RESUMEN
Cell therapy products are frequently developed and produced without incorporating cost considerations into process development, contributing to prohibitively costly products. Herein we contextualize individual process development decisions within a broad framework for cost-efficient therapeutic manufacturing. This roadmap guides the analysis of cost of goods (COG) arising from tissue procurement, material acquisition, facility operation, production, and storage. We present the specific COG considerations related to each of these elements as identified through a 2013 International Society for Cellular Therapy COG survey, highlighting the differences between autologous and allogeneic products. Planning and accounting for COG at each step in the production process could reduce costs, allowing for more affordable market pricing to improve the long-term viability of the cell therapy product and facilitate broader patient access to novel and transformative cell therapies.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/economía , Comercio , Costos y Análisis de Costo , HumanosRESUMEN
The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , HumanosRESUMEN
The lineages of both astrocytes and oligodendrocytes have been popular areas of research in the last decade. The source of these cells in the mature CNS is relevant to the study of the cellular response to CNS injury. A significant amount of evidence exists to suggest that resident precursor cells proliferate and differentiate into mature glial cells that facilitate tissue repair and recovery. Additionally, the re-entry of mature astrocytes into the cell cycle can also contribute to the pool of new astrocytes that are observed following CNS injury. In order to better understand the glial response to injury in the adult CNS we must revisit the astrocyte-oligodendrocyte relationship. Specifically, we argue that there is a common glial precursor cell from which astrocytes and oligodendrocytes differentiate and that the microenvironment surrounding the injury determines the fate of the stimulated precursor cell. Ideally, better understanding the origin of new glial cells in the injured CNS will facilitate the development of therapeutics targeted to alter the glial response in a beneficial way.
Asunto(s)
Astrocitos/fisiología , Lesiones Encefálicas/fisiopatología , Sistema Nervioso Central/fisiología , Regeneración Nerviosa/fisiología , Oligodendroglía/fisiología , Animales , Astrocitos/citología , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Sistema Nervioso Central/citología , Gliosis/fisiopatología , Humanos , Oligodendroglía/citología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Transverse myelitis (TM) is an immune-mediated spinal cord disorder associated with inflammation, demyelination, and axonal damage. We investigated the soluble immune derangements present in TM patients and found that IL-6 levels were selectively and dramatically elevated in the cerebrospinal fluid and directly correlated with markers of tissue injury and sustained clinical disability. IL-6 was necessary and sufficient to mediate cellular injury in spinal cord organotypic tissue culture sections through activation of the JAK/STAT pathway, resulting in increased activity of iNOS and poly(ADP-ribose) polymerase (PARP). Rats intrathecally infused with IL-6 developed progressive weakness and spinal cord inflammation, demyelination, and axonal damage, which were blocked by PARP inhibition. Addition of IL-6 to brain organotypic cultures or into the cerebral ventricles of adult rats did not activate the JAK/STAT pathway, which is potentially due to increased expression of soluble IL-6 receptor in the brain relative to the spinal cord that may antagonize IL-6 signaling in this context. The spatially distinct responses to IL-6 may underlie regional vulnerability of different parts of the CNS to inflammatory injury. The elucidation of this pathway identifies specific therapeutic targets in the management of CNS autoimmune conditions.
Asunto(s)
Interleucina-6/líquido cefalorraquídeo , Mielitis Transversa/líquido cefalorraquídeo , Traumatismos de la Médula Espinal/líquido cefalorraquídeo , Adulto , Animales , Axones/metabolismo , Axones/patología , Femenino , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Interleucina-6/administración & dosificación , Masculino , Ratones , Mielitis Transversa/complicaciones , Mielitis Transversa/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Técnicas de Cultivo de Órganos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/patologíaRESUMEN
A major challenge to commercializing cell-based therapies is developing scalable manufacturing processes while maintaining the critical quality parameters (identity, potency, purity, safety) of the final live cell product. Process development activities such as extended passaging and serum reduction/elimination can facilitate the streamlining of cell manufacturing process as long as the biological functions of the product remain intact. Best practices in process development will be dependent on cell characterization; a thorough understanding of the cell-based product. Unique biological properties associated with different types of cell-based products are discussed. Cell characterization may be used as a tool for successful process development activities, which can promote a candidate cell therapy product through clinical development and ultimately to a commercialized product.
