[Show abstract][Hide abstract] ABSTRACT: Corneal dysfunction is the second leading cause of blindness. Approximately 10 million patients worldwide are affected by some form of corneal disease. More than 50,000 cornea transplants are performed every year, but this procedure is limited by cornea donation availability. Recently, new cell replacement procedures have been developed to treat a variety of corneal diseases. This review will focus on the recent advances in the use of limbal epithelial stem cells (LESCs) to treat corneal epithelial cell deficiency and improvements in replacing dysfunctional corneal endothelial cells (CECs) with exogenous CECs. Several protocols have been developed to differentiate pluripotent stem cells into LESC- or CEC-like cells, potentially yielding an unlimited source for the cell replacement therapy of corneal diseases.
Regenerative Medicine 05/2015; 10(4):495-504. DOI:10.2217/rme.15.3 · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ten-eleven translocation (TET) enzymes mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is enriched in brain, and its ultimate DNA demethylation. However, the influence of TET and 5hmC on gene transcription in brain remains elusive. We found that ten-eleven translocation protein 1 (TET1) was downregulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration, which enhanced behavioral responses to cocaine. We then identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. Together, these reveal a previously unknown epigenetic mechanism of cocaine action and provide new insight into how 5hmC regulates transcription in brain in vivo.
[Show abstract][Hide abstract] ABSTRACT: Here we use a systems biology approach to comprehensively assess the conservation of gene networks in naive pluripotent stem cells (PSCs) with preimplantation embryos. While gene networks in murine naive and primed pluripotent states are reproducible across data sets, different sources of human stem cells display high degrees of variation, partly reflecting disparities in culture conditions. Finally, naive gene networks between human and mouse PSCs are not well conserved and better resemble their respective blastocysts.
[Show abstract][Hide abstract] ABSTRACT: Immunodeficiency, centromeric instability and facial anomalies type I (ICF1) syndrome is a rare genetic disease caused by
mutations in DNA methyltransferase (DNMT) 3B, a de novo DNA methyltransferase. However, the molecular basis of how DNMT3B deficiency leads to ICF1 pathogenesis is unclear. Induced
pluripotent stem cell (iPSC) technology facilitates the study of early human developmental diseases via facile in vitro paradigms. Here, we generate iPSCs from ICF Type 1 syndrome patient fibroblasts followed by directed differentiation of ICF1-iPSCs
to mesenchymal stem cells (MSCs). By performing genome-scale bisulfite sequencing, we find that DNMT3B-deficient iPSCs exhibit
global loss of non-CG methylation and select CG hypomethylation at gene promoters and enhancers. Further unbiased scanning
of ICF1-iPSC methylomes also identifies large megabase regions of CG hypomethylation typically localized in centromeric and
subtelomeric regions. RNA sequencing of ICF1 and control iPSCs reveals abnormal gene expression in ICF1-iPSCs relevant to
ICF syndrome phenotypes, some directly associated with promoter or enhancer hypomethylation. Upon differentiation of ICF1
iPSCs to MSCs, we find virtually all CG hypomethylated regions remained hypomethylated when compared with either wild-type
iPSC-derived MSCs or primary bone-marrow MSCs. Collectively, our results show specific methylome and transcriptome defects
in both ICF1-iPSCs and differentiated somatic cell lineages, providing a valuable stem cell system for further in vitro study of the molecular pathogenesis of ICF1 syndrome. GEO accession number: GSE46030.
Human Molecular Genetics 07/2014; 23(24). DOI:10.1093/hmg/ddu365 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using expression profiles from postmortem prefrontal cortex samples of 624 dementia patients and non-demented controls, we investigated global disruptions in the co-regulation of genes in two neurodegenerative diseases, late-onset Alzheimer's disease (AD) and Huntington's disease (HD). We identified networks of differentially co-expressed (DC) gene pairs that either gained or lost correlation in disease cases relative to the control group, with the former dominant for both AD and HD and both patterns replicating in independent human cohorts of AD and aging. When aligning networks of DC patterns and physical interactions, we identified a 242-gene subnetwork enriched for independent AD/HD signatures. This subnetwork revealed a surprising dichotomy of gained/lost correlations among two inter-connected processes, chromatin organization and neural differentiation, and included DNA methyltransferases, DNMT1 and DNMT3A, of which we predicted the former but not latter as a key regulator. To validate the inter-connection of these two processes and our key regulator prediction, we generated two brain-specific knockout (KO) mice and show that Dnmt1 KO signature significantly overlaps with the subnetwork (P = 3.1 × 10−12), while Dnmt3a KO signature does not (P = 0.017).
