Separate Cell Populations Research Articles

Many routes to the same destination: lessons from skeletal muscle development

The development and differentiation of vertebrate skeletal muscle provide an important paradigm to understand the inductive signals and molecular events controlling differentiation of specific cell types. Recent findings show that a core transcriptional network, initiated by the myogenic regulatory factors (MRFs; MYF5, MYOD, myogenin and MRF4), is activated by separate populations of cells in embryos in response to various signalling pathways. This review will highlight how cells from multiple distinct starting points can converge on a common set of regulators to generate skeletal muscle.

Single‐Cell Isolation and Cloning of Tetrahymena thermophila Cells with a Fluorescence‐Activated Cell Sorter

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.

Immunohistochemical Localization of Calbindin-D28k and Calretinin in the Spinal Cord of Lungfishes

A common pattern of distribution of neurons and fibers containing the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the spinal cord of terrestrial vertebrates has been recently demonstrated. Lungfishes are considered the closest living relatives of tetrapods, but practically no experimental data exist on the organization of their spinal cord. By means of immunohistochemical techniques, the localization of CB and CR was investigated in the spinal cord of the African (Protopterus dolloi) and Australian (Neoceratodus forsteri) lungfishes. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. A large population of immunoreactive cells was found in the dorsal column of the gray matter in both species, and abundant cells were distributed in the lateral and ventral columns. Ventrolateral motoneurons and multipolar cells were only intensely CB and CR immunoreactive in Neoceratodus. For the most part, separate cell populations contained either CB or CR, but a small subset of dorsally located neurons contained both in the two lungfishes. Colocalization was found in motoneurons and in ventrolaterally located cells only in Neoceratodus. Fiber labeling showed a predominance of CR-containing axons in the lateral and ventral funiculi of presumed supraspinal origin. These results show that lung-fishes and tetrapods have many features in common, suggesting that primitive anatomical, and likely functional, organization of the spinal cord of tetrapods is present in lungfishes.

Stem cell proliferation during in vitro development of the model cestode Mesocestoides corti from larva to adult worm

BackgroundIn free-living flatworms somatic differentiated cells do not divide, and a separate population of stem cells (called neoblasts) is responsible for cell proliferation and renewal. In cestodes, there is evidence that similar mechanisms of cell renewal exist.ResultsIn this work, we have characterized proliferative cells during the development of the model cestode Mesocestoides corti from larva (tetrathyridium) to young segmented worm. This was done by two complementary strategies with congruent results: characterizing cells in S phase and their progeny by incorporation of 5-bromo-2'-deoxyuridine, and characterizing cells in M phase by arresting mitotic cells with colchicine and studying their morphology and distribution. Proliferative cells are localized only in the inner parenchyma, particularly in close proximity to the inner muscle layer, but not in the cortical parenchyma nor in the sub-tegumental tissue. After proliferation some of these cells migrate to the outer regions were they differentiate. In the larvae, proliferative cells are more abundant in the anterior regions (scolex and neck), and their number diminishes in an antero-posterior way. During the development of adult segments periodic accumulation of proliferative cells are observed, including a central mass of cells that constitutes the genital primordium, which grows at least in part due to in situ proliferation. In later segments, the inner cells of genital primordia cease to proliferate and adopt a compact distribution, and proliferative cells are also found in the testes primordia.ConclusionsProliferative cells have a characteristic localization and morphology throughout development from larva to adult of Mesocestoides corti, which is similar, and probably evolutionary conserved, to that described in other model cestodes. The characteristics of proliferative cells suggest that these consist of undifferentiated stem cells.

Leukemia Cell–Selective Uptake of Cytarabine and Daunorubicin in the Bone Marrow Compartment Mediated by CPX-351 Liposome Injection

