Publications by authors named "Huck-Hui Ng"

104 Publications

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease.

Gastroenterology 2020 Oct 15;159(4):1471-1486.e12. Epub 2020 Jun 15.

Stem Cell and Regenerative Biology, Genome Institute of Singapore, Singapore. Electronic address:

Background & Aims: There are few in vitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells.

Methods: We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging.

Results: Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions.

Conclusions: We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.
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http://dx.doi.org/10.1053/j.gastro.2020.06.010DOI Listing
October 2020

Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson's disease modeling.

Stem Cells 2020 06 28;38(6):727-740. Epub 2020 Feb 28.

School of Biotechnology and Healthcare, Wuyi University, Jiangmen, People's Republic of China.

Recent studies have demonstrated the generation of midbrain-like organoids (MOs) from human pluripotent stem cells. However, the low efficiency of MO generation and the relatively immature and heterogeneous structures of the MOs hinder the translation of these organoids from the bench to the clinic. Here we describe the robust generation of MOs with homogeneous distribution of midbrain dopaminergic (mDA) neurons. Our MOs contain not only mDA neurons but also other neuronal subtypes as well as functional glial cells, including astrocytes and oligodendrocytes. Furthermore, our MOs exhibit mDA neuron-specific cell death upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, indicating that MOs could be a proper human model system for studying the in vivo pathology of Parkinson's disease (PD). Our optimized conditions for producing homogeneous and mature MOs might provide an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD.
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http://dx.doi.org/10.1002/stem.3163DOI Listing
June 2020

Potassium channel dysfunction in human neuronal models of Angelman syndrome.

Science 2019 12;366(6472):1486-1492

Signature Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore.

Disruptions in the ubiquitin protein ligase E3A () gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.
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http://dx.doi.org/10.1126/science.aav5386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735558PMC
December 2019

Large-Scale Whole-Genome Sequencing of Three Diverse Asian Populations in Singapore.

Cell 2019 10;179(3):736-749.e15

Department of Epidemiology and Biostatistics, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China; Computational and Systems Biology, Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore 138672, Singapore. Electronic address:

Underrepresentation of Asian genomes has hindered population and medical genetics research on Asians, leading to population disparities in precision medicine. By whole-genome sequencing of 4,810 Singapore Chinese, Malays, and Indians, we found 98.3 million SNPs and small insertions or deletions, over half of which are novel. Population structure analysis demonstrated great representation of Asian genetic diversity by three ethnicities in Singapore and revealed a Malay-related novel ancestry component. Furthermore, demographic inference suggested that Malays split from Chinese ∼24,800 years ago and experienced significant admixture with East Asians ∼1,700 years ago, coinciding with the Austronesian expansion. Additionally, we identified 20 candidate loci for natural selection, 14 of which harbored robust associations with complex traits and diseases. Finally, we show that our data can substantially improve genotype imputation in diverse Asian and Oceanian populations. These results highlight the value of our data as a resource to empower human genetics discovery across broad geographic regions.
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http://dx.doi.org/10.1016/j.cell.2019.09.019DOI Listing
October 2019

Lgr5 and Col22a1 Mark Progenitor Cells in the Lineage toward Juvenile Articular Chondrocytes.

Stem Cell Reports 2019 10 12;13(4):713-729. Epub 2019 Sep 12.

School of Biomedical Sciences, The University of Hong Kong, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China; The University of Hong Kong - Shenzhen Institute of Research and Innovation (HKU- SIRI), Hi-Tech Industrial Park, Nanshan, Shenzhen, China. Electronic address:

The synovial joint forms from a pool of progenitor cells in the future region of the joint, the interzone. Expression of Gdf5 and Wnt9a has been used to mark the earliest cellular processes in the formation of the interzone and the progenitor cells. However, lineage specification and progression toward the different tissues of the joint are not well understood. Here, by lineage-tracing studies we identify a population of Lgr5 interzone cells that contribute to the formation of cruciate ligaments, synovial membrane, and articular chondrocytes of the joint. This finding is supported by single-cell transcriptome analyses. We show that Col22a1, a marker of early articular chondrocytes, is co-expressed with Lgr5 cells prior to cavitation as an important lineage marker specifying the progression toward articular chondrocytes. Lgr5 cells contribute to the repair of a joint defect with the re-establishment of a Col22a1-expressing superficial layer.
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http://dx.doi.org/10.1016/j.stemcr.2019.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829785PMC
October 2019

A Chemically Defined Feeder-free System for the Establishment and Maintenance of the Human Naive Pluripotent State.