Molecular Systems Biology 07/2014; 10(7). DOI:10.15252/msb.20145304 · 10.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pilocytic astrocytoma (World Health Organization grade I) is one of the most common brain tumors in children and generally has a benign clinical course. However, it occasionally presents with more aggressive behavior. We report a case of pilocytic astrocytoma with malignant features on histology. An 8-month-old boy presented with a 10-day history of a right-sided facial paresis. Initial neuroimaging detected a pilocytic astrocytoma in multiple locations of the supratentorial and the infratentorial regions. Surgery was performed and histology revealed features of a pilocytic astrocytoma. No adjuvant therapy was administered, and a further magnetic resonance imaging was repeated 3 months postoperatively. The follow-up magnetic resonance imaging revealed that the lesion had increased in size after the partial resection. This tumor therefore presented a more aggressive behavior than usually observed with pilocytic astrocytoma.
[Show abstract][Hide abstract] ABSTRACT: Using the paradigm of in vitro differentiation of hESCs/iPSCs into retinal pigment epithelial (RPE) cells, we have recently profiled mRNA and miRNA transcriptomes to define a set of RPE mRNA and miRNA signature genes implicated in directed RPE differentiation. In this study, in order to understand the role of DNA methylation in RPE differentiation, we profiled genome-scale DNA methylation patterns using the method of reduced representation bisulfite sequencing (RRBS). We found dynamic waves of de novo methylation and demethylation in four stages of RPE differentiation. Integrated analysis of DNA methylation and RPE transcriptomes revealed a reverse-correlation between levels of DNA methylation and expression of a subset of miRNA and mRNA genes that are important for RPE differentiation and function. Gene Ontology (GO) analysis suggested that genes undergoing dynamic methylation changes were related to RPE differentiation and maturation. We further compared methylation patterns among human ESC- and iPSC-derived RPE as well as primary fetal RPE (fRPE) cells, and discovered that specific DNA methylation pattern is useful to classify each of the three types of RPE cells. Our results demonstrate that DNA methylation may serve as biomarkers to characterize the cell differentiation process during the conversion of human pluripotent stem cells into functional RPE cells.
PLoS ONE 03/2014; 9(3):e91416. DOI:10.1371/journal.pone.0091416 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.
PLoS ONE 02/2014; 9(2):e89464. DOI:10.1371/journal.pone.0089464 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Whether human induced pluripotent stem cells (hiPSCs) are epigenetically identical to human embryonic stem cells (hESCs) has been debated in the stem cell field. In this study, we analyzed DNA methylation patterns in a large number of hiPSCs (n = 114) and hESCs (n = 155), and identified a panel of 82 CpG methylation sites that can distinguish hiPSCs from hESCs with high accuracy. We show that 12 out of the 82 CpG sites were subject to hypermethylation in part by DNMT3B. Notably, DNMT3B contributes directly to aberrant hypermethylation and silencing of the signature gene, TCERG1L. Overall, we conclude that DNMT3B is involved in a wave of de novo methylation during reprogramming, a portion of which contributes to the unique hiPSC methylation signature. These 82 CpG methylation sites may be useful as biomarkers to distinguish between hiPSCs and hESCs.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation has critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single base-resolution DNA methylome from adult mouse dentate neurons consists of both CpG (∼75%) and CpH (∼25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in regions of low CpG density, depleted at protein-DNA interaction sites and anticorrelated with gene expression. Functionally, both methylated CpGs (mCpGs) and mCpHs can repress transcription in vitro and are recognized by methyl-CpG binding protein 2 (MeCP2) in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNA methyltransferase 3A (DNMT3A) for active maintenance in postmitotic neurons. These characteristics of CpH methylation suggest that a substantially expanded proportion of the neuronal genome is under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.