Abstract Background Anticancer drug combinations such as cytarabine plus daunorubicin can act synergistically, additively or antagonistically depending on the ratio of the agents being combined. We have shown that delivering synergistic cytarabine-to-daunorubicin drug ratios in vivo using nanoscale liposomes (CPX-351 liposome injection) provides dramatic efficacy improvements compared to the free drug cocktail in a wide range of preclinical leukemia models without exacerbating myelosuppressive effects. This enhanced efficacy is associated with increased and prolonged delivery of the synergistic drug ratio to bone marrow. Promising signs of efficacy have been observed with CPX-351 in previously treated patients with AML with poor prognosis. We aim to investigate the pharmacodynamic basis for the potent therapeutic activity of CPX-351. Patients and Methods A bone marrow–engrafting CCRF-CEM human leukemia xenograft model was developed. CPX-351 was injected intravenously (I.V.), and the bone marrows of femurs from tumor bearing mice were subsequently harvested. Cells were analyzed by flow cytometry, and leukemia cells were separated from normal bone marrow cells using anti-CD45 coated magnetic nanoparticles. Cells were analyzed for intracellular cytarabine-to-daunorubicin and liposome content using 3 H-Cyt, HPLC, and 14 C-lipid, respectively. Results Twenty-eight days after I.V. tumor inoculation, femurs contained approximately equal numbers of CCRF-CEM and normal bone marrow cell populations that could be readily separated and quantitatively harvested by antibody-coated magnetic nanoparticle-mediated isolation. Eighteen hours after CPX-351 I.V. injection, bone marrows of the tumor bearing mice contained 82.6 ng cytarabine, 63.0 ng daunorubicin, and 1.2 μg liposomal lipid per total femur aspirate. Within the separated cell populations, leukemia cells contained 5.9 ng cytarabine, 8.2 ng daunorubicin, and 0.16 μg liposomal lipid per 10 6 cells. These intracellular levels were 9.5-, 2.2-, and 1.9-fold higher than those observed in the normal bone marrow cell population. Confocal microscopy on cells incubated with CPX-351 in vitro demonstrated that CPX-351 liposomes are taken up intact by human leukemia cells and subsequently release the drugs intracellularly. Conclusion CPX-351 was designed to enhance the antitumor efficacy of cytarabine-to-daunorubicin combination therapy by encapsulating both agents within a drug carrier that maintains the synergistic 5:1 molar ratio for extended times after injection. The potent anti-leukemic activity obtained in the absence of significant nonhematologic toxicity with this formulation may be due, in part, to the selective accumulation of CPX-351 into leukemia cells.

Parallel but separate inputs from limbic cortices to the mammillary bodies and anterior thalamic nuclei in the rat.

The proposal that separate populations of subicular cells provide the direct hippocampal projections to the mammillary bodies and anterior thalamic nuclei was tested by placing two different fluorescent tracers in these two sites. In spite of varying the injection locations within the mammillary bodies and within the three principal anterior thalamic nuclei and the lateral dorsal thalamic nucleus, the overall pattern of results remained consistent. Neurons projecting to the thalamus were localized to the deepest cell populations within the subiculum while neurons projecting to the mammillary bodies consisted of more superficially placed pyramidal cells within the subiculum. Even when these two cell populations become more intermingled, e.g., in parts of the intermediate subiculum, almost no individual cells were found to project to both diencephalic targets. In adjacent limbic areas, i.e., the retrosplenial cortex, postsubiculum, and entorhinal cortex, populations of cells that project to the anterior thalamic nuclei and mammillary bodies were completely segregated. This segregated pattern included afferents to those nuclei comprising the head-direction system. The sole exception was a handful of double-labeled cells, mainly confined to the ventral subiculum, that were only found after pairs of injections in the anteromedial thalamic nucleus and mammillary bodies. The projections to the anterior thalamic nuclei also had a septal-temporal gradient with relatively fewer cells projecting from the ventral (temporal) subiculum. These limbic projections to the mammillary bodies and anterior thalamus comprise a circuit that is vital for memory, within which the two major components could convey parallel, independent information.

Transepithelial Ion Transport Properties of Human Native Small Airways

Small airways are the crucial target in the pathogenesis of cystic fibrosis and other airway diseases. Characterization of transepithelial ion transport properties of small airways have been limited by their relative inaccessibility, mainly in native human tissue. We used our recently introduced micro‐Ussing chamber (area=1 mm2) to obtain the first measurements of ion transport properties of human native small airways specimens (<2 mm i.d.). Spontaneous open circuit transepithelial potentials (Vt), conductance (Gt) and equivalent short circuit current were measured. In presence of Cl− gradient Vt hyperpolarized and Gt decreased significantly. In symmetric NaCl, activating Cl− conductance with cAMP mediators (Fsk/IBMX) and purinergic agonist (UTP) evoked distinctly opposite effects on Vt, depolarizing and hyperpolarizing, respectively. Luminal amiloride depolarized Vt and decreased Gt significantly. Results suggest that human small airway epithelia express very high constitutively active Cl− conductance that support both secretory as well as absorptive functions. Small airways may consist of separate populations of secretory and absorptive cells that recycle airway surface fluids locally to maintain appropriate airway hydration and hygiene.Supported by the Nancy Olmsted Trust, NIH (R01‐HL084042) and American Lung Association California.

