The transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular ...mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation.
The human placenta is a dynamic and heterogeneous organ critical in the establishment of the fetomaternal interface and the maintenance of gestational well-being. It is also the major source of ...cell-free fetal nucleic acids in the maternal circulation. Placental dysfunction contributes to significant complications, such as preeclampsia, a potentially lethal hypertensive disorder during pregnancy. Previous studies have identified significant changes in the expression profiles of preeclamptic placentas using whole-tissue analysis. Moreover, studies have shown increased levels of targeted RNA transcripts, overall and placental contributions in maternal cell-free nucleic acids during pregnancy progression and gestational complications, but it remains infeasible to noninvasively delineate placental cellular dynamics and dysfunction at the cellular level using maternal cell-free nucleic acid analysis. In this study, we addressed this issue by first dissecting the cellular heterogeneity of the human placenta and defined individual cell-type–specific gene signatures by analyzing more than 24,000 nonmarker selected cells from full-term and early preeclamptic placentas using large-scale microfluidic single-cell transcriptomic technology. Our dataset identified diverse cellular subtypes in the human placenta and enabled reconstruction of the trophoblast differentiation trajectory. Through integrative analysis with maternal plasma cell-free RNA, we resolved the longitudinal cellular dynamics of hematopoietic and placental cells in pregnancy progression. Furthermore, we were able to noninvasively uncover the cellular dysfunction of extravillous trophoblasts in early preeclamptic placentas. Our work showed the potential of integrating transcriptomic information derived from single cells into the interpretation of cell-free plasma RNA, enabling the noninvasive elucidation of cellular dynamics in complex pathological conditions.
Embryonic stem cell (ESC) culture conditions are important for maintaining long-term self-renewal, and they influence cellular pluripotency state. Here, we report single cell RNA-sequencing of mESCs ...cultured in three different conditions: serum, 2i, and the alternative ground state a2i. We find that the cellular transcriptomes of cells grown in these conditions are distinct, with 2i being the most similar to blastocyst cells and including a subpopulation resembling the two-cell embryo state. Overall levels of intercellular gene expression heterogeneity are comparable across the three conditions. However, this masks variable expression of pluripotency genes in serum cells and homogeneous expression in 2i and a2i cells. Additionally, genes related to the cell cycle are more variably expressed in the 2i and a2i conditions. Mining of our dataset for correlations in gene expression allowed us to identify additional components of the pluripotency network, including Ptma and Zfp640, illustrating its value as a resource for future discovery.
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•mESCs grown in three different conditions show distinct transcriptomes•Global intercellular variation is at similar levels in all three conditions•2i and a2i conditions maintain pluripotency with metabolic variation•Correlation analysis identifies additional pluripotency network genes
Teichmann, Marioni, and colleagues report full-transcript single cell RNA-sequencing of mESCs cultured in three different conditions: serum, 2i, and the alternative ground state a2i. They find that overall levels of intercellular heterogeneity are comparable across the three conditions, but different sets of genes are variably expressed.
Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow ...using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment.
In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a (+/+) and Bcl11a (-/-)). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a (-/-) HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a (-/-) HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a (-/-) HSCs are myeloerythroid-restricted and defective in self-renewal.
Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment.
We have previously reported an antigen-specific protocol to induce transplant tolerance and linked suppression to human embryonic stem cell (hESC)-derived tissues in immunocompetent mice through ...coreceptor and costimulation blockade. However, the exact mechanisms of acquired immune tolerance in this model have remained unclear.
We utilize the NOD.Foxp3
reporter mouse line and an ablative anti-hCD2 antibody to ask if CD4
FOXP3
regulatory T cells (Treg) are required for coreceptor and costimulation blockade-induced immune tolerance. We also perform genome-wide single-cell RNA-sequencing to interrogate Treg during immune rejection and tolerance and to indicate possible mechanisms involved in sustaining Treg function.
We show that Treg are indispensable for tolerance induced by coreceptor and costimulation blockade as depletion of which with an anti-hCD2 antibody resulted in rejection of hESC-derived pancreatic islets in NOD.Foxp3
mice. Single-cell transcriptomic profiling of 12,964 intragraft CD4
T cells derived from rejecting and tolerated grafts reveals that Treg are heterogeneous and functionally distinct in the two outcomes of transplant rejection and tolerance. Treg appear to mainly promote chemotactic and ubiquitin-dependent protein catabolism during transplant rejection while seeming to harness proliferative and immunosuppressive function during tolerance. We also demonstrate that this form of acquired transplant tolerance is associated with increased proliferation and PD-1 expression by Treg. Blocking PD-1 signaling with a neutralizing anti-PD-1 antibody leads to reduced Treg proliferation and graft rejection.
Our results suggest that short-term coreceptor and costimulation blockade mediates immune tolerance to hESC-derived pancreatic islets by promoting Treg proliferation through engagement of PD-1. Our findings could give new insights into clinical development of hESC-derived pancreatic tissues, combined with immunotherapies that expand intragraft Treg, as a potentially sustainable alternative treatment for T1D.
