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Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease threatening human health, yet no medicine is developed to treat this disease. In this study, we first discovered that Leptin mutant rats ( Lep ΔI14/ΔI14 ) exhibit characteristic NASH phenotypes including steatosis, lymphocyte infiltration, and ballooning after postnatal week 16. We then examined NASH progression by performing an integrated analysis of hepatic transcriptome in Leptin -deficient rats from postnatal 4 to 48 weeks. Initially, simple steatosis in Lep ΔI14/ΔI14 rats were observed with increased expression of the genes encoding for rate-limiting enzymes in lipid metabolism such as acetyl-CoA carboxylase and fatty acid synthase. When NASH phenotypes became well developed at postnatal week 16, we found gene expression changes in insulin resistance, inflammation, reactive oxygen species and endoplasmic reticulum stress. As NASH phenotypes further progressed with age, we observed elevated expression of cytokines and chemokines including C-C motif chemokine ligand 2, tumor necrosis factor ɑ, interleukin-6, and interleukin-1β together with activation of the c-Jun N-terminal kinase and nuclear factor-κB pathways. Histologically, livers in Lep ΔI14/ΔI14 rats exhibited increased cell infiltration of MPO + neutrophils, CD8 + T cells, CD68 + hepatic macrophages, and CCR2 + inflammatory monocyte-derived macrophages associated with macrophage polarization from M2 to M1. Subsequent cross-species comparison of transcriptomes in human, rat, and mouse NASH models indicated that Leptin -deficient rats bear more similarities to human NASH patients than previously established mouse NASH models. Taken together, our study suggests that Lep ΔI14/ΔI14 rats are a valuable pre-clinical rodent model to evaluate NASH drug safety and efficacy.

Single-cell transcriptome and single-cell methylome analysis have successfully revealed the heterogeneity in transcriptome and DNA methylome between single cells, and have become powerful tools to understand the dynamics of transcriptome and DNA methylome during the complicated biological processes, such as differentiation and carcinogenesis.Inspired by the success of using these single-cell -omics methods to understand the regulation of a particular "-ome," more interests have been put on elucidating the regulatory relationship among multiple-omics at single-cell resolution. The simultaneous profiling of multiple-omics from the same single cell would provide us the ultimate power to understand the relationship among different "-omes," but this idea is not materialized for decades due to difficulties to assay extremely tiny amount of DNA or RNA in a single cell.To address this technical challenge, we have recently developed a novel method named scMT-seq that can simultaneously profile both DNA methylome and RNA transcriptome from the same cell. This method enabled us to measure, from a single cell, the DNA methylation status of the most informative 0.5-1 million CpG sites and mRNA level of 10,000 genes, of which 3200 genes can be further analyzed with both promoter DNA methylation and RNA transcription. Using the scMT-seq data, we have successfully shown the regulatory relationship between DNA methylation and transcriptional level in a single dorsal root ganglion neuron (Hu et al., Genome Biol 17:88, 2016). We believe the scMT-seq would be a powerful technique to uncover the regulatory mechanism between transcription and DNA methylation, and would be of wide interest beyond the epigenetics community.

Fragile X syndrome (FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1 (FMR1), leading to decreased or absent expression of its encoded fragile X mental retardation protein (FMRP). However, the global transcriptional alteration by FMRP deficiency has not been well characterized at single nucleotide resolution, i.e., RNA-seq. Here, we performed in-vitro neuronal differentiation of human induced pluripotent stem (iPS) cells that were derived from fibroblasts of a FXS patient (FXS-iPSC). We then performed RNA-seq and examined the transcriptional misregulation at each intermediate stage during in-vitro differentiation of FXS-iPSC into neurons. After thoroughly analyzing the transcriptomic data and integrating them with those from other platforms, we found up-regulation of many genes encoding TFs for neuronal differentiation (WNT1, BMP4, POU3F4, TFAP2C, and PAX3), down-regulation of potassium channels (KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9, and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, which will help to discover potential targeting candidates for the cure of FXS symptoms.

Corneal endothelial cells (CECs) are a monolayer of cells covering the inner-side of cornea, playing a pivotal role in keeping the cornea transparent. Because adult CECs have no proliferative capacity, the loss of CECs during aging or under pathological conditions would lead to corneal edema, eventually leading to the blindness. Clinically, donated CECs have been successfully transplanted to treat the diseases of CEC deficiency; however, the source of CEC donation is very limited. As an alternative cell source for CEC transplantation, CEC-like cells can be obtained via in vitro differentiation of human pluripotent stem cells. In this study, we introduced a modified two-stage differentiation method to convert H9 human embryonic stem cells (hESCs) to neural crest cells (NCCs), then further into CEC-like cells. The CEC-like cells treated with bovine CEC conditional medium morphologically best resembled primary CECs among all the culture conditions. By whole transcriptome analysis, we found that the typical markers of CECs, such as Na+-K+-ATPase, AQP1, Col8a and ZO-1, are highly expressed in hESC-derived CEC-like cells. By comparing RNA transcriptome of hESC-derived CEC-like cells with human primary fetal and adult CECs, we further identified shared molecular markers such as TRIT1, HSPB11, CRY1 that can be used to quality control CEC derivatives from hESCs. Our study paves the way for the quality-control and future application of hESC-derived CECs in the treatment of CEC deficiency disorders., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Single-cell transcriptome and single-cell methylome technologies have become powerful tools to study RNA and DNA methylation profiles of single cells at a genome-wide scale. A major challenge has been to understand the direct correlation of DNA methylation and gene expression within single-cells. Due to large cell-to-cell variability and the lack of direct measurements of transcriptome and methylome of the same cell, the association is still unclear., Results: Here, we describe a novel method (scMT-seq) that simultaneously profiles both DNA methylome and transcriptome from the same cell. In sensory neurons, we consistently identify transcriptome and methylome heterogeneity among single cells but the majority of the expression variance is not explained by proximal promoter methylation, with the exception of genes that do not contain CpG islands. By contrast, gene body methylation is positively associated with gene expression for only those genes that contain a CpG island promoter. Furthermore, using single nucleotide polymorphism patterns from our hybrid mouse model, we also find positive correlation of allelic gene body methylation with allelic expression., Conclusions: Our method can be used to detect transcriptome, methylome, and single nucleotide polymorphism information within single cells to dissect the mechanisms of epigenetic gene regulation.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G1-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans.

Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons.

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).

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.