RESUMEN
Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.
Asunto(s)
Trasplante de Tejido Encefálico , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/trasplante , Células Madre Embrionarias/citología , Enfermedad de Parkinson/terapia , Animales , Diferenciación Celular , Línea Celular , Supervivencia Celular , Femenino , Humanos , Macaca mulatta , Mesencéfalo/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratas , Ratas Sprague-DawleyRESUMEN
Remyelination of chronically demyelinated axons in multiple sclerosis (MS) requires the recruitment of endogenous cells or their replacement by transplanted, exogenous oligodendrocyte progenitor cells (OPCs). We have previously shown that an OPC line, CG4, preferentially migrates after transplantation toward focal areas of inflammatory demyelination and axon loss created by injection of zymosan in the rat spinal cord. Here we show that many transplanted CG4 cells had already migrated into the inflammatory lesion after 1 day. We demonstrate that a large number of CG4 cells that had migrated, expressed the adhesion protein, CD44, and that CD44's main ligand, hyaluronic acid (HA) was robustly expressed in the inflammatory lesion. In an in vitro migration assay, migration declined significantly following blocking of CD44 expression on CG4 cells. Likewise, migration of CG4 cells toward a zymosan lesion in vivo was inhibited when transplanted cells were exposed to a CD44 blocking antibody prior to transplantation. These findings suggest that CD44 is a key molecule in the migration of OPCs toward the focal inflammatory demyelinated lesion induced by zymosan, and may be an important in OPC repair in MS.
Asunto(s)
Movimiento Celular/fisiología , Enfermedades Desmielinizantes/metabolismo , Receptores de Hialuranos/metabolismo , Células-Madre Neurales/metabolismo , Oligodendroglía/metabolismo , Médula Espinal/metabolismo , Animales , Enfermedades Desmielinizantes/patología , Femenino , Receptores de Hialuranos/genética , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Vaina de Mielina/trasplante , Regeneración Nerviosa/fisiología , Células-Madre Neurales/citología , Células-Madre Neurales/trasplante , Oligodendroglía/citología , Oligodendroglía/trasplante , Ratas , Ratas Sprague-Dawley , Médula Espinal/patologíaRESUMEN
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Exogenous cell replacement in MS lesions has been proposed as a means of achieving remyelination when endogenous remyelination has failed. However, the ability of exogenous cells to remyelinate axons in the presence of inflammation remains uncertain. We have explored the remyelinating capacity of an oligodendrocyte progenitor cell line CG-4 transduced with the GFP gene and transplanted adjacent to a zymosan-induced focal demyelination model in the rat spinal cord. The resulting zymosan-induced lesions were characterized by persistent macrophage/microglia activation, focal demyelination, degeneration of axons, and reactive astrogliosis. GFP(+) CG-4 cells were found to migrate preferentially toward the inflammatory lesion and survive inside the lesion. A proportion of GFP(+) CG-4 cells differentiated into mature oligodendrocytes and remyelinated axons within the lesion. These findings suggest that grafted oligodendrocyte progenitors may migrate toward areas of inflammation in the adult rat spinal cord, where they can survive and differentiate into myelinating oligodendrocytes.
Asunto(s)
Movimiento Celular/fisiología , Vaina de Mielina/fisiología , Mielitis/fisiopatología , Células-Madre Neurales/trasplante , Oligodendroglía/trasplante , Animales , Diferenciación Celular/fisiología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/fisiopatología , Femenino , Vaina de Mielina/patología , Mielitis/metabolismo , Mielitis/patología , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Oligodendroglía/citología , Oligodendroglía/fisiología , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/fisiopatologíaRESUMEN
Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.
Asunto(s)
Neuronas Dopaminérgicas , Células Madre Embrionarias Humanas , Animales , Diferenciación Celular , Mesencéfalo , Ratas , Vía de Señalización WntRESUMEN
Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.
