Small Groups Of Cells Research Articles

Glomus tumours in the skin and subcutis of three horses

Three horses presented with variably painful, nonulcerated masses of the head or neck that were diagnosed as glomus tumours. Grossly, they were fleshy, pink to tan masses ranging from 0.4 to 9 cm in diameter, involving either the deep dermis and subcutis or the subcutis and underlying skeletal muscle. Microscopically, neoplastic epithelioid cells were arranged in sheets, cords and packets within lobules. The neoplastic cells frequently abutted and formed nodular bulges into large endothelium-lined vascular spaces, especially around the tumour periphery. Large nerve branches were associated with each tumour. As determined by immunohistochemistry, the neoplastic cells consistently expressed α-smooth muscle actin and vimentin, and some cells in two of the cases expressed desmin. A laminin- or collagen IV-positive basement membrane was demonstrated around individual tumour cells or small groups of cells in all three cases. Morphological features and immunohistochemistry supported the diagnosis of glomus tumour, most consistent with the solid type in humans. Applying a classification system used in humans, two of these tumours met criteria of malignancy (glomangiosarcomas). One horse was euthanized due to complications associated with recurrence and treatment-related necrosis and secondary infection.

A rare case of malignant pediatric ectomesenchymoma arising from the falx cerebri

Malignant ectomesenchymoma is a rare tumor arising from mature ganglion cells with immature myogenous elements, with only 4 pediatric intracranial cases having been previously reported. The authors report a rare case of intracranial malignant ectomesenchymoma originating from the falx cerebri in a 10-year-old boy. The patient presented with a 2-week history of headache, nausea, and blurry vision, with mild lateral gaze diplopia. A CT scan revealed a solitary 7.2 × 3.8-cm dural-based mass that extended along the falx. No metastatic disease was identified, and the lesion was grossly resected without complication. Pathological investigation identified single and small groups of cells in a myxoid background, with polygonal or spindle-shaped cells containing eccentric nuclei and prominent nucleoli. Immunohistochemical staining of some cells was positive for smooth-muscle actin, CD99, and vimentin, whereas other cells (often process forming) were positive for S100 protein, synaptophysin, and neurofilament protein. Staining was negative for CD138, CD45, α-fetoprotein, CK AE1/3, glial fibrillary acidic protein, CK7, CK20, CD31, CD34, myoD, and desmin. Normal immunopositivity was seen for INI-1. The Ki 67 immunostaining had < 25% reactivity. The patient was treated with a sarcoma-based chemotherapy regimen and radiation to the craniospinal axis, and was found to be without recurrence or metastatic disease at 20 months.

Oldest fossil basidiomycete clamp connections

A rachis of the fossil filicalean fern Botryopteris antiqua containing abundant septate hyphae with clamp connections is preserved in a late Visean (Mississippian; ~330 Ma) chert from Esnost (Autun Basin) in central France. Largely unbranched tubular hyphae pass from cell to cell, but may sometimes produce a branch from a clamp connection. Other clamp-bearing hyphae occur clustered in individual cells or small groups of adjacent host cells. These hyphae may be tubular, catenulate with numerous hyphal swellings, or they may display a combination of both. The Visean hyphae with clamp connections predate Palaeancistrus martinii, the heretofore oldest direct fossil evidence of Basidiomycota, by some 25 Ma.

Oldest fossil basidiomycete clamp connections

A rachis of the fossil filicalean fern Botryopteris antiqua containing abundant septate hyphae with clamp connections is preserved in a late Visean (Mississippian; ~330Ma) chert from Esnost (Autun Basin) in central France. Largely unbranched tubular hyphae pass from cell to cell, but may sometimes produce a branch from a clamp connection. Other clamp-bearing hyphae occur clustered in individual cells or small groups of adjacent host cells. These hyphae may be tubular, catenulate with numerous hyphal swellings, or they may display a combination of both. The Visean hyphae with clamp connections predate Palaeancistrus martinii, the heretofore oldest direct fossil evidence of Basidiomycota, by some 25Ma.

