Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells.

When human cord blood-derived CD34+ cells are induced to differentiate, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. In particular, the cells pass through a transitory phase known as "multilineage-primed" state. These cells are characterized by a mixed gene expression profile, different in each cell, with the coexpression of many genes characteristic for concurrent cell lineages. The aim of our study is to understand the mechanisms of the establishment and the exit from this transitory state. We investigated this issue using single-cell RNA sequencing and ATAC-seq. Two phases were detected. The first phase is a rapid and global chromatin decompaction that makes most of the gene promoters in the genome accessible for transcription. It results 24 h later in enhanced and pervasive transcription of the genome leading to the concomitant increase in the cell-to-cell variability of transcriptional profiles. The second phase is the exit from the multilineage-primed phase marked by a slow chromatin closure and a subsequent overall down-regulation of gene transcription. This process is selective and results in the emergence of coherent expression profiles corresponding to distinct cell subpopulations. The typical time scale of these events spans 48 to 72 h. These observations suggest that the nonspecificity of genome decompaction is the condition for the generation of a highly variable multilineage expression profile. The nonspecific phase is followed by specific regulatory actions that stabilize and maintain the activity of key genes, while the rest of the genome becomes repressed again by the chromatin recompaction. Thus, the initiation of differentiation is reminiscent of a constrained optimization process that associates the spontaneous generation of gene expression diversity to subsequent regulatory actions that maintain the activity of some genes, while the rest of the genome sinks back to the repressive closed chromatin state.

Differentiation is accompanied by a progressive loss in transcriptional memory.

BACKGROUND: Cell differentiation requires the integration of two opposite processes, a stabilizing cellular memory, especially at the transcriptional scale, and a burst of gene expression variability which follows the differentiation induction. Therefore, the actual capacity of a cell to undergo phenotypic change during a differentiation process relies upon a modification in this balance which favors change-inducing gene expression variability. However, there are no experimental data providing insight on how fast the transcriptomes of identical cells would diverge on the scale of the very first two cell divisions during the differentiation process. RESULTS: In order to quantitatively address this question, we developed different experimental methods to recover the transcriptomes of related cells, after one and two divisions, while preserving the information about their lineage at the scale of a single cell division. We analyzed the transcriptomes of related cells from two differentiation biological systems (human CD34+ cells and T2EC chicken primary erythrocytic progenitors) using two different single-cell transcriptomics technologies (scRT-qPCR and scRNA-seq). CONCLUSIONS: We identified that the gene transcription profiles of differentiating sister cells are more similar to each other than to those of non-related cells of the same type, sharing the same environment and undergoing similar biological processes. More importantly, we observed greater discrepancies between differentiating sister cells than between self-renewing sister cells. Furthermore, a progressive increase in this divergence from first generation to second generation was observed when comparing differentiating cousin cells to self renewing cousin cells. Our results are in favor of a gradual erasure of transcriptional memory during the differentiation process.

Investigative power of genomic informational field theory relative to genome-wide association studies for genotype-phenotype mapping.

Identifying associations between phenotype and genotype is the fundamental basis of genetic analyses. Inspired by frequentist probability and the work of R. A. Fisher, genome-wide association studies (GWAS) extract information using averages and variances from genotype-phenotype datasets. Averages and variances are legitimated upon creating distribution density functions obtained through the grouping of data into categories. However, as data from within a given category cannot be differentiated, the investigative power of such methodologies is limited. Genomic informational field theory (GIFT) is a method specifically designed to circumvent this issue. The way GIFT proceeds is opposite to that of GWAS. Although GWAS determines the extent to which genes are involved in phenotype formation (bottom-up approach), GIFT determines the degree to which the phenotype can select microstates (genes) for its subsistence (top-down approach). Doing so requires dealing with new genetic concepts, a.k.a. genetic paths, upon which significance levels for genotype-phenotype associations can be determined. By using different datasets obtained in Ovis aries related to bone growth (dataset 1) and to a series of linked metabolic and epigenetic pathways (dataset 2), we demonstrate that removing the informational barrier linked to categories enhances the investigative and discriminative powers of GIFT, namely that GIFT extracts more information than GWAS. We conclude by suggesting that GIFT is an adequate tool to study how phenotypic plasticity and genetic assimilation are linked.NEW & NOTEWORTHY The genetic basis of complex traits remains challenging to investigate using classic genome-wide association studies (GWASs). Given the success of gene editing technologies, this point needs to be addressed urgently since there can only be useful editing technologies whether precise genotype-phenotype mapping information is available initially. Genomic informational field theory (GIFT) is a new mapping method designed to increase the investigative power of biological/medical datasets suggesting, in turn, the need to rethink the conceptual bases of quantitative genetics.

