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ABSTRACT: Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner.
Stem cell research 07/2012; 9(3):192-195. · 3.39 Impact Factor
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ABSTRACT: Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.
Cryobiology 12/2011; 63(3):298-305. · 2.06 Impact Factor
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Katherine Amps,
Peter W Andrews,
George Anyfantis,
Lyle Armstrong,
Stuart Avery,
Hossein Baharvand,
Julie Baker, Duncan Baker,
Maria B Munoz,
Stephen Beil, [......],
Kazutoshi Takahashi,
Timo Tuuri,
Parvathy Venu,
Yuri Verlinsky,
Dorien Ward-van Oostwaard,
Daniel J Weisenberger,
Yue Wu,
Shinya Yamanaka,
Lorraine Young,
Qi Zhou
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ABSTRACT: The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.
Nature Biotechnology 11/2011; 29(12):1132-44. · 29.50 Impact Factor
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ABSTRACT: Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study, we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters, including the number of cells in a colony, colony area and shape, intensity of nuclear staining, and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60), as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries, and identified 17 that promoted differentiation, as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug, pinacidil, which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2, ROCK, MNK1, RSK1, and MSK1 kinases may contribute to the survival of hESCs.
Stem cell research 09/2010; 5(2):104-19. · 3.39 Impact Factor
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ABSTRACT: Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) bear a great potential in regenerative medicine. In addition to optimized clinical grade culture conditions, efficient clinical grade cryopreservation methods for these cells are needed. Obtaining good survival after thawing has been problematic.
We used a novel, chemically defined effective xeno-free cryopreservation system for cryostorage and banking of hESCs and iPSCs. The earlier established slow freezing protocols have, even after recent improvements, resulted in low viability and thawed cells had a high tendency to differentiate. The medium is a completely serum and animal substance free product containing dimethylsulfoxide, anhydrous dextrose and a polymer as cryoprotectants. The cells were directly frozen at -70 degrees C, without a programmed freezer.
The number of frozen colonies versus the number of surviving colonies differed significantly for both HS293 (chi(2) = 9.616 with one degree of freedom and two-tailed P = 0.0019) and HS306 (chi(2) = 8.801 with one degree of freedom and two-tailed P = 0.0030). After thawing, the cells had a high viability (90-96%) without any impact on proliferation and differentiation, compared with the standard freezing procedure where viability was much lower (49%). The frozen-thawed hESCs and iPSCs had normal karyotype and maintained properties of pluripotent cells with corresponding morphological characteristics, and expressed pluripotency markers after 10 passages in culture. They formed teratomas containing tissue components of the three germ layers.
The defined freezing-thawing system described here offers an excellent simple option for banking of hESCs and iPSCs.
Human Reproduction 03/2010; 25(5):1271-9. · 4.47 Impact Factor
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ABSTRACT: The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs.
In Vitro Cellular & Developmental Biology - Animal 03/2010; 46(3-4):236-41. · 1.31 Impact Factor
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Elisa Närvä,
Reija Autio,
Nelly Rahkonen,
Lingjia Kong,
Neil Harrison,
Danny Kitsberg,
Lodovica Borghese,
Joseph Itskovitz-Eldor,
Omid Rasool,
Petr Dvorak, [......],
Timo Tuuri,
Wei Cui,
Oliver Brüstle, Duncan Baker,
Edna Maltby,
Harry D Moore,
Nissim Benvenisty,
Peter W Andrews,
Olli Yli-Harja,
Riitta Lahesmaa
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ABSTRACT: Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb-3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.
Nature Biotechnology 03/2010; 28(4):371-7. · 29.50 Impact Factor
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ABSTRACT: The long-term culture of human embryonic stem (ES) cells is inevitably subject to evolution, since any mutant that arises with a growth advantage will be selectively amplified. However, the evolutionary influences of population size, mutation rate, and selection pressure are frequently overlooked. We have constructed a Monte Carlo simulation model to predict how changes in these factors can influence the appearance and spread of mutant ES cells, and verified its applicability by comparison with in vitro data. This simulation provides an estimate for the expected rate of generation of culture-adapted ES cells under different assumptions for the key parameters. In particular, it highlights the effect of population size, suggesting that the maintenance of cells in small populations reduces the likelihood that abnormal cultures will develop.
