Author
Paul J. Gokhale
Other affiliations: Mount Sinai Hospital, Toronto
Bio: Paul J. Gokhale is an academic researcher from University of Sheffield. The author has contributed to research in topics: Embryonic stem cell & Stem cell. The author has an hindex of 25, co-authored 49 publications receiving 5149 citations. Previous affiliations of Paul J. Gokhale include Mount Sinai Hospital, Toronto.
Papers
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National Institute for Biological Standards and Control1, University of Sheffield2, Karolinska Institutet3, Technion – Israel Institute of Technology4, Leeds Teaching Hospitals NHS Trust5, Monash University6, Hebrew University of Jerusalem7, Thermo Fisher Scientific8, National Institutes of Health9, University of Cambridge10, University of Kansas11, University of Toronto12, Masaryk University13, Academy of Sciences of the Czech Republic14, Uppsala University15, Stanford University16, Newcastle University17, Pierre-and-Marie-Curie University18, WiCell19, University of Helsinki20, King's College London21, Kyoto University22, Hudson Institute23
TL;DR: The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide and found that despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers ofhuman embryonic stem cells.
Abstract: The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue- nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.
1,064 citations
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TL;DR: It is suggested that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.
Abstract: We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.
1,046 citations
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University of Sheffield1, Newcastle upon Tyne Hospitals NHS Foundation Trust2, Agency for Science, Technology and Research3, Royan Institute4, Stanford University5, Boston Children's Hospital6, University of Nottingham7, University of Southern California8, Hebrew University of Jerusalem9, Tel Aviv Sourasky Medical Center10, Cedars-Sinai Medical Center11, University of Geneva12, Manchester Academic Health Science Centre13, University of Manchester14, Genome Institute of Singapore15, Seoul National University16, Harvard University17, University of Edinburgh18, Masaryk University19, WiCell20, University of São Paulo21, Central South University22, University College London23, Karolinska Institutet24, Jawaharlal Nehru Centre for Advanced Scientific Research25, Kyoto University26, Shanghai Jiao Tong University27, Kurchatov Institute28, Russian Academy of Sciences29, Vrije Universiteit Brussel30, King's College London31, Leiden University32, University of Helsinki33, Yale University34, Hospital for Sick Children35, University of New South Wales36, University of Tampere37, Commonwealth Scientific and Industrial Research Organisation38
TL;DR: Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells, and single-nucleotide polymorphism analysis revealed that they included representatives of most major ethnic groups.
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.
506 citations
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TL;DR: RNA interference is used to downregulate β2‐microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells.
Abstract: We have used RNA interference (RNAi) to downregulate β2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not β2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.
302 citations
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TL;DR: It is shown that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes.
Abstract: Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression.
295 citations
Cited by
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TL;DR: It is demonstrated that iPS cells can be generated from adult human fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc.
18,175 citations
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TL;DR: Insight is provided into the transcriptional regulation of stem cells and how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal and how they collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops.
4,447 citations
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TL;DR: The first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA-protein interaction for several key regulatory factors were presented in this article.
Abstract: DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA-protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.
4,266 citations
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TL;DR: This work generated induced pluripotent stem cells capable of germline transmission from murine somatic cells by transd, and demonstrated the ability of these cells to reprogram into patient-specific and disease-specific stem cells.
Abstract: If it were possible to reprogram differentiated human somatic cells into a pluripotent state, patient-specific and disease-specific stem cells could be developed. Previous work generated induced pluripotent stem (iPS) cells capable of germline transmission from murine somatic cells by transd
4,034 citations
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TL;DR: The data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.
Abstract: Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.
3,035 citations