Showing papers by "Clive N. Svendsen published in 2016"
TL;DR: It is shown that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation in rats that model severe autosomal dominant retinitis pigmentosa.
260 citations
TL;DR: Common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques are discussed and how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues are suggested.
Abstract: Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease.
235 citations
TL;DR: This approach produced a maturation scale revealing that iPSC-derived spMNs were more similar to fetal spinal tissue than to adult sp MNs, and suggesting that developing strategies to further mature and age iPSCs will provide more effectiveiPSC models of ALS pathology.
Abstract: Modeling amyotrophic lateral sclerosis (ALS) with human induced pluripotent stem cells (iPSCs) aims to reenact embryogenesis, maturation and aging of spinal motor neurons (spMNs) in vitro. As the maturity of spMNs grown in vitro compared to spMNs in vivo remains largely unaddressed, it is unclear to what extent this in vitro system captures critical aspects of spMN development and molecular signatures associated with ALS. Here, we compared transcriptomes among iPSC-derived spMNs, fetal spinal tissues and adult spinal tissues. This approach produced a maturation scale revealing that iPSC-derived spMNs were more similar to fetal spinal tissue than to adult spMNs. Additionally, we resolved gene networks and pathways associated with spMN maturation and aging. These networks enriched for pathogenic familial ALS genetic variants and were disrupted in sporadic ALS spMNs. Altogether, our findings suggest that developing strategies to further mature and age iPSC-derived spMNs will provide more effective iPSC models of ALS pathology.
88 citations
Journal Article•
TL;DR: The aim was to develop a protocol for freezing neurons differentiated from induced pluripotent stem cells (iMNs) from a patient affected by spinal muscular atrophy, and it was found that while flash-frozen iMNs are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved cells.
Abstract: In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it has proven difficult to perform this technique on frozen samples because freezing cells before extracting nuclei impairs nuclear integrity and alters chromatin structure. We describe a protocol for freezing cells that is compatible with ATAC-Seq, producing results that compare well with those generated from fresh cells. We found that while flash-frozen samples are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved samples. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved samples agree quantitatively. We developed our protocol on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells from a patient affected by spinal muscular atrophy.
35 citations
TL;DR: In this paper, the authors developed a protocol for freezing neuronal cells that is compatible with ATAC-Seq; they focused on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells (iMNs) from a patient affected by spinal muscular atrophy.
Abstract: In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it is difficult to perform this technique on frozen samples because freezing cells before extracting nuclei can impair nuclear integrity and alter chromatin structure, especially in fragile cells such as neurons. Our aim was to develop a protocol for freezing neuronal cells that is compatible with ATAC-Seq; we focused on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells (iMNs) from a patient affected by spinal muscular atrophy. We found that while flash-frozen iMNs are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved cells. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved iMNs quantitatively agree.
34 citations
TL;DR: This review summarizes the current state of regenerative stem cell transplantation divided into seven conditions for which trials are currently active: demyelinating diseases/spinal cord injury, amyotrophic lateral sclerosis, stroke, Parkinson's disease, Huntington's Disease, macular degeneration and peripheral nerve diseases.
33 citations
TL;DR: It is concluded that fetal-derived human neural progenitor cells can survive well in the aging spinal cord, protect motor neurons and mature faster into astrocytes when compared to transplantation into the young spinal cord.
25 citations
TL;DR: The concussion model of repeat, mild, bilateral TBI induced long-lasting deficits in motor function, decreased cortical thickness, shrinkage of the corpus callosum, increased brain tauopathy, and earlier onset of ALS symptoms in SOD1 rats may allow for a greater understanding of the complex relationship between TBI and neurodegenerative diseases.
Abstract: BACKGROUNDConcussion injury is the most common form of traumatic brain injury (TBI). How recurrent concussions alter long-term outcomes is poorly understood, especially as related to the development of neurodegenerative disease. We evaluated the functional and pathological consequences of repeated T
23 citations
TL;DR: This study validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs), and shows that TGFβ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs.
Abstract: Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye, and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGFβ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGFβ-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis, and development of regenerative medicine approaches.
22 citations
TL;DR: It is found that increased hypointensity on MRI images could establish hNPCFer location, and another molecular imaging technique must be employed, including perhaps multimodal imaging, which would utilize MRI along with another imaging modality, for cell survival to be monitored in preclinical and clinical settings.
20 citations
TL;DR: The production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS is described for use in the development of safe and efficient gene therapy for neurological disorders.
Abstract: Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone’s ability to cross the blood–brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders.
TL;DR: The protocol of ex vivo delivery of growth factors via lentiviral vector-mediated genetic modification of hMSCs and hMSC transplantation into the skeletal muscle of a familial ALS rat model is described.
