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.

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.

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.

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.

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.

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.

Systemic epigenetic response to recombinant lentiviral vectors independent of proviral integration.

BACKGROUND: Lentiviral vectors (LV) are widely used for various gene transfer or gene therapy applications. The effects of LV on target cells are expected to be limited to gene delivery. Yet, human hematopoietic CD34+ cells respond to functional LVs as well as several types of non-integrating LVs by genome-wide DNA methylation changes. RESULTS: A new algorithm for the analysis of 450K Illumina data showed that these changes were marked by de novo methylation. The same 4126 cytosines located in islands corresponding to 1059 genes were systematically methylated. This effect required cellular entry of the viral particle in the cells but not the genomic integration of the vector cassette. Some LV preparations induced only mild sporadic changes while others had strong effects suggesting that LV batch heterogeneity may be related to the extent of the epigenetic response. CONCLUSION: These findings identify a previously uncharacterized but consistent cellular response to viral components and provide a novel example of environmentally modified epigenome.

Stochastic fluctuations and distributed control of gene expression impact cellular memory.

Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.

Lentiviral transduction of CD34(+) cells induces genome-wide epigenetic modifications.

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34(+) cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34(+) cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34(+) cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.

Rapid turnover of DNA methylation in human cells.

Recent studies demonstrated that cytosine methylation in the genome can be reversed without DNA replication by enzymatic mechanisms based on base excision-repair pathways. Both enzymatic methylation and demethylation mechanisms are active in the cell nucleus at the same time. One can hypothesize that the actual level of CpG methylation could be the result of a balance between the two antagonistic processes with a rapid turnover. In the present study, we used mass spectrometry to measure the total methyl-cytosine content of the genome in cultured human cells after short incubation with the known methyltransferase inhibitor 5-deoxy-azacytidine. A significant decrease of the DNA methylation was observed. Indeed, the inhibition of the methylation can only result in a rapid reduction of the overall methyl-cytosine level if the process of demethylation is simultaneous. These observations suggest that the enzymatic mechanisms responsible of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and rapid turnover of methylated cytosines. This conclusion is supported by the observation that 5-deoxy-azacytidine was incorporated in the genomic DNA of non-dividing cells and could be detected as soon as after two hours of incubation, hence providing a mechanistic explanation to the inhibition of methyltransferases. The observations are compatible with the idea that the enzymatic mechanisms that bring together of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and unsuspected rapid turnover of DNA methylation. This conclusion is at odds with the generally accepted view of high stability of cytosine methylation where the role of enzymatic demethylation is considered as limited to some special situations such as transcription. It places DNA methylation in the same category as other epigenetic modifications with covalent modifications dynamically added to and removed from the chromatin with high turnover rate.

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.

Epigenetic processes in a tetraploid mammal.

Polyploidy has played a most important role in speciation and evolution of plants and animals. It is thought that low frequency of polyploidy in mammals is due to a dosage imbalance that would interfere with proper development in mammalian polyploids. The first tetraploid mammal, Tympanoctomys barrerae (Octodontidae), appears to be an exception to this rule. In this study we investigated X chromosome inactivation (XCI) and genomic imprinting in T. barrerae, two epigenetic processes usually involved in dosage control in mammalian genomes. The imprinting status of the Peg1 gene was determined by Peg1 allelic expression studies. The inactive X chromosome was identified on interphase nuclei by immunofluorescence using specific antisera raised against Met3H3K27 and macroH2A1. Quantitative PCR was used to compare the Peg1/Dmd ratio in T. barrerae and in its most closely related diploid species, Octomys mimax. Our data demonstrate that parental-specific silencing of at least one gene and normal X chromosomal dosage mechanism are conserved in the tetraploid genome. We hypothesize a concerted action of genetic and epigenetic mechanisms during the process of functional diploidization of this tetraploid genome.

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.

Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.

BACKGROUND: Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines. METHODS: The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis. RESULTS: The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy. CONCLUSION: Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation.

Essential role for poly (ADP-ribosyl)ation in mouse preimplantation development.

BACKGROUND: Poly (ADP-ribosyl)ation is a covalent modification of many nuclear proteins. It has a strong chromatin modifying potential involved in DNA repair, transcription and replication. Its role during preimplantation development is unknown. RESULTS: We have observed strong but transient synthesis of poly ADP-ribose polymers on decondensing chromosomes of fertilized and parthenogenetically activated mouse oocytes. Inhibition of this transient upregulation with a specific enzyme inhibitor, 3-aminobenzamide, has long-term effects on the postimplantation development of the embryos. In addition, inhibition of poly (ADP-ribosyl)ation at the 4-8 cell stage selectively blocks morula compaction. CONCLUSION: These observations suggest that poly (ADP-ribosyl)ation is involved in the epigenetic chromatin remodeling in the zygote.