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WO2010046876A1 - Procédé pour l’identification fonctionnelle d’oligodendrocytes dérivés de svz basé sur la réponse sélective à la thrombine et utilisation de celui-ci - Google Patents

Procédé pour l’identification fonctionnelle d’oligodendrocytes dérivés de svz basé sur la réponse sélective à la thrombine et utilisation de celui-ci Download PDF

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WO2010046876A1
WO2010046876A1 PCT/IB2009/054679 IB2009054679W WO2010046876A1 WO 2010046876 A1 WO2010046876 A1 WO 2010046876A1 IB 2009054679 W IB2009054679 W IB 2009054679W WO 2010046876 A1 WO2010046876 A1 WO 2010046876A1
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svz
oligodendrocytes
thrombin
cells
derived
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Sofia Cristina Soares De Morais Grade
João José OLIVEIRA MALVA
Liliana INÁCIO BERNARDINO
Fabienne Agasse
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Centro de Neurociencias e Biologia Celular
Universidade de Coimbra
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Centro de Neurociencias e Biologia Celular
Universidade de Coimbra
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells

Definitions

  • the present invention discloses a method for the functional identification of SVZ- derived oligodendrocytes based on selective response to thrombin. Evaluation of functional SVZ-derived oligodendrocytes in living cultures is of major interest as it allows the subsequent use of these cells for grafting strategies in demyelinated diseased brains. The method described in the present work may open new perspective for the identification of new pro-oligodendrogenic factors. Summary of the Invention
  • oligodendrocytes are the cells responsible for myelination of axons in the CNS. Wrapping of axons by myelin sheaths ensure the high-speed conduction of nervous impulses. Therefore axonal conduction is dramatically impaired as oligodendrocytes degenerate in multiple sclerosis disease.
  • SVZ cells In the rodent brain, SVZ cells continuously produce olfactory bulb interneurons involved in odor discrimination (for review, see Lledo et al., 2006). Nevertheless, macroglial cells, namely astrocytes and oligodendrocytes, continue to be generated in the perinatal and intact adult SVZ (Levison and Goldman, 1993; Levison et al., 1999; Menn et al. , 2006) as well as in the lesioned adult SVZ upon demyelinating injuries (Nait-Oumesmar et al., 1999 and 2007; Picard-Riera et al., 2002; Menn et al., 2006; Cayre et al., 2006).
  • oligodendrocytes are generated in SVZ cell cultures (Levison and Goldman, 1997; Luskin et al., 1997; Levison et al., 2003), although in a lesser extent as compared to astrocytes, and neurons (Young and Levison, 1996; Weiss et al., 1996; Pluchino et al., 2003).
  • mitotically active progenitors less ramified and expressing the cell surface marker NG2, a sulfated proteoglycan evolve towards post-mitotic oligodendrocytes acquiring the expression of stagespecific markers concomitantly with morphological changes.
  • Glycosphingolipids such as Gal C (galactocerebrosides) and the sulfatide 04 are expressed in immature and mature oligodendrocytes whereas glycoproteins such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin-associated glycoprotein (MAG), are only present in mature oligodendrocytes.
  • oligodendrocytes maturation involves increase in the complexity of the ramification network (for review see Baumann and Pham-Dinh, 2001).
  • the present work describes a rapid method to functionally evaluate oligodendroglial differentiation in SVZ cell cultures.
  • the method is based on monitoring [Ca 2+ J 1 variations following stimulation with 50 mM KCl, 100 ⁇ M histamine or 0.1 U/ml thrombin.
  • increase of [Ca 2+ J 1 following KCl depolarization is a feature of cells belonging to the neuronal lineage whereas histamine induces [Ca 2+ J 1 rises in immature cells (Agasse et al., 2008).
  • the serine protease thrombin is a key player of the blood coagulation process as it converts the soluble fibrinogen into fibrin, triggering blood clothing.
  • thrombin is released as a consequence of stroke, head trauma as well as inflammatory and neurodegenerative processes. Excess of thrombin is deleterious.
  • thrombin signalling is coupled to [Ca 2+ J 1 increase through activation of G protein-coupled protease- activated receptors (PARs).
  • PARs G protein-coupled protease- activated receptors
  • rat oligodendrocytes from OLN93 cell line were shown to increase [Ca 2+ J 1 following thrombin stimulation, an effect mediated through PAR-I activation (Wang et al., 2004).
