Symmetric vs. Asymmetric Stem Cell Divisions: An Adaptation against Cancer?

Traditionally, it has been held that a central characteristic of stem cells is their ability to divide asymmetrically. Recent advances in inducible genetic labeling provided ample evidence that symmetric stem cell divisions play an important role in adult mammalian homeostasis. It is well understood that the two types of cell divisions differ in terms of the stem cells'' flexibility to expand when needed. On the contrary, the implications of symmetric and asymmetric divisions for mutation accumulation are still poorly understood. In this paper we study a stochastic model of a renewing tissue, and address the optimization problem of tissue architecture in the context of mutant production. Specifically, we study the process of tumor suppressor gene inactivation which usually takes place as a consequence of two “hits”, and which is one of the most common patterns in carcinogenesis. We compare and contrast symmetric and asymmetric (and mixed) stem cell divisions, and focus on the rate at which double-hit mutants are generated. It turns out that symmetrically-dividing cells generate such mutants at a rate which is significantly lower than that of asymmetrically-dividing cells. This result holds whether single-hit (intermediate) mutants are disadvantageous, neutral, or advantageous. It is also independent on whether the carcinogenic double-hit mutants are produced only among the stem cells or also among more specialized cells. We argue that symmetric stem cell divisions in mammals could be an adaptation which helps delay the onset of cancers. We further investigate the question of the optimal fraction of stem cells in the tissue, and quantify the contribution of non-stem cells in mutant production. Our work provides a hypothesis to explain the observation that in mammalian cells, symmetric patterns of stem cell division seem to be very common.     

The small intestine as a model for evaluating adult tissue stem cell drug targets

Adult tissue stem cells are defined and some current controversies are discussed. These crucial cells are responsible for all cell production in renewing tissues, and play a vital role in tissue regeneration. Although reliable stem cell markers are generally unavailable for adult epithelial tissues, the small intestinal crypts are an excellent in vivo model system to study stem cells. Within this tissue, the stem cells have a very well-defined cell position, allowing accurate definition of stem cell specific events. Clonal regeneration assays for the small intestine allow stem cell survival and functional competence to be studied. The ultimate lineage ancestor stem cells are extremely efficiently protected from genetic damage, which accounts for the low cancer incidence in this tissue. Some of the regulatory networks governing stem and transit cell behaviour are beginning to be understood and it is postulated that p53 plays a crucial role in these processes.     

Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord

Despite great insight into the molecular mechanisms that specify neuronal cell type in the spinal cord, cell behaviour underlying neuron production in this tissue is largely unknown. In other neuroepithelia, divisions with a perpendicular cleavage plane at the apical surface generate symmetrical cell fates, whereas a parallel cleavage plane generates asymmetric daughters, a neuron and a progenitor in a stem cell mode, and has been linked to the acquisition of neuron-generating ability. Using a novel long-term imaging assay, we have monitored single cells in chick spinal cord as they transit mitosis and daughter cells become neurons or divide again. We reveal new morphologies accompanying neuron birth and show that neurons are generated concurrently by asymmetric and terminal symmetric divisions. Strikingly, divisions that generate two progenitors or a progenitor and a neuron both exhibit a wide range of cleavage plane orientations and only divisions that produce two neurons have an exclusively perpendicular orientation. Neuron-generating progenitors are also distinguished by lengthening cell cycle times, a finding supported by cell cycle acceleration on exposure to fibroblast growth factor (FGF), an inhibitor of neuronal differentiation. This study provides a novel, dynamic view of spinal cord neurogenesis and supports a model in which cleavage plane orientation/mitotic spindle position does not assign neuron-generating ability, but functions subsequent to this step to distinguish stem cell and terminal modes of neuron production.     

Cell cycles in cell hierarchies

In the replacing tissues of the body, namely the bone marrow, testis, and the surface epithelia with their appendages, cell replacement would appear to be achieved using an hierarchically organized proliferative compartment with relatively few ultimate stem cells producing dividing transit cells which eventually differentiate and mature into the functional cells of the tissue. The cell cycle times of the various constituents of the hierarchy differ, and the stem cells apparently have a longer cell cycle than the transit cells. There may be variations in the cell cycle as cells pass through the transit population in some cases, e.g. in the bone marrow, while in others the cycle time remains fairly constant, e.g. in the testis. The difference in the cell cycle time between stem cells and transit cells is not completely unequivocal, and there is little or no difference in cycle time in the epithelium on the dorsal surface of the tongue while in other cases the experimental evidence for long stem-cell cycles is somewhat imprecise. However, the epithelium in the small intestine and the spermatogonia in the testis have been fairly extensively studied and here the evidence clearly shows a lengthening of the cell cycle as more primitive cells are considered.     