Stem Cell Reports 2019 10 12;13(4):612-626. Epub 2019 Sep 12.

Stem Cell and Regenerative Biology, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore. Electronic address:

The distinct states of pluripotency in the pre- and post-implantation embryo can be captured in vitro as naive and primed pluripotent stem cell cultures, respectively. The study and application of the naive state remains hampered, particularly in humans, partially due to current culture protocols relying on extraneous undefined factors such as feeders. Here we performed a small-molecule screen to identify compounds that facilitate chemically defined establishment and maintenance of human feeder-independent naive embryonic (FINE) stem cells. The expression profile in genic and repetitive elements of FINE cells resembles the 8-cell-to-morula stage in vivo, and only differs from feeder-dependent naive cells in genes involved in cell-cell/cell-matrix interactions. FINE cells offer several technical advantages, such as increased amenability to transfection and a longer period of genomic stability, compared with feeder-dependent cells. Thus, FINE cells will serve as an accessible and useful system for scientific and translational applications of naïve pluripotent stem cells.
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http://dx.doi.org/10.1016/j.stemcr.2019.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829768PMC
October 2019

New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation.

Gastroenterology 2019 12 22;157(6):1615-1629.e17. Epub 2019 Aug 22.

Department of Pathology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Background & Aims: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms.

Methods: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4 cells). We compared gene expression patterns of SALL4 cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4 SNU-398 or HCC26.1 cells or from SALL4 patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured.

Results: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4 cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4 hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4 cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4 SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4 PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4 and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP.

Conclusions: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4 cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.
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http://dx.doi.org/10.1053/j.gastro.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309153PMC
December 2019

Translational potential of human brain organoids.

Ann Clin Transl Neurol 2018 02 20;5(2):226-235. Epub 2018 Jan 20.

Department of Neurology National Neuroscience Institute 20 College Road Singapore 169856 Singapore.

The recent technology of 3D cultures of cellular aggregates derived from human stem cells have led to the emergence of tissue-like structures of various organs including the brain. Brain organoids bear molecular and structural resemblance with developing human brains, and have been demonstrated to recapitulate several physiological and pathological functions of the brain. Here we provide an overview of the development of brain organoids for the clinical community, focusing on the current status of the field with an critical evaluation of its translational value.
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http://dx.doi.org/10.1002/acn3.505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817829PMC
February 2018

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation.

Sci Rep 2017 12 7;7(1):17156. Epub 2017 Dec 7.

Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China.

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.
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http://dx.doi.org/10.1038/s41598-017-16009-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719399PMC
December 2017

Lab-grown mini-brains upgraded.

Nat Cell Biol 2017 Aug;19(9):1010-1012

Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.

Three-dimensional brain organoid models have come into the spotlight as in vitro tools to recapitulate complex features of the brain. Four recent papers now leverage current technologies to generate new flavours of brain organoids and address aspects of brain biology which, to date, have been challenging to explore.
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http://dx.doi.org/10.1038/ncb3601DOI Listing
August 2017

High-resolution RNA allelotyping along the inactive X chromosome: evidence of RNA polymerase III in regulating chromatin configuration.

Sci Rep 2017 04 3;7:45460. Epub 2017 Apr 3.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive 637551, Singapore.

We carried out padlock capture, a high-resolution RNA allelotyping method, to study X chromosome inactivation (XCI). We examined the gene reactivation pattern along the inactive X (Xi), after Xist (X-inactive specific transcript), a prototype long non-coding RNA essential for establishing X chromosome inactivation (XCI) in early embryos, is conditionally deleted from Xi in somatic cells (Xi). We also monitored the behaviors of X-linked non-coding transcripts before and after XCI. In each mutant cell line, gene reactivation occurs to ~6% genes along Xi in a recognizable pattern. Genes with upstream regions enriched for SINEs are prone to be reactivated. SINE is a class of retrotransposon transcribed by RNA polymerase III (Pol III). Intriguingly, a significant fraction of Pol III transcription from non-coding regions is not subjected to Xist-mediated transcriptional silencing. Pol III inhibition affects gene reactivation status along Xi, alters chromatin configuration and interferes with the establishment XCI during in vitro differentiation of ES cells. These results suggest that Pol III transcription is involved in chromatin structure re-organization during the onset of XCI and functions as a general mechanism regulating chromatin configuration in mammalian cells.
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http://dx.doi.org/10.1038/srep45460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377358PMC
April 2017

The metabolic programming of stem cells.