  • histamine and thrombin we aimed at develop a novel method able to functionally discriminate the major cell types differentiating from SVZ neural stem cell cultures, namely immature cells, progenitors, astrocytes, neurons and oligodendrocytes.
  • oligodendrocytes are rare cells in a SVZ cell culture
  • T3 hormone as a pro-oligodendrogenic factor is quite well described. Indeed, Barres et al.
  • T3 stops cell division of bipotential precursors of both astrocytes type 2 and oligodendrocytes, named oligodendrocyte type 2 astrocytes (O-2A) allowing them to enter in the differentiation stage of oligodendrocytic lineage. Accordingly, T3 favors oligodendrocyte fate in rat embryonic and adult neural stem cells (Johe et al., 1996) as well as in human embryonic stem cells (Murray and Dubois-Dalcq, 1997; Kang et al 2007).
  • T3 not only accelerates the rate of oligodendrocyte precursors generation from O-2A precursors but also the complexity of their morphology towards a mature oligodendrocyte (Murray and Dubois-Dalcq 1997) and the synthesis of myelin proteins (Baas et al., 2002; Franco et al., 2008). As expected, we observed much more 04 positive oligodendrocytes in the T3 treated cultures as compared to the non-treated cultures.
  • the cell surface 04 sulfatide is expressed largely throughout the oligodendrocyte lineage, being the preoligodendrocyte progenitors the only cells that do not present this antigen (Baumann and Pham-Dinh, 2001; Mokry et al., 2008).
  • MAP-2 cells 10 DCX cells, 38 GFAP cells, 8 Nestin cells as well as in 14 04 positive cells.
  • Ratios displayed by MAP-2 and GFAP positive cells are significantly different from the one displayed by 04 oligodendrocytes. Indeed, MAP-2 neurons and GFAP astrocytes present a ratio close to 1 accordingly to the absence of [Ca 2+ ], rises under thrombin or histamine stimulations. 04 oligodendrocytes display a ratio -1.43, consistent with a increase of [Ca 2+ J 1 following thrombin but not histamine perfusion. Nestin positive cells present a low ratio consistent with its strong response to histamine but not to thrombin.
  • thrombin stimulation proves to be a reliable tool to discriminate oligodendrocyte in SVZ cell cultures.
  • Some rare cells were observed to respond both to histamine and thrombin.
  • Increase of [Ca 2+ J 1 following histamine simulation is a characteristic of immature cells such as nestin positive cells.
  • pre-oligodendrocyte progenitors, 04 negative may increase [Ca 2+ ], under both thrombin and histamine stimulations.
  • thrombin triggered-Ca 2+ increase in SVZ- derived oligodendrocytes is mediated by activation of PAR- 1 receptor.
  • the PAR-I agonist TFLLR-NH 2 triggers massive Ca 2+ increase in T3-treated SVZ cells in a similar way as thrombin.
  • specific PAR- 1 antagonist blocks thrombin- induced Ca 2+ increase demonstrating that Ca 2+ increase triggered by thrombin is mediated by PAR-I receptor activation.
  • Ca 2+ increase triggered by TFLLR-NH 2 stimulation is maintained in free calcium perfusion indicating that Ca 2+ is recruited from internal stores in SVZ-derived oligodendrocytes.
  • TFLLR-NH 2 activated signaling involves G q/ ⁇ or Gi 2/ i 3 , that are associated with a subsequent activation of PLC- ⁇ or PLC- ⁇ respectively, both leading to an ultimate increase of [Ca 2 + ⁇ .
  • Co-perfusion of TFLLR-NH 2 and U73122, a general PLC inhibitor suppresses Ca 2 + increase in SVZ-derived oligodendrocytes.
  • FIG. 16 Figure 1 - Experimental protocols performed in SCCI analysis aiming the functional identification/quantification of oligodendrocytes derived from SVZ cell cultures (A), as well as the clarification of the cellular mechanism triggered by activation of thrombin PAR-I receptor (B-E).
  • A oligodendrocytes derived from SVZ cell cultures
  • B-E the clarification of the cellular mechanism triggered by activation of thrombin PAR-I receptor
  • Cells loaded with the Ca 2+ probe FURA-2AM were continuously perfused in Krebs solution and stimuli were applied at different time intervals as shown by the time sequences.