Asunto(s)
Dopamina , Células Madre Embrionarias Humanas , Animales , Diferenciación Celular , Neuronas Dopaminérgicas , Mesencéfalo , Ratones , Ratas , Distribución TisularRESUMEN
BACKGROUND: With the enhanced use of chemotherapy and the advent of increased patient survival rates, there are an increasing number of cancer survivors living with chemotherapy-induced cognitive impairment. A growing number of clinical studies have brought to light the association of agents like methotrexate in generating these neurological sequelae, although mechanisms remain unclear. METHODS: Here, we use a clinically relevant regimen of several cycles of methotrexate and leucovorin rescue to develop a model of chemotherapy-induced cognitive impairment, and investigate the in vivo long-term (16 mo) impact of high-dose systemic methotrexate on white matter cellular dynamics as assessed by stereology, animal behavior, and diffusion tensor imaging. RESULTS: Our results indicate that at 6 and 16 months post-chemotherapy, methotrexate-treated rats exhibit a significant and permanent decrease in the number of oligodendrocytes and their progenitors in the white matter, in corpus callosum volumes, and myelin basic protein. These findings are associated with mostly delayed deficits in performance on Morris Water Maze and Novel Object Recognition tasks. Diffusion tensor imaging demonstrates significantly decreased fractional anisotropy values in the callosum genu, body, and splenium, as well as previously unassessed areas like the fimbria. Interestingly, these white matter changes are preceded by an earlier, transient decrement in white matter microglia at 3 months, and hippocampal neural progenitors at 3 and 6 months. CONCLUSION: These results demonstrate a significant negative impact of methotrexate on the oligodendrocyte compartment and white matter, associated with cognitive impairment. The data also support the use of diffusion tensor imaging in monitoring white matter integrity in this context.
Asunto(s)
Disfunción Cognitiva , Metotrexato/efectos adversos , Sustancia Blanca , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/efectos adversos , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/diagnóstico por imagen , Disfunción Cognitiva/patología , Imagen de Difusión Tensora , Modelos Animales de Enfermedad , Femenino , Masculino , Metotrexato/administración & dosificación , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/efectos de los fármacos , Sustancia Blanca/patologíaRESUMEN
There is heterogeneity in neural stem and progenitor cell characteristics depending on their species and regional origin. In search for potent in vitro-expanded human neural precursor cells and cell therapy methods to repair the injured human spinal cord, the possible influence exerted by intrinsic cellular heterogeneity has to be considered. Data available on in vitro-expanded human spinal cord-derived cells are sparse and it has previously been difficult to establish long-term neurosphere cultures showing multipotentiality. In the present paper, human spinal cord-derived neurospheres were cultured in the presence of EGF, bFGF and CNTF for up to 25 passages (>350 days) in vitro. In contrast to the human first trimester subcortical forebrain, spinal cord tissue>9.5 weeks of gestation could not serve as a source for long-term neurosphere cultures under the present conditions. After withdrawal of mitogens, cultured neurospheres (at 18 passages) gave rise to cells with neuronal, astrocytic and oligodendrocytic phenotypes in vitro. After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immuno-deficient adult rats, large numbers of cells survived at least up to 6 weeks, expressing neuronal and astrocytic phenotypes. These results demonstrate that it is possible to expand and maintain multipotent human spinal cord-derived neurospheres in vitro for extended time-periods and that they have promising in vivo potential after engraftment to the injured spinal cord.
Asunto(s)
Trasplante de Tejido Fetal/fisiología , Supervivencia de Injerto/fisiología , Neuronas/trasplante , Esferoides Celulares/trasplante , Traumatismos de la Médula Espinal/cirugía , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Células Madre Fetales/citología , Células Madre Fetales/trasplante , Trasplante de Tejido Fetal/métodos , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Células Madre Multipotentes/citología , Células Madre Multipotentes/trasplante , Neuroglía/citología , Neuroglía/trasplante , Neuronas/citología , Prosencéfalo/citología , Prosencéfalo/embriología , Prosencéfalo/trasplante , Ratas , Ratas Desnudas , Esferoides Celulares/citología , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/trasplante , Trasplante de Células Madre/métodos , Trasplante HeterólogoRESUMEN
Human pluripotent stem cells (hPSCs) provide an unlimited cell source for regenerative medicine. Hormone-producing cells are particularly suitable for cell therapy, and hypopituitarism, a defect in pituitary gland function, represents a promising therapeutic target. Previous studies have derived pituitary lineages from mouse and human ESCs using 3D organoid cultures that mimic the complex events underlying pituitary gland development in vivo. Instead of relying on unknown cellular signals, we present a simple and efficient strategy to derive human pituitary lineages from hPSCs using monolayer culture conditions suitable for cell manufacturing. We demonstrate that purified placode cells can be directed into pituitary fates using defined signals. hPSC-derived pituitary cells show basal and stimulus-induced hormone release in vitro and engraftment and hormone release in vivo after transplantation into a murine model of hypopituitarism. This work lays the foundation for future cell therapy applications in patients with hypopituitarism.
