RESUMEN

Cell replacement therapy is expected as a new and more radical treatment against brain damage. We previously reported that transplanted human cerebral organoids extend their axons along the corticospinal tract in rodent brains. The axons reached the spinal cord but were still sparse. Therefore, this study optimized the host brain environment by the adeno-associated virus (AAV)-mediated expression of axon guidance proteins in mouse brain. Among netrin-1, SEMA3, and L1CAM, only L1CAM significantly promoted the axonal extension of mouse embryonic brain tissue-derived grafts. L1CAM was also expressed by donor neurons, and this promotion was exerted in a haptotactic manner by their homophilic binding. Primary cortical neurons cocultured on L1CAM-expressing HEK-293 cells supported this mechanism. These results suggest that optimizing the host environment by the AAV-mediated expression of axon guidance molecules enhances the effect of cell replacement therapy.

Asunto(s)

Molécula L1 de Adhesión de Célula Nerviosa , Animales , Ratones , Humanos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/farmacología , Células HEK293 , Axones/metabolismo , Tractos Piramidales , Encéfalo/metabolismo , Netrina-1/metabolismo , Netrina-1/farmacología

RESUMEN

The transplantation of dopaminergic (DA) progenitors derived from pluripotent stem cells improves the behavior of Parkinson's disease model animals. However, the survival of DA progenitors is low, and the final yield of DA neurons is only approximately 0.3%-2% the number of transplanted cells. Zonisamide (ZNS) increases the number of survived DA neurons upon the transplantation of mouse-induced pluripotent stem (iPS) cell-derived DA progenitors in the rat striatum. In this study, we induced DA progenitors from human iPS cells and transplanted them into the striatum of female rats with daily administration of ZNS. The number of survived DA neurons was evaluated 1 and 4 months after transplantation by immunohistochemistry, which revealed that the number of survived DA neurons was significantly increased with the administration of ZNS. To assess the mechanism of action of ZNS, we performed a gene expression analysis to compare the gene expression profiles in striatum treated with or without ZNS. The analysis revealed that the expression of SLIT-and NTRK-like protein 6 (SLITRK6) was upregulated in rat striatum treated with ZNS. In conclusion, ZNS promotes the survival of DA neurons after the transplantation of human-iPS cell-derived DA progenitors in the rat striatum. SLITRK6 is suggested to be involved in this supportive effect of ZNS by modulating the environment of the host brain.

Asunto(s)

Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/trasplante , Zonisamida/farmacología , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Femenino , Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratas , Ratas Endogámicas F344

RESUMEN

The cerebral cortical tissue of murine embryo and pluripotent stem cell-derived neurons can survive in the adult brain and extend axons to the spinal cord. These features suggest that cell transplantation can be a strategy to reconstruct the corticospinal tract (CST). It is unknown, however, which cell population makes for safe and effective donor cells. To address this issue, we grafted the cerebral cortex of E14.5 mouse to the brain of adult mice and found that the cells in the graft extending axons along the CST expressed CTIP2. By using CTIP2:GFP knock-in mouse embryonic stem cells (mESCs), we identified L1CAM as a cell surface marker to enrich CTIP2+ cells. We sorted L1CAM+ cells from E14.5 mouse brain and confirmed that they extended a larger number of axons along the CST compared to L1CAM- cells. Our results suggest that sorting L1CAM+ cells from the embryonic cerebral cortex enriches subcortical projection neurons to reconstruct the CST.

RESUMEN

BACKGROUND: Cell transplantation is expected to be a promising treatment for Parkinson's disease (PD), in which re-innervation of the host striatum by grafted dopamine (DA) neurons is essential. In particular, the dorsolateral part of the striatum is important because it is the target of midbrain A9 DA neurons, which are degenerated in PD pathology. The effect of exercise on the survival and maturation of grafted neurons has been reported in several neurological disease models, but never in PD models. OBJECTIVE: We investigated how exercise influences cell transplantation for PD, especially from the viewpoint of cell survival and neurite extensions. METHODS: Ventral mesencephalic neurons from embryonic (E12.5) rats were transplanted into the striatum of adult 6-OHDA-lesioned rats. The host rats then underwent treadmill training as exercise after the transplantation. Six weeks after the transplantation, they were sacrificed, and the grafts in the striatum were analyzed. RESULTS: The addition of exercise post-transplantation significantly increased the number of surviving DA neurons. Moreover, it promoted neurite extensions from the graft toward the dorsolateral part of the striatum. CONCLUSIONS: This study indicates a beneficial effect of exercise after cell transplantation in PD.

