Pluripotent stem cells in regenerative medicine: challenges and recent progress
TL;DR: Recent progress in directed differentiation, some of the new technologies that have facilitated the success of hPSC therapies and the remaining hurdles on the road towards developing hPSc-based cell therapies are discussed.
Abstract: The derivation of disease-relevant cell types from pluripotent stem cells holds much promise for disease therapy. The recent progress in directed differentiation and the challenges ahead are discussed in this Review. After years of incremental progress, several recent studies have succeeded in deriving disease-relevant cell types from human pluripotent stem cell (hPSC) sources. The prospect of an unlimited cell source, combined with promising preclinical data, indicates that hPSC technology may be on the verge of clinical translation. In this Review, we discuss recent progress in directed differentiation, some of the new technologies that have facilitated the success of hPSC therapies and the remaining hurdles on the road towards developing hPSC-based cell therapies.
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TL;DR: A simple and reproducible 3D culture approach for generating a laminated cerebral cortex–like structure, named human cortical spheroids (hCSs), from pluripotent stem cells, which demonstrate that cortical neurons participate in network activity and produce complex synaptic events.
Abstract: The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.
1,104 citations
TL;DR: Three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons are generated and it is found that in Timothy syndrome—a neurodevelopmental disorder caused by mutations in the CaV1.2 calcium channel—interneurons display abnormal migratory saltations.
Abstract: The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.
837 citations
TL;DR: An overview of Wnt-β-catenin signaling highlighting its key functions during development and adult tissue homeostasis is provided.
Abstract: The Wnt-β-catenin signaling pathway is an evolutionarily conserved cell-cell communication system that is important for stem cell renewal, cell proliferation and cell differentiation both during embryogenesis and during adult tissue homeostasis. Genetic or epigenetic events leading to hypo- or hyper-activation of the Wnt-β-catenin signaling cascade have also been associated with human diseases such as cancer. Understanding how this pathway functions is thus integral for developing therapies to treat diseases or for regenerative medicine approaches. Here, and in the accompanying poster, we provide an overview of Wnt-β-catenin signaling and briefly highlight its key functions during development and adult tissue homeostasis.
467 citations
Cites background from "Pluripotent stem cells in regenerat..."
...In line with its role in embryonic development, the further differentiation of ESCs into different germ layers requires the temporal activation of Wnt-β-catenin signaling to steer cells towards the mesendoderm lineage or inactivation to obtain neuroectoderm (Tabar and Studer, 2014)....
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TL;DR: Techniques for membrane disruption-based intracellular delivery from 1911 until the present achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution.
Abstract: Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo types—small molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications a...
420 citations
TL;DR: It is found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro, they transition from a predominantly fetal to an increasingly matureAstrocyte state.
385 citations
Cites background from "Pluripotent stem cells in regenerat..."
...Human induced pluripotent stem cells (iPSCs) provide a unique platform to investigate neural development in vitro (Dolmetsch and Geschwind, 2011; Paşca et al., 2014; Tabar and Studer, 2014) and to elucidate mechanisms of astrocyte development and dysfunction....
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...Human induced pluripotent stem cells (iPSCs) provide a unique platform to investigate neural development in vitro (Dolmetsch and Geschwind, 2011; Paşca et al., 2014; Tabar and Studer, 2014) and to elucidate mechanisms of astrocyte development and dysfunction....
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References
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TL;DR: In this article, the authors used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4p16.3 as the likely location of the defect, which is expanded and unstable on HD chromosomes.
7,224 citations
Journal Article•
TL;DR: The Huntington's disease mutation involves an unstable DNA segment, similar to those described in fragile X syndrome, spino-bulbar muscular atrophy, and myotonic dystrophy, acting in the context of a novel 4p16.3 gene to produce a dominant phenotype.
6,992 citations
TL;DR: A human pluripotent stem cell-derived three-dimensional organoid culture system that develops various discrete, although interdependent, brain regions that include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes is developed.
Abstract: The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue
3,508 citations
TL;DR: A review of achievements made possible by site-specific nuclease technologies and applications of these reagents for genetic analysis and manipulation, including the therapeutic potential of ZFNs and TALENs, and future prospects for the field are discussed.
3,235 citations
TL;DR: Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.
Abstract: Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.
3,152 citations