Genes Dev 2017 02 17;31(4):336-346. Epub 2017 Mar 17.

Genome Institute of Singapore, Singapore 138675.

Advances in metabolomics have deepened our understanding of the roles that specific modes of metabolism play in programming stem cell fates. Here, we review recent metabolomic studies of stem cell metabolism that have revealed how metabolic pathways can convey changes in the extrinsic environment or their niche to program stem cell fates. The metabolic programming of stem cells represents a fine balance between the intrinsic needs of a cellular state and the constraints imposed by extrinsic conditions. A more complete understanding of these needs and constraints will afford us greater mastery over our control of stem cell fates.
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http://dx.doi.org/10.1101/gad.293167.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358754PMC
February 2017

Direct Induction and Functional Maturation of Forebrain GABAergic Neurons from Human Pluripotent Stem Cells.

Cell Rep 2016 08 4;16(7):1942-53. Epub 2016 Aug 4.

Molecular Neurophysiology Laboratory, Signature Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore. Electronic address:

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.
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http://dx.doi.org/10.1016/j.celrep.2016.07.035DOI Listing
August 2016

Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.

Cell Stem Cell 2016 08 28;19(2):248-257. Epub 2016 Jul 28.

Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore; Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 639798, Singapore. Electronic address:

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.
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http://dx.doi.org/10.1016/j.stem.2016.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510242PMC
August 2016

CTRL+INSERT: retrotransposons and their contribution to regulation and innovation of the transcriptome.

EMBO Rep 2016 08 11;17(8):1131-44. Epub 2016 Jul 11.

Gene Regulation Laboratory, Genome Institute of Singapore, Singapore Department of Biochemistry, National University of Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore School of Biological Sciences, Nanyang Technological University, Singapore.

The human genome contains millions of fragments from retrotransposons-highly repetitive DNA sequences that were once able to "copy and paste" themselves to other regions in the genome. However, the majority of retrotransposons have lost this capacity through acquisition of mutations or through endogenous silencing mechanisms. Without this imminent threat of transposition, retrotransposons have the potential to act as a major source of genomic innovation. Indeed, large numbers of retrotransposons have been found to be active in specific contexts: as gene regulatory elements and promoters for protein-coding genes or long noncoding RNAs, among others. In this review, we summarise recent findings about retrotransposons, with implications in gene expression regulation, the expansion of gene isoform diversity and the generation of long noncoding RNAs. We highlight key examples that demonstrate their role in cellular identity and their versatility as markers of cell states, and we discuss how their dysregulation may contribute to the formation of and possibly therapeutic response in human cancers.
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http://dx.doi.org/10.15252/embr.201642743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967949PMC
August 2016

Looping around Reprogramming: The Topological Memory of Induced Pluripotency.

Cell Stem Cell 2016 05;18(5):557-9

Gene Regulation Laboratory, Genome Institute of Singapore, Singapore 138672; Department of Biochemistry, National University of Singapore, Singapore 117597; Department of Biological Sciences, National University of Singapore, Singapore 117597; School of Biological Sciences, Nanyang Technological University, Singapore 639798. Electronic address:

Genome architecture is associated with cellular identity, but how this organization changes during reprogramming is not well understood. Now in Cell Stem Cell, Krijger et al. (2016) and Beagan et al. (2016) report 3D chromatin interaction maps before and after reprogramming, providing evidence for topological memory in induced pluripotent stem cells.
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http://dx.doi.org/10.1016/j.stem.2016.04.006DOI Listing
May 2016

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters.

Genome Res 2016 05 8;26(5):612-23. Epub 2016 Mar 8.

Computational and Systems Biology, Genome Institute of Singapore, Singapore 138672, Singapore;

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation.
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http://dx.doi.org/10.1101/gr.201038.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864461PMC
May 2016

Induced pluripotent stem cells in Parkinson's disease: scientific and clinical challenges.

J Neurol Neurosurg Psychiatry 2016 07 1;87(7):697-702. Epub 2016 Feb 1.

National Neuroscience Institute, Singapore, Singapore Genome Institute of Singapore, Singapore, Singapore Department of Neurology, Singapore General Hospital, Singapore, Singapore Duke-NUS Graduate Medical School, Singapore, Singapore.