  • E Throm/Hist ratios for each cell phenotype. *** p ⁇ 0.001 using unpaired Student's t test, as compared to MAP-2, DCX, GFAP cultured astrocytes and Nestin.
  • FIG. 3 Calcium increase in oligodendrocyte-like cells upon thrombin stimulation is mediated by PAR-I receptor activation.
  • A Bar graph represents means of peaks of normalized ratios of fluorescence at 340/380 following stimulation with thrombin, or TFLLR-NH 2 , or thrombin together with TFLLR-NH 2 . *** p ⁇ 0.001 using unpaired Student's t test, as compared to thrombin.
  • B Representative profiles of response elicited by oligodendrocyte-like cells upon perfusion accordingly to the previously presented protocol (see Figure 1 A and B).
  • FIG. 4 Calcium signalling downstream PAR-I activation in SVZ-derived oligodendrocytes. Bar graph represents means of peaks of normalized ratios of fluorescence at 340/380 displayed by SVZ-derived oligodendrocytes following perfusion accordingly to the previously presented protocol (see Figure 1 A, C, D, E).
  • FIG. 20 Figure 5 - Functional evaluation of the percentage of oligodendrocyte-like responding cells.
  • A Bar graph depicts percentage of oligodendrocyte-like responding cells in SVZ control cultures and in controls exposed to 30 nM T3 for 7-10 days. Negative controls were performed in hippocampal neuronal cultures as well as cortical astrocyte cultures. Data are show as means ⁇ SEM. *** p ⁇ 0.001 using unpaired Student's t test as compared to control cultures. In neuronal and astrocytic cultures, no oligodendrocyte-like responding cells were found.
  • B Representative profiles of response elicited upon perfusion accordingly to the previously presented protocol (see Figure 1 A). Detailed Description of the Invention
  • SVZ cells were obtained from 1 to 3-day old C57B1/6 donor mice. Briefly, brains were removed following decapitation and placed in Hanks' balanced salt solution (HBSS). Meninges were gently removed and the brains sectioned in 450 ⁇ m coronal slices from where fragments of SVZ were dissected out.
  • HBSS Hanks' balanced salt solution
  • the cell suspension was diluted in serum- free culture medium (SFM) composed of Dulbecco's modified Eagle's medium/Ham's F- 12 medium with GlutaMAX-I, supplemented with 100 U/ml penicillin, 100 ⁇ g ⁇ il streptomycin, 1% B27, 10 ng ⁇ il epidermal growth factor (EGF) and 10 ng ⁇ il basic fibroblast growth factor (FGF-2) (all from Gibco).
  • SFM serum- free culture medium
  • EGF epidermal growth factor
  • FGF-2 basic fibroblast growth factor
  • the neurospheres were allowed to develop during 7 to 10 days at 37 oC in the absence or the presence of 30 nM 3,3',5-triiodo-L-thyronine (T3; Fluka) or 20 ng/ml stem cell factor (SCF; Chemicon International, Temecula, CA), to direct the differentiation of the progenitor cells towards an oligodendroglial or a neuronal fate, respectively, as seen previousl y by others (Murray and Dubois-Dalcq, 1997, Jin et al. 2002). Following the treatment period, SCCI experiments and immunostaining protocols were performed. Hippocampal neuronal culture
  • cells were mechanically dissociated in HBSS.
  • the cells were maintained in serum-free Neurobasal medium, supplemented with 2% B27 supplement, 25 ⁇ M glutamate, 200 ⁇ M glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • Cells were plated on poly-D-lysine coated coverslips placed within 12- well cell culture plates (75 000 cells/well). The cells were kept in a humidified incubator of 95% air-5% CO 2 , for 7 to 8 days before SCCI analysis.
  • Neurobasal medium supplemented with 2% B27, 200 ⁇ M glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin, without glutamate.
  • Cortical astrocytic culture was changed every four days for Neurobasal medium supplemented with 2% B27, 200 ⁇ M glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin, without glutamate.
  • KCl causes neuronal depolarization and subsequent increase of [Ca 2 + ]j in neurons, histamine stimulation leads to an increase of [Ca 2+ ], in stem/progenitors cells (Agasse et al., 2008), and thrombin induces an intracellular rise of calcium in oligodendroglial cells (Wang et al., 2004).
