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Article ; Online: A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

Shelley, Brandon C / Gowing, Geneviève / Svendsen, Clive N

Journal of visualized experiments : JoVE

2014 , Issue 88

Abstract: A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving ... ...

Abstract	A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
MeSH term(s)	Brain/cytology ; Brain/embryology ; Cell Aggregation/physiology ; Cell Communication/physiology ; Cell Growth Processes/physiology ; Cytological Techniques/methods ; Humans ; Neural Stem Cells/cytology ; Pluripotent Stem Cells/cytology
Language	English
Publishing date	2014-06-15
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/51219
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: A cgmp-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue

Shelley, Brandon C / Gowing, Geneviève / Svendsen, Clive N

Journal of visualized experiments. 2014 June 15, , no. 88

2014

Abstract	A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
Keywords	automation ; brain ; chopping ; clinical trials ; dissociation ; good manufacturing practices ; neural stem cells ; tissue culture
Language	English
Dates of publication	2014-0615
Size	p. e51219.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/51219
Database	NAL-Catalogue (AGRICOLA)

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Article: Human Neural Progenitor Transplantation Rescues Behavior and Reduces α-Synuclein in a Transgenic Model of Dementia with Lewy Bodies.

Goldberg, Natalie R S / Marsh, Samuel E / Ochaba, Joseph / Shelley, Brandon C / Davtyan, Hayk / Thompson, Leslie M / Steffan, Joan S / Svendsen, Clive N / Blurton-Jones, Mathew

Stem cells translational medicine

2017 Volume 6, Issue 6, Page(s) 1477–1490

Abstract: Synucleinopathies are a group of neurodegenerative disorders sharing the common feature of misfolding and accumulation of the presynaptic protein α-synuclein (α-syn) into insoluble aggregates. Within this diverse group, Dementia with Lewy Bodies (DLB) is ...

Abstract	Synucleinopathies are a group of neurodegenerative disorders sharing the common feature of misfolding and accumulation of the presynaptic protein α-synuclein (α-syn) into insoluble aggregates. Within this diverse group, Dementia with Lewy Bodies (DLB) is characterized by the aberrant accumulation of α-syn in cortical, hippocampal, and brainstem neurons, resulting in multiple cellular stressors that particularly impair dopamine and glutamate neurotransmission and related motor and cognitive function. Recent studies show that murine neural stem cell (NSC) transplantation can improve cognitive or motor function in transgenic models of Alzheimer's and Huntington's disease, and DLB. However, examination of clinically relevant human NSCs in these models is hindered by the challenges of xenotransplantation and the confounding effects of immunosuppressant drugs on pathology and behavior. To address this challenge, we developed an immune-deficient transgenic model of DLB that lacks T-, B-, and NK-cells, yet exhibits progressive accumulation of human α-syn (h-α-syn)-laden inclusions and cognitive and motor impairments. We demonstrate that clinically relevant human neural progenitor cells (line CNS10-hNPCs) survive, migrate extensively and begin to differentiate preferentially into astrocytes following striatal transplantation into this DLB model. Critically, grafted CNS10-hNPCs rescue both cognitive and motor deficits after 1 and 3 months and, furthermore, restore striatal dopamine and glutamate systems. These behavioral and neurochemical benefits are likely achieved by reducing α-syn oligomers. Collectively, these results using a new model of DLB demonstrate that hNPC transplantation can impact a broad array of disease mechanisms and phenotypes and suggest a cellular therapeutic strategy that should be pursued. Stem Cells Translational Medicine 2017;6:1477-1490.
MeSH term(s)	Animals ; Astrocytes/cytology ; Astrocytes/metabolism ; Cells, Cultured ; Humans ; Lewy Body Disease/therapy ; Memory ; Mice ; Neural Stem Cells/cytology ; Neural Stem Cells/metabolism ; Neural Stem Cells/transplantation ; Neurogenesis ; Stem Cell Transplantation/methods ; alpha-Synuclein/metabolism
Chemical Substances	alpha-Synuclein
Language	English
Publishing date	2017-02-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2642270-0
ISSN	2157-6580 ; 2157-6564
ISSN (online)	2157-6580
ISSN	2157-6564
DOI	10.1002/sctm.16-0362
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells.

Akhtar, Aslam Abbasi / Gowing, Genevieve / Kobritz, Naomi / Savinoff, Steve E / Garcia, Leslie / Saxon, David / Cho, Noell / Kim, Gibum / Tom, Colton M / Park, Hannah / Lawless, George / Shelley, Brandon C / Mattis, Virginia B / Breunig, Joshua J / Svendsen, Clive N

Stem cell reports

2018 Volume 10, Issue 6, Page(s) 1696–1704

Abstract: Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a promising therapy for neurodegenerative diseases. Glial cell ... ...