Induced pluripotent stem cells (iPSCs), which greatly circumvent the ethical issue of human embryonic stem cells (ESCs), can be induced to differentiate to dopaminergic (DA) neurons, and hence be used as a human disease model for Parkinson's disease (PD). iPSCs can be also utilised to probe the mechanism, and serve as an 'in vivo' platform for drug screening and for cell-replacement therapies. However, any clinical trial approaches should be extensively supported by validated robust biological evidence (based on previous experience with fetal mesencephalic transplantation), in particular, the production and selection of the 'ideal' neurons (functional units with no oncological risk), together with the careful screening of appropriate candidates (such as genetic carriers), with inbuilt safeguards (safety studies) in the evaluation and monitoring (functional neuroimaging of both DA and non-DA system) of trial subjects. While iPSCs hold great promise for PD, there are still numerous scientific and clinical challenges that need to be surmounted before any clinical application can be safely introduced.
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http://dx.doi.org/10.1136/jnnp-2015-312036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941130PMC
July 2016

Biological Networks Governing the Acquisition, Maintenance, and Dissolution of Pluripotency: Insights from Functional Genomics Approaches.

Cold Spring Harb Symp Quant Biol 2015 18;80:189-98. Epub 2015 Nov 18.

Stem Cell and Regenerative Biology, Genome Institute of Singapore, Singapore 138672, Singapore Department of Biochemistry, National University of Singapore, Singapore 117597, Singapore Department of Biological Sciences, National University of Singapore, Singapore 117597, Singapore School of Biological Sciences, Nanyang Technological University, Singapore 639798, Singapore.

The repertoire of transcripts encoded by the genome contributes to the diversity of cellular states. Functional genomics aims to comprehensively uncover the roles of these transcripts to reconstruct biological networks and transform this information into useful knowledge. High-throughput functional screening has served as a powerful genetic discovery tool by enabling massively parallel implementation of biological assays. In recent years, high-throughput screening has unearthed crucial players in the regulation of different aspects of pluripotency, which is a unique property that enables a cell to differentiate into multiple cell types of the three major lineages. Pluripotency thus represents an interesting biological paradigm for studying the acquisition, maintenance, and dissolution of cellular states. In this review, we highlight the major findings of high-throughput studies to dissect these three aspects of pluripotency for the mouse and human systems. Collectively, they provide new insights into cell fate maintenance and transition. In addition, we also discuss the opportunities and challenges awaiting high-throughput screening in the future.
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http://dx.doi.org/10.1101/sqb.2015.80.027326DOI Listing
January 2018

ELABELA Is an Endogenous Growth Factor that Sustains hESC Self-Renewal via the PI3K/AKT Pathway.

Cell Stem Cell 2015 Oct 17;17(4):435-47. Epub 2015 Sep 17.

Institute of Medical Biology, Human Genetics and Embryology Laboratory, A(∗)STAR, Singapore 138648; Institute of Molecular and Cell Biology, A(∗)STAR, Singapore 138673. Electronic address:

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFβ pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.
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http://dx.doi.org/10.1016/j.stem.2015.08.010DOI Listing
October 2015

Deterministic Restriction on Pluripotent State Dissolution by Cell-Cycle Pathways.

Cell 2015 Jul;162(3):564-79

Stem Cell and Regenerative Biology, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore; Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 639798, Singapore. Electronic address:

During differentiation, human embryonic stem cells (hESCs) shut down the regulatory network conferring pluripotency in a process we designated pluripotent state dissolution (PSD). In a high-throughput RNAi screen using an inclusive set of differentiation conditions, we identify centrally important and context-dependent processes regulating PSD in hESCs, including histone acetylation, chromatin remodeling, RNA splicing, and signaling pathways. Strikingly, we detected a strong and specific enrichment of cell-cycle genes involved in DNA replication and G2 phase progression. Genetic and chemical perturbation studies demonstrate that the S and G2 phases attenuate PSD because they possess an intrinsic propensity toward the pluripotent state that is independent of G1 phase. Our data therefore functionally establish that pluripotency control is hardwired to the cell-cycle machinery, where S and G2 phase-specific pathways deterministically restrict PSD, whereas the absence of such pathways in G1 phase potentially permits the initiation of differentiation.
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http://dx.doi.org/10.1016/j.cell.2015.07.001DOI Listing
July 2015

Layered polymeric capsules inhibiting the activity of RNases for intracellular delivery of messenger RNA.

J Mater Chem B 2015 Jul 22;3(28):5842-5848. Epub 2015 Jun 22.

Institute of Materials Research and Engineering, A*STAR, 3 Research Link, Singapore, 117602, Singapore.

