artigo - neurosferas

Upload: clarissa-fernandes

Post on 07-Apr-2018

217 views

Category:

Documents


0 download

TRANSCRIPT

  • 8/6/2019 artigo - neurosferas

    1/16

    Author contributions: B.C.-T.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; K.P.:Conception and design, Collection and/or assembly of data; I.B.: Collection and/or assembly of data; P.K.: Conception and design, Collection and/orassembly of data; C.M.: Conception and design, Financial Support, Data analysis and interpretation, Final approval of manuscript; P.Z.: Conception anddesign, Financial Support, Data analysis and interpretation, Final approval of manuscript; D.van der Kooy: Conception and design, Financial Support,Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

    Contact Information: Derek van der Kooy, University of Toronto, Dept Molecular Genetics, TD-CCBR, Rm 1102, 160 College Street, Toronto, Ontario,M5S 3E1, Canada. Phone (416) 978-1960, Fax: (416) 978-2666, email: [email protected]. Received June 09, 2008; accepted forpublication August 11, 2008; first published online in Stem Cells Express August 28, 2008. AlphaMed Press 1066-5099/2008/$30.00/0 doi:10.1634/stemcells.2008-0558

    STEMCELLS

    TISSUE-SPECIFIC STEM CELLS

    Dont Look: Growing clonal versus non-clonal neural stem cell colonies

    Brenda L.K. Coles-Takabe1, Ian Brain2, Kelly A. Purpura3,4, Phillip Karpowicz1, Peter W. Zandstra3,4,Cindi M. Morshead2, Derek van der Kooy1.

    1Department of Molecular Genetics, University of Toronto; 2Department of Surgery, Division of Anatomy, University ofToronto; 3Institute of Biomaterials and Biomedical Engineering, University of Toronto; 4Department of ChemicalEngineering and Applied Chemistry, University of Toronto

    Key words. Stem Cell Clonality Tissue Culture Fluorescent Mice

    ABSTRACT

    Recent reports have challenged the clonality of theneurosphere assay in assessing neural stem cell (NSC)

    numbers quantitatively. We tested the clonality of the

    neurosphere assay by culturing mixtures of differently

    labeled neural cells, watching single neural cells

    proliferate using video microscopy, and by

    encapsulating single NSCs and their progeny. The

    neurosphere assay was clonal when using primary cells

    plated at 10 cells/l or less, however when using

    passaged NSCs the spheres were only clonal if plated

    at 1 cells/l. Most important, moving the plates during

    the growth phase (to look at cultures microscopically)greatly increased the incidence of non-clonal colonies.

    To ensure clonal sphere formation, and investigate

    non-autonomous effects on clonal sphere formation

    frequencies, single NSCs were encapsulated in agarose

    and proliferated as clonal free-floating spheres. We

    demonstrate that clonal neurospheres can be grown by

    avoiding movement-induced aggregation, by single cell

    tracking and by encapsulation of single cells.

    INTRODUCTION

    There are two cardinal features of stem cells;they must demonstrate long-term self-renewaland must be multipotential the progeny ofsingle stem cells comprise multiple cell types ofthe tissue in which they reside. In order to assessthe presence of stem cells many researchers havetaken advantage of the in vitro proliferation ofneural stem cells to form clonal colonies ineither monolayer cultures [1] or in a free-floating

    3D colony known as the neurosphere assay [2].Recently, the validity of the in vitroneurosphere-formation assay as a measure of

    clonality and multipotentiality [3,4,5], and as anaccurate predictor of the in vivo number of stemcells in a tissue [6], has been called intoquestion. Many labs have used sphere-formingassays to identify stem cells in various tissuetypes including brain [2,7,8,9,10], retina[11,12,13], cornea [14] olfactoryneuroepithelium [15], pancreas [16], skin [17],muscle [18], bone marrow [19] and embryonicstem cells [20].