Gene expression heterogeneities in embryonic stem cell populations: origin and function

Stem and progenitor cells are populations of cells that retain the capacity to populate specific lineages and to transit this capacity through cell division. However, attempts to define markers for stem cells have met with limited success. Here we consider whether this limited success reflects an intrinsic requirement for heterogeneity with stem cell populations. We focus on Embryonic Stem (ES) cells, in vitro derived cell lines from the early embryo that are considered both pluripotent (able to generate all the lineages of the future embryo) and indefinitely self renewing. We examine the relevance of recently reported heterogeneities in ES cells and whether these heterogeneities themselves are inherent requirements of functional potency and self renewal.     

The disparity between human cell senescence in vitro and lifelong replication in vivo

Cultured human fibroblasts undergo senescence (a loss of replicative capacity) after a uniform, fixed number of approximately 50 population doublings, commonly termed the Hayflick limit. It has been long known from clonal and other quantitative studies, however, that cells decline in replicative capacity from the time of explantation and do so in a stochastic manner, with a half-life of only approximately 8 doublings. The apparent 50-cell doubling limit reflects the expansive propagation of the last surviving clone. The relevance of either figure to survival of cells in the body is questionable, given that stem cells in some renewing tissues undergo >1,000 divisions in a lifetime with no morphological sign of senescence. Oddly enough, these observations have had little if any effect on general acceptance of the Hayflick limit in its original form. The absence of telomerase in cultured human cells and the shortening of telomeres at each population doubling have suggested that telomere length acts as a mitotic clock that accounts for their limited lifespan. This concept assumed an iconic character with the report that ectopic expression of telomerase by a vector greatly extended the lifespan of human cells. That something similar might occur in vivo seemed consistent with initial reports that most human somatic tissues lack telomerase activity. More careful study, however, has revealed telomerase activity in stem cells and some dividing transit cells of many renewing tissues and even in dividing myocytes of repairing cardiac muscle. It now seems likely that telomerase is active in vivo where and when it is needed to maintain tissue integrity. Caution is recommended in applying telomerase inhibition to kill telomerase-expressing cancer cells, because it would probably damage stem cells in essential organs and even increase the likelihood of secondary cancers. The risk may be especially high in sun-exposed skin, where there are usually thousands of p53-mutant clones of keratinocytes predisposed to cancer.     

Problems of somatic mutation and cancer

Somatic mutation plays a key role in transforming normal cells into cancerous cells. The analysis of cancer progression therefore requires the study of how point mutations and chromosomal mutations accumulate in cellular lineages. The spread of somatic mutations depends on the mutation rate, the number of cell divisions in the history of a cellular lineage, and the nature of competition between different cellular lineages. We consider how various aspects of tissue architecture and cellular competition affect the pace of mutation accumulation. We also discuss the rise and fall of somatic mutation rates during cancer progression.     

Epidermal stem cells: interactions in developmental environments

Homeostasis of continuously renewing adult tissues, such as the epidermis of the skin, is maintained by epidermal stem cells (EpiSC), which are a small population of undifferentiated, self-renewing basal keratinocyte cells that produce daughter transit amplifying (TA) cells to make up the majority of the proliferative basal cell population in the epidermis. We have isolated EpiSC from neonatal and adult skin, and shown that these cells can regenerate an epidermis that lasts long term in vitro and in vivo, and that permanently expresses a recombinant gene in the regenerated tissue (Bickenbach and Dunnwald, 2000; Dunnwald et al., 2001). When we injected murine EpiSC into the developing blastocyst environment of the mouse, we found that both neonatal and adult EpiSC retained some ability to participate in the formation of tissues from all three germ layers (Liang and Bickenbach, 2002; Bickenbach and Chinnathambi, 2004; Liang et al., 2004). Although it appears evident that EpiSC act as pluripotent stem cells, how this reprogramming takes place is not understood. EpiSC might directly transdifferentiate into other cell types or they might first dedifferentiate into a more primitive cell type, and then proceed to develop along a cell lineage pathway. To begin to unravel this, we co-cultured EpiSC with embryonic stem (ES) cells, and found that EpiSC could alter their cell lineage protein expression to that of a more primitive cell type. We also placed EpiSC in a wounded environment and found that EpiSC interacted with the mesenchymal cells repopulating the wound bed. Our findings indicate that the population of cells that we isolate as EpiSC has a pluripotent capability. This has led us to postulate a paradigm shift for somatic stem cells. We propose that tissues maintain a sequestered population of uncommitted stem cells that retain a regenerative response which is enhanced when the cells are exposed to developmental or stress influences.     