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment.

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned).

Combination of imaging flow cytometry and time-lapse microscopy for the study of label-free morphology dynamics of hematopoietic cells.

Cell differentiation is a longitudinal and dynamic process. Studying and quantifying such a process require tools combining precise time resolution and statistical power. Imaging flow cytometry (IFC) provides statistically significant number of microscopy images of individual cells in a sample at a given time point. Time-lapse microscopy (TLM) is the method of choice for studying the dynamics of cell processes at a high temporal, but low statistical resolution. In this work, we show that the dynamic changes of cord-blood derived CD34+ cells in response to cytokine stimulation can be successfully studied, in a label-free way, by the combination of the IFCs statistical power and the TLM's high time resolution. Cell morphology phenotypes were quantified through roundness and surface area, measured both in IFC and with a homemade segmentation algorithm in TLM. Two distinct morphologies-polarized and round-were observed in cord-blood derived CD34+. We show that some cells have the ability to fluctuate between these morphologies, suggesting that the apparent stable composition of round and polarized cells may actually represent a dynamic equilibrium. This example demonstrates that the different resolutions and modalities of IFC and TLM are complementary and allow the study of complex dynamic biological processes. © 2017 International Society for Advancement of Cytometry.

Hypoxic culture conditions enhance the generation of regulatory T cells.

The generation of large amounts of induced CD4+  CD25+  Foxp3+ regulatory T (iTreg) cells is of great interest for several immunotherapy applications, therefore a better understanding of signals controlling iTreg cell differentiation and expansion is required. There is evidence that oxidative metabolism may regulate several key signalling pathways in T cells. This prompted us to investigate the effects of oxygenation on iTreg cell generation by comparing the effects of atmospheric (21%) or of low (5%) O2 concentrations on the phenotype of bead-stimulated murine splenic CD4+ T cells from Foxp3-KI-GFP T-cell receptor transgenic mice. The production of intracellular reactive oxygen species was shown to play a major role in the generation of iTreg cells, a process characterized by increased levels of Sirt1, PTEN and Glut1 on the committed cells, independently of the level of oxygenation. The suppressive function of iTreg cells generated either in atmospheric or low oxygen levels was equivalent. However, greater yields of iTreg cells were obtained under low oxygenation, resulting from a higher proliferative rate of the committed Treg cells and higher levels of Foxp3, suggesting a better stability of the differentiation process. Higher expression of Glut1 detected on iTreg cells generated under hypoxic culture conditions provides a likely explanation for the enhanced proliferation of these cells as compared to those cultured under ambient oxygen. Such results have important implications for understanding Treg cell homeostasis and developing in vitro protocols for the generation of Treg cells from naive T lymphocytes.

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.

Understanding Cell Differentiation Through Single-Cell Approaches: Conceptual Challenges of the Systemic Approach.

The cells of a multicellular organism are derived from a single zygote and genetically almost identical. Yet, they are phenotypically very different. This difference is the result of a process commonly called cell differentiation. How the phenotypic diversity emerges during ontogenesis or regeneration is a central and intensely studied but still unresolved issue in biology. Cell biology is facing conceptual challenges that are frequently confused with methodological difficulties. How to define a cell type? What stability or change means in the context of cell differentiation and how to deal with the ubiquitous molecular variations seen in the living cells? What are the driving forces of the change? We propose to reframe the problem of cell differentiation in a systemic way by incorporating different theoretical approaches. The new conceptual framework is able to capture the insights made at different levels of cellular organization and considered previously as contradictory. It also provides a formal strategy for further experimental studies.

Metabolic adaptation pilots the differentiation of human hematopoietic cells.