Asunto(s)
Corticotrofos/metabolismo , Células Madre Embrionarias/metabolismo , Hipopituitarismo/terapia , Células Madre Pluripotentes/metabolismo , Tirotrofos/metabolismo , Hormona Adrenocorticotrópica/biosíntesis , Hormona Adrenocorticotrópica/metabolismo , Animales , Benzamidas/farmacología , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos , Corticotrofos/citología , Corticotrofos/efectos de los fármacos , Dioxoles/farmacología , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Hormona Folículo Estimulante/biosíntesis , Hormona Folículo Estimulante/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Expresión Génica , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hipopituitarismo/genética , Hipopituitarismo/metabolismo , Hipopituitarismo/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Hipófisis/metabolismo , Hipófisis/patología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Tirotrofos/citología , Tirotrofos/efectos de los fármacos , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ability to expand human neural precursor cells in vitro offers new possibilities for future cell therapies. However, concern over immunologically based rejection of in vitro-expanded human neural cells confounds their use as donor cells. Here, we demonstrate that the expression of human leukocyte antigen (HLA) class I and II molecules, but not the co-stimulatory proteins CD40, CD80 and CD86, substantially increase during expansion of neurospheres. Furthermore, peripheral lymphocytes were unresponsive when co-cultured with in vitro-expanded neural cells. Taken together, these results suggest a low immunogenicity of these cultured human neural cells despite HLA incompatibility and high HLA expression.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Complejo Mayor de Histocompatibilidad/fisiología , Neuronas/inmunología , Prosencéfalo/citología , Médula Espinal/citología , Análisis de Varianza , Antígenos CD/metabolismo , Recuento de Células/métodos , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Embrión de Mamíferos , Femenino , Feto , Citometría de Flujo/métodos , Humanos , Inmunohistoquímica/métodos , Activación de Linfocitos/fisiología , Embarazo , Prosencéfalo/embriología , Médula Espinal/embriología , Células Madre/metabolismo , Factores de TiempoRESUMEN
Radiation therapy to the brain is a powerful tool in the management of many cancers, but it is associated with significant and irreversible long-term side effects, including cognitive decline and impairment of motor coordination. Depletion of oligodendrocyte progenitors and demyelination are major pathological features that are particularly pronounced in younger individuals and severely limit therapeutic options. Here we tested whether human ESC-derived oligodendrocytes can functionally remyelinate the irradiated brain using a rat model. We demonstrate the efficient derivation and prospective isolation of human oligodendrocyte progenitors, which, upon transplantation, migrate throughout the major white matter tracts resulting in both structural and functional repair. Behavioral testing showed complete recovery of cognitive function while additional recovery from motor deficits required concomitant transplantation into the cerebellum. The ability to repair radiation-induced damage to the brain could dramatically improve the outlook for cancer survivors and enable more effective use of radiation therapies, especially in children.
Asunto(s)
Encéfalo/citología , Encéfalo/efectos de la radiación , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/terapia , Células Madre Embrionarias Humanas/citología , Vaina de Mielina/metabolismo , Oligodendroglía/citología , Animales , Encéfalo/fisiopatología , Trastornos del Conocimiento/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Oligodendroglía/trasplante , Ratas , Ratas Desnudas , Rayos XRESUMEN
To validate human neural precursor cells (NPCs) as potential donor cells for transplantation therapy after spinal cord injury (SCI), we investigated the effect of NPCs, transplanted as neurospheres, in two different rat SCI models. Human spinal cord-derived NPCs (SC-NPCs) transplanted 9 days after spinal contusion injury enhanced hindlimb recovery, assessed by the BBB locomotor test. In spinal compression injuries, SC-NPCs transplanted immediately or after 1 week, but not 7 weeks after injury, significantly improved hindlimb recovery compared to controls. We could not detect signs of mechanical allodynia in transplanted rats. Four months after transplantation, we found more human cells in the host spinal cord than were transplanted, irrespective of the time of transplantation. There was no focal tumor growth. In all groups the vast majority of NPCs differentiated into astrocytes. Importantly, the number of surviving rat spinal cord neurons was highest in groups transplanted acutely and subacutely, which also showed the best hindlimb function. This suggests that transplanted SC-NPCs improve the functional outcome by a neuroprotective effect. We conclude that SC-NPCs reliably enhance the functional outcome after SCI if transplanted acutely or subacutely, without causing allodynia. This therapeutic effect is mainly the consequence of a neuroprotective effect of the SC-NPCs.