Asunto(s)
Bioensayo/métodos , Bioensayo/normas , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Control de Calidad , HumanosRESUMEN
Neuroadapted Sindbis virus (NSV) is a neuronotropic virus that causes a fulminant encephalomyelitis in susceptible mice due to death of motor neurons in the brain and spinal cord. We and others have found that uninfected motor neurons die in response to NSV infection, at least in part due to disrupted astrocytic glutamate transport, resulting in excitotoxic motor neuron death. Here, we examined the mechanisms of astrocyte dysregulation associated with NSV infection. Treatment of organotypic slice cultures with NSV results in viral replication, cell death, altered astrocyte morphology, and the downregulation of the astrocytic glutamate transporter, GLT-1. We have found that TNF-alpha can mediate GLT-1 downregulation. Furthermore, TNF-alpha deficient mice infected with NSV exhibit neither GLT-1 downregulation nor neuronal death of brainstem and cervical spinal cord motor neurons and have markedly reduced mortality. These findings have implications for disease intervention and therapeutic development for the prevention of CNS damage associated with inflammatory responses.
Asunto(s)
Infecciones por Alphavirus/metabolismo , Encéfalo/metabolismo , Encefalomielitis/metabolismo , Ácido Glutámico/metabolismo , Virus Sindbis/fisiología , Médula Espinal/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Infecciones por Alphavirus/virología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/virología , Muerte Celular , Regulación hacia Abajo , Encefalomielitis/patología , Encefalomielitis/virología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/citología , Neuronas/metabolismo , Neuronas/virología , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Médula Espinal/virología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Astrocytes play important roles in normal CNS function; however, following traumatic injury or during neurodegeneration, astrocytes undergo changes in morphology, gene expression and cellular function known as reactive astrogliosis, a process that may also include cell proliferation. At present, the role of astrocyte proliferation is not understood in disease etiology of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder that is characterized by a relatively rapid degeneration of upper and lower motor neurons. Therefore, the role of astrocyte proliferation was assessed in both acute and chronic mouse models of motor neuron degeneration, neuroadapted sindbis virus (NSV)-infected mice and SOD1(G93A) mice, respectively. While astrocytes proliferated in the lumbar spinal cord ventral horn of both disease models, they represented only a small percentage of the dividing population in the SOD1(G93A) spinal cord. Furthermore, selective ablation of proliferating GFAP(+) astrocytes in 1) NSV-infected transgenic mice in which herpes simplex virus-thymidine kinase is expressed in GFAP(+) cells (GFAP-TK) and in 2) SOD1(G93A)xGFAP-TK mice did not affect any measures of disease outcome such as animal survival, disease onset, disease duration, hindlimb motor function or motor neuron loss. Ablation of dividing astrocytes also did not alter overall astrogliosis in either model. This was likely due to the finding that proliferation of NG2(+) glial progenitors were unaffected. These findings demonstrate that while normal astrocyte function is an important factor in the etiology of motor neuron diseases such as ALS, astrocyte proliferation itself does not play a significant role.
Asunto(s)
Astrocitos/citología , Proliferación Celular , Modelos Animales de Enfermedad , Neuronas Motoras/citología , Degeneración Nerviosa/patología , Enfermedad Aguda , Animales , Astrocitos/patología , Astrocitos/fisiología , Células Cultivadas , Enfermedad Crónica , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Degeneración Nerviosa/genéticaRESUMEN
The focus of most neurodegenerative disease studies has been on neuronal death in particular subpopulations of the central nervous system. The associated response of glial populations has been ascribed the term "reactive astrocytosis." This has been defined as the proliferation of astrocytes accompanied by cellular hypertrophy and changes in gene expression following injury to the central nervous system. Yet the significance of that response to disease course is debated. In both human ALS and in the SOD1G93A mouse model of ALS, reactive astrocytosis is a hallmark of the disease--particularly at endstage. The brain also harbors immature progenitors which have the capacity for differentiation into both glial and neuronal lineages. We examined whether glial progenitors in the adult spinal cord of SOD1G93A mice become activated and contribute the astroglial response observed in this model. We found that the glial progenitor proteoglycan NG2 is increased in parallel with GFAP during the symptomatic phase of the disease and that there is a differential in vitro response of SOD1G93A glial progenitors to inflammatory cytokines when compared to wildtype mouse glial progenitors. This response was accompanied by the proliferation of glial progenitors but not mature GFAP+ astrocytes, through the translocation of the transcription factor Olig2 from the nucleus to the cytoplasm-resulting in astrocyte differentiation. These data suggest that adult glial progenitors from SOD1G93A mice differentially respond to inflammatory cytokines and contribute to the observed reactive astrocytosis observed in SOD1G93A mouse lumbar spinal cord.