Neuronal cell death during metamorphosis of Hydractina echinata (Cnidaria, Hydrozoa)

In planula larvae of the invertebrate Hydractinia echinata (Cnidaria, Hydrozoa), peptides of the GLWamide and the RFamide families are expressed in distinct subpopulations of neurons, distributed in a typical spatial pattern through the larval body. However, in the adult polyp GLWamide or RFamide-expressing cells are located at body parts that do not correspond to the prior larval regions. Since we had shown previously that during metamorphosis a large number of cells are removed by programmed cell death (PCD), we aimed to analyze whether cells of the neuropeptide-expressing larval nerve net are among those sacrificed. By immunohistochemical staining and in situ hybridization, we labeled GLWamide- and RFamide-expressing cells. Double staining of neuropeptides and degraded DNA (TUNEL analysis) identified some neurosensory cells as being apoptotic. Derangement of the cytoplasm and rapid destruction of neuropeptide precursor RNA indicated complete death of these particular sensory cells in the course of metamorphosis. Additionally, a small group of RFamide-positive sensory cells in the developing mouth region of the primary polyp could be shown to emerge by proliferation. Our results support the idea that during metamorphosis, specific parts of the larval neuronal network are subject to neurodegeneration and therefore not used for construction of the adult nerve net. Most neuronal cells of the primary polyp arise by de novo differentiation of stem cells commited to neural differentiation in embryogenesis. At least some nerve cells derive from proliferation of progenitor cells. Clarification of how the nerve net of these basal eumetazoans degenerates may add information to the understanding of neurodegeneration by apoptosis as a whole in the animal kingdom.

Establishment of the embryonic shoot apical meristem in Arabidopsis thaliana

In higher plants, shoot organs such as leaves, branches, and flowers are generated from the shoot apical meristem (SAM), a small group of undifferentiated cells located at the tip of the shoot. The SAM maintains its pluripotency and simultaneously produces lateral organs at its periphery. The SAM arises during embryogenesis and its positioning requires axis-dependent embryo patterning and compartmentalization of the embryo apex. Here, we introduce major factors involved in these processes in Arabidopsis thaliana and discuss how the embryonic SAM is established.

Quantitative In vivo Imaging of the Effects of Inhibiting Integrin Signaling via Src and FAK on Cancer Cell Movement: Effects on E-cadherin Dynamics

Most cancer-related deaths are due to the development of metastatic disease, and several new molecularly targeted agents in clinical development have the potential to prevent disease progression. However, it remains difficult to assess the efficacy of antimetastatic agents in the clinical setting, and an increased understanding of how such agents work at different stages of the metastatic cascade is important in guiding their clinical use. We used optical window chambers combined with photobleaching, photoactivation, and photoswitching to quantitatively measure (a) tumor cell movement and proliferation by tracking small groups of cells in the context of the whole tumor, and (b) E-cadherin molecular dynamics in vivo following perturbation of integrin signaling by inhibiting focal adhesion kinase (FAK) and Src. We show that inhibition of Src and FAK suppresses E-cadherin-dependent collective cell movement in a complex three-dimensional tumor environment, and modulates cell-cell adhesion strength and endocytosis in vitro. This shows a novel role for integrin signaling in the regulation of E-cadherin internalization, which is linked to regulation of collective cancer cell movement. This work highlights the power of fluorescent, direct, in vivo imaging approaches in the preclinical evaluation of chemotherapeutic agents, and shows that inhibition of the Src/FAK signaling axis may provide a strategy to prevent tumor cell spread by deregulating E-cadherin-mediated cell-cell adhesions.

Emergence and patterning of the five cell types of the Zea mays anther locule

One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. In flowering plants, the sexual organs (stamens and carpels) are composed almost entirely of somatic cells, a small subset of which switch to meiosis; however, the mechanism of meiotic cell fate acquisition is a long-standing botanical mystery. In the maize ( Zea mays) anther microsporangium, the somatic tissues consist of four concentric cell layers that surround and support reproductive cells as they progress through meiosis and pollen maturation. Male sterility, defined as the absence of viable pollen, is a common phenotype in flowering plants, and many male sterile mutants have defects in somatic and reproductive cell fate acquisition. However, without a robust model of anther cell fate acquisition based on careful observation of wild-type anther ontogeny, interpretation of cell fate mutants is limited. To address this, the pattern of cell proliferation, expansion, and differentiation was tracked in three dimensions over 30 days of wild-type (W23) anther development, using anthers stained with propidium iodide (PI) and/or 5-ethynyl-2′-deoxyuridine (EdU) (S-phase label) and imaged by confocal microscopy. The pervading lineage model of anther development claims that new cell layers are generated by coordinated, oriented cell divisions in transient precursor cell types. In reconstructing anther cell division patterns, however, we can only confirm this for the origin of the middle layer (ml) and tapetum, while young anther development appears more complex. We find that each anther cell type undergoes a burst of cell division after specification with a characteristic pattern of both cell expansion and division. Comparisons between two inbreds lines and between ab- and adaxial anther florets indicated near identity: anther development is highly canalized and synchronized. Three classical models of plant organ development are tested and ruled out; however, local clustering of developmental events was identified for several processes, including the first evidence for a direct relationship between the development of ml and tapetal cells. We speculate that small groups of ml and tapetum cells function as a developmental unit dedicated to the development of a single pollen grain.

