARS for a new biological application in the field of spinal cord analysis. We have indeed compared several parameters of spinal cord myelin sheath in three different species, i.e ., mouse, lemur, and human using a label-free method. In all species, we analyzed the dorsal and the lateral funiculi of the adult thoracic spinal cord. We identified g-ratio differences between species. Indeed, in both funiculi, g-ratio was higher in mice than in the two primate species, and the myelin g-ratio in lemurs was higher than in humans. We also detected a difference in g-ratio between the dorsal and the lateral funiculi only in humans. Furthermore, species differences between axon and fiber diameters as well as myelin thickness were observed. These data may reflect species specificities of conduction velocity of myelin fibers. A comparison of data obtained by CARS imaging and fluoromyelin staining, a method that, similar to CARS, does not require resin embedding and dehydration, displays similar results. CARS is, therefore, a label-free alternative to other microscopy techniques to characterize myelin in healthy and neurological disorders affecting the spinal cord

Persistence of FoxJ1+ Pax6+ Sox2+ ependymal cells throughout life in the human spinal cord

International audienceEpendymal cells lining the central canal of the spinal cord play a crucial role in providing a physical barrier and in the circulation of cerebrospinal fluid. These cells express the FOXJ1 and SOX2 transcription factors in mice and are derived from various neural tube populations, including embryonic roof and floor plate cells. They exhibit a dorsal-ventral expression pattern of spinal cord developmental transcription factors (such as MSX1, PAX6, ARX, and FOXA2), resembling an embryonic-like organization. Although this ependymal region is present in young humans, it appears to be lost with age. To re-examine this issue, we collected 17 fresh spinal cords from organ donors aged 37-83 years and performed immunohistochemistry on lightly fixed tissues. We observed cells expressing FOXJ1 in the central region in all cases, which co-expressed SOX2 and PAX6 as well as RFX2 and ARL13B, two proteins involved in ciliogenesis and cilia-mediated sonic hedgehog signaling, respectively. Half of the cases exhibited a lumen and some presented portions of the spinal cord with closed and open central canals. Co-staining of FOXJ1 with other neurodevelopmental transcription factors (ARX, FOXA2, MSX1) and NESTIN revealed heterogeneity of the ependymal cells. Interestingly, three donors aged > 75 years exhibited a fetal-like regionalization of neurodevelopmental transcription factors, with dorsal and ventral ependymal cells expressing MSX1, ARX, and FOXA2. These results provide new evidence for the persistence of ependymal cells expressing neurodevelopmental genes throughout human life and highlight the importance of further investigation of these cells

Microbial contamination and tissue procurement location: A conventional operating room is not mandatory. An observational study.

BackgroundStandard operating rooms (SOR) are assumed to be the best place to prevent microbial contamination when performing tissue procurement. However, mobilizing an operating room is time and cost consuming if no organ retrieval is performed. In such case, non-operating dedicated rooms (NODR) are usually recommended by European guidelines for tissue harvesting. Performing the tissue retrieval in the Intensive care unit (ICU) when possible might be considered as it allows a faster and simpler procedure.ObjectiveOur primary objective was to study the relationship between the risk of microbial contamination and the location (ICU, SOR or NODR) of the tissue retrieval in heart-beating and non-heart-beating deceased donors.Materials and methodWe retrospectively reviewed all deceased donors' files of the local tissue banks of Montpellier and Marseille from January 2007 to December 2014. The primary endpoint was the microbial contamination of the grafts. We built a multivariate regression model and used a GEE (generalized estimating equations) allowing us to take into account the clustered structure of our data.Results2535 cases were analyzed involving 1027 donors. The retrieval took place for 1189 in a SOR, for 996 in a hospital mortuary (NODR) and for 350 in an ICU. 285 (11%) microbial contaminations were revealed. The multivariate analysis found that the location in a hospital mortuary was associated with a lower risk of contamination (OR 0.43, 95% CI [0.2-0.91], p = 0.03). A procurement performed in the ICU was not associated with a significant increased risk (OR 0.62, 95% CI [0.26-1.48], p = 0.4).ConclusionAccording to our results, performing tissue procurement in dedicated non-sterile rooms could decrease the rate of allograft tissue contamination. This study also suggests that in daily clinical practice, transferring patients from ICU to SOR for tissue procurement could be avoided as it does not lead to less microbial contamination

The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

International audienceThe anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non-small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions

A Cellular Taxonomy of the Adult Human Spinal Cord

The mammalian spinal cord functions as a community of glial and neuronal cell types to accomplish sensory processing, autonomic control, and movement; conversely, the dysfunction of these cell types following spinal cord injury or disease states can lead to chronic pain, paralysis, and death. While we have made great strides in understanding spinal cellular diversity in animal models, it is crucial to characterize human biology directly to uncover specialized features of basic function and to illuminate human pathology. Here, we present a cellular taxonomy of the adult human spinal cord using single nucleus RNA-sequencing with spatial transcriptomics and antibody validation. We observed 29 glial clusters, including rare cell types such as ependymal cells, and 35 neuronal clusters, which we found are organized principally by anatomical location. To demonstrate the potential of this resource for understanding human disease, we analyzed the transcriptome of spinal motoneurons that are prone to degeneration in amyotrophic lateral sclerosis (ALS) and other diseases. We found that, compared with all other spinal neurons, human motoneurons are defined by genes related to cell size, cytoskeletal structure, and ALS, thereby supporting a model of a specialized motoneuron molecular repertoire that underlies their selective vulnerability to disease. We include a publicly available browsable web resource with this work, in the hope that it will catalyze future discoveries about human spinal cord biology