Mécanismes d'action de la thérapie cellulaire par cellules souches mésenchymateuses après infarctus cérébral chez le rat. Développement d'un médicament de thérapie innovante

Stroke is the leading cause of disability in adult. Less than 10% of patients can be treated with thrombolysis. Except rehabilitation, no effective treatment exists to improve functional recovery after the acute phase. Therefore, there is a wide need to develop an effective therapy applicable after several days or weeks following stroke. Using a multiparametric approach (microvascular MRI, analysis of angiogenic genes expression and behavioral study) in rat ischemic stroke model, we defined a transition stage (D3-D7) followed by a subacute phase (D7-D25) during post-stroke remodeling. These two phases represent an interesting target time-window for administration of pro-angiogenic therapies. Since 20 years, cell therapy, notably by human mesenchymal stem/stromal cells (hMSC), emerged as a “regenerative treatment” with threefold increase in clinical trial during the last 10 years. However, still limited data are available regarding the mechanisms by which hMSC benefit, especially at the subacute phase. We progressed in understanding the microvascular plasticity that occurs after an intravenous injection of hMSC in a rat model of transient focal cerebral ischemia. Our preclinical studies were carried out simultaneously with a phase II clinical trial that currently goes on in Grenoble (ISIS: Intravenous Stem Cells After Ischemic Stroke). We reported a sustained functional and cognitive long-term benefit of hMSC IV injected at the subacute stage correlated to an increase of angiogenesis. Ang2, Ang1, SDF-1 and TGFβ1, whose endogenous level tends to be overexpressed by hMSC, would enhance stabilization and survival of newborn vessels, accounting for benefit of these cells. As part of the hMSC development as an advanced therapy medicinal product, we realized an in vivo tumorigenicity assay and showed the absence of tumor development after hMSC injection. We also retrospectively analyzed hMSC produced for the phase II clinical trial. We confirmed the feasibility to produce hMSC, conformed to specifications and in adequate quantity, in the Cell Therapy Unit. In addition, we showed that ex vivo expanded hMSC can present, non clonal, erratic chromosomal abnormalities. Such chromosomal abnormalities appeared to be more related to the maintenance in culture than to the manufacturing process. A “donor” component may also contribute to emergence of such abnormalities.L'accident vasculaire cérébral (AVC) représente la première cause de handicap acquis de l'adulte. A l'heure actuelle, moins de 10% des patients peuvent bénéficier de la thrombolyse, et aucun traitement, en dehors de la rééducation, ne permet de réduire efficacement le handicap. Il existe donc un réel besoin de disposer de nouvelles thérapeutiques permettant d'améliorer la récupération et pouvant être administrées dans un délai élargi par rapport à celui de la thrombolyse. Nos travaux expérimentaux chez le rat, associant imagerie IRM de la microvascularisation, analyse de l'expression des gènes de l'angiogenèse et étude comportementale, ont permis de définir une phase de transition (J3-J7) suivie d'une phase subaigüe (J7-J25) post-AVC. Ces deux phases sont apparues comme des fenêtres thérapeutiques potentielles pour l'administration de traitement pro-angiogéniques. Depuis près de 20 ans, de nombreuses équipes se sont tournées vers la thérapie cellulaire, notamment par cellules souches/stromales mésenchymateuses humaines (CSMh), comme thérapie réparatrice dans les AVC avec un triplement du nombre d'essais cliniques au cours des 10 dernières années. Cependant, les données de la littérature ne permettent pas de bien comprendre le mécanisme d'action des CSMh, particulièrement après une administration à la phase subaigüe. Nos travaux ont permis de progresser dans la compréhension de l'effet microvasculaire des CSMh, administrées dans les conditions d'un essai clinique de phase II qui se déroule actuellement à Grenoble (ISIS : Intravenous Stem Cells After Ischemic Stroke). Nous avons montré que la récupération sensori-motrice et cognitive post-ischémique observée après administration intraveineuse de CSMh était liée à une augmentation de l'angiogenèse. Les facteurs angiogéniques Ang2, Ang1, SDF-1 et TGFβ1, dont la sécrétion endogène est augmentée par les CSMh, semblent participer à une meilleure stabilisation vasculaire et pourrait expliquer l'effet bénéfique de ces cellules. Dans le cadre du développement des CSMh en tant que médicament de thérapie innovante, nous avons montré l'absence de potentiel tumorigène des CSMh par une étude toxicologique de tumorigénicité in vivo. Par analyse rétrospective des CSMh produites dans le cadre de l'essai clinique de phase II, nous avons montré la faisabilité de la production de CSMh conformes aux spécifications et en quantité suffisante par l'Unité de Thérapie Cellulaire. Par ailleurs, ces CSMh cultivées ex vivo peuvent présenter des anomalies caryotypiques erratiques, non clônales. Ces anomalies semblent être liées au maintien en culture, plus qu'au procédé lui-même. Une composante "donneur" semble également contribuer à l'apparition de ces anomalies