A continuous supply of energy is an essential prerequisite for survival and represents the highest priority for the cell. We hypothesize that cell differentiation is a process of optimization of energy flow in a changing environment through phenotypic adaptation. The mechanistic basis of this hypothesis is provided by the established link between core energy metabolism and epigenetic covalent modifications of chromatin. This theory predicts that early metabolic perturbations impact subsequent differentiation. To test this, we induced transient metabolic perturbations in undifferentiated human hematopoietic cells using pharmacological inhibitors targeting key metabolic reactions. We recorded changes in chromatin structure and gene expression, as well as phenotypic alterations by single-cell ATAC and RNA sequencing, time-lapse microscopy, and flow cytometry. Our observations suggest that these metabolic perturbations are shortly followed by alterations in chromatin structure, leading to changes in gene expression. We also show that these transient fluctuations alter the differentiation potential of the cells.

Investigative power of Genomic Informational Field Theory (GIFT) relative to GWAS for genotype-phenotype mapping.

Identifying associations between phenotype and genotype is the fundamental basis of genetic analyses. Inspired by frequentist probability and the work of R.A. Fisher, genome-wide association studies (GWAS) extract information using averages and variances from genotype-phenotype datasets. Averages and variances are legitimated upon creating distribution density functions obtained through the grouping of data into categories. However, as data from within a given category cannot be differentiated, the investigative power of such methodologies is limited. Genomic Informational Field Theory (GIFT) is a method specifically designed to circumvent this issue. The way GIFT proceeds is opposite to that of GWAS. Whilst GWAS determines the extent to which genes are involved in phenotype formation (bottom-up approach), GIFT determines the degree to which the phenotype can select microstates (genes) for its subsistence (top-down approach). Doing so requires dealing with new genetic concepts, a.k.a. genetic paths, upon which significance levels for genotype-phenotype associations can be determined. By using different datasets obtained in ovis aries related to bone growth (Dataset-1) and to a series of linked metabolic and epigenetic pathways (Dataset-2), we demonstrate that removing the informational barrier linked to categories enhances the investigative and discriminative powers of GIFT, namely that GIFT extracts more information than GWAS. We conclude by suggesting that GIFT is an adequate tool to study how phenotypic plasticity and genetic assimilation are linked.

Single-cell transcriptional uncertainty landscape of cell differentiation.

Background: Single-cell studies have demonstrated the presence of significant cell-to-cell heterogeneity in gene expression. Whether such heterogeneity is only a bystander or has a functional role in the cell differentiation process is still hotly debated. Methods: In this study, we quantified and followed single-cell transcriptional uncertainty - a measure of gene transcriptional stochasticity in single cells - in 10 cell differentiation systems of varying cell lineage progressions, from single to multi-branching trajectories, using the stochastic two-state gene transcription model. Results: By visualizing the transcriptional uncertainty as a landscape over a two-dimensional representation of the single-cell gene expression data, we observed universal features in the cell differentiation trajectories that include: (i) a peak in single-cell uncertainty during transition states, and in systems with bifurcating differentiation trajectories, each branching point represents a state of high transcriptional uncertainty; (ii) a positive correlation of transcriptional uncertainty with transcriptional burst size and frequency; (iii) an increase in RNA velocity preceding the increase in the cell transcriptional uncertainty. Conclusions: Our findings suggest a possible universal mechanism during the cell differentiation process, in which stem cells engage stochastic exploratory dynamics of gene expression at the start of the cell differentiation by increasing gene transcriptional bursts, and disengage such dynamics once cells have decided on a particular terminal cell identity. Notably, the peak of single-cell transcriptional uncertainty signifies the decision-making point in the cell differentiation process.

An image-guided microfluidic system for single-cell lineage tracking.

Cell lineage tracking is a long-standing and unresolved problem in biology. Microfluidic technologies have the potential to address this problem, by virtue of their ability to manipulate and process single-cells in a rapid, controllable and efficient manner. Indeed, when coupled with traditional imaging approaches, microfluidic systems allow the experimentalist to follow single-cell divisions over time. Herein, we present a valve-based microfluidic system able to probe the decision-making processes of single-cells, by tracking their lineage over multiple generations. The system operates by trapping single-cells within growth chambers, allowing the trapped cells to grow and divide, isolating sister cells after a user-defined number of divisions and finally extracting them for downstream transcriptome analysis. The platform incorporates multiple cell manipulation operations, image processing-based automation for cell loading and growth monitoring, reagent addition and device washing. To demonstrate the efficacy of the microfluidic workflow, 6C2 (chicken erythroleukemia) and T2EC (primary chicken erythrocytic progenitors) cells are tracked inside the microfluidic device over two generations, with a cell viability rate in excess of 90%. Sister cells are successfully isolated after division and extracted within a 500 nL volume, which was demonstrated to be compatible with downstream single-cell RNA sequencing analysis.