[Show abstract][Hide abstract] ABSTRACT: The newly developed next-generation sequencing platforms, in combination with genome-scale amplification methods, provide a powerful tool to study genomics from a single cell. This mini-review summarizes the technologies of single cell genomics and their applications in several areas of biomedical research including stem cells, cancer biology and reproductive medicine. Particularly, it highlights recent advances in single cell exome sequencing, RNA-seq, and genome sequencing. The application of these powerful techniques will shed new light on the fundamental principles of gene transcription and genome organization at single-cell level and improve our understanding of cellular heterogeneity and diversity in multicellular organisms.
[Show abstract][Hide abstract] ABSTRACT: Mammalian pre-implantation development is a complex process involving dramatic changes in the transcriptional architecture. We report here a comprehensive analysis of transcriptome dynamics from oocyte to morula in both human and mouse embryos, using single-cell RNA sequencing. Based on single-nucleotide variants in human blastomere messenger RNAs and paternal-specific single-nucleotide polymorphisms, we identify novel stage-specific monoallelic expression patterns for a significant portion of polymorphic gene transcripts (25 to 53%). By weighted gene co-expression network analysis, we find that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. This result indicates a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a step-wise fashion from cleavage to morula. Cross-species comparisons with mouse pre-implantation embryos reveal that the majority of human stage-specific modules (7 out of 9) are notably preserved, but developmental specificity and timing differ between human and mouse. Furthermore, we identify conserved key members (or hub genes) of the human and mouse networks. These genes represent novel candidates that are likely to be key in driving mammalian pre-implantation development. Together, the results provide a valuable resource to dissect gene regulatory mechanisms underlying progressive development of early mammalian embryos.
[Show abstract][Hide abstract] ABSTRACT: Primordial germ cells (PGCs) undergo dramatic rearrangements to their methylome during embryogenesis, including initial genome-wide DNA demethylation that establishes the germline epigenetic ground state. The role of the 5-methylcytosine (5mC) dioxygenases Tet1 and Tet2 in the initial genome-wide DNA demethylation process has not been examined directly. Using PGCs differentiated from either control or Tet2(-/-); Tet1 knockdown embryonic stem cells (ESCs), we show that in vitro PGC (iPGC) formation and genome-wide DNA demethylation are unaffected by the absence of Tet1 and Tet2, and thus 5-hydroxymethylcytosine (5hmC). However, numerous promoters and gene bodies were hypermethylated in mutant iPGCs, which is consistent with a role for 5hmC as an intermediate in locus-specific demethylation. Altogether, our results support a revised model of PGC DNA demethylation in which the first phase of comprehensive 5mC loss does not involve 5hmC. Instead, Tet1 and Tet2 have a locus-specific role in shaping the PGC epigenome during subsequent development.
[Show abstract][Hide abstract] ABSTRACT: The corneal endothelium is composed of a monolayer of corneal endothelial cells (CECs), which is essential for maintaining corneal transparency. In order to better characterize CECs in different developmental stages, we profiled mRNA transcriptomes in human fetal and adult corneal endothelium with the goal to identify novel molecular markers in these cells. By comparing CECs with 12 other tissue types, we identified 245 and 284 signature genes that are highly expressed in fetal and adult CECs, respectively. Functionally, these genes are enriched in pathways characteristic of CECs, including inorganic anion transmembrane transporter, extracellular matrix structural constituent and cyclin-dependent protein kinase inhibitor activity. Importantly, several of these genes are disease target genes in hereditary corneal dystrophies, consistent with their functional significance in CEC physiology. We also identified stage-specific markers associated with CEC development, such as specific members in the TGF-beta and Wnt signaling pathways only expressed in fetal, but not in adult CECs. Lastly, by immunohistochemistry of ocular tissues, we demonstrated the unique protein localization for Wnt5a, S100A4, S100A6, and IER3, the four novel markers for fetal and adult CECs. The identification of a new panel of stage-specific markers for CECs would be very useful for characterizing CECs derived from stem cells or ex vivo expansion for cell replacement therapy.