  • SVZ cultures were loaded for 40 min, at 37 0 C, with 5 ⁇ M Fura-2 AM (Molecular Probes, Eugene, OR, USA) in Krebs solution (132 mM NaCl, 4 mM KCl, 1.4 mM MgCl 2 , 2.5 mM CaCl 2 , 6 mM glucose, 10 mM HEPES, pH 7.4) plus 0.1% fatty acid free bovine serum albumin (BSA) and 0.02% pluronic acid F- 127.
  • BSA bovine serum albumin
  • the coverslips were then washed for 10 min at room temperature in Krebs solution to remove the probe that remained outside the cells, and subsequently mounted on RC-20 chamber in a PH3 platform (Warner Instruments) on the stage of an inverted fluorescence microscope Axiovert 200 (Carl Zeiss). Cells were continuously perfused with Krebs solution and stimulated at defined periods of time by 50 mM KCl, 100 ⁇ M histamine and 0,1 U/ml thrombin. Solutions were applied to the cells in the chamber by a fast-pressurized system (95% air-5% CO 2 atmosphere) (AutoMate Scientific, Inc).
  • [29] [Ca 2+ ] was assessed by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation (750 msec) at 340 nm and 380 nm, using a Lambda DG4 apparatus (Sutter Instruments Company), and a 510 nm bandpass filter (Carl Zeiss ref. 21). Fluorescence acquisition was given by a 4Ox objective and a Coll SNAP digital camera (Roper Scientific). Approximately 100 cells per field were analyzed and the corresponding acquired values were processed using the MetaFluor software (Universal Imaging Corporation).
  • mice monoclonal anti-O4 (1:100, Chemicon International
  • mouse monoclonal anti-MAP-2 (1:200; Sigma)
  • rabbit monoclonal anti-GFAP (1:100; Sigma
  • mouse monoclonal anti- nestin (1:200; Chemicon International)
  • rabbit polyclonal anti-doublecortin (1:200, Cell Signaling, Danvers; MA) and goat polyclonal PAR-I (1:200; Santa Cruz Biotechnology).
  • coverslips were washed 2-3 times in PBS, and subsequently incubated for Ih at room temperature (RT) with the proper secondary antibodies diluted in PBS: goat anti-mouse Alexa Fluor 594 (1:200, Molecular Probes), goat anti-rabbit Alexa Fluor 488 (1:200, Molecular Probes) and rabbit anti-goat Alexa Fluor 594.
  • the cells were incubated for 5 minutes at RT, with Hoechst 33342 (2 ⁇ g/ml, Molecular Probes) in PBS, for nuclear staining.
  • the coverslips were mounted in Dakocytomation fluorescent medium (Dakocytomation Inc., CA). Fluorescent images were recorded using a digital camera coupled to an Axioskop microscope (Carl Zeiss, Gottingen, Germany). Data Analysis
  • KCl evokes a [Ca 2+ ], increase in neurons but not in immature cells whilst histamine leads to an [Ca 2+ ], increase in immature cells but not in neurons. It was also shown in the same work that neither KCl nor histamine lead to [Ca 2+ ], rise in astrocytes. Nonetheless, in this study, we didn't search for the effect of KCl or histamine pulses on intracellular calcium variations in oligodendrocytes. Herein, we characterize the intracellular calcium profile of oligodendrocytes upon perfusion with KCl and histamine.
  • thrombin as a possible Ca2 + -associated stimulus for SVZ-derived oligodendrocytes as this protease was described to induce transient intracellular calcium rises in OLN-93 cells, an oligodendrocytic cell lineage (Wang et al., 2004). We therefore hypothesize that thrombin may work as a tool for the functional identification of oligodendrocytes derived from SVZ cells.
  • SVZ neurospheres obtained from newborn mice.
  • the spheres were grown over 6 to 8 days in proliferative conditions, and subsequently seeded on poly-D-lysine coverslips, remaining in the absence of growth factors for 7 to 10 days.
  • cells emerge continuously from the spheres and differentiate in neurons, astrocytes, and oligodendrocytes although in a minor extent, forming a heterogeneous pseudo-monolayer of diverse cells in various stages of maturation.