Warfarin pharmacogenetics: development of a dosing algorithm for Omani patients.

The objective of our present study was to develop a warfarin dosing algorithm for the Omani patients, as performances of warfarin dosing algorithms vary across populations with impact on the daily maintenance dose. We studied the functional polymorphisms of CYP2C9, CYP4F2 and VKORC1 genes to evaluate their impact on the warfarin maintenance dose in an admixed Omani patient cohort with Caucasian, African and Asian ancestries. We observed a 64-fold inter-patient variability for warfarin to achieve stable international normalized ratio in these patients. Univariate analysis revealed that age, gender, weight, atrial fibrillation, deep vein thrombosis/pulmonary embolism and variant genotypes of CYP2C9 and VKORC1 loci were significantly associated with warfarin dose in the studied patient population. However, multiple regression model showed that only the atrial fibrillation, and homozygous CYP2C9 variant genotypes (*2/*3 and *3/*3) and VKORC1 GA and AA genotypes remained significant. A multivariate model, which included demographic, clinical and pharmacogenetic variables together explained 63% of the overall inter-patient variability in warfarin dose requirement in this microgeographically defined, ethnically admixed Omani patient cohort on warfarin. This locally developed model performed much better than the International Warfarin Pharmacogenetics Consortium (IWPC) model as the latter could only explain 34% of the inter-patient variability in Omani patients. VKORC1 3673G>A polymorphism emerged as the single most important predictor of warfarin dose variability, even in this admixed population (partial R(2)=0.45).

Warfarin pharmacogenetics: polymorphisms of the CYP2C9, CYP4F2, and VKORC1 loci in a genetically admixed Omani population.

This is the first study to evaluate the spectrum and prevalence of dose-predictive genetic polymorphisms of the CYP2C9, CYP4F2 and VKORC1 loci together, in a geographically defined, ethnically admixed healthy adult Omani population sharing common lifestyle/environmental factors. Since the present-day Omani population is the result of an admixture of Caucasian, African and Asian ancestries, we compared the pharmacogenetic profile of these three loci in this population. Interestingly, the Omani pharmacogenetic profile, in terms of allele and genotype distribution, has values that are intermediate between Caucasians and African Americans, the African admixture further substantiated by the presence of the CYP2C9*8 allele. However, limitations and usefulness of such comparisons warrant caution, as the data from pharmacogenetic literature do not always represent bona fide population categories. Furthermore, definition of study population based on microgeographical scale would be more appropriate in pharmacogenetic research rather than the flawed racial, ethnic, or social categorizations since pharmacogenetic variation is clinal, and genetic influences will be further altered by lifestyle and environmental factors.

Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice.

There is increasing evidence in both plants and animals that epigenetic marks are not always cleared between generations. Incomplete erasure at genes associated with a measurable phenotype results in unusual patterns of inheritance from one generation to the next, termed transgenerational epigenetic inheritance. The Agouti viable yellow (A(vy)) allele is the best-studied example of this phenomenon in mice. The A(vy) allele is the result of a retrotransposon insertion upstream of the Agouti gene. Expression at this locus is controlled by the long terminal repeat (LTR) of the retrotransposon, and expression results in a yellow coat and correlates with hypomethylation of the LTR. Isogenic mice display variable expressivity, resulting in mice with a range of coat colours, from yellow through to agouti. Agouti mice have a methylated LTR. The locus displays epigenetic inheritance following maternal but not paternal transmission; yellow mothers produce more yellow offspring than agouti mothers. We have analysed the DNA methylation in mature gametes, zygotes, and blastocysts and found that the paternally and maternally inherited alleles are treated differently. The paternally inherited allele is demethylated rapidly, and the maternal allele is demethylated more slowly, in a manner similar to that of nonimprinted single-copy genes. Interestingly, following maternal transmission of the allele, there is no DNA methylation in the blastocyst, suggesting that DNA methylation is not the inherited mark. We have independent support for this conclusion from studies that do not involve direct analysis of DNA methylation. Haplo-insufficiency for Mel18, a polycomb group protein, introduces epigenetic inheritance at a paternally derived A(vy) allele, and the pedigrees reveal that this occurs after zygotic genome activation and, therefore, despite the rapid demethylation of the locus.

