RESUMO
Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.
Assuntos
Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Complemento C1q/biossíntese , Complemento C3a/biossíntese , Células-Tronco Neurais/fisiologia , Neutrófilos/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Complemento C1q/antagonistas & inibidores , Complemento C1q/genética , Complemento C1q/imunologia , Complemento C3a/antagonistas & inibidores , Complemento C3a/genética , Complemento C3a/imunologia , Meios de Cultivo Condicionados , Humanos , Imunidade Inata , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/imunologia , Neutrófilos/imunologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Neural stem cell (NSC) cultures have been considered technically challenging for time-lapse analysis due to high motility, photosensitivity, and growth at confluent densities. We have tested feasibility of long-term live-cell time-lapse analysis for NSC migration and differentiation studies. Here, we describe a method to study the dynamics of cell cycle, migration, and lineage selection in cultured multipotent mouse or human NSCs using single-cell tracking during a long-term, 7-14â¯day live-cell time-lapse analysis. We used in-house made PDMS inserts with five microwells on a glass coverslip petri-dish to constrain NSC into the area of acquisition during long-term live-cell imaging. In parallel, we have defined image acquisition settings for single-cell tracking of cell cycle dynamics using Fucci-reporter mouse NSC for 7â¯days as well as lineage selection and migration using human NSC for 14â¯days. Overall, we show that adjustments of live-cell analysis settings can extend the time period of single-cell tracking in mouse or human NSC from 24-72â¯h up to 7-14â¯days and potentially longer. However, we emphasize that experimental use of repeated fluorescence imaging will require careful consideration of controls during acquisition and analysis.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Neurais/citologia , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Rastreamento de Células/métodos , Humanos , Células-Tronco Neurais/fisiologiaRESUMO
Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected the ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery. These data identify a successful strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.
RESUMO
Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery vs. control and hNSC transplant alone. These data identify a successful novel strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.
RESUMO
C1q plays a key role as a recognition molecule in the immune system, driving autocatalytic complement cascade activation and acting as an opsonin. We have previously reported a non-immune role of complement C1q modulating the migration and fate of human neural stem cells (hNSC); however, the mechanism underlying these effects has not yet been identified. Here, we show for the first time that C1q acts as a functional hNSC ligand, inducing intracellular signaling to control cell behavior. Using an unbiased screening strategy, we identified five transmembrane C1q signaling/receptor candidates in hNSC (CD44, GPR62, BAI1, c-MET, and ADCY5). We further investigated the interaction between C1q and CD44 , demonstrating that CD44 mediates C1q induced hNSC signaling and chemotaxis in vitro, and hNSC migration and functional repair in vivo after spinal cord injury. These results reveal a receptor-mediated mechanism for C1q modulation of NSC behavior and show that modification of C1q receptor expression can expand the therapeutic window for hNSC transplantation.
Assuntos
Complemento C1q/metabolismo , Receptores de Hialuronatos/metabolismo , Glicoproteínas de Membrana/metabolismo , Células-Tronco Neurais/metabolismo , Receptores de Complemento/metabolismo , Animais , Diferenciação Celular , Transplante de Células , Células Cultivadas , Camundongos , Transdução de Sinais , Traumatismos da Medula EspinalRESUMO
We previously showed the efficacy of multiple research cell lines (RCLs) of human CNS neural stem cells (HuCNS-SCs) in mouse and rat models of thoracic spinal cord injury (SCI), supporting a thoracic SCI clinical trial. Experts recommend in vivo preclinical testing of the intended clinical cell lot/line (CCL) in models with validity for the planned clinical target. We therefore tested the efficacy of two HuCNS-SC lines in cervical SCI: one RCL, and one CCL intended for use in the Pathway Study of cervical SCI in man. We assessed locomotor recovery and sensory function, as well as engraftment, migration, and fate. No evidence of efficacy of the CCL was observed; some data suggested a negative impact of the CCL on outcomes. These data raise questions about the development and validation of potency/comparability assays for clinical testing of cell products, and lack of US Food and Drug Administration requirements for in vivo testing of intended clinical cell lines.
Assuntos
Medula Cervical/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Transdução de Sinais , Traumatismos da Medula Espinal/metabolismo , Transplante de Células-Tronco , Animais , Diferenciação Celular , Linhagem da Célula , Medula Cervical/patologia , Modelos Animais de Doenças , Feminino , Sobrevivência de Enxerto , Humanos , Locomoção , Camundongos , Atividade Motora , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/reabilitação , Traumatismos da Medula Espinal/terapia , Fatores de Tempo , Resultado do TratamentoRESUMO
Multipotent human central nervous system-derived neural stem cells transplanted at doses ranging from 10,000 (low) to 500,000 (very high) cells differentiated predominantly into the oligodendroglial lineage. However, while the number of engrafted cells increased linearly in relationship to increasing dose, the proportion of oligodendrocytic cells declined. Increasing dose resulted in a plateau of engraftment, enhanced neuronal differentiation, and increased distal migration caudal to the transplantation sites. Dose had no effect on terminal sensory recovery or open-field locomotor scores. However, total human cell number and decreased oligodendroglial proportion were correlated with hindlimb girdle coupling errors. Conversely, greater oligodendroglial proportion was correlated with increased Ab step pattern, decreased swing speed, and increased paw intensity, consistent with improved recovery. These data suggest that transplant dose, and/or target niche parameters can regulate donor cell engraftment, differentiation/maturation, and lineage-specific migration profiles.