Continuous separation of cells of high osteoblastic differentiation potential from mesenchymal stem cells on an antibody-immobilized column

Here, we report that two distinctive cell populations with osteoblastic differentiation ability were found in adherent cell populations from bone marrow. Mesenchymal stem cells (MSCs) were conventionally isolated by using adherent property of bone marrow cells onto a plastic culture dish. MSCs enriched on the basis of their adherent property were considered phenotypically and functionally heterogeneous. We developed a ligand-immobilized surface for separating subpopulation of adherent cells derived from bone marrow by the cell rolling process. We successfully isolate two cell populations with high differentiation ability for osteoblasts in adherent bone marrow cells by using the anti-CD34 antibody-immobilized column. The antibody was covalently conjugated with polyacrylic acid and introduced onto the inner surface of a silicone tube. When cell suspension of MSCs was injected into the antibody-immobilized column, different cell populations were isolated. After the cultivation of isolated cells in the osteoblastic differentiation medium for 1 week, few sub-populations were strongly induced to form osteoblastic cells. This study revealed that the ligand-immobilized surface can be used to continually separate cell populations under a labeling-free condition.

Microfluidic platform for chemotaxis in gradients formed by CXCL12 source-sink cells.

Chemokine CXCL12 promotes CXCR4-dependent chemotaxis of cancer cells to characteristic organs and tissues, leading to metastatic disease. This study was designed to investigate how cells expressing CXCR7 regulate chemotaxis of a separate population of CXCR4 cells under physiologic conditions in which cells are exposed to gradients of CXCL12. We recapitulated a cancer-stroma microenvironment by patterning CXCR4-expressing cancer cells in microchannels at spatially defined positions relative to CXCL12-producing cells and CXCR7-expressing cells. CXCR7 scavenges and degrades CXCL12, which has been proposed to facilitate CXCR4-dependent chemotaxis through a source-sink model. Using the microchannel device, we demonstrated that chemotaxis of CXCR4 cells depended critically on the presence and location of CXCR7 cells (sink) relative to chemokine secreting cells (source). Furthermore, inhibiting CXCR4 on migrating cells or CXCR7 on sink cells blocked CXCR4-dependent chemotaxis toward CXCL12, showing that the device can identify new therapeutic agents that block migration by targeting chemoattractant scavenging receptors. Our system enables efficient chemotaxis under much shallower yet more physiological chemoattractant gradients by generating an in vitro microenvironment where combinations of cellular products may be secreted along with formation of a chemoattractant gradient. In addition to elucidating mechanisms of CXCL-12 mediated chemotaxis, this simple and robust method can be broadly useful for engineering multiple microenvironments to investigate intercellular communication.

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

IntroductionNucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.MethodsMicroarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip® Bovine Genome Arrays. Differential expression levels for a number of genes were confirmed by quantitative real time polymerase chain reaction (qRT-PCR) on bovine, AC, AF and NP cells, as well as separated bovine NP and notochordal (NC) cells. Expression of these novel markers were further tested on normal human AC, AF and NP cells, and degenerate AF and NP cells.ResultsMicroarray comparisons between NP/AC&AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed ≥100 fold. A subset of these were verified by qRT-PCR and shown to be expressed in bovine NC cells. Eleven genes (SNAP25, KRT8, KRT18, KRT19, CDH2, IBSP, VCAN, TNMD, BASP1, FOXF1 & FBLN1) were also differentially expressed in normal human NP cells, although to a lesser degree. Four genes (SNAP25, KRT8, KRT18 and CDH2) were significantly decreased in degenerate human NP cells, while three genes (VCAN, TNMD and BASP1) were significantly increased in degenerate human AF cells. The IVD negative marker FBLN1 was significantly increased in both degenerate human NP and AF cells.ConclusionsThis study has identified a number of novel genes that characterise the bovine and human NP and IVD transcriptional profiles, and allows for discrimination between AC, AF and NP cells. Furthermore, the similarity in expression profiles of the separated NP and NC cell populations suggests that these two cell types may be derived from a common lineage. Although interspecies variation, together with changes with IVD degeneration were noted, use of this gene expression signature will benefit tissue engineering studies where defining the NP phenotype is paramount.