Epigenetic gene expression noise and phenotypic diversification of clonal cell populations.

Spontaneous emergence of phenotypic heterogeneity in cultures of genetically identical cells is a frequently observed phenomenon that provides a simple in vitro experimental system to model the problems of in vivo differentiation. In the present study, we have investigated whether stochastic variation of gene expression levels could contribute to phenotypic change in human cells. We have applied the two fluorescence-coding gene method and the expression variability of the two reporter genes to human cells in culture. We have quantified the portion of gene expression variation determined by global, promoter-specific, or by epigenetic sources. These two types of variation appear to contribute, in different ways, to the phenotypic diversification of clonal cell populations. Global, or promoter-specific, gene expression noise increases with cellular stress and contributes to the emergence of cellular diversity by diversifying the gene-expression levels. Epigenetic mechanisms act to increase the robustness of the cellular state by stabilizing gene transcription levels or by reinforcing the silenced state.

Constraints on Human CD34+ Cell Fate due to Lentiviral Vectors Can Be Relieved by Valproic Acid.

The initial stages following the in vitro cytokine stimulation of human cord blood CD34+ cells overlap with the period when lentiviral gene transfer is typically performed. Single-cell transcriptional profiling and time-lapse microscopy were used to investigate how the vector-cell crosstalk impacts on the fate decision process. The single-cell transcription profiles were analyzed using a new algorithm, and it is shown that lentiviral transduction during the early stages of stimulation modifies the dynamics of the fate choice process of the CD34+ cells. The cells transduced with a lentiviral vector are biased toward the common myeloid progenitor lineage. Valproic acid, a histone deacetylase inhibitor known to increase the grafting potential of the CD34+ cells, improves the transduction efficiency to almost 100%. The cells transduced in the presence of valproic acid can subsequently undergo normal fate commitment. The higher gene transfer efficiency did not alter the genomic integration profile of the vector. These observations open the way to substantially improving lentiviral gene transfer protocols.

Effect of CYP4F2, VKORC1, and CYP2C9 in Influencing Coumarin Dose: A Single-Patient Data Meta-Analysis in More Than 15,000 Individuals.

The cytochrome P450 (CYP)4F2 gene is known to influence mean coumarin dose. The aim of the present study was to undertake a meta-analysis at the individual patients level to capture the possible effect of ethnicity, gene-gene interaction, or other drugs on the association and to verify if inclusion of CYP4F2*3 variant into dosing algorithms improves the prediction of mean coumarin dose. We asked the authors of our previous meta-analysis (30 articles) and of 38 new articles retrieved by a systematic review to send us individual patients' data. The final collection consists of 15,754 patients split into a derivation and validation cohort. The CYP4F2*3 polymorphism was consistently associated with an increase in mean coumarin dose (+9% (95% confidence interval (CI) 7-10%), with a higher effect in women, in patients taking acenocoumarol, and in white patients. The inclusion of the CYP4F2*3 in dosing algorithms slightly improved the prediction of stable coumarin dose. New pharmacogenetic equations potentially useful for clinical practice were derived.

Conceptual Challenges of the Systemic Approach in Understanding Cell Differentiation.

The cells of a multicellular organism are derived from a single zygote and genetically identical. Yet, they are phenotypically very different. This difference is the result of a process commonly called cell differentiation. How the phenotypic diversity emerges during ontogenesis or regeneration is a central and intensely studied but still unresolved issue in biology. Cell biology is facing conceptual challenges that are frequently confused with methodological difficulties. How to define a cell type? What stability or change means in the context of cell differentiation and how to deal with the ubiquitous molecular variations seen in the living cells? What are the driving forces of the change? We propose to reframe the problem of cell differentiation in a systemic way by incorporating different theoretical approaches. The new conceptual framework is able to capture the insights made at different levels of cellular organization and considered previously as contradictory. It also provides a formal strategy for further experimental studies.

The constant variation: DNA methylation changes during preimplantation development.

Studies on the DNA methylation changes in the mouse preimplantation embryo suggested a simple and attractive model explaining the process believed to be general in mammals. However, recent reports revealed marked differences between different species that abrogates the universal validity of the model. In order to find an explanation to the differences, we have analyzed the published mouse data and compared them to the observations available in other species. The emerging common theme is the high variability of the methylation at all scales of observation and all levels of organization. This variability is the likely consequence of a dynamic and active redistribution process of the cytosine methylation in the genome.