Establishment of Mouse Embryonic Stem Cell-Derived Erythroid Progenitor Cell Lines Able to Produce Functional Red Blood Cells

BACKGROUND: The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells. METHODOLOGY/PRINCIPAL FINDINGS: We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells. CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells

Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction

On the spontaneous stochastic dynamics of a single gene: complexity of the molecular interplay at the promoter

International audienceBACKGROUND: Gene promoters can be in various epigenetic states and undergo interactions with many molecules in a highly transient, probabilistic and combinatorial way, resulting in a complex global dynamics as observed experimentally. However, models of stochastic gene expression commonly consider promoter activity as a two-state on/off system. We consider here a model of single-gene stochastic expression that can represent arbitrary prokaryotic or eukaryotic promoters, based on the combinatorial interplay between molecules and epigenetic factors, including energy-dependent remodeling and enzymatic activities. RESULTS: We show that, considering the mere molecular interplay at the promoter, a single-gene can demonstrate an elaborate spontaneous stochastic activity (eg. multi-periodic multi-relaxation dynamics), similar to what is known to occur at the gene-network level. Characterizing this generic model with indicators of dynamic and steady-state properties (including power spectra and distributions), we reveal the potential activity of any promoter and its influence on gene expression. In particular, we can reproduce, based on biologically relevant mechanisms, the strongly periodic patterns of promoter occupancy by transcription factors (TF) and chromatin remodeling as observed experimentally on eukaryotic promoters. Moreover, we link several of its characteristics to properties of the underlying biochemical system. The model can also be used to identify behaviors of interest (eg. stochasticity induced by high TF concentration) on minimal systems and to test their relevance in larger and more realistic systems. We finally show that TF concentrations can regulate many aspects of the stochastic activity with a considerable flexibility and complexity. CONCLUSIONS: This tight promoter-mediated control of stochasticity may constitute a powerful asset for the cell. Remarkably, a strongly periodic activity that demonstrates a complex TF concentration-dependent control is obtained when molecular interactions have typical characteristics observed on eukaryotic promoters (high mobility, functional redundancy, many alternate states/pathways). We also show that this regime results in a direct and indirect energetic cost. Finally, this model can constitute a framework for unifying various experimental approaches. Collectively, our results show that a gene - the basic building block of complex regulatory networks - can itself demonstrate a significantly complex behavior

Single Cell Dynamics Causes Pareto-Like Effect in Stimulated T Cell Populations

International audienceCell fate choice during the process of differentiation may obey to deterministic or stochastic rules. In order to discriminate between these two strategies we used time-lapse microscopy of individual murine CD4 + T cells that allows investigating the dynamics of proliferation and fate commitment. We observed highly heterogeneous division and death rates between individual clones resulting in a Pareto-like dominance of a few clones at the end of the experiment. Commitment to the Treg fate was monitored using the expression of a GFP reporter gene under the control of the endogenous Foxp3 promoter. All possible combinations of proliferation and differentiation were observed and resulted in exclusively GFP-, GFP+ or mixed phenotype clones of very different population sizes. We simulated the process of proliferation and differentiation using a simple mathematical model of stochastic decision-making based on the experimentally observed parameters. The simulations show that a stochastic scenario is fully compatible with the observed Pareto-like imbalance in the final population

Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer

International audienceUnexpectedly, the synthetic antioxidant MnTBAP was found to cause a rapid and reversible downregulation of CD4 on T cells in vitro and in vivo. This effect resulted from the internalization of membrane CD4 T cell molecules into clathrin-coated pits and involved disruption of the CD4/p56(Lck) complex. The CD4 deprivation induced by MnTBAP had functional consequences on CD4-dependent infectious processes or immunological responses as shown in various models, including gene therapy. In cultured human T cells, MnTBAP-induced downregulation of CD4 functionally suppressed gp120- mediated lentiviral transduction in a model relevant for HIV infection. The injection of MnTBAP in mice reduced membrane CD4 on lymphocytes in vivo within 5 days of treatment, preventing OVA peptide T cell immunization while allowing subsequent immunization once treatment was stopped. In a mouse gene therapy model, MnTBAP treatment at the time of adenovirus-associated virus (AAV) vector administration, successfully controlled the induction of anti-transgene and anti-capsid immune responses mediated by CD4(+) T cells, enabling the redosing mice with the same vector. These functional data provide new avenues to develop alternative therapeutic immunomodulatory strategies based on temporary regulation of CD4. These could be particularly useful for AAV gene therapy in which novel strategies for redosing are needed

Dynamic analysis of stochastic transcription cycles

In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly increased the cyclicity. Stochastically timed bursts of transcription in an apparently random subset of cells in a tissue may thus produce an overall coordinated but heterogeneous phenotype capable of acute responses to stimuli