Interstitial stem cells in Hydra: multipotency and decision-making

Interstitial stem cells in Hydra constitute a population of multipotent cells, which continuously give rise to differentiated products during the growth and budding of Hydra polyps. They also give rise to germ cells in animals undergoing sexual differentiation. Cloning experiments have shown that interstitial stem cells are multipotent. In vivo tracing of stem cell lineages has revealed that stem cells divide symmetrically to yield two stem cells or asymmetrically to yield one stem cell daughter and one daughter cell which initiates nerve or nematocyte differentiation. Following commitment, some nerve cell precursors migrate from the body column into the head or foot region, thus giving rise to the high density of nerve cells observed in these regions. Stem cell proliferation is regulated by changes in the self-renewal probability and is controlled by stem cell density. Nerve cell commitment is controlled by several peptides including the Head Activator. Factors affecting nematocyte commitment are not known, but wnt and notch signaling are both required for differentiation of committed precursors.     

Myogenic stem cell commitment probability remains constant as a function of organismal and mitotic age

Chicken myogenic stem cells can undergo symmetric and asymmetric cell divisions. Symmetric divisions produce two stem cells or two cells committed to terminal muscle differentiation. Asymmetric divisions produce one stem cell and one committed cell. Committed cells undergo four divisions, and their progeny differentiate into postmitotic, biochemically distinct muscle cells, which can be identified immunocytochemically. The control of stem cell commitment was investigated in vitro by means of cell cloning and subcloning experiments, and computer modeling. We found that stem cell commitment is a process which can be modeled as a stochastic event, with a central tendency or probability of 0.2 +/- 0.1. This value is independent of organismal or mitotic age of the stem cells, cell density, or growth in a mitogen-poor environment. Myogenic stem cells stop dividing after approximately 30 divisions in vitro. Since the probability of commitment to terminal differentiation remains below 0.5, clonal senescence and terminal differentiation are separate processes in this system.     

Aproliferin: a modulator of proliferative potential

Cellular differentiation proceeds through a series of steps in which cells undergo modifications in cellular phenotype and proliferative potential. Differentiation has been extensively studied in 3T3-T mesenchymal stem cells and growth arrest, non-terminal and terminal differentiation have been identified as three distinct stages in the adipocyte differentiation of these cells. The terminal stage of differentiation is associated with irreversible loss of proliferative potential and commitment to the expression of the adipocyte phenotype. A protein has been partially purified from human plasma that can induce the transition of 3T3-T adipocytes from the non-terminal to the terminal state of differentiation. This protein, designated aproliferin, has a mol. wt of approximately 45,000 and is trypsin, acid and heat labile. Induction of terminal differentiation by aproliferin is associated with changes in the synthesis of a limited number of cellular proteins. The ability of aproliferin to induce terminal differentiation in non-terminally differentiated cells is highly specific as a wide variety of pharmacological and biochemical agents do not mimic the effects of this agent. Apoliferin may be one of an emerging class of molecules which can affect differentiation and induce irreversible changes in cell function.     

Commitment to erythroid differentiation by friend erythroleukemia cells: a stochastic analysis

A method for the clonal analysis of murine erythroleukemia cells has been developed which allows the precise characterization of the number of progeny produced by each cell and the degree of differentiation of each progeny cell. The potential of almost every cell in the culture can be monitored because a plating efficiency close to 100% has been achieved. The effects of treatment with an inducer of differentiation (DMSO) on the proliferative capacity of the treated cells have been studied with this technique. Cells from a mass culture treated with inducer give rise to colonies of differentiated progeny when subsequently cloned in the absence of inducer. Colonies exhibiting this phenotype represent the progeny of cells committed to the differentiation pathway by treatment with inducer. We observe that the commitment decision limits the subsequent proliferative capacity of the cell to four additional cell divisions. A quantitative analysis suggests that the commitment decision for each cell is made in a stochastic manner. Irreversible commitment to the expression of differentiated functions occurs with discrete probability per cell generation for many cell generations. The value for this probability is a function of the concentration of inducer (DMSO). A correlative biochemical study suggests that an irreversible commitment decision by a significant proportion of the population precedes or accompanies increases in cytoplasmic globin mRNA levels, one of the earliest detectable biochemical markers for erythroid differentiation in this system.A specific kinetic model based on these considerations has been developed to predict clonal phenotypes as a function of time and probability of commitment. Quantitative predictions based on this model are in excellent agreement with experimental observations. The effectiveness of a stochastic model in predicting the behavior of this system is discussed in relation to the stochastic behavior of normal hematopoiesis and the biochemical mechanisms which control these differentiation programs.     

Spatio-temporal organization of DNA replication in murine embryonic stem, primary, and immortalized cells

The extent to which chromosomal domains are reorganized within the nucleus during differentiation is central to our understanding of how cells become committed to specific developmental lineages. Spatio-temporal patterns of DNA replication are a reflection of this organization. Here, we demonstrate that the temporal order and relative duration of these replication patterns during S-phase are similar in murine pluripotent embryonic stem (ES) cells, primary adult myoblasts, and an immortalized fibroblast line. The observed patterns were independent of fixation and denaturation techniques. Importantly, the same patterns were detected when fluorescent nucleotides were introduced into living cells, demonstrating their physiological relevance. These data suggest that heritable gene silencing during commitment to specific cell lineages is not mediated by global changes in the sub-nuclear organization and replication timing of chromosome domains.     