  • spheres were treated with 30 nM 3,3',5-triiodo-L-thyronine (T3) during the differentiation period in order to obtain a SVZ culture highly enriched in oligodendroglia among the progeny.
  • T3 3,3',5-triiodo-L-thyronine
  • Throm/Hist values between 04 oligodendrocytes and other cell types, as well as between nestin immature cells and other cell types were statistically different. According to these findings, SVZ-derived oligodendrocytes can be functionally discriminated among other cells in SVZ cell cultures. Based on these data we concluded that, in our cultures, cells that present Throm/Hist above 1.3 are oligodendrocytes. Thrombin-induced Ca 2+ increase in SVZ-derived oligodendrocytes is mediated by PAR-I activation
  • Thrombin receptors are included in the protease-activated receptors (PARs) family, characterized by a particularly interesting process of activation, that involves 1) recognition of the receptor by the enzyme, 2) irreversible cleavage of the receptor exodomain at a specific enzymatic site, 3) consequent exposure of a new N-terminus that acts as a tethered ligand, 4) intramolecular binding of the tethered ligand, activating the body of the receptor 5) initiation of PAR signalling.
  • PARs protease-activated receptors
  • the PARs family is composed of four receptors, PAR-I, PAR-2, PAR-3 and PAR-4.
  • PAR-I, PAR-3 and PAR-4 are activated upon cleavage by the serine protease thrombin.
  • Many of the effects of thrombin are mediated by PAR-I, being the most extensively studied subtype and the prototype of the family.
  • PAR-4 exodomain lacks the carboxyl-terminal sequence that confers high- affinity binding to thrombin, consequently PAR-4 requires higher concentrations of thrombin for activation compared with the other thrombin receptors (Kahn et al., 1999).
  • PAR-3 does not appear to signal by itself having a possible cofactor role in the cleavage and activation of PAR-4 (Nakanishi-Matsui et al., 2000). Based on this knowledge we firstly focused on PAR-I expression and function in our SVZ cultures. Our hypothesis was also supported by the finding that PAR- 1 is the only thrombin receptor functionally expressed in the oligodendrocyte cell line OLN-93, where it was associated with an increase of [Ca 2+ J 1 evoked by thrombin. Besides, the same authors showed that primary cultured oligodendrocytes express high levels of PAR-I but almost undetectable levels of PAR-3 (Wang et al., 2004).
  • PAR-I is a transmembrane metabotropic G protein-coupled receptor (GPCR). Particularly, PAR-I can couple to members of the G12/13, G q/ ⁇ and G l/o families of large heterotrimeric GTPases (for review see Coughlin 2000). To clarify the role of these GTPases in the thrombin-induced [Ca 2+ J 1 rise, we incubated T3 treated SVZ cells with 200 ng/ml PTX, well known to inactivate of Gi/Go proteins, the last 24h before the SCCI ( Figure 1 D).
  • GPCR G protein-coupled receptor
  • the signalling cascade triggered by TFLLR- NH 2 includes the PTX-insensitive proteins G q/ ⁇ or G 12 /G 13 .
  • G q/ ii activates phospholipase C isoform ⁇ (PLC ⁇ ) that catalyzes phosphoinositide hydrolysis, leading to Ca 2+ mobilization from the intracellular compartments, and protein kinase C (PKC) activation.
  • PLC protein kinase C
  • G 12 /G 13 binds small monomeric GTPases of the Ras and Rho families that dually activate PLC isoform ⁇ (PLC ⁇ ), also resulting in Ca 2+ recruitment and PKC activation (Seifert et al., 2008).
  • SVZ culture with a T3 3OnM treated culture both after 7-10 days after deposition.
  • a control culture presents -4% of cells responding with a thrombin/hist ratio above 1.3 (3.65 ⁇ 1.54; 14 coverslips analyzed) whereas a T3 treated culture contains -35% of these cells (34.62 + 8.22; 10 coverslips analyzed) ( Figure 5).
  • SCCI experiments including the three subsequent pulses, KCl, histamine and thrombin were performed in defined cultures of hippocampal neurons and also in cultures of cortical astrocytes. Both cultures presented no cells with the established ratio Throm/Hist>1.3.