Patterns of Low-grade Serous Carcinoma With Emphasis on the Nonepithelial-lined Spaces Pattern of Invasion and the Disorganized Orphan Papillae

Low-grade serous carcinoma is a relatively recent recognized entity. The cytologic features of this type of carcinoma have been described in detail; however, the different histologic patterns of this carcinoma have not been discussed yet. We reviewed the hematoxylin and eosin-stained slides and studied the different patterns of invasion and the histologic features of areas that were not obviously invasive of 40 cases of ovarian pure low-grade serous carcinoma. Destructive areas of invasion were present in all cases; however, in 25 of the 40 cases obvious invasive areas were mixed with foci that were difficult to recognize as invasive. The obvious areas of invasion were characterized by the presence of tumor cells within nonepithelial-lined spaces, which are clefts in the stroma surrounding the tumor cells. The different patterns of the tumor cells within the nonepithelial-lined spaces and the frequency that they are found were: groups or nests (nidi pattern) in 100% of the cases, micropapillae in 70%, these 2 patterns frequently were mixed, macropapillae in 25%, large solid groups in 5%, and single cells in 3%. The 2 patterns difficult to recognize as invasive because of the absence of nonepithelial-lined spaces were disorganized orphan papillae within epithelial-lined spaces present in 40% of the cases, and small irregular glands with a haphazard distribution infiltrating the tissue eliciting desmoplastic stromal reaction present in 22% of the cases. Calcifications were present in all cases, and mucin in 70% of the cases. Areas of stromal invasion characterized by tumor cells within nonepithelial-lined spaces or clefts are seen in all low-grade carcinomas. Within these spaces, small groups of tumor cells are found frequently mixed with micropapillae. In 62% of the cases, in addition to obvious areas of invasion, there were 2 patterns difficult to recognize as invasive, disorganized orphan papillae, and irregular well-developed glands.

Retinal Ganglion Cell Layer of the Caspian Seal Pusa caspica: Topography and Localization of the High-Resolution Area

Retinal topography, cell density and sizes of ganglion cells in the Caspian seal (Pusa caspica) were analyzed in retinal whole mounts stained with cresyl-violet. The topographic distribution of ganglion cells displayed an area of high cell density located in the temporal quadrant of the retina and was similar to the area centralis of terrestrial carnivores. It extended nasally, above the optic disk, as a streak of increased cell density. In different whole mounts, the peak cell density in the high-density area ranged from 1,684 to 1,844 cells/mm² (mean 1,773 cells/mm²). The cell density data predict a retinal resolution of around 8.5 cycles/degree in water. A distinctive feature of the Caspian seal’s retina is the large size of ganglion cells and the low cell density compared to terrestrial mammals. The ganglion cell diameter ranged from 10 to 58 µm. Cell size histograms featured bimodal patterns with groups of small and large ganglion cells. The large cells appeared similar to α-cells of terrestrial mammals and constituted 7% of the total ganglion cell population.