Retinoic acid receptors exhibit cell‐autonomous functions in cranial neural crest cells

Previous work has emphasized the crucial role of retinoic acid (RA) in the ontogenesis of the vast majority of mesenchymal structures derived from the neural crest cells (NCC), which migrate through, or populate, the frontonasal process and branchial arches. Using somatic mutagenesis in the mouse, we have selectively ablated two or three retinoic acid receptors (i.e., RARalpha/RARbeta, RARalpha/RARgamma and RARalpha/RARbeta/RARgamma) in NCC. By rigorously analyzing these mutant mice, we found that survival and migration of NCC is normal until gestational day 10.5, suggesting that RAR-dependent signaling is not intrinsically required for the early steps of NCC development. However, ablation of Rara and Rarg genes in NCC yields an agenesis of the median portion of the face, demonstrating that RARalpha and RARgamma act cell-autonomously in postmigratory NCC to control the development of structures derived from the frontonasal process. In contrast, ablation of the three Rar genes in NCC leads to less severe defects of the branchial arches derived structures compared with Rar compound null mutants. Therefore, RARs exert a function in the NCC as well as in a separated cell population. This work demonstrates that RARs use distinct mechanisms to pattern cranial NCC.

Ectopic retinal ON bipolar cell synapses in the OFF inner plexiform layer: Contacts with dopaminergic amacrine cells and melanopsin ganglion cells

A key principle of retinal organization is that distinct ON and OFF channels are relayed by separate populations of bipolar cells to different sublaminae of the inner plexiform layer (IPL). ON bipolar cell axons have been thought to synapse exclusively in the inner IPL (the ON sublamina) onto dendrites of ON-type amacrine and ganglion cells. However, M1 melanopsin-expressing ganglion cells and dopaminergic amacrine (DA) cells apparently violate this dogma. Both are driven by ON bipolar cells, but their dendrites stratify in the outermost IPL, within the OFF sublamina. Here, in the mouse retina, we show that some ON cone bipolar cells make ribbon synapses in the outermost OFF sublayer, where they costratify with and contact the dendrites of M1 and DA cells. Whole-cell recording and dye filling in retinal slices indicate that type 6 ON cone bipolars provide some of this ectopic ON channel input. Imaging studies in dissociated bipolar cells show that these ectopic ribbon synapses are capable of vesicular release. There is thus an accessory ON sublayer in the outer IPL.

The Edinger–Westphal Nucleus Represents Different Functional Cell Groups in Different Species

In all vertebrates, including humans, the Edinger-Westphal nucleus (EW) forms a circumscribed cell group dorsomedial to the oculomotor nucleus (nIII). Traditionally the EW is considered the location of parasympathetic preganglionic neurons of the ciliary ganglion, mediating pupillary constriction and accommodation. In a comparative study in rat, ferret, monkey, and human, the location of cholinergic neurons within and around the nIII, which includes motoneurons of the extra-ocular muscles and the preganglionic neurons of the ciliary ganglion, was compared to the location of urocortin-positive neurons. Irrespective of the species, the cholinergic and urocortin-positive neurons form largely separated cell populations adjacent to each other. Only in monkey, cholinergic putative preganglionic neurons were found within the cytoarchitecturally defined EW, whereas in rat, ferret, and human the EW is almost exclusively composed of urocortin-positive neurons. In humans, the presumed preganglionic neurons are located as an inconspicuous group of choline acetyltransferase-positive neurons dorsal to the urocortin-positive EW.