Stem cell research 09/2009; 4(1):50-6. · 3.39 Impact Factor
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ABSTRACT: Human embryonic stem cells undergo adaptive changes that can increase their growth capacity upon prolonged culture in vitro. This is frequently associated with nonrandom karyotypic changes, commonly involving amplification of genetic material from chromosomes 12, 17, and X. A recent study suggested that the karyotypically abnormal cells can be identified by their expression of CD30, which confers resistance to apoptosis. We have now investigated CD30 expression and apoptosis in karyotypically normal and abnormal sublines of the human ES cell line, H7, but our results were contrary to those previously observed. In this cell line, CD30 expression did not segregate the normal and abnormal cells, and abnormal cells were not protected from apoptosis. These data suggest that culture adaptation can occur through a variety of mechanisms.
Stem Cells 03/2009; 27(5):1057-65. · 7.78 Impact Factor
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ABSTRACT: The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in regenerative medicine. We report here the proteomic profile of 293T epithelial cells reprogrammed to a pluripotent state using undifferentiated embryonal carcinoma (NCCIT) cellular extracts. 293T cells were reversibly permeabilized with streptolysin O, incubated in an extract of NCCIT cells or a control extract of 293T cells for 1 h, resealed with CaCl(2), and cultured. OCT4 and SOX2 gene expression were up-regulated in NCCIT extract-treated cells relative to control cells, whereas there was no alteration in DNMT3B gene expression. Thirty percent of NCCIT extract-treated cells were positive for SSEA-4, and karyotyping confirmed their 293T origin, excluding the possibility of contamination from NCCIT cells. Two-dimensional PAGE revealed approximately 400 protein spots for each cell type studied. At least 10 protein spots in the proteome of NCCIT extract-treated cells had an expression profile similar to that of NCCIT and remained unaltered in control cells. Using tandem mass spectrometry, we identified these proteins, which include 78-kDa glucose-regulated protein precursor and tropomyosin alpha-3 chain. This investigation provides the first evidence that proteins are altered in a specific manner in NCCIT extract-treated cells. This is the first report on the proteomic characterization of the nuclear reprogramming process.
Molecular & Cellular Proteomics 03/2009; 8(6):1401-12. · 7.40 Impact Factor
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ABSTRACT: A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serum-free medium, hESF9, for the culture of HESCs on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), l-ascorbic acid-2-phosphate (0.1 microg/ml), and heparin. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/ml although higher concentrations were deleterious in the presence of heparin.
Proceedings of the National Academy of Sciences 10/2008; 105(36):13409-14. · 9.68 Impact Factor
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ABSTRACT: Teratocarcinomas are a subset of tumours that result from the neoplastic transformation of primordial germ cells. Such germ cell tumours (GCT) are histologically heterogeneous, reflecting a capacity for differentiation (pluripotency) of their embryonal carcinoma (EC) stem cells. However, malignant evolution of these tumours may ultimately correlate with a decrease in pluripotency, because this would tend to increase the propensity of EC cells for self-renewal. Human embryonic stem (ES) cells, derived from early blastocysts, closely resemble EC cells and, on prolonged culture in vitro, acquire progressive genetic changes that show striking similarity to those seen in GCT (e.g. gain of material from chromosome 12). In parallel, these abnormal ES cells show enhanced population growth rates and plating efficiencies, indicative of their adaptation to culture conditions. Understanding the mechanisms that drive such culture adaptation of ES cells may also provide insights into the development and progression of GCT.
International Journal of Andrology 09/2007; 30(4):275-81; discussion 281. · 3.59 Impact Factor
