Preserving the genetic integrity of human embryonic stem cells

Mitalipova, Maisam; Rao, Raja R; Hoyer, Deborah M; Johnson, Julie A.; Meisner, Lorraine F.; Jones, Karen L.; Dalton, Stephen; Stice, S.L.

doi:10.1038/nbt0105-19

Cited by 380 publications

(327 citation statements)

References 8 publications

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“…Prior studies highlighted chromosomes 12, 17, 20 and X as involved in most of the genomic aberrations 12,13 and both chromosomal gains and losses were frequently reported in cells from different sources. It is notable that, trisomy 12 is known to take over the culture in a few passages 15,34 but in our study, aneuploid cells carrying additional copy of chromosome 12 do not seem to be enriched in culture over time. Taken together, since chromosome gain and loss appears to be stochastic, with no significantly recurrent aneuploidy of specific chromosome, genome-wide analyses using CGH array or SNP array (and at a later stage -whole-genome sequencing and single-nucleotide polymorphism genotyping) 7 may have some advantages over multi-FISH hybridizations as an improved standardized genetic screening of human ESCs and hiPSCs.…”

Section: Aneuploidy Screeningcontrasting

confidence: 61%

“…11 Karyotypic abnormalities of hESCs in long-term cultures have been reported before. [12][13][14][15] In hESCs, most of the alteration have been attributed to chromosomes 12 and 17, but chromosomes X and 20 were also repeatedly described. 12,16 G-banding technique is extremely laborious, requires dividing cells in culture and metaphases of sufficient quality for analysis, which have already proven difficulties to obtain.…”

Section: Introductionmentioning

confidence: 99%

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Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

et al. 2012

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Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.

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Section: Aneuploidy Screeningcontrasting

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

et al. 2012

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“…18 This technique may reduce the possibility of immortalization events seen in human embryonic stem cells associated with repetitive trypsin treatment and dissociation to single cells. 45 We have previously shown that hNPC grown as neurospheres without dissociation during passage do not express telomerase required for immortalization 46 and therefore have a finite lifespan in culture (Wright et al, in preparation). We have also shown that transplanted hNPC express Ki67 and divide in vivo for only a limited time.…”

Section: Discussionmentioning

confidence: 99%

Human neural progenitors deliver glial cell line-derived neurotrophic factor to parkinsonian rodents and aged primates

et al. 2005

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Glial cell line-derived neurotrophic factor (GDNF) has been shown to increase the survival and functioning of dopamine neurons in a variety of animal models and some recent human trials. However, delivery of any protein to the brain remains a challenge due to the blood/brain barrier. Here we show that human neural progenitor cells (hNPC) can be genetically modified to release glycosylated GDNF in vitro under an inducible promoter system. hNPC-GDNF were transplanted into the striatum of rats 10 days following a partial lesion of the dopamine system. At 2 weeks following transplantation, the cells had migrated within the striatum and were releasing physiologically relevant levels of GDNF. This was sufficient to increase host dopamine neuron survival and fiber outgrowth. At 5 weeks following grafting there was a strong trend towards functional improvement in transplanted animals and at 8 weeks the cells had migrated to fill most of the striatum and continued to release GDNF with transport to the substantia nigra. These cells could also survive and release GDNF 3 months following transplantation into the aged monkey brain. No tumors were found in any animal. hNPC can be genetically modified, and thereby represent a safe and powerful option for delivering growth factors to specific targets within the central nervous system for diseases such as Parkinson's.

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“…Genetic evaluation of therapeutic hESC E. Stephenson et al damage caused by reactive oxygen species and free radicals, which are generated during normal oxidative cell metabolism. Indeed, undifferentiated hESCs have a much lower expression of Cu/Zn-superoxide dismutase, glutathione peroxidase 1, and peroxiredoxin 1 and 2, which are enzymes that normally protect cells against such damage (Cho et al 2006 (Draper et al 2004;Hoffman & Carpenter 2005;Mitalipova et al 2005). Comparison of mechanical cutting of colonies and the use of enzymes for passage in the same hESC line suggested that mechanical cutting better supports maintenance of a normal karyotype in long-term culture; the enzyme-treated cell line was more likely to develop karyotype abnormalities (most often chromosome 12 and 17 trisomy).…”

Section: Extrinsic Factorsmentioning

confidence: 99%

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

Stephenson

Ogilvie

Patel

et al. 2010

J. R. Soc. Interface.

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The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.

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Preserving the genetic integrity of human embryonic stem cells

Cited by 380 publications

References 8 publications

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

Human neural progenitors deliver glial cell line-derived neurotrophic factor to parkinsonian rodents and aged primates

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

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