Caractérisation des précurseurs neuraux dérivés de cellules pluripotentes induites in vitro et in vivo après transplantation dans le système nerveux central démyélinisé

Induced pluripotent stem cell-derived neural precursor cells (iPS-NPCs) may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases and can serve as suitable tools to model myelin disorders or to test the potential of pharmacological compounds. So far the therapeutic potential of these cells was approached in neonatal conditions. However, the repair efficacy and safety of these cells in the demyelinated adult central nervous system (CNS), a condition associated with decreased cell plasticity and scaring, remains to be well addressed. Moreover, whether the therapeutic behavior of these pluripotent-derived cells resembles that of physiologically committed CNS-derived precursors remains elusive. First, I used mouse iPS-NPCs and compared them side-by-side to embryonic CNS-derived cells, in vitro and in vivo after engraftment in models of adult spinal cord demyelination. My data revealed the prominent capacity of survival, safe integration, migration and timely differentiation of the grafted cells into mature oligodendrocytes. Grafted cells generated compact myelin around host axons, restoring nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Second, to validate the functionality of human iPS-NPC-derived glial precursors, I transplanted them in newborn and adult models of dys/demyelination. My data showed widespread migration, integration and extensive generation of functional oligodendrocytes ensheathing host axons, forming compact myelin while reconstructing nodes of Ranvier both in newborn grafted and adult demyelination contexts.Les précurseurs neuraux dérivés de cellules souches pluripotentes induites (iPS-NPCs) peuvent représenter la source cellulaire autologue idéale pour la thérapie cellulaire visant à promouvoir la remyélinisation et la neuroprotection des maladies de la myéline. Jusqu'à présent, le potentiel thérapeutique de ces cellules a été abordé dans des conditions néonatales. Cependant, l'efficacité de la réparation et de la sécurité de ces cellules dans le système nerveux central (SNC), une condition associée à une diminution de la plasticité cellulaire et effarouchement, reste à être bien traités. D'ailleurs, il reste à démontrer si le comportement de ces cellules ressemble à celle des NPCs du SNC. D'abord, j'ai comparé des iPS-NPCs de souris avec des cellules embryonnaires du SNC, in vitro et après greffe dans des modèles de démyélinisation de la moelle épinière de souris adulte. Nos données ont révélé la capacité de survie, intégration, migration et différenciation rapide des cellules greffées en oligodendrocytes matures. Les cellules greffées ont généré de la myéline compacte autour des axones, la restauration de n¿uds de Ranvier et la vitesse de conduction aussi efficacement que les précurseurs du SNC dérivés tandis supplantant cellules endogènes. Ensuite, pour valider la fonctionnalité des précurseurs gliaux humains dérivés des iPS-NPC, je les ai transplantés dans des modèles nouveau-nés et adultes de dys/démyélinisation. Mes données ont montré la migration généralisée, l'intégration et génération de oligodendrocytes fonctionnels, la formation de la myéline compacte tout en reconstruisant n¿uds de Ranvier dans chez les nouveau-nés et les adultes greffés

Stem cell derived oligodendrocytes to study myelin diseases

International audienceOligodendroglial pathology is central to de‐ and dysmyelinating, but also contributes to neurodegenerative and psychiatric diseases as well as brain injury. The understanding of oligodendroglial biology in health and disease has been significantly increased during recent years by experimental in vitro and in vivo preclinical studies as well as histological analyses of human tissue samples. However, for many of these diseases the underlying pathology is still not fully understood and treatment options are frequently lacking. This is at least partly caused by the limited access to human oligodendrocytes from patients to perform functional studies and drug screens. The induced pluripotent stem cell technology (iPSC) represents a possibility to circumvent this obstacle and paves new ways to study human disease and to develop new treatment options for so far incurable central nervous system (CNS) diseases. In this review, we summarize the differences between human and rodent oligodendrocytes, provide an overview of the different techniques to generate oligodendrocytes from human progenitor or stem cells and describe the results from studies using iPSC derived oligodendroglial lineage cells. Furthermore, we discuss future perspectives and challenges of the iPSC technology with respect to disease modeling, drug screen, and cell transplantation approaches

High Dose Pharmaceutical Grade Biotin (MD1003) Accelerates Differentiation of Murine and Grafted Human Oligodendrocyte Progenitor Cells In Vivo