Mechanisms of MSC as a cellular therapy in stroke - clinical of an advanced therapy medicinal product

L'accident vasculaire cérébral (AVC) représente la première cause de handicap acquis de l'adulte. A l'heure actuelle, moins de 10% des patients peuvent bénéficier de la thrombolyse, et aucun traitement, en dehors de la rééducation, ne permet de réduire efficacement le handicap. Il existe donc un réel besoin de disposer de nouvelles thérapeutiques permettant d'améliorer la récupération et pouvant être administrées dans un délai élargi par rapport à celui de la thrombolyse. Nos travaux expérimentaux chez le rat, associant imagerie IRM de la microvascularisation, analyse de l'expression des gènes de l'angiogenèse et étude comportementale, ont permis de définir une phase de transition (J3-J7) suivie d'une phase subaigüe (J7-J25) post-AVC. Ces deux phases sont apparues comme des fenêtres thérapeutiques potentielles pour l'administration de traitement pro-angiogéniques. Depuis près de 20 ans, de nombreuses équipes se sont tournées vers la thérapie cellulaire, notamment par cellules souches/stromales mésenchymateuses humaines (CSMh), comme thérapie réparatrice dans les AVC avec un triplement du nombre d'essais cliniques au cours des 10 dernières années. Cependant, les données de la littérature ne permettent pas de bien comprendre le mécanisme d'action des CSMh, particulièrement après une administration à la phase subaigüe. Nos travaux ont permis de progresser dans la compréhension de l'effet microvasculaire des CSMh, administrées dans les conditions d'un essai clinique de phase II qui se déroule actuellement à Grenoble (ISIS : Intravenous Stem Cells After Ischemic Stroke). Nous avons montré que la récupération sensori-motrice et cognitive post-ischémique observée après administration intraveineuse de CSMh était liée à une augmentation de l'angiogenèse. Les facteurs angiogéniques Ang2, Ang1, SDF-1 et TGFβ1, dont la sécrétion endogène est augmentée par les CSMh, semblent participer à une meilleure stabilisation vasculaire et pourrait expliquer l'effet bénéfique de ces cellules. Dans le cadre du développement des CSMh en tant que médicament de thérapie innovante, nous avons montré l'absence de potentiel tumorigène des CSMh par une étude toxicologique de tumorigénicité in vivo. Par analyse rétrospective des CSMh produites dans le cadre de l'essai clinique de phase II, nous avons montré la faisabilité de la production de CSMh conformes aux spécifications et en quantité suffisante par l'Unité de Thérapie Cellulaire. Par ailleurs, ces CSMh cultivées ex vivo peuvent présenter des anomalies caryotypiques erratiques, non clônales. Ces anomalies semblent être liées au maintien en culture, plus qu'au procédé lui-même. Une composante "donneur" semble également contribuer à l'apparition de ces anomalies.Stroke is the leading cause of disability in adult. Less than 10% of patients can be treated with thrombolysis. Except rehabilitation, no effective treatment exists to improve functional recovery after the acute phase. Therefore, there is a wide need to develop an effective therapy applicable after several days or weeks following stroke. Using a multiparametric approach (microvascular MRI, analysis of angiogenic genes expression and behavioral study) in rat ischemic stroke model, we defined a transition stage (D3-D7) followed by a subacute phase (D7-D25) during post-stroke remodeling. These two phases represent an interesting target time-window for administration of pro-angiogenic therapies. Since 20 years, cell therapy, notably by human mesenchymal stem/stromal cells (hMSC), emerged as a “regenerative treatment” with threefold increase in clinical trial during the last 10 years. However, still limited data are available regarding the mechanisms by which hMSC benefit, especially at the subacute phase. We progressed in understanding the microvascular plasticity that occurs after an intravenous injection of hMSC in a rat model of transient focal cerebral ischemia. Our preclinical studies were carried out simultaneously with a phase II clinical trial that currently goes on in Grenoble (ISIS: Intravenous Stem Cells After Ischemic Stroke). We reported a sustained functional and cognitive long-term benefit of hMSC IV injected at the subacute stage correlated to an increase of angiogenesis. Ang2, Ang1, SDF-1 and TGFβ1, whose endogenous level tends to be overexpressed by hMSC, would enhance stabilization and survival of newborn vessels, accounting for benefit of these cells. As part of the hMSC development as an advanced therapy medicinal product, we realized an in vivo tumorigenicity assay and showed the absence of tumor development after hMSC injection. We also retrospectively analyzed hMSC produced for the phase II clinical trial. We confirmed the feasibility to produce hMSC, conformed to specifications and in adequate quantity, in the Cell Therapy Unit. In addition, we showed that ex vivo expanded hMSC can present, non clonal, erratic chromosomal abnormalities. Such chromosomal abnormalities appeared to be more related to the maintenance in culture than to the manufacturing process. A “donor” component may also contribute to emergence of such abnormalities