  • SCF treated SVZ cultures displayed neuronal profile, given by a low ratio Hist/KCl established by Agasse et al. 2008, having -1.4% cells with a Thromb/Hist ratio (1.4 ⁇ 0.98; 5 coverslips analyzed), characteristic of oligodendrocytes (data not shown).
  • Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 103(6):879-887. [55] Kang S, Cho MS, Seo H, Yoon CJ, Oh SK, Choi YM, Kim D. (2007) Efficient induction of oligodendrocytes from human embryonic stem cells. Embryonic Stem
  • Neural stem cells in the subventricular zone are a source of astrocytes and oligodendrocytes, but not microglia.
  • Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J. Neurosci. 16:7599-609. [76] Young GM, Levison SW. (1996) Persistence of multipotential progenitors in the juvenile rat subventricular zone. Dev Neurosci. 18:255-65.

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Abstract

Les cellules souches neurales de la zone sous-ventriculaire (SVZ) conduisent à des progéniteurs ayant la capacité à se différencier en les principaux types de cellules du SNC, comprenant les oligodendrocytes formant de la myéline. Dans les présents travaux, nous avons examiné si des oligodendrocytes dérivés de SVZ vivants pouvaient être fonctionnellement discriminés sur la base des variations du taux de calcium libre intracellulaire ([Ca2+]i) après des stimulations par KCl 50 mM, histamine 100 μM ou thrombine 0,1 U/ml. À cette fin, des cultures de cellules SVZ, dérivées de souris donneuses P 1-3 C57B1/6 néonatales, sont traitées avec l’hormone tri-iodo-L-thyronine (T3) afin d’augmenter la différenciation en oligodendrocytes. Notre groupe a précédemment démontré que des neurones dérivés de SVZ et des cellules immatures peuvent être discriminés par leur augmentation sélective de ([Ca2+]i) déclenchée par des stimulations par KCl 50 mM et histamine 100 μM respectivement. Dans la présente étude, nous démontrons que des oligodendrocytes 04-positifs, comme observé précédemment dans des astrocytes, ne répondent pas à l’un quelconque de ces stimuli. Cependant, les oligodendrocytes 04-positifs présentent une augmentation spécifique de ([Ca2+]i) après stimulation avec la thrombine. Les taux de ([Ca2+]i) ne sont pas modifiés par perfusion de thrombine dans des cellules nestine-immatures, des astrocytes et des neurones. Par conséquent, les oligodendrocytes peuvent être différenciés des autres types de cellules dérivées de SVZ sur la base du rapport sélectif de réponse après stimulation avec la thrombine et l’histamine. L’augmentation de calcium induite par la thrombine dans des oligodendrocytes dérivés de SVZ est véhiculée par le récepteur activé par la protéase 1 (PAR-1) et met en œuvre l’activation en aval de phospholipase C (PLC) avec recrutement consécutif de calcium à partir des réserves internes. La différenciation oligodendrogliale dans des cultures de cellules SVZ est principalement analysée par immunocytochimie étant donné qu’il n’existe pas d’outil fiable permettant l’évaluation de leur différenciation fonctionnelle. L’évaluation d’oligodendrocytes dérivés de SVZ fonctionnels dans des cultures vivantes est d’un intérêt majeur étant donné qu’elle permet l’utilisation consécutive de ces cellules pour des stratégies de greffage dans des cerveaux ayant une maladie de démyélinisation. Le procédé décrit dans la présente invention peut ouvrir de nouvelles perspectives pour l’identification de nouveaux facteurs pro-oligodendrogéniques.
PCT/IB2009/054679 2008-10-22 2009-10-22 Procédé pour l’identification fonctionnelle d’oligodendrocytes dérivés de svz basé sur la réponse sélective à la thrombine et utilisation de celui-ci Ceased WO2010046876A1 (fr)

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WO2013021196A2 (fr) 2011-08-08 2013-02-14 University Of Coimbra Différenciation d'oligodendrocytes

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WO2001085797A1 (fr) * 2000-05-10 2001-11-15 Mayo Foundation For Medical Education & Research Anticorps igm humains permettant de declencher la remyelinisation, et utilisations de ceux-ci a des fins diagnostiques et therapeutiques, plus particulierement dans le systeme nerveux central
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013021196A2 (fr) 2011-08-08 2013-02-14 University Of Coimbra Différenciation d'oligodendrocytes

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