Accumulating evidences suggest a strong correlation between metabolic changes and neurodegeneration in CNS demyelinating diseases such as multiple sclerosis (MS). Biotin, an essential cofactor for five carboxylases, is expressed by oligodendrocytes and involved in fatty acid synthesis and energy production. The metabolic effect of biotin or high-dose-biotin (MD1003) has been reported on rodent oligodendrocytes in vitro, and in neurodegenerative or demyelinating animal models. However, clinical studies, showed mild or no beneficial effect of MD1003 in amyotrophic lateral sclerosis (ALS) or MS. Here, we took advantage of a mouse model of myelin deficiency to study the effects of MD1003 on the behavior of murine and grafted human oligodendrocytes in vivo. We show that MD1003 increases the number and the differentiation potential of endogenous murine oligodendroglia over time. Moreover, the levels of MD1003 are increased in the plasma and brain of pups born to treated mothers, indicating that MD1003 can pass through the mother’s milk. The histological analysis of the grafted animals shows that MD1003 increased proliferation and accelerates differentiation of human oligodendroglia, but without enhancing their myelination potential. These findings provide important insights into the role of MD1003 on murine and human oligodendrocyte maturation/myelination that may explain the mitigated outcome of ALS/MS clinical trials

Beneficial contribution of induced pluripotent stem cell-progeny to Connexin 47 dynamics during demyelination-remyelination

International audienceOligodendrocytes are extensively coupled to astrocytes, a phenomenon ensuring glial homeostasis and maintenance of central nervous system myelin. Molecular disruption of this communication occurs in demyelinating diseases such as multiple sclerosis. Less is known about the vulnerability and reconstruction of the panglial network during adult demyelination‐remyelination. Here, we took advantage of lysolcithin‐induced demyelination to investigate the expression dynamics of the oligodendrocyte specific connexin 47 (Cx47) and to some extent that of astrocyte Cx43, and whether this dynamic could be modulated by grafted induced pluripotent stem cell (iPSC)‐neural progeny. Our data show that disruption of Cx43‐Cx47 mediated hetero‐cellular gap‐junction intercellular communication following demyelination is larger in size than demyelination. Loss of Cx47 expression is timely rescued during remyelination and accelerated by the grafted neural precursors. Moreover, mouse and human iPSC‐derived oligodendrocytes express Cx47, which co‐labels with astrocyte Cx43, indicating their integration into the panglial network. These data suggest that in rodents, full lesion repair following transplantation occurs by panglial reconstruction in addition to remyelination. Targeting panglial elements by cell therapy or pharmacological compounds may help accelerating or stabilizing re/myelination in myelin disorders

Early Netrin-1 Expression Impairs Central Nervous System Remyelination

International audienceObjective: Chronically demyelinated multiple sclerosis (MS) lesions are frequently characterized by scarce undifferentiated oligodendrocyte progenitor cells (OPCs), suggesting the exhaustion of a local OPC pool followed by failure of recruitment and differentiation. Stimulating prompt OPC recruitment following demyelination could improve myelin repair by providing sufficient numbers of remyelinating cells during the repair-permissive period. Understanding mechanisms that determine this process may have important therapeutic implications. We therefore investigated the role of the guidance molecule netrin-1 in OPC recruitment and central nervous system (CNS) remyelination. Methods: Netrin-1 expression was analyzed immunohistochemically in different types of MS lesions and in the murine lysolecithin model of demyelination. The influence of netrin-1 on CNS remyelination was examined using gain and loss of function experiments. Results: We show that in MS lesions, astrocytes upregulate netrin-1 expression early during demyelination and netrin-1 receptors are expressed by OPCs. In contrast, in the efficiently repairing lysolecithin model of demyelination (astrocyte-free), netrin-1 expression is absent during early phases and detected concomitant with completion of OPC recruitment. In vitro migration assays demonstrated that netrin-1 is a chemorepellent for migrating adult OPCs. In mouse lesions, antibody-mediated disruption of netrin-1 function at the peak phase of recruitment increased OPC numbers. Conversely, lentiviral-mediated induction of netrin-1 expression prior to OPC recruitment reduced the number of cells recruited and impaired remyelination. Interpretation: Our findings support the conclusion that netrin-1 expression within demyelinating MS plaques blocks OPC recruitment, which with repeated demyelinating episodes contributes to permanent remyelination failure