Évaluation du procédé de mise en banqued unités de sang placentaire à usage thérapeutique au sein de la banque EFS Rhône-Alpes site de Saint Ismier

Les banques de cellules souches hématopoïétiques (CSH) de sang de cordon à usage thérapeutique ou unités de sang placentaire (USP) doivent respecter les spécifications, imposées par la réglementation française, au cours du procédé de mise en banque. En vue d une demande d accréditation, nous avons évalué le procédé de la banque de Saint Ismier EFS Rhône Alpes. Nous avons réalisé une étude rétrospective de l impact des critères cliniques/biologiques, de la qualité des prélèvements et du procédé de transformation sur la conservation des USP, durant les 18 premiers mois d activité de cette banque. Le procédé utilisé permet d obtenir, à la décongélation, une viabilité des cellules mononucléées et une récupération cellulaire conformes. Par contre, le nombre de cellules nucléées totales (CNT) avant congélation n était pas corrélé à celui des CSH après décongélation, soutenant la faible pertinence du nombre de CNT comme critère majeur de sélection des USP par les centres greffeurs. La décision d entrée en production sur le seul critère CNT a causé la destruction de 10% des USP après miniaturisation, motivant la numération systématique des CSH à réception pour limiter les coûts de miniaturisation inutile. 50,2% des prélèvements ont été éliminés à cause d un volume et/ou d un nombre de CNT insuffisant. Le taux de conformité des USP congelées (33%) était dans la moyenne nationale et leur qualité égalait celle de banques accréditées. La non comparution des mères donneuses à la visite de sécurisation retardait l inscription sur le registre des donneurs de MO d un 1/3 des USP. La remise en cause de cette étape pourrait donc augmenter les chances d allogreffe pour de nombreux patients.GRENOBLE1-BU Médecine pharm. (385162101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Intravenous Administration of Human Adipose Derived-Mesenchymal Stem Cells Is Not Efficient in Diabetic or Hypertensive Mice Subjected to Focal Cerebral Ischemia

Copyright © 2019 Mangin, Cogo, Moisan, Bonnin, Maïer and Kubis.As the second cause of death and cognitive decline in industrialized countries, stroke is a major burden for society. Vascular risks factors such as hypertension and diabetes are involved in most stroke patients, aggravate stroke severity, but are still poorly taken into account in preclinical studies. Microangiopathy and sustained inflammation are exacerbated, likely explaining the severity of stroke in those patients. We sought to demonstrate that intravenous administration of human adipose derived-mesenchymal stem cells (hADMSC) that have immunomodulatory properties, could accelerate sensorimotor recovery, prevent long-term spatial memory impairment and promote neurogenesis, in diabetic or hypertensive mice, subjected to permanent middle cerebral artery occlusion (pMCAo). Diabetic (streptozotocin IP) or hypertensive (L-NAME in drinking water) male C57Bl6 mice subjected to pMCAo, were treated by hADMSC (500,000 cells IV) 2 days after cerebral ischemia induction. Infarct volume, neurogenesis, microglial/macrophage density, T-lymphocytes density, astrocytes density, and vessel density were monitored 7 days after cells injection and at 6 weeks. Neurological sensorimotor deficit and spatial memory were assessed until 6 weeks post-stroke. Whatever the vascular risk factor, hADMSC showed no effect on functional sensorimotor recovery or cognitive decline prevention at short or long-term assessment, nor significantly modified neurogenesis, microglial/macrophage, T-lymphocytes, astrocytes, and vessel density. This work is part of a European program (H2020, RESSTORE). We discuss the discrepancy of our results with those obtained in rats and the optimal cell injection time frame, source and type of cells according to the species stroke model. A comprehensive understanding of the mechanisms preventing recovery should help for successful clinical translation, but first could allow identifying good and bad responders to cell therapy in stroke.The RESSTORE project received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Grant Agreement No. 681044 RESSTORE project (www.resstore.eu) and GM was directly funded by the RESSTORE project

Biomaterial Applications in Cell‐Based Therapy in Experimental Stroke

International audienceStroke is an important health issue corresponding to the second cause of mortality and first cause of severe disability with no effective treatments after the first hours of onset. Regenerative approaches such as cell therapy provide an increase in endogenous brain structural plasticity but they are not enough to promote a complete recovery. Tissue engineering has recently aroused a major interesting development of biomaterials for use into the central nervous system. Many biomaterials have been engineered based on natural compounds, synthetic compounds, or a mix of both with the aim of providing polymers with specific properties. The mechanical properties of biomaterials can be exquisitely regulated forming polymers with different stiffness, modifiable physical state that polymerizes in situ, or small particles encapsulating cells or growth factors. The choice of biomaterial compounds should be adapted for the different applications, structure target, and delay of administration. Biocompatibilities with embedded cells and with the host tissue and biodegradation rate must be considerate. In this paper, we review the different applications of biomaterials combined with cell therapy in ischemic stroke and we explore specific features such as choice of biomaterial compounds and physical and mechanical properties concerning the recent studies in experimental stroke