631 Mislocalization of the apoptosome protein Apaf-1 is a strong marker of drug resistance in B cell lymphomas

We report 4 cases of pseudomesotheliomatous carcinoma of the lung, which has clinical and microscopic features similar to malignant mesothelioma, but with ultrastructural, immunohistochemical, and molecular characteristics suggestive of a histogenesis from type II pneumocytes. Neoplasm grows as a diffuse or solid pattern of large polygonal cells with sharply defined borders. Hale's colloidal iron is positive in the cytoplasm of small groups of cells and, focally, in some intercellular spaces. Ultrastructure showed short microvilli in the surface. Immunohistochemically, tumor cells were positive for thyroid transcription factor–1, podoplanin, mesothelin, pan-cytokeratin, CK-7, CK-19, Ber-EP4, epithelial membrane antigen, apoprotein surfactant A, epidermal growth factor receptor, Leu-M1, carcinoembryonic antigen, E-cadherin, and CD-44 and negative for mesothelioma markers thrombomodulin and calretinin. In some areas, there were small cysts which contained a concentric fibrilar basophilic material apoprotein surfactant A positive. Chromosomal imbalances with comparative genomic hybridization technique were identified with a median of 15 abnormalities per case (range, 1-26): 51 gains, 6 losses, and 1 high-level amplification. The most frequent aberrations among the cases were gains on chromosomes regions 1q, 3q, 5p, 8q, 16p, and 18q and losses in 17p11-13 and 17q 22-q25. High-level amplifications were detected on 7p13-p21. In all cases, there was a characteristic association between the gains on 16p and those on 18q. The 4 cases resulted in death in less than 14 months, in spite of complete surgery and chemotherapy in 2 cases. Our aim is to complement the current understanding of this pseudomesotheliomatous “pneumocytic” carcinoma and alert pathologists to this rare entity to avoid misdiagnosis.

289 3D culture systems for cancer drug evaluation

We report 4 cases of pseudomesotheliomatous carcinoma of the lung, which has clinical and microscopic features similar to malignant mesothelioma, but with ultrastructural, immunohistochemical, and molecular characteristics suggestive of a histogenesis from type II pneumocytes. Neoplasm grows as a diffuse or solid pattern of large polygonal cells with sharply defined borders. Hale's colloidal iron is positive in the cytoplasm of small groups of cells and, focally, in some intercellular spaces. Ultrastructure showed short microvilli in the surface. Immunohistochemically, tumor cells were positive for thyroid transcription factor–1, podoplanin, mesothelin, pan-cytokeratin, CK-7, CK-19, Ber-EP4, epithelial membrane antigen, apoprotein surfactant A, epidermal growth factor receptor, Leu-M1, carcinoembryonic antigen, E-cadherin, and CD-44 and negative for mesothelioma markers thrombomodulin and calretinin. In some areas, there were small cysts which contained a concentric fibrilar basophilic material apoprotein surfactant A positive. Chromosomal imbalances with comparative genomic hybridization technique were identified with a median of 15 abnormalities per case (range, 1-26): 51 gains, 6 losses, and 1 high-level amplification. The most frequent aberrations among the cases were gains on chromosomes regions 1q, 3q, 5p, 8q, 16p, and 18q and losses in 17p11-13 and 17q 22-q25. High-level amplifications were detected on 7p13-p21. In all cases, there was a characteristic association between the gains on 16p and those on 18q. The 4 cases resulted in death in less than 14 months, in spite of complete surgery and chemotherapy in 2 cases. Our aim is to complement the current understanding of this pseudomesotheliomatous “pneumocytic” carcinoma and alert pathologists to this rare entity to avoid misdiagnosis.

Cancer stem cell, niche and EGFR decide tumor development and treatment response: A bio-computational simulation study

Recent research in cancer biology has suggested the hypothesis that tumors are initiated and driven by a small group of cancer stem cells (CSCs). Furthermore, cancer stem cell niches have been found to be essential in determining fates of CSCs, and several signaling pathways have been proven to play a crucial role in cellular behavior, which could be two important factors in cancer development. To better understand the progression, heterogeneity and treatment response of breast cancer, especially in the context of CSCs, we propose a mathematical model based on the cell compartment method. In this model, three compartments of cellular subpopulations are constructed: CSCs, progenitor cells (PCs), and terminal differentiated cells (TCs). Moreover, (1) the cancer stem cell niche is, considered by modeling its effect on division patterns (symmetric or asymmetric) of CSCs, and (2) the EGFR signaling pathway is integrated by modeling its role in cell proliferation, apoptosis. Our simulation results indicate that (1) a higher probability for symmetric division of CSC may result in a faster expansion of tumor population, and for a larger number of niches, the tumor grows at a slower rate, but the final tumor volume is larger; (2) higher EGFR expression correlates to tumors with larger volumes while a saturation function is observed, and (3) treatments that inhibit tyrosine kinase activity of EGFR may not only repress the tumor volume, but also decrease the CSCs percentages by shifting CSCs from symmetric divisions to asymmetric divisions. These findings suggest that therapies should be designed to effectively control or eliminate the symmetric division of CSCs and to reduce or destroy the CSC niches.

Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm-Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.

The tensor-based model for growth and cell divisions of the root apex. II. Lateral root formation

In this work, the formation of the virtual lateral root (VLR) is shown. The VLR is formed using the 2D simulation model of growth and cell divisions based on the concept of growth tensor, specified for radish. Growth is generated by the field of growth rates of an unsteady type (GT field). Principal directions of growth (PDGs) are assumed to define the orientation of cell divisions. Temporal sequences of the VLR formation are a result of an application of the GT field to the polygon meshwork representing cell pattern of already initiated primordium. The computer-generated lateral root (LR) develops realistically, and its cell pattern is vivid and similar to that observed in anatomical sections. The real and virtual LRs show similar cellular organization, both originate from a small group of cells situated in two-cell layers of the pericycle and both layers are engaged in the LR development. The LR formation seems to be controlled at the tensor level and individual cells presumably detect PDGs and obey them in the course of the cell divisions. PDGs are postulated to affect the cellular organization of the LR. Using the method of computer simulations, cellular aspects of the LR morphogenesis are discussed.

Velocity and pattern of ice propagation and deep supercooling in woody stems of Castanea sativa, Morus nigra and Quercus robur measured by IDTA

Infrared differential thermal analysis (IDTA) was used to monitor the velocity and pattern of ice propagation and deep supercooling of xylem parenchyma cells (XPCs) during freezing of stems of Castanea sativa L., Morus nigra L. and Quercus robur L. that exhibit a macro- and ring-porous xylem. Measurements were conducted on the surface of cross- and longitudinal stem sections. During high-temperature freezing exotherms (HTEs; -2.8 to -9.4°C), initial freezing was mainly observed in the youngest year ring of the sapwood (94%), but occasionally elsewhere (older year rings: 4%; bark: 2%). Initially, ice propagated rapidly in the largest xylem conduits. This resulted in a distinct freezing pattern of concentric circles in C. sativa and M. nigra. During HTEs, supercooling of XPCs became visible in Q. robur stems, but not in the other species that have narrower pith rays. Intracellular freezing of supercooled XPCs of Q. robur became visible by IDTA during low-temperature freezing exotherms (<-17.4 °C). Infrared differential thermal analysis revealed the progress and the two-dimensional pattern of XPC freezing. XPCs did not freeze at once, but rather small cell groups appeared to freeze at random anywhere in the xylem. By IDTA, ice propagation and deep supercooling in stems can be monitored at meaningful spatial and temporal resolutions.

Electrophysiological and Chemical Properties in Subclassified Acutely Dissociated Cells of Rat Trigeminal Ganglion by Current Signatures

In the present study, we subclassified acutely dissociated trigeminal ganglion (TRG) cells of rats using a current signature method in whole cell patch-clamp recordings. Using modified criteria for cell classification for the dorsal root ganglion (DRG), TRG cells were subclassified into nine cell types: 1-5, 7-9, and 13. Types 1, 3, and 7 were in the small cell groups (15-24 μm); types 4, 5, and 8-13 were in the medium cell groups (25-38 μm); and type 2 was a mixed group of both cell sizes. Types 1-3, 5, and 7 showed high-input resistance and types 1, 2, and 7 showed more depolarized resting membrane potentials. Types 1, 2, and 5-13 expressed long-duration action potentials (APs), but types 3 and 4 expressed short-duration APs. Sensitivities to capsaicin, protons, and adenosine 5'-triphosphate (ATP) in TRG cell types largely corresponded to DRG cell types. However, different from the matched DRG types, half of TRG type 1 cells were capsaicin insensitive, showing desensitizing proton-induced currents, and types 5, 7, and 9 exhibited slow-desensitizing ATP-induced currents. Types 4, 5, and 8-13 had nicotine sensitivity, but the other cell types were insensitive. These results indicate that the "current signatures" classification is a useful means to separate TRG cells into internally homogeneous subpopulations that were distinct from other cell types. Furthermore, the data suggest some specific differences in the chemical responsiveness of some cell types between the TRG and DRG.

Doublesex/mab3 related-1 (dmrt1) is essential for development of anterior neural plate derivatives in Ciona.

