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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., (© 2017 International Society for Advancement of Cytometry.)

The increasing popularity of stem cells in life science research has at least two major causes. On one hand, the study of stem cells may provide insights into one of the major secrets of biology: the mechanisms of cell differentiation. On the other hand, stem cells are potentially promising tools for regenerative therapy. The understanding of how environmental stimuli are translated into phenotypic differentiation through gene expression changes and how the same stimuli at the same time may perturb the normal process of cellular differentiation, growth and maintenance is a central issue for fundamental research but is also essential for the development of efficient and safe procedures for therapeutic use. This article assembles the known facts, as pieces of a puzzle, into a coherent picture around the idea of why stem cells are so sensitive to their culture environment and what practical consequences this implies.

In the present paper, I propose a hypothesis whereby the necessity to maintain the permanent energy-dissipating metabolic flux represents the primary force that determines the eukaryotic cell's choice to grow, divide and/or differentiate. This view is based on the universal structure and the strict redox neutrality of the core metabolic network. I propose that the direct substrate level coupling between metabolism and gene expression through epigenetic mechanisms provides a mechanistic explanation of how this control is implemented., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

Background: The spontaneous emergence of phenotypic heterogeneity in clonal populations of mammalian cells in vitro is a rule rather than an exception. We consider two simple, mutually non-exclusive models that explain the generation of diverse cell types in a homogeneous population. In the first model, the phenotypic switch is the consequence of extrinsic factors. Initially identical cells may become different because they encounter different local environments that induce adaptive responses. According to the second model, the phenotypic switch is intrinsic to the cells that may occur even in homogeneous environments., Principal Findings: We have investigated the "extrinsic" and the "intrinsic" mechanisms using computer simulations and experimentation. First, we simulated in silico the emergence of two cell types in a clonal cell population using a multiagent model. Both mechanisms produced stable phenotypic heterogeneity, but the distribution of the cell types was different. The "intrinsic" model predicted an even distribution of the rare phenotype cells, while in the "extrinsic" model these cells formed small clusters. The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line., Conclusions: The observations emphasize the importance of the "ecological" context and suggest that, consistently with the "extrinsic" model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch. Nevertheless, the "intrinsic" model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells.

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., ((c) 2007 International Society for Analytical Cytology.)

Genomic imprinting is traditionally defined as an epigenetic process leading to parental origin-dependent monoallelic expression of some genes. The current paradigm considers this unusual expression mode as the biological raison d être of imprinting. The present chapter proposes a critical review of our ideas about genomic imprinting in light of more recent investigatory progress. Many observations are difficult to explain on the basis of the current paradigm. Studies of allelic expression of many imprinted genes and other characteristics of chromatin domains containing clustered imprinted genes, such as replication and chromatin structure, revealed an unexpectedly complex situation that challenged the role of genomic imprinting as a mechanism of transcriptional regulation. The emerging picture is that parental imprinting is a feature of large chromatin domains with their own domain-wide characteristics. The primary biological function of imprinting may reside in the differential chromatin structure of the parental chromosomal regions and not in the monoallelic expression of some of the genes contained within them.

Both X chromosome inactivation and autosomal genomic imprinting generate a functional hemizygosity. Here we consider models that explain the evolution of genomic imprinting and X chromosome inactivation from novel perspectives. Specifically, we suggest that random (in)activation events are common in genes and gene clusters with a low probability of transcription. These generate variability that natural selection has acted on to evolve stable monoallelic expression. Possible selection forces might include a need for dosage compensation and the prevention of biallelic silencing where a total switch off would be lethal. Two different mechanisms can accomplish regular monoallelic expression - genomic imprinting and gene counting.

The H19 and insulin-like growth factor 2 (Igf2) genes in the mouse are models for genomic imprinting during development. The genes are located only 90 kb apart in the same transcriptional orientation [1], but are reciprocally imprinted: Igf2 is paternally expressed while H19 is maternally expressed. It has been suggested that expression of H19 and repression of Igf2 (or the converse) on a given chromosome are mechanistically linked and that the parental imprint operates at the level of transcription [2]. Although expression of Igf2 and H19 is thought to be monoallelic, the data have so far been obtained exclusively by looking at steady-state RNA levels using techniques that reflect the average activity of the genes in a cell population [3] [4]. Here, we have adapted a fluorescent in situ hybridisation (FISH) method to detect nascent RNA molecules of Igf2 and H19 at the initial transcription sites in the nuclei of wild-type mouse embryonic liver cells. Nine different transcription patterns were observed, reflecting a high heterogeneity of transcription at the single-cell level. Our observations suggest that regulation of Igf2 and H19 by parental imprinting is much more complex than previously proposed and acts at both transcriptional and post-transcriptional levels.

Problem: Immunologic recognition of pregnancy is important for normal gestation. Successful pregnancy is characterized by a Th2 dominance, whereas there is a Th1 dominance in failed pregnancies. We assume that a signal given by the fertilized ovum induces a Th2 shift, necessary for a normal outcome. In vitro fertilization provides a tool for testing this possibility., Method of Study: Phytohemagglutinin-stimulated lymphocytes were incubated for 48 hr in the presence of culture media from in vitro fertilized eggs, as well as in follicular fluid (FF) and control supernatants. Total RNA was isolated from the lymphocytes by the guanidine-isothiocyanate method and interleukin (IL)-10 mRNA expression was detected by reverse transcription-polymerase chain reaction., Results: Ten percent of the activated lymphocytes incubated with FF expressed IL-10 mRNA, whereas 88% of the lymphocytes activated with supernatants of sperm + oocytes gave a positive signal. Significantly (P < 0.05) fewer (50%) lymphocytes stimulated in the presence of control supernatants also expressed mRNA for IL-10. In supernatants of activated lymphocytes incubated with the culture medium of spermia + oocytes, the concentration of IL-10 was significantly higher than in the lymphocytes incubated with FF., Conclusion: These data suggest that the presence of the fertilized ovum induces a Th2 shift, which enables pregnancy to proceed to term.

In the present study, we have examined the origin and growth pattern of the beta cells in pancreatic islets, to determine whether a single progenitor cell gave rise to all the precursors of the islets, or if each of a few progenitor cells is the founder of a different islet, or if each islet is a mixture of cells originating from a pool of progenitor cells. Aggregation mouse chimaeras where the pancreatic beta cells derived from each embryo can be identified in the islets on histological sections were analyzed. In two chimaeras, all the islets contained cells from both the aggregated embryo. This clearly demonstrates that each islet resulted from several independent cells. In addition, the beta cells derived from either embryo component were in very small clusters in the islets, suggesting that in situ cell division did not account significantly for islet growth.