Regulation of differentiation in normal and transformed erythroid cells.

Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.     

The CDK inhibitor p27 enhances neural differentiation in pluripotent NTERA2 human EC cells, but does not permit differentiation of 2102Ep nullipotent human EC cells

Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are the malignant counterparts of pluripotent embryonic stem (ES) cells, but commonly exhibit a reduced ability to differentiate, presumably because of continual selection for genetic changes that alter the balance between self-renewal, differentiation and apoptosis in favour of self-renewal. To explore the nature of the genetic changes that promote nullipotency, we have compared two human EC cell lines, a 'nullipotent' line, 2102Ep, and a 'pluripotent' line, NTERA2. A hybrid derived by fusion of these cells differentiates in response to retinoic acid but, unlike the parental NTERA2 line, does not form terminally differentiated neurons. This implies that the nullipotent EC cell line, 2102Ep, differs in expression of at least two functions in comparison with the NTERA2 pluripotent line, one affecting commitment to differentiation, and one affecting terminal neural differentiation. We have now investigated the possible role of the CDK inhibitor, p27kip1 (p27) in commitment and terminal differentiation. In NTERA2, but not in 2102Ep cells, retinoic acid induces up-regulation of p27 expression, suggesting that 2102Ep cells lack this capacity. However, constitutive expression of a p27 transgene does not overcome the block to differentiation in the 2102Ep parental cells; commitment to differentiation must be blocked elsewhere. On the other hand, constitutive over-expression of p27 from a transgene enhances the neural differentiation of NTERA2 cells. Our results suggest that p27 plays a role in terminal neuronal differentiation of human EC cells, but not in their initial commitment to differentiation, and that other factors, possibly Cyclin D2, specifically limit its ability to promote neural differentiation.     

Chromatin organization and differentiation in embryonic stem cell models

Embryonic stem cells derived from mammalian embryos represent indispensable tools for mammalian genetics. Their key features--self-renewal and pluripotency--enable them, on the one hand, to be propagated in culture almost indefinitely and, on the other, to be used to study the molecular details of cell commitment and differentiation. In the past few years, it has become clear that chromatin and epigenetic modifications have a central role in maintaining the gene expression programs that are important for both self-renewal and cell commitment. Therefore, studies focused on the chromatin profiles of embryonic stem cells are likely to be very informative for understanding pluripotency and the process of differentiation, and ultimately for using embryonic stem cells as a tool for cell replacement therapy or as models for the study of genetic diseases, cancer progression or drug testing.     

Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation

Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC). However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications.     

Role of Sonic Hedgehog Signaling in Oligodendrocyte Differentiation

During development, the secreted molecule Sonic Hedgehog (Shh) is required for lineage specification and proliferation of oligodendrocyte progenitors (OLPs), which are the glia cells responsible for the myelination of axons in the central nervous system (CNS). Shh signaling has been implicated in controlling both the generation of oligodendrocytes (OLGs) during embryonic development and their production in adulthood. Although, some evidence points to a role of Shh signaling in OLG development, its involvement in OLG differentiation remains to be fully determined. The objective of this study was to assess whether Shh signaling is involved in OLG differentiation after neural stem cell commitment to the OLG lineage. To address these questions, we manipulated Shh signaling using cyclopamine, a potent inhibitor of Shh signaling activator Smoothened (Smo), alone or combined with the agonist SAG in OLG primary cultures and assessed expression of myelin-specific markers. We found that inactivation of Shh signaling caused a dose-dependent decrease in myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in differentiating OLGs. Co-treatment of the cells with SAG reversed the inhibitory effect of cyclopamine on both myelin-specific protein levels and morphological changes associated with it. Further experiments are required to elucidate the molecular mechanism by which Shh signaling regulates OLG differentiation.     

Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole

The effect of imidazole on DMSO-induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO-induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO-induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.     

Coordinated involvement of cathepsins S, D and cystatin C in the commitment of hematopoietic stem cells to dendritic cells

The identity of biochemical players which underpin the commitment of CD34(+) hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14(+) cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34(+) stem cells from peripheral and cord blood. As shown by CD4(+) T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity.     

Procaine inhibits the erythroid differentiation of MEL cells by blocking commitment: Possible involvement of calcium metabolism

The action of procaine on the terminal erythroid differentiation of murine erythroleukemia (MEL) cells has been investigated at the level of individual cells. At concentrations (7 × 10^?4 M) which had no inhibitory effect on cell growth, pretreatment of these cells with procaine for 12–24 hr caused a pronounced inhibition (> 90%) of commitment to terminal erythroid differentiation of dimethyl sulfoxide (DMSO)-treated cells. Simultaneous treatment of MEL cells with DMSO and procaine, however, resulted to only slight inhibition (< 20%) of commitment. Blockade of commitment by procaine pretreatment appears to be general since it was observed in cells treated with other inducers (6-thioguanine, dimethylformamide). Procaine pretreatment did not abolish the ability of MEL cells to complete the “latent period” and commit upon the removal of the block. Reversal of procaine inhibition of commitment was obtained by the addition of either CaCl₂ (1.0 mM), calcium ionophore A23817 (1 μg/ml), but not of MgCl₂ (1.0 mM). From these data we conclude that procaine inhibits the terminal erythroid differentiation of MEL cells by blocking an event or process required for commitment which occurs prior to commitment itself. Our results suggest that this process involves calcium metabolism.