An investigation of the specification of unequal cleavages in leech embryos.

Leech embryos develop via stereotyped cell divisions, many of which are unequal. The first division generates identifiable cells, blastomeres AB and CD, which normally follow distinct developmental pathways. When these two cells are dissociated and cultured in isolation, their fates remain distinct and are reminiscent of normal development, but their typical cleavage patterns are disrupted; cell AB undergoes relatively few cell divisions, giving rise to a variable number of macromeres and micromeres, while cell CD cleaves many times, usually forming a poorly organized set of macromeres, embryonic stem cells (teloblasts), and micromeres. We have investigated the hypothesis that the abnormal cleavage pattern of isolated CD blastomeres is due to removal of mechanical constraints normally imposed by cell AB. We find that when cell CD is constrained in vitro to mimic its in vivo shape, it cleaves more normally.     

Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event we term 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.     

Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt

We consider some of the problems involved in current discussions on stem cells in adult mammalian tissues. The present concepts involve a number of pitfalls, weaknesses and logical, semantic and classification problems. This indicates the necessity for new and well-defined concepts that are amenable to experimental analysis. One of the major difficulties in considering stem cells is that they are defined in terms of their functional capabilities which can only be assessed by testing the abilities of the cells, which itself may alter their characteristics during the assay procedure: a situation similar to the uncertainty principle in physics. The terms that describe stem cell functions are often not well defined and are used loosely, which can lead to confusion. If such context-dependent interactions exist between the manipulation and measurement process and the challenged stem cells, the question of, for example, the number of stem cells, in a tissue has to be posed in a new way. Rather than obtaining a single number one might end up with various different numbers under different circumstances, all being complementary. This might suggest that stemness is not a property but a spectrum of capabilities from which to choose. This concept might facilitate a reconciliation between the different and sometimes opposing experimental results. Given certain experimental evidence, we have attempted to provide a novel concept to describe structured cell populations in tissues involving stem cells, transit cells and mature cells. It is based on the primary assumption that the proliferation and differentiation/maturation processes are in principle independent entities in the sense that each may proceed without necessarily affecting the other. Stem cells may divide without maturation while cells approaching functional competence may mature but do not divide. In contrast, transit cells divide and mature showing intermediate properties between stem cells and mature functional cells. The need to describe this transition process and the variable coupling between proliferation and maturation leads us to formulate a spiral model of cell and tissue organisation. This concept is illustrated for the intestinal epithelium. It is concluded that the small intestinal crypts contain 4-16 actual stem cells in steady state but up to 30-40 potential stem cells (clonogenic cells) which may take over stem cell properties following perturbations. This implies that transit cells can under certain circumstances behave like actual stem cells while they undergo maturation under other conditions. There is also evidence that the proliferation and differentiation/maturation processes are subject to controls that ultimately lead to a change in the spiral trajectories.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stochastic elimination of cancer cells

Tissues of multicellular organisms consist of stem cells and differentiated cells. Stem cells divide to produce new stem cells or differentiated cells. Differentiated cells divide to produce new differentiated cells. We show that such a tissue design can reduce the rate of fixation of mutations that increase the net proliferation rate of cells. It has, however, no consequence for the rate of fixation of neutral mutations. We calculate the optimum relative abundance of stem cells that minimizes the rate of generating cancer cells. There is a critical fraction of stem cell divisions that is required for a stochastic elimination ('wash out') of cancer cells.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Molecular mechanisms of asymmetric divisions in mammary stem cells