Nogo Receptor Inhibition Enhances Functional Recovery following Lysolecithin-Induced Demyelination in Mouse Optic Chiasm

<div><p>Background</p><p>Inhibitory factors have been implicated in the failure of remyelination in demyelinating diseases. Myelin associated inhibitors act through a common receptor called Nogo receptor (NgR) that plays critical inhibitory roles in CNS plasticity. Here we investigated the effects of abrogating NgR inhibition in a non-immune model of focal demyelination in adult mouse optic chiasm.</p><p>Methodology/Principal Findings</p><p>A focal area of demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. To knock down <i>NgR</i> levels, siRNAs against NgR were intracerebroventricularly administered via a permanent cannula over 14 days, Functional changes were monitored by electrophysiological recording of latency of visual evoked potentials (VEPs). Histological analysis was carried out 3, 7 and 14 days post demyelination lesion. To assess the effect of NgR inhibition on precursor cell repopulation, BrdU was administered to the animals prior to the demyelination induction. Inhibition of NgR significantly restored VEPs responses following optic chiasm demyelination. These findings were confirmed histologically by myelin specific staining. siNgR application resulted in a smaller lesion size compared to control. NgR inhibition significantly increased the numbers of BrdU+/Olig2+ progenitor cells in the lesioned area and in the neurogenic zone of the third ventricle. These progenitor cells (Olig2+ or GFAP+) migrated away from this area as a function of time.</p><p>Conclusions/Significance</p><p>Our results show that inhibition of NgR facilitate myelin repair in the demyelinated chiasm, with enhanced recruitment of proliferating cells to the lesion site. Thus, antagonizing NgR function could have therapeutic potential for demyelinating disorders such as Multiple Sclerosis.</p></div

siRNA-mediated knockdown of <i>NgR</i>.

<p>qPCR of third ventricle samples show levels of <i>NgR</i> expression at 24, 48 and 72 hrs after single siRNA injection. Gene expression was normalized with <i>Gapdh</i>. Data are pooled from three independent experiments providing similar results (total number of mice for each data point  = 6). Graph shows fold changes in <i>NgR</i> mRNA using qPCR analysis. Boxes represent the fifth to ninety-fifth percentiles around the median with whiskers for minimum and maximum values. Statistical analysis used two-way ANOVA and Bonferroni's post -test. Treatment had significant effects on the level of NgR gene expression. NgR gene expression was decreased significantly 24 h after siNgR injection compared to siControl, the asterisk indicates that the difference between the pairs denoted are significant at the confidence levels ***p<0.001.</p

Increased numbers of BrdU+/GFAP+ cells within the lesion site following NgR inhibition.

<p>(A–C) Immunofluorescent images of Optic chiasm in LPC-treated animals at 3, 7 and 14 dpi. The number of BrdU+/GFAP+ cells at LPC 3 dpi (A), LPC 7 dpi (B) and 14 dpi (C) was increased. (D–F) Optic chiasm images of LPC+siNgR treated animals at 3 (D), 7 (E) and 14 dpi (F). The number of BrdU+/GFAP+ cells at 7 and 14 dpi in the lesion site was increased. (G) The number of BrdU+/GFAP+ cells in optic chiasm of saline-treated animals (Control) at 7 dpi was low. Arrows indicate double-labeled cells and Square shows the cells magnified in inset. (H) BrdU+/GFAP+ cells per area/mm² are quantified and averaged from the counts of nine sections of each chiasm. Statistical analysis of the differences between numbers of BrdU+/GFAP+ cells in the optic chiasm of all groups was done by two-way ANOVA followed by Bonferroni's post-test. Differences between groups were significant (p<0.001). Post-test showed that the number of BrdU+/GFAP+ cells in LPC treated animals at 7 dpi and 14 dpi was increased significantly compared to Control (both, p<0.05). In LPC+siNgR treated animals the number of BrdU+/GFAP+ cells at 7 and 14 dpi in the lesion site was increased significantly compared to Control (both, ^***p<0.001). NgR inhibition enhance the number of BrdU+/GFAP+ cells in optic chiasm compered to LPC groups and this change was significant between siNgR 14 dpi and LPC 14 dpi (^***p<0.001). Data are expressed as Mean ± SEMs, N = 3, Bars: 100 µm.</p