Tunable Microfluidic Devices for Dynamically Controlling Sub-Cellular Environments

Microfluidic devices with the versatility of precisely controlling the immediate micro-environment of a single cell or a small population of cells have resulted in a paradigm shift in studying cellular development, local sub-cellular cell signaling, and ligand-activated dynamic cellular responses. In this paper, we report the development of multi-layered PDMS microfluidic devices that are dynamically tunable to provide on-demand, different spatio-temporally regulated micro-environments to in-vitro cultures of primary cells, like neurons and myotubes. The cell culture chamber was made in PDMS by exclusion-molding from a SU-8 master. The pneumatic actuator channel was molded separately, aligned and plasma-bonded on top of the cell-culture chamber. The roof of the cell-chamber contained elastomeric actuators designed to generate a dynamically-addressable, isolated compartment within the chamber. The separation of fluidic micro-domains was characterized with dyes. This tunable device can be used to (a) co-culture spatially separated cell populations, (b) dynamically separate axonal and somal compartments of a neuron, and (c) focally incubate sub-cellular regions of myotubes with extra-cellular biomolecules and ligands. We demonstrate time-lapse imaging and simulated models using the finite-element method, to show the dynamic tuning of the microfluidic channels, in the absence as well as in the presence of cells.

Computational Influence of Adult Neurogenesis on Memory Encoding

Adult neurogenesis in the hippocampus leads to the incorporation of thousands of new granule cells into the dentate gyrus every month, but its function remains unclear. Here, we present computational evidence that indicates that adult neurogenesis may make three separate but related contributions to memory formation. First, immature neurons introduce a degree of similarity to memories learned at the same time, a process we refer to as pattern integration. Second, the extended maturation and change in excitability of these neurons make this added similarity a time-dependent effect, supporting the possibility that temporal information is included in new hippocampal memories. Finally, our model suggests that the experience-dependent addition of neurons results in a dentate gyrus network well suited for encoding new memories in familiar contexts while treating novel contexts differently. Taken together, these results indicate that new granule cells may affect hippocampal function in several unique and previously unpredicted ways.

TheDrosophilahomolog of vertebrateIslet1is a key component in early cardiogenesis

In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. We have analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, we believe it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate.

FACS as useful tool to study distinct hyalocyte populations

Hyalocytes, the cells of the vitreous body, are assumed to be involved in physiological as well as patho-physiological processes within the eye. However, current knowledge about the cells is still limited. As different morphological types of hyalocytes are described in the literature, it seems reasonable to try to isolate individual populations prior to characterization of single cell types. To achieve this, the present study investigated the utility of fluorescence activated cell sorting (FACS) for hyalocyte separation. Subsequent to digestion of vitreous bodies using collagenase, the resulting cell suspension was analyzed and separated using FACS without any additional staining. Two-parameter dot plots of forward scatter (indicating size) against sideward scatter (indicating granularity) showed two distinct cell populations; staining with propidium iodide confirmed that both populations represent living cells. After sorting, cells of both populations were seeded on tissue culture plastic (tissue culture treated polystyrene). Only one population attached and proliferated, whereas the other population was non-adherent. Even when seeding the native cell mix, only one population of cells was observed after two passages, as indicated by FACS. Furthermore, ascorbic acid increased proliferation of these cells similarly to the proliferation of the separated cell population. These data point out that only one of the two populations adheres and proliferates on tissue culture plastic. To conclude, the established isolation technique allows for separation of clearly defined hyalocyte populations. Moreover, clear hints were obtained that only one of the two populations adheres and proliferates under the commonly applied culture conditions.

Deregulated Bcl-2 Family Expression Drives Chronic Myeloid Leukemia Cancer Stem Cell Survival.