Asunto(s)

Neuronas Dopaminérgicas/trasplante , Terapia por Ejercicio , Supervivencia de Injerto/fisiología , Neostriado/cirugía , Neuritas/fisiología , Enfermedad de Parkinson/rehabilitación , Enfermedad de Parkinson/cirugía , Condicionamiento Físico Animal/fisiología , Animales , Modelos Animales de Enfermedad , Embrión de Mamíferos , Oxidopamina , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas

RESUMEN

Seckel syndrome (SS) is a rare spectrum of congenital severe microcephaly and dwarfism. One SS-causative gene is Ataxia Telangiectasia and Rad3-Related Protein (ATR), and ATR (c.2101 A>G) mutation causes skipping of exon 9, resulting in a hypomorphic ATR defect. This mutation is considered the cause of an impaired response to DNA replication stress, the main function of ATR, contributing to the pathogenesis of microcephaly. However, the precise behavior and impact of this splicing defect in human neural progenitor cells (NPCs) is unclear. To address this, we established induced pluripotent stem cells (iPSCs) from fibroblasts carrying the ATR mutation and an isogenic ATR-corrected counterpart iPSC clone. SS-patient-derived iPSCs (SS-iPSCs) exhibited cell type-specific splicing; exon 9 was dominantly skipped in fibroblasts and iPSC-derived NPCs, but it was included in undifferentiated iPSCs and definitive endodermal cells. SS-iPSC-derived NPCs (SS-NPCs) showed distinct expression profiles from ATR non-mutated NPCs with negative enrichment of neuronal genesis-related gene sets. In SS-NPCs, abnormal mitotic spindles occurred more frequently than in gene-corrected counterparts, and the alignment of NPCs in the surface of the neurospheres was perturbed. Finally, we tested several splicing-modifying compounds and found that TG003, a CLK1 inhibitor, could pharmacologically rescue the exon 9 skipping in SS-NPCs. Treatment with TG003 restored the ATR kinase activity in SS-NPCs and decreased the frequency of abnormal mitotic events. In conclusion, our iPSC model revealed a novel effect of the ATR mutation in mitotic processes of NPCs and NPC-specific missplicing, accompanied by the recovery of neuronal defects using a splicing rectifier.

Asunto(s)

Empalme Alternativo , Proteínas de la Ataxia Telangiectasia Mutada , Enanismo , Facies , Células Madre Pluripotentes Inducidas , Microcefalia , Modelos Biológicos , Mutación , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular , Enanismo/enzimología , Enanismo/genética , Enanismo/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Masculino , Microcefalia/enzimología , Microcefalia/genética , Microcefalia/patología

RESUMEN

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1+ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.

Asunto(s)

Neuronas Dopaminérgicas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas/metabolismo , Animales , Diferenciación Celular/genética , Separación Celular/métodos , Células Cultivadas , Neuronas Dopaminérgicas/citología , Femenino , Humanos , Macaca fascicularis , Masculino , Mesencéfalo/citología , Mesencéfalo/metabolismo , Ratones Endogámicos C57BL , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/terapia , Proteínas/genética , Ratas Sprague-Dawley , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Trasplante de Células Madre/métodos , Trasplante Heterólogo

RESUMEN

For cell transplantation therapy for Parkinson's disease (PD) to be realized, the grafted neurons should be integrated into the host neuronal circuit to restore the lost neuronal function. Here, using wheat-germ agglutinin-based transsynaptic tracing, we show that integrin α5 is selectively expressed in striatal neurons that are innervated by midbrain dopaminergic (DA) neurons. In addition, we found that integrin α5ß1 was activated by the administration of estradiol-2-benzoate (E2B) in striatal neurons of adult female rats. Importantly, we observed that the systemic administration of E2B into hemi-parkinsonian rat models facilitates the functional integration of grafted DA neurons derived from human induced pluripotent stem cells into the host striatal neuronal circuit via the activation of integrin α5ß1. Finally, methamphetamine-induced abnormal rotation was recovered earlier in E2B-administered rats than in rats that received other regimens. Our results suggest that the simultaneous administration of E2B with stem cell-derived DA progenitors can enhance the efficacy of cell transplantation therapy for PD.