Biomaterial Applications in Cell-Based Therapy in Experimental Stroke

Stroke is an important health issue corresponding to the second cause of mortality and first cause of severe disability with no effective treatments after the first hours of onset. Regenerative approaches such as cell therapy provide an increase in endogenous brain structural plasticity but they are not enough to promote a complete recovery. Tissue engineering has recently aroused a major interesting development of biomaterials for use into the central nervous system. Many biomaterials have been engineered based on natural compounds, synthetic compounds, or a mix of both with the aim of providing polymers with specific properties. The mechanical properties of biomaterials can be exquisitely regulated forming polymers with different stiffness, modifiable physical state that polymerizes in situ, or small particles encapsulating cells or growth factors. The choice of biomaterial compounds should be adapted for the different applications, structure target, and delay of administration. Biocompatibilities with embedded cells and with the host tissue and biodegradation rate must be considerate. In this paper, we review the different applications of biomaterials combined with cell therapy in ischemic stroke and we explore specific features such as choice of biomaterial compounds and physical and mechanical properties concerning the recent studies in experimental stroke

Intracerebral injection of human mesenchymal stem cells impacts cerebral microvasculature after experimental stroke: MRI study.

International audienceStroke, the leading cause of disability, lacks treatment beyond thrombolysis. The acute injection of human mesenchymal stem cells (hMSCs) provides a benefit which could be mediated by an enhancement of angiogenesis. A clinical autologous graft requires an hMSC culture delay incompatible with an acute administration. This study evaluates the cerebral microvascular changes after a delayed injection of hMSCs. At day 8 after middle cerebral artery occlusion (MCAo), two groups of rats received an intracerebral injection in the damaged brain of either 10 μL of cell suspension medium (MCAo-PBS, n = 4) or 4 × 10(5) hMSCs (MCAo-hMSC, n = 5). Two control groups of healthy rats underwent the same injection procedures in the right hemisphere (control-PBS, n = 6; control-hMSC, n = 5). The effect of hMSCs on the microvasculature was assessed by MRI using three parameters: apparent diffusion coefficient (ADC), cerebral blood volume (CBV) and vessel size index (VSI). At day 9, eight additional rats were euthanised for a histological study of the microvascular parameters (CBV, VSI and vascular fraction). No ADC difference was observed between MCAo groups. One day after intracerebral injection, hMSCs abolished the CBV increase observed in the lesion (MCAo-hMSC: 1.7 ± 0.1% versus MCAo-PBS: 2.2 ± 0.2%) and delayed the VSI increase (vasodilation) secondary to cerebral ischaemia. Histological analysis at day 9 confirmed that hMSCs modified the microvascular parameters (CBV, VSI and vascular fraction) in the lesion. No ADC, CBV or VSI differences were observed between control groups. At the stroke post-acute phase, hMSC intracerebral injection rapidly and transiently modifies the cerebral microvasculature. This microvascular effect can be monitored in vivo by MRI. Copyright © 2012 John Wiley & Sons, Ltd

Multiparametric MRI including oxygenation mapping of experimental ischaemic stroke

International audienceRecent advances in MRI methodology, such as microvascular and brain oxygenation (StO2) imaging, may prove useful in obtaining information about the severity of the acute stroke. We assessed the potential of StO2 to detect the ischaemic core in the acute phase compared to apparent diffusion coefficient and to predict the final necrosis. Sprague-Dawley rats (n = 38) were imaged during acute stroke (D0) and 21 days after (D21). A multiparametric MRI protocol was performed at 4.7T to characterize brain damage within three region of interest: 'LesionD0' (diffusion), 'Mismatch' representing penumbra (perfusion/diffusion) and 'Hypoxia' (voxels < 40% of StO2 within the region of interest LesionD0). Voxel-based analysis of stroke revealed heterogeneity of the region of interest LesionD0, which included voxels with different degrees of oxygenation decrease. This finding was supported by a dramatic decrease of vascular and perfusion parameters within the region of interest hypoxia. This zone presented the lowest values of almost all parameters analysed, indicating a higher severity. Our study demonstrates the potential of StO2 magnetic resonance imaging to more accurately detect the ischaemic core without the inclusion of any reversible ischaemic damage. Our follow-up study indicates that apparent diffusion coefficient imaging overestimated the final necrosis while StO2 imaging did not