Intracellular delivery of messenger RNA (mRNA) is a promising approach for experimental and therapeutic manipulation of cellular activity. However, environmental RNase hinders reliable handling of mRNA for experimental and therapeutic use. In this study, biodegradable capsules composed of dextran sulfate and poly-l-arginine in the layer-by-layer (LbL) fashion are employed for the protection and delivery of mRNA. Our results demonstrate that addition of RNase inhibitors to mRNA while co-precipitation with CaCO and subsequent LbL encapsulation are both crucial to preserve the integrity of mRNA. The expression of functional luciferase enzyme in HEK293T human embryonic kidney cells after incubation with synthetic luciferase-encoding mRNA capsules indicates the reliability of the encapsulating system and cellular intake of functional mRNAs. These improvements in mRNA encapsulation should provide essential basis for microcapsule-based mRNA delivery for further applications.
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http://dx.doi.org/10.1039/c5tb00615eDOI Listing
July 2015

Dynamic transcription of distinct classes of endogenous retroviral elements marks specific populations of early human embryonic cells.

Cell Stem Cell 2015 Feb;16(2):135-41

Gene Regulation Laboratory, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore; Department of Biochemistry, National University of Singapore, Singapore 117559, Singapore.

About half of the human genome consists of highly repetitive elements, most of which are considered dispensable for human life. Here, we report that repetitive elements originating from endogenous retroviruses (ERVs) are systematically transcribed during human early embryogenesis in a stage-specific manner. Our analysis highlights that the long terminal repeats (LTRs) of ERVs provide the template for stage-specific transcription initiation, thereby generating hundreds of co-expressed, ERV-derived RNAs. Conversion of human embryonic stem cells (hESCs) to an epiblast-like state activates blastocyst-specific ERV elements, indicating that their activity dynamically reacts to changes in regulatory networks. In addition to initiating stage-specific transcription, many ERV families contain preserved splice sites that join the ERV segment with non-ERV exons in their genomic vicinity. In summary, we find that ERV expression is a hallmark of cellular identity and cell potency that characterizes the cell populations in early human embryos.
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http://dx.doi.org/10.1016/j.stem.2015.01.005DOI Listing
February 2015

Regulatory crosstalk between lineage-survival oncogenes KLF5, GATA4 and GATA6 cooperatively promotes gastric cancer development.

Gut 2015 May 22;64(5):707-19. Epub 2014 Jul 22.

Cancer and Stem Cell Biology program, Duke-NUS Graduate Medical School Singapore, Singapore, Singapore Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Cellular and Molecular Research, National Cancer Centre, Singapore, Singapore Genome Institute of Singapore, Singapore, Singapore.

Objective: Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications.

Design: KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice.

Results: KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs.

Conclusions: KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells.
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http://dx.doi.org/10.1136/gutjnl-2013-306596DOI Listing
May 2015

Sox2: masterminding the root of cancer.

Cancer Cell 2014 Jul;26(1):3-5

Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456, Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599, Singapore. Electronic address:

The transcription factor Sox2 is a master regulator that maintains stemness in embryonic stem cells and neural stem cells. Using elegant lineage tracing strategies and genetic reporter mouse models, two studies (one of which is by Vanner and colleagues in this issue of Cancer Cell) now demonstrate that rare Sox2-expressing cells are the founding cancer stem cell population driving tumor initiation and therapy resistance.
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http://dx.doi.org/10.1016/j.ccr.2014.06.024DOI Listing
July 2014

Nanoscale chromatin profiling of gastric adenocarcinoma reveals cancer-associated cryptic promoters and somatically acquired regulatory elements.

Nat Commun 2014 Jul 10;5:4361. Epub 2014 Jul 10.

1] Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, 60 Biopolis Street, Genome #02-01, Singapore 138672, Singapore [2] Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore [3] Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, #12-01, Singapore 117599, Singapore [4] Cellular and Molecular Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore.

Chromatin alterations are fundamental hallmarks of cancer. To study chromatin alterations in primary gastric adenocarcinomas, we perform nanoscale chromatin immunoprecipitation sequencing of multiple histone modifications in five gastric cancers and matched normal tissues. We identify hundreds of somatically altered promoters and predicted enhancers. Many cancer-associated promoters localize to genomic sites lacking previously annotated transcription start sites (cryptic promoters), driving expression of nearby genes involved in gastrointestinal cancer, embryonic development and tissue specification. Cancer-associated promoters overlap with embryonic stem cell regions targeted by polycomb repressive complex 2, exhibiting promoter bivalency and DNA methylation loss. We identify somatically acquired elements exhibiting germline allelic biases and non-coding somatic mutations creating new promoters. Our findings demonstrate the feasibility of profiling chromatin from solid tumours with limited tissue to identify regulatory elements, transcriptional patterns and regulatory genetic variants associated with cancer.
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http://dx.doi.org/10.1038/ncomms5361DOI Listing
July 2014

Klf2 is an essential factor that sustains ground state pluripotency.