Asunto(s)

Neuronas Dopaminérgicas/efectos de los fármacos , Estradiol/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Integrina alfa5beta1/metabolismo , Células-Madre Neurales/efectos de los fármacos , Anciano , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cuerpo Estriado/citología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/trasplante , Estradiol/análogos & derivados , Femenino , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Integrina alfa5beta1/genética , Masculino , Metanfetamina , Ratones Endogámicos C57BL , Microscopía Confocal , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/trasplante , Oxidopamina , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/metabolismo , Enfermedad de Parkinson Secundaria/terapia , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células Madre/métodos , Trasplante Heterólogo

RESUMEN

BACKGROUND: To evaluate the in vivo function of human dopaminergic (DA) neurons, Parkinson's disease (PD) model rats made by the hemi-lateral injection of 6-hydroxydopamine (6-OHDA) are widely used as host animals. In the case of such xeno-transplantation, however, immunosuppression is needed for good survival of the grafted cells. NEW METHODS: In order to determine whether human mature neurons can survive in X-linked severe combined immunodeficiency (X-SCID) rats without immunosuppression, we grafted human embryonic stem cell (ESC)-derived DA neurons into the striatum of X-SCID rats. We next treated the X-SCID rats with 6-OHDA and grafted mouse fetal DA neurons or human induced pluripotent stem cell (iPSC)-derived DA neurons to examine whether these rats can be used as PD model rats. RESULTS: X-SCID rats did not elicit immune responses against human ESC-derived DA neurons and consequently resulted in good survival of the cells without immunosuppression. Furthermore, 6-OHDA-lesioned X-SCID rats exhibited rotational behavior, which was recovered by grafting mouse fetal DA neurons or human iPSC-derived DA neurons. COMPARISON WITH EXISTING METHODS: Immunosuppression by drugs such as Cyclosporine A requires daily injection, which is stressful for rats and moreover may cause renal or hepatic failure. Furthermore, blood levels of the drug may not be stable, which weakens the reliability of the data. CONCLUSIONS: Our results provide a more accessible and reliable method to evaluate the in vivo function of human DA neurons, potentially offering a pre-clinical study for the application of pluripotent stem cells.

Asunto(s)

Neuronas Dopaminérgicas/trasplante , Actividad Motora , Trastornos Parkinsonianos/fisiopatología , Ratas Mutantes , Trasplante Heterólogo , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/fisiología , Células Madre Embrionarias/fisiología , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Oxidopamina , Trastornos Parkinsonianos/patología , Putamen/fisiopatología , Putamen/cirugía , Ratas Endogámicas F344 , Ratas Desnudas , Ratas Transgénicas , Rotación , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/patología , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/fisiopatología

RESUMEN

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson's disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN(+) cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN(+) cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN(+) cells in a NURR1(+) cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

Asunto(s)

Diferenciación Celular , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/trasplante , Células Madre Pluripotentes Inducidas/citología , Animales , Conducta Animal , Técnicas de Cultivo de Célula , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mesencéfalo/citología , Mesencéfalo/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Ratas , Serina Endopeptidasas/metabolismo , Factores de Tiempo

RESUMEN

Cell replacement therapy using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is a promising strategy for the treatment of neurologic diseases such as Parkinson's disease (PD). However, a limiting factor for effective cell transplantation is the low survival rate of grafted cells, especially neurons. In this study, we modified the host environment and investigated whether the simultaneous administration of soluble factors can improve the survival and differentiation of murine iPSC-derived dopaminergic (DA) neurons in host brains. With the goal of applying this technology in clinical settings in the near future, we selected drugs that were already approved for clinical use. The drugs included two commonly used anti-convulsants, valproic acid (VPA) and zonisamide (ZNS), and estradiol (E2), also known as biologically active estrogen. Following neural induction of murine iPSCs, we collected neural progenitor cells (NPCs) by sorting PSA-NCAM(+) cells, then treated the PSA-NCAM(+) cells with drugs for 4 days. An immunofluorescence study revealed that 0.01 mM and 0.1 mM of VPA and 10 nM of E2 increased the percentage of tyrosine hydroxylase(+) (TH: a DA neuron marker) cells in vitro. Furthermore, 0.1 mM of VPA increased the percentage of TH(+) cells that simultaneously express the midbrain markers FOXA2 and NURR1. Next, in order to determine the effects of the drugs in vivo, the iPSC-derived NPCs were transplanted into the striata of intact SD rats. The animals received intraperitoneal injections of one of the drugs for 4 weeks, then were subjected to an immunofluorescence study. VPA administration (150 mg/kg/daily) increased the number of NeuN(+) post-mitotic neurons and TH(+) DA neurons in the grafts. Furthermore, VPA (150 mg/kg/daily) and ZNS (30 mg/kg/daily) increased the number of TH(+)FOXA2(+) midbrain DA neurons. These results suggest that the systemic administration of VPA and ZNS may improve the efficiency of cell replacement therapy using iPSCs to treat PD.