Asunto(s)
Células-Madre Neurales/fisiología , Células-Madre Neurales/trasplante , Traumatismos de la Médula Espinal/cirugía , Médula Espinal/citología , Animales , Modelos Animales de Enfermedad , Femenino , Feto , Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico HSP27/metabolismo , Miembro Posterior/fisiopatología , Humanos , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/metabolismo , Umbral del Dolor/fisiología , Ratas , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Factores de TiempoRESUMEN
Cranial placodes are embryonic structures essential for sensory and endocrine organ development. Human placode development has remained largely inaccessible despite the serious medical conditions caused by the dysfunction of placode-derived tissues. Here, we demonstrate the efficient derivation of cranial placodes from human pluripotent stem cells. Timed removal of the BMP inhibitor Noggin, a component of the dual-SMAD inhibition strategy of neural induction, triggers placode induction at the expense of CNS fates. Concomitant inhibition of fibroblast growth factor signaling disrupts placode derivation and induces surface ectoderm. Further fate specification at the preplacode stage enables the selective generation of placode-derived trigeminal ganglia capable of in vivo engraftment, mature lens fibers, and anterior pituitary hormone-producing cells that upon transplantation produce human growth hormone and adrenocorticotropic hormone in vivo. Our results establish a powerful experimental platform to study human cranial placode development and set the stage for the development of human cell-based therapies in sensory and endocrine disease.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias/citología , Células Endocrinas/citología , Neuronas/citología , Células Madre Pluripotentes/citología , Hormona Adrenocorticotrópica/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Células Endocrinas/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Estratos Germinativos/citología , Hormona del Crecimiento/metabolismo , Humanos , Cristalino/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neuronas/metabolismo , Periferinas/genética , Periferinas/metabolismo , Hipófisis/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplante , Ganglio del Trigémino/citologíaRESUMEN
In vitro expanded neural precursor cells (NPCs) may provide a stable source for cell therapy. In search of the optimal cell source for spinal cord repair, we investigated influences of gestational age, regional heterogeneity, and long-term in vitro propagation. The cellular content of neurosphere cultures prior to and after in vitro differentiation was studied by immunocytochemistry and flow cytometry. Human forebrain and spinal cord NPCs deriving from first-trimester tissue were cultured as neurospheres in the presence of epidermal growth factor, basic fibroblast growth factor, and ciliary neurotrophic factor. Proteins characteristic for embryonic stem cells, i.e., Tra-1-60, Tra-1-81, and SSEA-4, were present in approximately 0.5% of the cells in donor tissues and neurospheres. The proportions of nestin- and proliferating cell nuclear antigen-immunoreactive (IR) cells were also maintained, whereas the CD133-IR population increased in vitro. Glial fibrillary acidic protein-IR cells increased in number, and in contrast the fraction of beta-tubulin III-IR cells decreased, at and beyond passage 5 in spinal cord but not forebrain cultures. However, dissociated and in vitro-differentiated forebrain- and spinal cord-derived neurospheres generated similar proportions of neurons, astrocytes, and oligodendrocytes. Gestational age of the donor tissue, which ranged from 4.5 to 12 weeks for forebrain and from 4.5 to 9.5 weeks for spinal cord, did not affect the proportion of cells with different phenotypes in culture. Thus, cellular composition of human neurosphere cultures differs as a result of long-term in vitro propagation and regional heterogeneity of source tissue, despite expansion under equal culture conditions. This could in turn imply that human spinal cord and forebrain NPCs present different repair potentials in in vivo settings.