Cell Stem Cell 2014 Jun;14(6):864-72

Genome Institute of Singapore, 60 Biopolis Street, #02-01 Genome Building, Singapore 138672, Singapore; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456, Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD6, Centre for Translational Medicine, 14 Medical Drive #14-01T, Singapore 117599, Singapore. Electronic address:

The maintenance of mouse embryonic stem cells (mESCs) requires LIF and serum. However, a pluripotent "ground state," bearing resemblance to preimplantation mouse epiblasts, can be established through dual inhibition (2i) of both prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. While Gsk3 inhibition has been attributed to the transcriptional derepression of Esrrb, the molecular mechanism mediated by Mek inhibition remains unclear. In this study, we show that Krüppel-like factor 2 (Klf2) is phosphorylated by Erk2 and that phospho-Klf2 is proteosomally degraded. Mek inhibition hence prevents Klf2 protein phosphodegradation to sustain pluripotency. Indeed, while Klf2-null mESCs can survive under LIF/Serum, they are not viable under 2i, demonstrating that Klf2 is essential for ground state pluripotency. Importantly, we also show that ectopic Klf2 expression can replace Mek inhibition in mESCs, allowing the culture of Klf2-null mESCs under Gsk3 inhibition alone. Collectively, our study defines the Mek/Erk/Klf2 axis that cooperates with the Gsk3/Tcf3/Esrrb pathway in mediating ground state pluripotency.
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http://dx.doi.org/10.1016/j.stem.2014.04.015DOI Listing
June 2014

The role of SON in splicing, development, and disease.

Wiley Interdiscip Rev RNA 2014 Sep-Oct;5(5):637-46. Epub 2014 Apr 30.

Gene Regulation Laboratory, Genome Institute of Singapore, Singapore, Singapore.

SON is a nuclear protein involved in multiple cellular processes including transcription, pre-messenger RNA (mRNA) splicing, and cell cycle regulation. Although SON was discovered 25 years ago, the importance of SON's function was only realized recently when its roles in nuclear organization and pre-mRNA splicing as well as the influence of these activities in maintaining cellular health were unveiled. Furthermore, SON was implicated to have a key role in stem cells as well as during the onset of various diseases such as cancer, influenza, and hepatitis. Here we review the progress that has been made in studying this multifunctional protein and discuss questions that remain to be answered about SON.
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http://dx.doi.org/10.1002/wrna.1235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4138235PMC
April 2015

Identifiability and privacy in pluripotent stem cell research.

Cell Stem Cell 2014 Apr;14(4):427-30

Centre of Genomics and Policy, Faculty of Medicine, Department of Human Genetics, McGill University, Montreal, QC H3A 0G1, Canada.

Data sharing is an essential element of research; however, recent scientific and social developments have challenged conventional methods for protecting privacy. Here we provide guidance for determining data sharing thresholds for human pluripotent stem cell research aimed at a wide range of stakeholders, including research consortia, biorepositories, policy-makers, and funders.
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http://dx.doi.org/10.1016/j.stem.2014.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815907PMC
April 2014

The retrovirus HERVH is a long noncoding RNA required for human embryonic stem cell identity.

Nat Struct Mol Biol 2014 Apr 30;21(4):423-5. Epub 2014 Mar 30.

1] Gene Regulation Laboratory, Genome Institute of Singapore, Singapore. [2] Department of Biochemistry, National University of Singapore, Singapore. [3] Department of Biological Sciences, National University of Singapore, Singapore. [4] School of Biological Sciences, Nanyang Technological University, Singapore.

Human endogenous retrovirus subfamily H (HERVH) is a class of transposable elements expressed preferentially in human embryonic stem cells (hESCs). Here, we report that the long terminal repeats of HERVH function as enhancers and that HERVH is a nuclear long noncoding RNA required to maintain hESC identity. Furthermore, HERVH is associated with OCT4, coactivators and Mediator subunits. Together, these results uncover a new role of species-specific transposable elements in hESCs.
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http://dx.doi.org/10.1038/nsmb.2799DOI Listing
April 2014