Human Molecular Genetics 12/2012; 22(7). DOI:10.1093/hmg/dds527 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic regulation of the genome is critical for the emergence of diverse cell lineages during development. To understand the role of DNA methylation during retinal network formation, we generated a mouse retinal-specific Dnmt1 deletion mutation from the onset of neurogenesis. In the hypomethylated Dnmt1-mutant retina, neural progenitor cells continue to proliferate, however, the cell cycle progression is altered, as revealed by an increased proportion of G1 phase cells. Despite production of all major retinal neuronal cell types in the Dnmt1-mutant retina, various postmitotic neurons show defective differentiation, including ectopic cell soma and aberrant dendritic morphologies. Specifically, the commitment of Dmnt1-deficient progenitors towards the photoreceptor fate is not affected by DNA hypomethylation, yet the initiation of photoreceptor differentiation is severely hindered, resulting in reduction and mislocalization of rhodopsin-expressing cells. In addition to compromised neuronal differentiation, Dnmt1 deficiency also leads to rapid cell death of photoreceptors and other types of neurons in the postnatal retina. These results indicate that Dnmt1-dependent DNA methylation is critical for expansion of the retinal progenitor pool, as well as for maturation and survival of postmitotic neurons.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early-passage neural stem cells (NSCs) derived from Dnmt3a-deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild-type counterparts. However, successive passaging to passage 6 (P6), when wild-type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a(-/-) NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1(-/-) NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a(-/-) NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a(-/-) NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC-derived-NSCs.
Journal of Neuroscience Research 10/2012; 90(10):1883-91. DOI:10.1002/jnr.23077 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.
PLoS ONE 07/2012; 7(7):e37224. DOI:10.1371/journal.pone.0037224 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the mammalian genome, DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA. During development, the pattern of DNA methylation in the genome changes as a result of a dynamic process involving both de novo DNA methylation and demethylation. As a consequence, differentiated cells develop a stable and unique DNA methylation pattern that regulates tissue-specific gene transcription. In this chapter, we will review the process of DNA methylation and demethylation in the nervous system. We will describe the DNA (de)methylation machinery and its association with other epigenetic mechanisms such as histone modifications and noncoding RNAs. Intriguingly, postmitotic neurons still express DNA methyltransferases and components involved in DNA demethylation. Moreover, neuronal activity can modulate their pattern of DNA methylation in response to physiological and environmental stimuli. The precise regulation of DNA methylation is essential for normal cognitive function. Indeed, when DNA methylation is altered as a result of developmental mutations or environmental risk factors, such as drug exposure and neural injury, mental impairment is a common side effect. The investigation into DNA methylation continues to show a rich and complex picture about epigenetic gene regulation in the central nervous system and provides possible therapeutic targets for the treatment of neuropsychiatric disorders.Neuropsychopharmacology Reviews advance online publication, 11 July 2012; doi:10.1038/npp.2012.112.
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 07/2012; 38(1). DOI:10.1038/npp.2012.112 · 7.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transcription factors (TFs) can direct cell fate by binding to DNA and regulating gene transcription. Controlling the intracellular levels of specific TFs can therefore enable reprogramming of cellular function and differentiation. Direct delivery of recombinant TFs to target cells can thus have widespread therapeutic value, but has remained challenging due to structural fragility of TFs and inefficient membrane transduction. Here we describe the functional delivery of TFs using degradable polymeric nanocapsules to drive cellular differentiation. The nanocapsules were synthesized with poly(ethylene) glycol (PEG)-based monomers and intracellularly-degradable crosslinkers. Physical properties and release kinetics of the nanocapsules were optimized through tuning of monomer and crosslinker ratios to achieve enhanced delivery of cargo destined for the nuclei. The nanocapsules did not display cytotoxicity in primary cell lines up to concentrations of 5 μm. A recombinant myogenic transcription factor, MyoD, was delivered to the nuclei of myoblast cells using degradable nanocapsules to induce myogenic differentiation. MyoD was confirmed to be delivered to the nuclei of myoblasts using confocal microscopy and was demonstrated to be active in transcription through a luciferase-based reporter assay. More importantly, delivered MyoD was able to drive myoblast differentiation as evidenced by the hallmark elongated and multinuclear morphology of myotubes. The activation of downstream cascade was also confirmed through immunostaining of late myogenic markers myogenin and My-HC. The efficiency of differentiation achieved via nanocapsule delivery is significantly higher than that of native MyoD, and is comparable to that of plasmid transfection. The encapsulated MyoD can also withstand prolonged protease treatment and remain functional. The ease of preparation, biocompatibility and effective cargo delivery make the polymeric nanocapsule a useful tool to deliver a variety of recombinant TFs for therapeutic uses.