    The papers [3,4,5] that call into question theclonality of the sphere assay also challenge theconclusions of published papers in which the

    Stem Cells Express, published online August 28, 2008; doi:10.1634/stemcells.2008-0558

    Copyright 2008 AlphaMed Press

  • 8/6/2019 artigo - neurosferas

    2/16

  • 8/6/2019 artigo - neurosferas

    3/16

    Clonal vs Non-clonal Neurospheres

    3

    Encapsulation: To encapsulate single cellswithin an agarose matrix, 2.0% (w/v) SeaPrepagarose (Cambrex, Rockland ME) was preparedwith PBS. Methods similar to aggregateencapsulation [23] were employed. Briefly, 2-

    4.5 x 106 NS cells were suspended in 300 l

    serum-free media, mixed with 1.2 ml agarose, 75l Pluronic F-68 (Sigma) and allowed to

    equilibrate to 37 C. Small drops of the cellsuspension/agarose mixture were added to 20 ml

    emulsion oil heated to 37 C (200 ccsdimethylpolysiloxane; DMPS, Sigma) in ascintillation vial using a 1 ml pipetteMan. Themixture was emulsified using the CellSysMicrodrop maker set at 2100 rpm for 1 minute atroom temperature followed by 1 minute in an icebath and then set at 1100 rpm for 10 minutes in

    the ice bath. The encapsulation mixture wasdivided into two 15 ml conical tubes and 5 mlHanks Buffered Salt Solution (HBSS; Gibco)was overlaid prior to centrifugation at 600g for10 minutes. The oil phase was removed from thetop of each tube, and the aqueous phase wasaspirated. Both pellets were re-suspended in 2 mlof HBSS, and added to 10 ml fresh HBSS inanother 15 ml conical tube prior to centrifugationat 400g for 5 min. The now encapsulated cellswere resuspended in serum-free media and then

    cell counts and viability were assayed usingTrypan Blue exclusion. The cells were only usedif the final single-cell encapsulated cells wereover 98% of the cells encapsulated. Finally, thesingle encapsulated neural stem cells andprogeny were plated alone at low densities (5cells/l) or plated at these densities with a highdensity (100 cells /l) of non-encapsulateddissociated host neurosphere cells (neural stemcells and their progeny). The encapsulationexperiments were done three times and 4-6 wells

    counted per condition and per experiment.

    Passaging: Spheres were mechanicallydissociated using a small borehole fire-polishedpipette, cell strained (40um mesh) andresuspended in serum free media. Cell countsand viability were assayed using Trypan Blueexclusion. All passaging experiments were done

    3 times and at least 6 wells per condition and perexperiment were counted.

    Imaging: Cells were plated at 10 cell/l in both6-well (3125 cells/cm2) and 24-well (2632cells/cm2) plates and imaged every 10 minutes

    over the course of 7 days using a Zeiss Axiovert200 Imaging System [32,33].

    Statistical Analysis

    All data were compiled using Microsoft Exceland then statistical analysis was carried out usingANOVA (GB STAT).

    RESULTS

    Primary Neurosphere Formation

    We isolated neural stem cells from thesubependymal region of adult transgenic miceexpressing either EYFP or dsRed protein behindthe actin promoter. The single dissociated cellsfrom each mouse strain were plated together at a1:1 ratio over a range of cell densities rangingfrom a final cell density of 0.5 cells/l up to 50cells/l (Figure 1A). The cells proliferated andformed neurospheres, with a diameter of at least100 m, over the course of seven days (Figure 2A-F). We observed that spheres formed from

    primary cells were clonal (spheres that wereeither only dsRed derived or only EYFP derived)100% of the time at 0.5 cells/l whether the cellswere left undisturbed during the growth phase ofsphere formation or if the cells were taken out ofthe incubator and observed under the microscopedaily (Figure 3 A). The cells that were leftundisturbed (stationary) during the experimentgave rise to clonal spheres when plated at 10cells/l or less, giving rise to 50-60 spheres perwell, with an incidence of chimerism of only 4%

    at 10 cells/l. However, if the initial cell densitywas increased to 20 cells/l (greater than 75spheres per well) or if the plates were movedwhen the density was only 5 cells/l or higher,the incidence of non-clonal sphere formationwas always greater than 10% (Figure 3 A);similar increases in non-clonal sphere formationwere found when the plates were moved only onday three in vitro (data not shown). There was a

  • 8/6/2019 artigo - neurosferas

    4/16

  • 8/6/2019 artigo - neurosferas

    5/16

  • 8/6/2019 artigo - neurosferas

    6/16

  • 8/6/2019 artigo - neurosferas

    7/16

  • 8/6/2019 artigo - neurosferas

    8/16

  • 8/6/2019 artigo - neurosferas

    9/16

    Clonal vs Non-clonal Neurospheres

    9

    the dermis of mammalian skin. Nat. Cell Biol. 3: 778-784.

