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; Hurley, Amanda; Onorati, Marco; Castiglioni, Valentina; Patitucci, Teresa N.; Svendsen, Soshana; Mattis, Virginia B.; McGivern, Jered V.; Schwab, Andrew J.; Sareen, Dhruv; Kim, Ho Won; Cattaneo, Elena; Svendsen, Clive N.

doi:10.1016/j.scr.2013.01.009

Cited by 106 publications

(135 citation statements)

References 34 publications

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“…They were grown on Matrigel in feeder-free conditions. To generate neural progenitors, cells were accutased and lifted, then grown for up to 35 passages as floating neurospheres in epidermal growth factor and fibroblast growth factor (100 mg/ml) (68). Neural progenitors were then plated onto poly-ornithine/laminin-coated coverslips and differentiated toward striatal cultures for 42 days as previously described (33).…”

Section: Methodsmentioning

confidence: 99%

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington’s disease

et al. 2014

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Age-related neurodegenerative disorders including Alzheimer's disease and Huntington's disease (HD) consistently show elevated DNA damage, but the relevant molecular pathways in disease pathogenesis remain unclear. One attractive gene is that encoding the ataxia-telangiectasia mutated (ATM) protein, a kinase involved in the DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been demonstrated in Alzheimer's disease and in mouse models of other neurodegenerative diseases. We show that ATM signaling was consistently elevated in cells derived from HD mice and in brain tissue from HD mice and patients. ATM knockdown protected from toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we show that genetic reduction of Atm gene dosage by one copy ameliorated multiple behavioral deficits and partially improved neuropathology. Small-molecule ATM inhibitors reduced mHTT-induced death of rat striatal neurons and induced pluripotent stem cells derived from HD patients. Our study provides converging genetic and pharmacological evidence that reduction of ATM signaling could ameliorate mHTT toxicity in cellular and animal models of HD, suggesting that ATM may be a useful therapeutic target for HD.

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Section: Methodsmentioning

confidence: 99%

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington’s disease

et al. 2014

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“…There are other cells types that use this procedure for expansion. An example are EZ spheres, neural stem cells generated from pluripotent stem cells 41 . This technique has great potential for use with other types of tissue that require constant cell-to-cell contact during expansion.…”

Section: Future Applicationsmentioning

confidence: 99%

“…The provided chopping protocol allows for the production of large-scale banks from one fetal sample greater than passage 10, an unlikely feat using standard passaging methods. While this method for passaging hNPCs is unconventional, it is growing in popularity and was recently, published with other cell types such as neural stem cells derived from human embryonic and induced pluripotent stem cells, enabling large scale expansion for various applications including in vitro disease modeling [41][42][43][44][45][46] 1. The two most important factors to address before chopping is sphere diameter and media conditioning or color.…”

Section: Introductionmentioning

confidence: 99%

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Shelley

Gowing

Svendsen

2014

JoVE

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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. Video LinkThe video component of this article can be found at

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“…Likewise, although primary cell lines can provide fully differentiated cells that have a close approximation of the native function, the collection of all cell types is not possible because of the level of invasion required to collect them, and the limited proliferative activity of primary cells can make reproducing experiments difficult. 9 On the contrary, human-derived iPS and ES cell lines can overcome these disadvantages. After the expansion of these pluripotent cell lines, a sufficient number of the desired types of cells can be prepared by using appropriate differentiation protocols, that is, these technologies can provide an opportunity to maintain specific somatic cells on a large scale and in a renewable way.…”

Section: Ips Cells For In Vitro Modeling Of Native Physiological Condmentioning

confidence: 99%

“…It has been observed that 30% to 40% of all patients have mutations in NLRP3 in only a small number (≈10%) of somatic cells, 50,51 despite the fact that nearly half of all patients with CINCA syndrome carry heterozygous gain-of-function mutations of the gene. 9,52 Recently, Tanaka et al 44 generated both NLP3 mutant and nonmutant iPS cell lines from patients with CINCA syndrome with somatic mosaicism and described their differentiation into macrophages. In this case, they succeeded in recapitulating the disease-relevant phenotype using only mutant macrophages derived from iPS cells, demonstrating that NLRP3-mutant macrophages are responsible for the pathogenesis of mosaic CINCA syndrome.…”

Section: Somatic Mosaicism In Cinca Syndromementioning

confidence: 99%

Perspectives for Induced Pluripotent Stem Cell Technology

Nagata

Yamanaka

2014

Circulation Research

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Induced pluripotent stem cell technology makes in vitro reprogramming of somatic cells from individualswith various genetic backgrounds possible. By applying this technology, it is possible to produce pluripotent stem cells from biopsy samples of arbitrarily selected individuals with various genetic backgrounds and to subsequently maintain, expand, and stock these cells. From these induced pluripotent stem cells, target cells and tissues can be generated after certain differentiation processes. These target cells/tissues are expected to be useful in regenerative medicine, disease modeling, drug screening, toxicology testing, and proof-of-concept studies in drug development. Therefore, the number of publications concerning induced pluripotent stem cells has recently been increasing rapidly, demonstrating that this technology has begun to infiltrate many aspects of stem cell biology and medical applications. In this review, we discuss the perspectives of induced pluripotent stem cell technology for modeling human diseases. In particular, we focus on the cloning event occurring through the reprogramming process and its ability to let us analyze the development of complex disease-harboring somatic mosaicism. (Circ Res. 2014;114:505-510.)

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EZ spheres: A stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

Cited by 106 publications

References 34 publications

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington’s disease

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington’s disease

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Perspectives for Induced Pluripotent Stem Cell Technology

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