Introduction: A growing body of evidence suggests that a relatively rare subset of cells within a cancer subverts properties normally ascribed to stem cells in regenerating tissues, such as enhanced self-renewal and survival capacity. Recent studies suggest that these cancer stem cells (CSC) are resistant to treatments that target rapidly dividing cells. In blast crisis chronic myeloid leukemia (BC CML) and some forms of acute myelogenous leukemia (AML), research performed by ourselves and others indicates that CSC originate from the CD34+CD38+lineage- compartment of hematopoietic cells and can serially transplant blast crisis leukemia in immunodeficient mice. Despite abundant data indicating that Bcl-2 family proteins are involved in CML progression, the importance of these proteins in CSC survival remains to be elucidated. Clinical data have shown that CML stem cells become more resistant to therapies targeting BCR-ABL with progression to blast crisis. As BCR-ABL targeted therapy initiates apoptosis, these results suggest that CML CSC may become increasingly resistant to apoptosis with progression. Based on these findings and the results from our serial transplantation experiments, we hypothesized that CML CSC deregulate apoptosis pathways by differential expression of Bcl-2 family molecules and that these changes contribute to CSC ability to survive serial transplantation.Methods: Quantitative FACS Aria analysis of Bcl-2 protein levels was compared in blast crisis (n=5) and chronic phase CML (n=3) patient samples. Mean fluorescence intensity (MFI) of Bcl-2 staining was compared between different hematopoietic populations within the patient samples. For gene expression analysis, cDNA was made from RNA isolated from sorted progenitor populations (CD34+CD38+Lin −). Isoform specific RT-PCR was used to determine expression levels of Bcl-2, Bcl-X, and Mcl-1 isoforms. Mcl-1 expression was confirmed using qPCR. In addition, preliminary experiments were performed (n=2) to determine if CSC engraftment could be reduced in vivo by targeted inhibition of Bcl-2 family molecules using Apogossypol, a clinically tested Bcl-2-family inhibitor. Briefly, immunocompromised neonatal mice were transplanted intrahepatically with luciferase GFP transduced granulocytic sarcomas from mice transplanted with BC CSC using our previously published methodology (Geron et al, Cancer Cell 2008). Transplanted mice were treated for 15 days with Apogossypol by oral gavage and engraftment was monitored by weekly bioluminescent imaging. Engraftment levels were determined by FACS analysis of human CD45+ expression in mouse livers on week 11 post-transplant.Results: Comparing the MFI of Bcl-2 staining in the entire live mononuclear cell population, we detected no statistically significant difference in levels between the blast crisis and chronic phase samples. However, when we gated on separate cell populations, differences in the Bcl-2 MFI emerged. There was a statistically significant increase (P<0.03) in Bcl-2 MFI exclusively in the CD34+CD38+lineage- population of the blast crisis samples indicative of cell type and context specific deregulation of apoptosis in the CSC population. Further, we were interested in whether there were differences in Bcl-2 family expression at the transcriptional level. Notably, while we detected no difference in the levels of the isoforms of Bcl-2 and Bcl-X, splice isoform specific RT-PCR and qPCR revealed a decrease in the expression of the short isoform of Mcl-1, which encodes a pro-apoptotic protein, in serially transplanted BC CSC (CD34+CD38+). Together these results indicate that CML CSC may indeed deregulate the expression of several Bcl-2 family proteins. To test the therapeutic potential of inhibiting these deregulated apoptotic pathways in CML, we treated mice engrafted with CML CSC with Apogossypol, a broad-spectrum inhibitor of pro-survival Bcl-2 molecules. We saw a statistically significant decrease (P<0.05) in the number of CD45+ cells engrafted in the mouse liver after 3 weeks of Apogossypol treatment. Overall, our results suggest that the subversion of apoptosis plays an important role in allowing CML CSC to be serially transplanted and that apoptotic pathways may be a useful target for therapeutics aimed at inhibiting these cells.

Day‐length encoding through tonic photic effects in the retinorecipient SCN region

The circadian clock in the suprachiasmatic nucleus (SCN) plays a critical role in seasonal processes by sensing ambient photoperiod. To explore how it measures day-length, we assessed the state of SCN oscillators using markers for neuronal activity (c-FOS) and the clock protein (PER1) in Syrian hamsters housed in long (LD, 16 : 8 h light : dark) vs. short days (SD, 8 : 16 h light : dark). During SD, there was no detectable phase dispersion across the rostrocaudal extent of the nucleus. In contrast, during LD, rhythms in the caudal SCN phase led those in the mid- and rostral SCN by 4-8 h and 8-12 h, respectively. Importantly, some neurons in the retinorecipient core SCN were unique in that they were FOS-positive during the dark phase in LD, but not SD. Transfer of LD animals to constant darkness or skeleton photoperiod revealed that dark-phase FOS expression depends on tonic light exposure rather than on intrinsic clock properties. By transferring animals from SD to LD, we next discovered that there are two separate populations of SCN cells, one responding to acute and the other to tonic light exposure. The results suggest that the seasonal encoding of day-length by the SCN entails reorganization of its constituent oscillators by a subgroup of neurons in the SCN core that respond to tonic photic cues.

Cultured trout gill epithelia enriched in pavement cells or in mitochondria-rich cells provides insights into Na+ and Ca2+ transport

The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kOmega cm(2)) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na(+) fluxes and application of the Ussing flux ratio criterion demonstrated active Na(+) uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca(2+) uptake and Na(+)/K(+)-ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.