    18. Tamaki T., Okada Y., Uchiyama Y., Tono K., MasudaM., Wada M., Hoshi A., Ishikawa T. and Akatsuka A.(2007). Clonal multipotency of skeletal muscle-derivedstem cells between mesodermal and ectodermallineage. Stem Cells. 25(9): 2283-90.

    19. Hermann A., Gastl R., Liebau S., et al. (2004).

    Efficient generation of neural stem cell-like cells fromthe adult human bone marrow stromal cells. J Cell Sci.117(19): 4411-22.

    20. Makinodan E, Manabe T, Makinodan M, Yamauchi T,Matsuyoshi H, Sakumura R, Tatsumi K, Wanaka A..(2007). A novel role for Fyn: change in sphereformation ability in murine embryonic stem cells.Neuroscience. 147(1): 1-4.

    21. Heisenberg WZ. Quantum Theory and Measurement.Physik. 1927; 43:172198. doi: 10.1007/BF01397280.English translation in: Quantum Theory andMeasurement. Edited by Wheeler JA, Zurek WH.Princeton: Princeton University Press; 1983:6284.

    22. Dang S.M., Gerecht-Nir S., Chen J., Itskovitz-Eldor J.and Zandstra P.W. (2004). Controlled, scalableembryonic stem cell differentiation culture. Stem Cells.22: 275-282.

    23. Dang SM and Zandstra PW. (2005). Scalableproduction of embryonic stem cell-derived cells.Methods Mol Biol. 290:353-64.

    24. Shimomura A., Nomura R. & Senda T. (2003).Lithium inhibits apoptosis of mouse neural progenitorcells. NeuroReport. 14(14): 1779-1782.

    25. Louis S.A., Rietze R.L., Deleyrolle L., et al. (2008).Enumeration of neural stem and progenitor cells in theneural colony forming cell assay. Stem Cells. 26(4):

    988-96.

    26. Golmohammadi M.G., Blackmore D.G., Large B., etal. (2008). Comparative analysis of the frequency anddistribution of stem and progenitor cells in the adultmouse brain. Stem Cells. 26(4): 979-87.

    27. Trevisan M., Yan X-Q. and Iscove N.N. (1996). Cycleinitiation and colony formation in culture by murinemarrow cells with long-term reconstituting potential invivo. Blood. 88(11): 4149-4158.

    28. Sauvageau G., Iscove N.N. and Humphries R.K.(2004). In vitro and in vivo expansion of hematopoieticstem cells. Oncogene. 23(43):7223-32. Review.

    29. Morshead C.M., Reynolds B.A., Craig C.G.,McBurney M.W., Staines W.A., Morassutti D., WeissS., van der Kooy D. (1994). Neural stem cells in theadult mammalian forebrain: a relatively quiescentsubpopulation of subependymal cells. Neuron. 13(5):1071-1082.

    30. Morshead C.M., Craig C.G., van der Kooy D. (1998).In vivo clonal analyses reveal the properties ofendogenous neural stem cell proliferation in the adultmammalian forebrain. Development. 125(12): 2251-

    61.31. Lathia J.D., Rao M.S., Mattson M.P., Ffrench-Constant

    C. (2007). The microenvironment of the embryonicneural stem cell: Lessons from adult niches? Dev Dyn.236(12): 3267- 3282.

    32. Karpowicz P., Morshead C.M., Kam A., Jervis E.,Ramunas J., Cheng V., and van der Kooy D. (2005).Support for the immortal strand hypothesis: neuralstem cells partition DNA asymmetrically in vitro. JCell Biol. 170(5): 721732.

    33. Ramuna J., Illman M., Kam A., Farn K., Kelly L.,Morshead C.M. and Jervis E.J. (2006). Identification ofa behavioral phenotype in neural stem cell cultures:

    Lineage informatics of undifferentiated neurosphere-derived cells. Cytometry. 69: 1202-1211.

  • 8/6/2019 artigo - neurosferas

    10/16

    Clonal vs Non-clonal Neurospheres

    10

    Figure 1. Experimental design. Panel A is the methodology for testing the clonality of both theprimary and passaged neural stem cells. Briefly, the cells from the brain or passaged neurosphere cellsare dissociated, plated at a 1:1 ratio and counted seven days later. Panel B shows the basic method forthe encapsulation experiment. The passaged neurospheres are dissociated, placed in an agarosesolution, spun in a Microdrop maker to encapsulate the cells, plated and counted four days later.