Stem cells have the remarkable ability to undergo proliferative symmetric divisions and self‐renewing asymmetric divisions. Balancing of the two modes of division sustains tissue morphogenesis and homeostasis. Asymmetric divisions of Drosophila neuroblasts (NBs) and sensory organ precursor (SOP) cells served as prototypes to learn what we consider now principles of asymmetric mitoses. They also provide initial evidence supporting the notion that aberrant symmetric divisions of stem cells could correlate with malignancy. However, transferring the molecular knowledge of circuits underlying asymmetry from flies to mammals has proven more challenging than expected. Several experimental approaches have been used to define asymmetry in mammalian systems, based on daughter cell fate, unequal partitioning of determinants and niche contacts, or proliferative potential. In this review, we aim to provide a critical evaluation of the assays used to establish the stem cell mode of division, with a particular focus on the mammary gland system. In this context, we will discuss the genetic alterations that impinge on the modality of stem cell division and their role in breast cancer development.     

Dynamic Interplay of Spectrosome and Centrosome Organelles in Asymmetric Stem Cell Divisions

Stem cells have remarkable self-renewal ability and differentiation potency, which are critical for tissue repair and tissue homeostasis. Recently it has been found, in many systems (e.g. gut, neurons, and hematopoietic stem cells), that the self-renewal and differentiation balance is maintained when the stem cells divide asymmetrically. Drosophila male germline stem cells (GSCs), one of the best characterized model systems with well-defined stem cell niches, were reported to divide asymmetrically, where centrosome plays an important role. Utilizing time-lapse live cell imaging, customized tracking, and image processing programs, we found that most acentrosomal GSCs have the spectrosomes reposition from the basal end (wild type) to the apical end close to hub-GSC interface (acentrosomal GSCs). In addition, these apically positioned spectrosomes were mostly stationary while the basally positioned spectrosomes were mobile. For acentrosomal GSCs, their mitotic spindles were still highly oriented and divided asymmetrically with longer mitosis duration, resulting in asymmetric divisions. Moreover, when the spectrosome was knocked out, the centrosomes velocity decreased and centrosomes located closer to hub-GSC interface. We propose that in male GSCs, the spectrosome recruited to the apical end plays a complimentary role in ensuring proper spindle orientation when centrosome function is compromised.     

Isolation and Genetic Characterization of Cell-Lineage Mutants of the Nematode CAENORHABDITIS ELEGANS

Twenty-four mutants that alter the normally invariant post-embryonic cell lineages of the nematode Caenorhabditis elegans have been isolated and genetically characterized. In some of these mutants, cell divisions fail that occur in wild-type animals; in other mutants, cells divide that do not normally do so. The mutants differ in the specificities of their defects, so that it is possible to identify mutations that affect some cell lineages but not others. These mutants define 14 complementation groups, which have been mapped. The abnormal phenotype of most of the cell-lineage mutants results from a single recessive mutation; however, the excessive cell divisions characteristic of one strain, CB1322, require the presence of two unlinked recessive mutations. All 24 cell-lineage mutants display incomplete penetrance and/or variable expressivity. Three of the mutants are suppressed by pleiotropic suppressors believed to be specific for null alleles, suggesting that their phenotypes result from the complete absence of gene activity.     

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.     

Branching stochastic processes with immigration in analysis of renewing cell populations

This paper considers the utility of a new class of stochastic branching processes with non-homogeneous immigration in modeling complex renewing cell systems. Such systems typically include the population of stem cells that provides an inexhaustible supply of cells necessary for maintaining the cellular composition of a tissue. A stem cell may be induced to transform (differentiate) into a progenitor cell. Progenitor cells retain the ability to proliferate and their function is believed to provide a quick proliferative response to an increased demand for cells in the population. There may be several sub-types of progenitor cells. Terminally differentiated cells do not divide under normal conditions; they are responsible for maintaining tissue-specific functions. Recent advancements in experimental techniques offer considerable scope for quantitative studies of in vivo cell kinetics based on stochastic modeling of renewing cell populations. However, no ready-made theory is currently available to take full advantage of these advancements. This paper introduces such a theory with a special focus on its feasibility in biological applications.