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1.
Stem Cell Res ; 41: 101581, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31812872

RESUMO

Human pluripotent stem cells (hPSC) represent a unique opportunity to study fundamental biological processes in a human- and cell-specific setting. Its translational potential and the impact on human health makes this technology revolutionary. The possibility to generate stem cells from almost any somatic cell, and their capacity to be differentiated in virtually all cells of the body has been demonstrated extensively during the last decade of research. Target-centric as well as phenotypic screenings using differentiated cells have become a reality, while the use of these cells for "disease modelling" is still challenging due to the paucity of relevant and reproducible phenotypes. The combination of hPSCs with gene editing technologies aiming to e.g. reduce immunogenic response has enabled promising clinical trials that will eventually demonstrate their therapeutic potential in tissue regeneration and cancer treatment. Maximizing the therapeutic applications of hPSCs requires systematic data comparison, consensus between scientists and health care professionals, as well as a close collaboration between research labs, clinics, and regulators. The goal of this review is to provide a comprehensive outlook of the current use of hPSCs in drug development and regenerative medicine for the treatment of central nervous system (CNS) disorders. In the first part, we analyse how hPSCs are currently used in drug development and discuss their use in challenging paradigms such as neurodegeneration. In the second part we review the status of hPSCs in regenerative medicine. Finally, key challenges and pitfalls of the technology will be discussed, and strategies proposed to improve hPSC research and to benefit patients across different therapeutic areas.


Assuntos
Diferenciação Celular , Doenças do Sistema Nervoso Central/terapia , Células-Tronco Pluripotentes/citologia , Medicina Regenerativa , Transplante de Células-Tronco , Animais , Humanos
2.
Trends Biotechnol ; 35(7): 573-576, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28511937

RESUMO

Achieving consistency in standards of access to and quality of human induced pluripotent stem cells has lagged behind their use. In Europe, a network of academic and industrial partners has been established to overcome this challenge. The experience reveals the devil in the detail of worthy ambitions informing future efforts.


Assuntos
Bancos de Espécimes Biológicos , Células-Tronco Pluripotentes , Europa (Continente) , Humanos
3.
Neurochem Int ; 59(3): 432-44, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21762743

RESUMO

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.


Assuntos
Neurônios/citologia , Células-Tronco/citologia , Animais , Adesão Celular , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase/métodos , Transplante de Células-Tronco
4.
Cytotherapy ; 12(3): 313-25, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20370348

RESUMO

BACKGROUND AIMS: Spinal cord injury is a devastating injury that impacts drastically on the victim's quality of life. Stem cells have been proposed as a therapeutic strategy. Neural stem (NS) cells have been harvested from embryonic mouse forebrain and cultured as adherent cells. These NS cells express markers of neurogenic radial glia. METHODS: Mouse NS cells expressing green fluorescent protein (GFP) were transplanted into immunosupressed rat spinal cords following moderate contusion injury at T9. Animals were left for 2 and 6 weeks then spinal cords were fixed, cryosectioned and analyzed. Stereologic methods were used to estimate the volume and cellular environment of the lesions. Engraftment, migration and differentiation of NS cells were also examined. RESULTS: NS cells integrated well into host tissue and appeared to migrate toward the lesion site. They expressed markers of neurons, astrocytes and oligodendrocytes at 2 weeks post-transplantation and markers of neurons and astrocytes at the 6-week time-point. NS cells appeared to have a similar morphologic phenotype to radial glia, in particular at the pial surface. CONCLUSIONS: Although no functional recovery was observed using the Basso Beattie Bresnahan (BBB) locomotor rating scale, NS cells are a potential cellular therapy for treatment of injured spinal cord. They may be used as delivery vehicles for therapeutic proteins because they show an ability to migrate toward the site of a lesion. They may also be used to replace lost or damaged neurons and oligodendrocytes.


Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Neurônios , Traumatismos da Medula Espinal , Medula Espinal , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Comportamento Animal/fisiologia , Biomarcadores/metabolismo , Sobrevivência Celular , Células Cultivadas , Camundongos , Neurônios/citologia , Neurônios/fisiologia , Neurônios/transplante , Distribuição Aleatória , Ratos , Medula Espinal/citologia , Medula Espinal/patologia , Medula Espinal/transplante , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/cirurgia , Células-Tronco/citologia
5.
Methods Mol Biol ; 329: 353-69, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16846004

RESUMO

The process of bringing a new pharmacologically active drug to market is laborious, time consuming, and costly. From drug discovery to safety assessment, new methods are constantly sought to develop faster and more efficient procedures to eliminate drugs from further investigation because of their limited effectiveness or high toxicity. Because in vitro cell assays are an important arm of this discovery process, it is therefore somewhat unsurprising that there is an emerging contribution of embryonic stem (ES) cell technology to this area. This technology utilizes the in vitro differentiation of ES cells into somatic cell target populations that, when coupled to the use of "lineage selection" protocols, allows for the production of infinite numbers of pure populations of the desired cells for both bioactivity and toxicological screens. Unlike the use of transformed cell lines, ES-derived cells remain karyotypically normal and therefore better reflect the potential responses of cells in vivo, and when selected are more homogeneous than those obtained using primary cultures. In this chapter we discuss the use of ES cell-derived somatic cells in pharmacological screens, with particular emphasis on neural cells, and describe the methods and protocols associated with the development of ES cell-derived neural cell assays.


Assuntos
Embrião de Mamíferos/citologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Meios de Cultura , Proteínas de Ligação a DNA/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Marcação de Genes , Genes Reporter , Vetores Genéticos , Proteínas de Grupo de Alta Mobilidade/genética , Camundongos , Plasmídeos/genética , Fatores de Transcrição SOXB1
6.
Pharmacol Res ; 47(4): 269-78, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12644383

RESUMO

Established embryonic stem (ES) cell lines have been at the forefront of approaches to understand gene function during embryogenesis and in adult vertebrate organisms, principally due to exploitation of two essential attributes; their pluripotency or ability to contribute to all three germinal layers and germ line in mice and their ease of genetic modification. Endeavours to routinely establish ES cells from species other than mice have met with limited success, although with rapid progress being made in our understanding of their basic cell biology, the regular derivation of lines from pre-implantation embryos will become easier for many species including humans. With a recent growing awareness of how these cells can be made to grow in an unlimited, but regulated manner plus how their fate can be directed or manipulated into diverse, mature phenotypes in culture, it has become clear that the biological resource offers additional attractive features applicable for future biomedical research and therapy. Advanced mouse ES-based technologies are being used in the industry for pharmaceutical discovery and development, while it is also anticipated that human ES cell reagents will revolutionise aspects of regenerative medicine. This review will summarise the advantages, potential and great hope for ES cell based systems in these contexts.


Assuntos
Linhagem Celular/efeitos dos fármacos , Química Farmacêutica , Embrião de Mamíferos/citologia , Células-Tronco/citologia , Animais , Agregação Celular/efeitos dos fármacos , Agregação Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Avaliação de Medicamentos , Embrião de Mamíferos/efeitos dos fármacos , Engenharia Genética , Humanos , Camundongos , Células-Tronco/efeitos dos fármacos
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