Asunto(s)
Células Madre Adultas/fisiología , Encéfalo/citología , Proliferación Celular , Neuroglía/fisiología , Superóxido Dismutasa/genética , Células Madre Adultas/efectos de los fármacos , Factores de Edad , Animales , Antígenos/metabolismo , Bromodesoxiuridina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Recuento de Células/métodos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos , Proteoglicanos/metabolismoRESUMEN
Neuroadapted Sindbis Virus (NSV) is a neuronotropic virus that causes hindlimb paralysis in susceptible mice and rats. The authors and others have demonstrated that though death of infected motor neurons occurs, bystander death of uninfected neurons also occurs and both contribute to the paralysis that ensues following infection. The authors have previously shown that the treatment of NSV-infected mice with minocycline, an inhibitor that has many functions within the central nervous system (CNS), including inhibiting microglial activation, protects mice from paralysis and death. The authors, therefore, proposed that microglial activation may contribute to bystander death of motor neurons following NSV infection. Here, the authors tested the hypothesis using a conditional knock-out of activated macrophage-lineage cells, including endogenous CNS macrophage cells. Surprisingly, ablation of these cells resulted in more rapid death and similar weakness in the hind limbs of NSV-infected animals compared with that of control animals. Several key chemokines including IL-12 and monocyte chemoattractant protein-1 (MCP-1) did not become elevated in these animals, resulting in decreased infiltration of T lymphocytes into the CNS of the knock-down animals. Either because of the decreased macrophage activation directly or because of the reduced immune cell influx, viral replication persisted longer within the nervous system in knock-down mice than in wild type mice. The authors, therefore, conclude that although macrophage-lineage cells in the CNS may contribute to neurodegeneration in certain situations, they also serve a protective role, such as control of viral replication.
Asunto(s)
Infecciones por Alphavirus/inmunología , Enfermedades Virales del Sistema Nervioso Central/inmunología , Macrófagos/inmunología , Macrófagos/virología , Infecciones por Alphavirus/genética , Animales , Encéfalo/inmunología , Encéfalo/virología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Enfermedades Virales del Sistema Nervioso Central/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Virus Sindbis/genética , Virus Sindbis/inmunología , Médula Espinal/inmunología , Médula Espinal/virologíaRESUMEN
OBJECTIVE: We explored the potential of embryonic stem cell-derived motor neurons to functionally replace those cells destroyed in paralyzed adult rats. METHODS: We administered a phosphodiesterase type 4 inhibitor and dibutyryl cyclic adenosine monophosphate to overcome myelin-mediated repulsion and provided glial cell-derived neurotrophic factor within the sciatic nerve to attract transplanted embryonic stem cell-derived axons toward skeletal muscle targets. RESULTS: We found that these strategies significantly increased the success of transplanted axons extending out of the spinal cord into ventral roots. Furthermore, transplant-derived axons reached muscle, formed neuromuscular junctions, were physiologically active, and mediated partial recovery from paralysis. INTERPRETATION: We conclude that restoration of functional motor units by embryonic stem cells is possible and represents a potential therapeutic strategy for patients with paralysis. To our knowledge, this is the first report of the anatomical and functional replacement of a motor neuron circuit within the adult mammalian host.
Asunto(s)
Parálisis/terapia , Recuperación de la Función , Nervio Ciático/fisiología , Médula Espinal/fisiología , Trasplante de Células Madre/métodos , Factores de Edad , Animales , Recuento de Células , Supervivencia Celular , Células Cultivadas/trasplante , Electrofisiología , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Supervivencia de Injerto , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Músculo Esquelético/inervación , Regeneración Nerviosa/fisiología , Unión Neuromuscular/fisiología , Parálisis/fisiopatología , Ratas , Ratas Endogámicas Lew , Nervio Ciático/citología , Médula Espinal/citologíaRESUMEN
The immune system has a complex and dynamic relationship with the nervous system, both in health and disease. The immune system surveys the central and peripheral nervous systems, becoming activated in response to foreign substances, infectious particles or neoplasms. Conversely, the nervous system modulates immune system function both through the neuroendocrine axis and through vagus nerve efferents. In disease states, this dynamic relationship is perturbed, resulting in neuropsychiatric diseases. In this manuscript, we will summarize fundamental principles of the immune system and its interaction with the nervous system. We will describe the critical components of the adaptive and innate branches of the immune system and will describe important effectors and signalling pathways in each. By understanding the principles of the immune system and how these principles relate to nervous system function, the reader will be prepared to interpret subsequent manuscripts in this issue.