Abstract	Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a promising therapy for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is being used in a clinical trial for amyotrophic lateral sclerosis. However, chronic trophic factor delivery prohibits dose adjustment or cessation if side effects develop. To address this, we engineered a doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule. Human induced pluripotent stem cell (iPSC)-derived neural progenitors were stably transfected with the vector and transplanted into the adult mouse brain. Doxycycline can penetrate the graft, with addition and withdrawal providing inducible and reversible GDNF expression in vivo, over multiple cycles. Our findings provide proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.
MeSH term(s)	Cell- and Tissue-Based Therapy ; Gene Expression ; Gene Expression Regulation, Developmental ; Genes, Reporter ; Genetic Therapy ; Genetic Vectors/genetics ; Glial Cell Line-Derived Neurotrophic Factor/genetics ; Glial Cell Line-Derived Neurotrophic Factor/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology ; Induced Pluripotent Stem Cells/metabolism ; Neural Stem Cells/cytology ; Neural Stem Cells/metabolism ; Plants, Genetically Modified ; Stem Cell Transplantation/methods ; Transduction, Genetic ; Transgenes
Chemical Substances	Glial Cell Line-Derived Neurotrophic Factor
Language	English
Publishing date	2018-04-26
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2720528-9
ISSN	2213-6711 ; 2213-6711
ISSN (online)	2213-6711
ISSN	2213-6711
DOI	10.1016/j.stemcr.2018.03.024
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells.

Milani, Pamela / Escalante-Chong, Renan / Shelley, Brandon C / Patel-Murray, Natasha L / Xin, Xiaofeng / Adam, Miriam / Mandefro, Berhan / Sareen, Dhruv / Svendsen, Clive N / Fraenkel, Ernest

Scientific reports

2016 Volume 6, Page(s) 25474

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 ... ...

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.
Language	English
Publishing date	2016-05-05
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep25474
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Chromosome 7 and 19 trisomy in cultured human neural progenitor cells.

Sareen, Dhruv / McMillan, Erin / Ebert, Allison D / Shelley, Brandon C / Johnson, Julie A / Meisner, Lorraine F / Svendsen, Clive N

PloS one

2009 Volume 4, Issue 10, Page(s) e7630

Abstract: Background: Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the ...

Abstract	Background: Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations. Methods and findings: While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC(+7)) and trisomy 19 (hNPC(+19)), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC(+7) and hNPC(+19) cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (beta(III)-tubulin) in hNPC(+7) and hNPC(+19), using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50-60 population doublings and never showed neoplastic changes. Although hNPC(+7) and hNPC(+19) survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line. Conclusions: We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.
MeSH term(s)	Brain/embryology ; Cell Culture Techniques/methods ; Chromosome Aberrations ; Chromosomes, Human, Pair 19/ultrastructure ; Chromosomes, Human, Pair 7/ultrastructure ; Cytogenetics ; Embryonic Stem Cells/cytology ; ErbB Receptors/metabolism ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Neurons/cytology ; Oligonucleotide Array Sequence Analysis ; Stem Cells/cytology ; Trisomy
Chemical Substances	ErbB Receptors (EC 2.7.10.1)
Language	English
Publishing date	2009-10-29
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0007630
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

Ebert, Allison D / Shelley, Brandon C / Hurley, Amanda M / Onorati, Marco / Castiglioni, Valentina / Patitucci, Teresa N / Svendsen, Soshana P / Mattis, Virginia B / McGivern, Jered V / Schwab, Andrew J / Sareen, Dhruv / Kim, Ho Won / Cattaneo, Elena / Svendsen, Clive N

Stem cell research

2013 Volume 10, Issue 3, Page(s) 417–427

Abstract: We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, ... ...

Abstract	We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.
MeSH term(s)	Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry ; Epidermal Growth Factor/pharmacology ; Fibroblast Growth Factor 2/pharmacology ; Humans ; Induced Pluripotent Stem Cells/cytology ; Intermediate Filament Proteins/metabolism ; Multipotent Stem Cells/cytology ; Nerve Tissue Proteins/metabolism ; Nestin ; Neural Stem Cells/cytology ; Neural Stem Cells/metabolism ; Octamer Transcription Factor-3/metabolism ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Recombinant Proteins/pharmacology ; SOXB1 Transcription Factors/metabolism ; Up-Regulation
Chemical Substances	Culture Media ; Intermediate Filament Proteins ; NES protein, human ; Nerve Tissue Proteins ; Nestin ; Octamer Transcription Factor-3 ; Recombinant Proteins ; SOXB1 Transcription Factors ; Fibroblast Growth Factor 2 (103107-01-3) ; Epidermal Growth Factor (62229-50-9)
Language	English
Publishing date	2013-02-04
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2393143-7
ISSN	1876-7753 ; 1873-5061
ISSN (online)	1876-7753
ISSN	1873-5061
DOI	10.1016/j.scr.2013.01.009
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

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Article: A cgmp-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue

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Article: Human Neural Progenitor Transplantation Rescues Behavior and Reduces α-Synuclein in a Transgenic Model of Dementia with Lewy Bodies.

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Article ; Online: Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells.

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Article ; Online: Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells.

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Article ; Online: Chromosome 7 and 19 trisomy in cultured human neural progenitor cells.

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Article ; Online: EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

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