Abstract We report the first voxel-based morphometric (VBM) study to examine cerebral grey and white matter and cerebrospinal fluid (CSF) using computational morphometry in never-medicated, ...first-episode psychosis (FEP). Region-of-interest (ROI) analysis was also performed blind to group membership. 26 never-medicated individuals with FEP (23 with DSM-IV schizophrenia) and 38 healthy controls had MRI brain scans. Groups were balanced for age, sex, handedness, ethnicity, paternal socio-economic status, and height. Healthy controls were recruited from the local community by advertisement. Grey matter, white matter, and CSF: global brain volume ratios were significantly smaller in patients. Patients had significantly less grey matter volume in L and R caudate nuclei, cingulate gyri, parahippocampal gyri, superior temporal gyri, cerebellum and R thalamus, prefrontal cortex. They also had significantly less white matter volume in the R anterior limb of the internal capsule fronto-occipital fasciculus and L and R fornices, and significantly greater CSF volume especially in the R lateral ventricle. Excluding the 3 subjects with brief psychotic disorder did not alter our results. Our data suggest that fronto-temporal and subcortical-limbic circuits are morphologically abnormal in never-medicated, schizophrenia. ROI analysis comparing the schizophrenia group ( n = 23) with the healthy controls ( n = 38) confirmed caudate volumes were significantly smaller bilaterally by 11%, and lateral ventricular volume was significantly larger on the right by 26% in the patients. Caudate nuclei and lateral ventricular volume measurements were uncorrelated (Pearson correlation coefficient 0.30, p = 0.10), ruling out the possibility of segmentation artefact. Ratio of lateral ventricle to caudate volume was bilaterally significantly increased ( p < 0.005, 2-tailed), which could represent an early biomarker in first-episode, never-medicated schizophrenia.
Recent studies have suggested that single-stranded DNA (ssDNA) library preparation can enrich short DNA species from the plasma of healthy individuals, cancer patients, and transplant recipients. ...Based on previous observations that fetal DNA molecules in the maternal plasma are shorter than maternal DNA molecules, ssDNA library preparation may potentially enrich fetal DNA and provide substantial improvement in noninvasive prenatal testing.
We tested this hypothesis by comparing the maternal plasma DNA sequencing results using 2 types of ssDNA library preparation methods and a standard double-stranded DNA (dsDNA) library method using samples from first- and third-trimester pregnancies. We also evaluated the performance of ssDNA and dsDNA library methods in the noninvasive prenatal detection of trisomy 21 from maternal plasma.
Short DNA species were significantly enriched in ssDNA libraries. However, contrary to previous speculation, no significant enrichment was observed in the overall fetal fraction in maternal plasma collected in the first trimester. Our use of an ssDNA library did not reduce the variation in chromosomal representation when compared with a standard dsDNA library in the first-trimester plasma samples. ssDNA libraries also showed inferior performance in the noninvasive prenatal detection of trisomy 21 from maternal plasma. Detailed fetal fraction analysis using size-fractionated Y chromosome sequences and fetal-specific single-nucleotide polymorphisms (SNPs) revealed an unexpected finding that short maternal DNA was preferentially enriched over short fetal DNA in an ssDNA library irrespective of GC content.
Our findings have shown that ssDNA library preparation preferentially enriches short maternally derived DNA in maternal plasma.
Sustainable consumption and production is a critical issue in the chemical industry due to increasing public concerns on environmental and safety issues. Organizations are urged to improve the ...quality of chemical products while minimizing the environmental impacts during production. In current practice, chemists and formulators have to determine both the ingredients to be used and the machine parameter settings during product development and production. Without appropriate operations strategies for managing sustainable consumption and production, a significant portion of the ingredients, toxic materials and pollutants are wasted or emitted during the trial-and-error processes when developing chemical products. In addition, inappropriate machine parameter settings, such as blending speed and blending temperature, result in inefficient energy use. Motivated by these issues, this paper describes a recursive operations strategy (ROS) model for achieving sustainable consumption and production in the chemical industry. The ROS model first identifies the business strategy, and then defines operations strategies by assessing the competitive priorities and policies with the use of artificial intelligence, including case-based reasoning and fuzzy logic, so as to manage the operations functions. The effectiveness of the model is verified by means of a case study. The results indicate that the model can provide direct guidelines for the users to develop products based on previously developed products. By so doing, the number of trials for testing various ingredient formulae can be reduced, minimizing the ingredient waste. The proposed model is also capable of achieving continuous improvement and determining the optimal production process conditions for avoiding unnecessary energy consumption.
•To support the sustainable chemical product development and production.•To design policies in the selection of chemical ingredients.•To define operations strategy for appropriate production process conditions.•To minimize chemical waste by reducing the number of trials in product formulation.•To avoid unnecessary energy consumption by adopting the optimal process parameters.
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