Assuntos
Diferenciação Celular , Células-Tronco Neurais/transplante , Neurônios/citologia , Oligodendroglia/citologia , Traumatismos da Medula Espinal/terapia , Animais , Antígenos Nucleares/metabolismo , Linhagem da Célula , Movimento Celular , Células Cultivadas , Microambiente Celular , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Recuperação de Função Fisiológica , Nicho de Células-TroncoRESUMO
The effect of transplantation dose on the spatiotemporal dynamics of human neural stem cell (hNSC) engraftment has not been quantitatively evaluated in the central nervous system. We investigated changes over time in engraftment/survival, proliferation, and migration of multipotent human central nervous system-derived neural stem cells (hCNS-SCns) transplanted at doses ranging from 10,000 to 500,000 cells in spinal cord injured immunodeficient mice. Transplant dose was inversely correlated with measures of donor cell proliferation at 2 weeks post-transplant (WPT) and dose-normalized engraftment at 16 WPT. Critically, mice receiving the highest cell dose exhibited an engraftment plateau, in which the total number of engrafted human cells never exceeded the initial dose. These data suggest that donor cell expansion was inversely regulated by target niche parameters and/or transplantation density. Investigation of the response of donor cells to the host microenvironment should be a key variable in defining target cell dose in pre-clinical models of CNS disease and injury.
Assuntos
Células-Tronco Neurais/transplante , Traumatismos da Medula Espinal/terapia , Animais , Apoptose , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Hospedeiro Imunocomprometido , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neurais/citologia , Transplante HeterólogoRESUMO
Neural stem cell transplantation may have the potential to yield repair and recovery of function in central nervous system injury and disease, including spinal cord injury (SCI). Multiple pathological processes are initiated at the epicenter of a traumatic spinal cord injury; these are generally thought to make the epicenter a particularly hostile microenvironment. Conversely, the injury epicenter is an appealing potential site of therapeutic human central nervous system-derived neural stem cell (hCNS-SCns) transplantation because of both its surgical accessibility and the avoidance of spared spinal cord tissue. In this study, we compared hCNS-SCns transplantation into the SCI epicenter (EPI) versus intact rostral/caudal (R/C) parenchyma in contusion-injured athymic nude rats, and assessed the cell survival, differentiation, and migration. Regardless of transplantation site, hCNS-SCns survived and proliferated; however, the total number of hCNS-SCns quantified in the R/C transplant animals was twice that in the EPI animals, demonstrating increased overall engraftment. Migration and fate profile were unaffected by transplantation site. However, although transplantation site did not alter the proportion of human astrocytes, EPI transplantation shifted the localization of these cells and exhibited a correlation with calcitonin gene-related peptide fiber sprouting. Critically, no changes in mechanical allodynia or thermal hyperalgesia were observed. Taken together, these data suggest that the intact parenchyma may be a more favorable transplantation site than the injury epicenter in the subacute period post-SCI.
Assuntos
Células-Tronco Neurais/transplante , Neurônios/transplante , Traumatismos da Medula Espinal/cirurgia , Nicho de Células-Tronco , Transplante de Células-Tronco/métodos , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Comportamento Animal , Biomarcadores/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Humanos , Hiperalgesia/etiologia , Hiperalgesia/fisiopatologia , Hiperalgesia/psicologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/metabolismo , Neurônios/patologia , Limiar da Dor , Ratos , Ratos Nus , Esferoides Celulares , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/psicologia , Transplante de Células-Tronco/efeitos adversos , Fatores de TempoRESUMO
The spinal cord injury (SCI) microenvironment undergoes dynamic changes over time, which could potentially affect survival or differentiation of cells in early versus delayed transplantation study designs. Accordingly, assessment of safety parameters, including cell survival, migration, fate, sensory fiber sprouting, and behavioral measures of pain sensitivity in animals receiving transplants during the chronic postinjury period is required for establishing a potential therapeutic window. The goal of the study was assessment of safety parameters for delayed transplantation of human central nervous system-derived neural stem cells (hCNS-SCns) by comparing hCNS-SCns transplantation in the subacute period, 9 days postinjury (DPI), versus the chronic period, 60 DPI, in contusion-injured athymic nude rats. Although the number of surviving human cells after chronic transplantation was lower, no changes in cell migration were detected between the 9 and 60 DPI cohorts; however, the data suggest chronic transplantation may have enhanced the generation of mature oligodendrocytes. The timing of transplantation did not induce changes in allodynia or hyperalgesia measures. Together, these data support the safety of hCNS-SCns transplantation in the chronic period post-SCI.
Assuntos
Células-Tronco Neurais/transplante , Neurogênese , Oligodendroglia/transplante , Traumatismos da Medula Espinal/cirurgia , Medula Espinal/patologia , Transplante de Células-Tronco/métodos , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Marcha , Sobrevivência de Enxerto , Humanos , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Atividade Motora , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Limiar da Dor , Ratos , Ratos Nus , Recuperação de Função Fisiológica , Esferoides Celulares , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Transplante de Células-Tronco/efeitos adversos , Fatores de TempoRESUMO
Accurate automated cell fate analysis of immunostained human stem cells from 2- and 3-dimensional (2D-3D) images would improve efficiency in the field of stem cell research. Development of an accurate and precise tool that reduces variability and the time needed for human stem cell fate analysis will improve productivity and interpretability of the data across research groups. In this study, we have created protocols for high performance image analysis software Volocity® to classify and quantify cytoplasmic and nuclear cell fate markers from 2D-3D images of human neural stem cells after in vitro differentiation. To enhance 3D image capture efficiency, we optimized the image acquisition settings of an Olympus FV10i® confocal laser scanning microscope to match our quantification protocols and improve cell fate classification. The methods developed in this study will allow for a more time efficient and accurate software based, operator validated, stem cell fate classification and quantification from 2D and 3D images, and yield the highest ≥94.4% correspondence with human recognized objects.