Abstract: Therapeutic protein and molecule delivery to target sites by transplanted human stem cells holds great promise for ex vivo gene therapy. Our group has demonstrated the therapeutic benefits of ex vivo gene therapy targeting the skeletal muscles in a transgenic rat model of familial amyotrophic lateral sclerosis (ALS). We used human mesenchymal stem cells (hMSCs) and genetically modified them to release neuroprotective growth factors such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF). Intramuscular growth factor delivery via hMSCs can enhance neuromuscular innervation and motor neuron survival in a rat model of ALS (SOD1(G93A) transgenic rats). Here, we describe the protocol of ex vivo delivery of growth factors via lentiviral vector-mediated genetic modification of hMSCs and hMSC transplantation into the skeletal muscle of a familial ALS rat model.
Patent•
19 Oct 2016TL;DR: In this article, the authors proposed culturing brain endothelial cells, and optionally astrocytes and neurons in a fluidic device under conditions whereby the cells mimic the structure and function of the blood brain barrier.
Abstract: The invention relates to culturing brain endothelial cells, and optionally astrocytes and neurons in a fluidic device under conditions whereby the cells mimic the structure and function of the blood brain barrier. Culture of such cells in a microfluidic device, whether alone or in combination with other cells, drives maturation and/or differentiation further than existing systems.
Patent•
16 Jun 2016TL;DR: In this article, methods and compositions related to generation of induced pluripotent stem cells (iPSCs) are described. But they do not consider the use of non-integrating episomal plasmid vectors.
Abstract: Described herein are methods and compositions related to generation of induced pluripotent stem cells (iPSCs). Improved techniques for establishing highly efficient, reproducible reprogramming using non-integrating episomal plasmid vectors. Using the described reprogramming protocol, one is able to consistently reprogram non-T cells with close to 100% success from non-T cell or non-B cell sources. Further advantages include use of a defined reprogramming media E7 and using defined clinically compatible substrate recombinant human L-521. Generation of iPSCs from these blood cell sources allows for recapitulation of the entire genomic repertoire, preservation of genomic fidelity and enhanced genomic stability.
TL;DR: A protocol for freezing cells that is compatible with ATAC-Seq is described, producing results that compare well with those generated from fresh cells and verified that epigenetic results from fresh and cryopreserved samples agree quantitatively.
Abstract: In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it has proven difficult to perform this technique on frozen samples because freezing cells before extracting nuclei impairs nuclear integrity and alters chromatin structure. We describe a protocol for freezing cells that is compatible with ATAC-Seq, producing results that compare well with those generated from fresh cells. We found that while flash-frozen samples are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved samples. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved samples agree quantitatively. We developed our protocol on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells from a patient affected by spinal muscular atrophy.
Patent•
19 Oct 2016
TL;DR: In this paper, the authors concerne la culture de cellules endotheliales cerebrales, and eventuellement d'astrocytes et de neurones dans un dispositif fluidique dans des conditions dans lesquelles les cellules imitent la structure and la fonction de la barriere hemato-encephalique.
Abstract: L'invention concerne la culture de cellules endotheliales cerebrales, et eventuellement d'astrocytes et de neurones dans un dispositif fluidique dans des conditions dans lesquelles les cellules imitent la structure et la fonction de la barriere hemato-encephalique. La culture de telles cellules dans un dispositif microfluidique, soit seules soit en combinaison avec d'autres cellules, entraine une maturation et/ou une differenciation plus poussee que dans les systemes existants.
Patent•
16 Jun 2016TL;DR: In this article, methods and compositions related to generation of induced pluripotent stem cells (iPSCs) are described. But they do not consider the use of non-integrating episomal plasmid vectors.
Abstract: Described herein are methods and compositions related to generation of induced pluripotent stem cells (iPSCs). Improved techniques for establishing highly efficient, reproducible reprogramming using non-integrating episomal plasmid vectors. Using the described reprogramming protocol, one is able to consistently reprogram non-T cells with close to 100% success from non-T cell or non-B cell sources. Further advantages include use of a defined reprogramming media E7 and using defined clinically compatible substrate recombinant human L-521. Generation of iPSCs from these blood cell sources allows for recapitulation of the entire genomic repertoire, preservation of genomic fidelity and enhanced genomic stability.
Patent•
28 Oct 2016TL;DR: In this article, an ionizing radiation source and a digital radiation detector are coupled to opposing blades of a tissue retractor for performing a variety of medical procedures, including those involving introducing one or more substances into a subject's body.
Abstract: The present invention teaches apparatuses, systems and methods for performing a variety of medical procedures, including those involving introducing one or more substances into a subjects body. In some embodiments, the invention teaches an imaging system that includes an ionizing radiation source and a digital radiation detector that are coupled to opposing blades of a tissue retractor