  • 8/6/2019 artigo - neurosferas

    11/16

    Clonal vs Non-clonal Neurospheres

    11

    Figure 2. Neurosphere Formation. Primary stem cells plated at 10 cells/l in either plates that werestationary (not moved at all during the growth phase), A-C (A: YFP. B: dsRed and C: merged), ormoved (taken out daily and examined under the microscope); D-F (D: YFP, E: dsRed, and F: merged).A-C show the same field from a stationary experiment and demonstrate that the spheres are welldispersed and not touching after 7 days of growth. A1-C1 panels are enlargements of the one chimericdsRed sphere with a single YFP cell attached to the outside from panels (marked by white asterisks)

    A-C. D-F show (in a single field) that moving the plates forces the spheres closer together andincreases the rate of chimerism. D1-F 1 panels are enlargements of two chimeric spheres from D-F, onedsRed sphere with a single YFP cell on the outside of the middle of the sphere (marked by whiteasterisks) and one intermixed dsRed /YFP sphere. For panels A-F the arrows indicate clonally derivedspheres, the arrowheads indicate chimeric spheres, and the scale bars equal 200m.

  • 8/6/2019 artigo - neurosferas

    12/16

    Clonal vs Non-clonal Neurospheres

    12

  • 8/6/2019 artigo - neurosferas

    13/16

    Clonal vs Non-clonal Neurospheres

    13

    Figure 3. Clonality of Neurospheres. A: primary neural stem cells form clonal spheres at low densitiesin stationary wells (less that 5% chimeric spheres at 20 cells/l or less). However, if the plates aremoved during the growth phase there is a significantly higher incidence of chimerism. B: PassagedSpheres form clonal spheres only at densities lower than 1 cell/l (the incidence of chimerism less than5%); however if the plates are moved, then even at 0.5 cells/l many of the spheres are not clonal. C:graph of primary neurospheres grown in plates that were not moved, showing that the number of non-

    clonal spheres increases as the sphere density increases, which is exacerbated by movement. D: Graphof stationary, passaged neurospheres demonstrating that most of the cell densities plated give morethan 100 spheres, and hence there is an increase in the incidence of chimeric spheres. The moved,passaged neurospheres aggregated into one large mass when plated over 5 cells/l, therefore the datawas not included in the graph.

  • 8/6/2019 artigo - neurosferas

    14/16

    Clonal vs Non-clonal Neurospheres

    14

    Figure 4. Encapsulation of stem cells. A: Picture of a single encapsulated YFP cell with non-encapsulated dsRed cells. The white thin arrow is pointing to the YFP positive cell inside of theagarose capsule (indicated by the thick white arrow). The white arrowheads indicate the twounencapsulated dsRed cells. B: A picture of separate encapsulated and non-encapsulated neurospheres.The black arrowhead indicates a clonal encapsulated EYFP neurosphere, the black arrow shows a fewdsRed cells attached to the outside of a EYFP non-clonal neurosphere - presumably after it burst out of

    the encapsulating gel, the yellow arrow points to a dsRed neurosphere that is most likely non-clonaldue to the high plating, and the white arrowheads indicate a few of the many empty agarose capsules.The white arrow indicates a dead cell trapped in the agarose capsule that is not expressing anyfluorescence. (Several other dead cells not indicated also are present in spheres) The scale bar equals200m. C: graph of the numbers of clonal encapsulated EYFP spheres arising when plated alone at 5cells/l or when plated as encapsulated single cells (5 cells/l) with a high density (100 cells/l) ofsurrounding dsRed passaged neurosphere cells.

  • 8/6/2019 artigo - neurosferas

    15/16

    Clonal vs Non-clonal Neurospheres

    15

  • 8/6/2019 artigo - neurosferas

    16/16

    DOI:10.1634/stemcells.2008-0558published online Aug 28, 2008;Stem Cells

    Zandstra, Cindi M. Morshead and Derek van der KooyBrenda L.K. Coles-Takabe, Ian Brain, Kelly A. Purpura, Phillip Karpowicz, Peter W.

    Don't Look: Growing clonal versus non-clonal neural stem cell colonies

    This information is current as of September 7, 2008

    & ServicesUpdated Information

    http://www.StemCells.comincluding high-resolution figures, can be found at:

    http://www.stemcells.com/http://www.stemcells.com/http://www.stemcells.com/