Ascidian larvae have a hollow, dorsal central nervous system that shares many morphological features with vertebrate nervous systems yet is composed of very few cells. We show here that a null mutation in the gene dmrt1 in the ascidian Ciona savignyi results in profound abnormalities in the development of the sensory vesicle (brain), as well as other anterior ectodermal derivatives, including the palps and oral siphon primordium (OSP). Although the phenotype of the mutant embryos is variable, the majority have a complete loss of the most anterior structures (palps and OSP) and extensive disruption of sensory structures, such as the light-sensitive ocellus, in the sensory vesicle. dmrt1 is expressed early in the blastula embryo in a small group of presumptive ectodermal cells as they become restricted to anterior neural, OSP and palp fates. Despite the early and restricted expression of dmrt1, we were unable, using several independent criteria, to observe a defect in the mutant embryos until the early tailbud stage. We speculate that the variability and late onset in the phenotype may be due to partially overlapping activities of other gene products.

Morphometric analysis of the neuronal numbers and densities of the inferior olivary complex in the donkey ( Equus asinus)

The morphometric interrelations between the compartments of the inferior olivary complex (IOC) in the donkey ( Equus asinus) were ascertained by examining serial sections throughout the entire length of the IOC for both sides. Nissl-stained celloidin sections of four brainstems of donkeys were used. The IOC consisted of three major nuclei and four small cell groups. The total neuronal count in both sides of the IOC was 202,040 ± 8480 cells. The medial accessory olivary nucleus (MAO) had the largest relative area (46%) and the highest number of neurons (90,800 ± 7600). The dorsal accessory olivary nucleus (DAO) had the second largest relative area (33%), while the principal olivary nucleus (PO) had the lowest relative area (21%). However, the total neuron count in the PO was larger (60,840 ± 1840) than DAO (50,360 ± 4040). The average neuronal density was 2700 ± 400 cells/mm 3. The numerical values of the current study of the IOC in the donkey were similar to those of other mammals.

Abstract 5152: Cancer stem cells arise directly from differentiated cancer cells: A new model of dynamic equilibrium modulated by the Akt pathway

Abstract Introduction: Cancer stem cells (CSCs) are undifferentiated, highly tumorigenic, and therapy-resistant tumor cells which play a central role in cancer formation and progression. To date, CSCs have been widely conceptualized as a small population of self-renewing founder cells from which all other cancer cells arise, an intuitive but unproven model. Here we propose a novel alternative model based on our discovery of a dynamic two-way equilibrium: CSCs indeed generate differentiated cancer cells, but the differentiated cancer cells in turn de-differentiate and replenish the CSC population. Methods: Subpopulations of CSCs and differentiated cancer cells within the J82 human bladder cancer cell line were tracked over serial time points using FACS with Hoechst dye staining, a method that identified a small side population (SP) of Hoechst negative CSCs versus the majority of Hoechst positive differentiated cancer cells (non-SP). To rule out artifactual cross-contamination during sorting, the Hoechst/FACS experiments were also conducted with GFP labeled SP and non-SP subpopulations. Results: We identified an SP subpopulation and confirmed its CSC phenotype relative to the non-SP subpopulation (increased progenitor gene transcripts, clonogenicity, and tumorigenicity). Surprisingly, we observed that the SP subpopulation cycled in size over time, comprising ∼1% of all cells after seeding, then rising to &amp;gt;20% of all cells as the culture neared confluence, and then falling to ∼1% again after passaging to a new plate, and so on through serial passages. Moreover, non-SP cells that were isolated by FACS and cultured independently gave rise to a new SP subpopulation over several days. To validate this phenomenon, we grew GFP+ non-SP cells combined with GFP− SP cells (80:20 ratio) in vitro and in vivo. Remarkably, both in culture and in tumor xenografts the previously GFP− SP subpopulation turned progressively GFP+ as it expanded over time, confirming that the SP subpopulation was arising from conversion of non-SP cells. This conversion was significantly reduced in culture by LY294002, an Akt pathway inhibitor. Conclusion: Our studies demonstrate for the first time that CSCs are not a small and static group of founder cells; rather, they are continuously depleted and repleted in an ongoing state of dynamic equilibrium with differentiated cancer cells. Much like microbial populations, the cancer cells do not proliferate randomly but rather interact in a highly coordinated manner that is governed by environmental factors and mediated at least in part by the Akt pathway. Specifically, under permissive conditions, a rapidly-dividing, differentiated state predominates, but in response to space and nutrient depletion the differentiated cells reconstitute a resistant, dormant CSC population which preserves and eventually regenerates the cancer when favorable conditions return. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5152.