Cosa sono le cellule staminali?

Transcription

1 Cosa sono le cellule staminali?

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3 Cellule Staminali Embrionali: totipotenti o pluripotenti Provengono dalla inner cell mass di embrioni di 4-5 giorni (blastocisti) prodotti in vitro Adulte: multipotenti Cellule indifferenziate mescolate ad altre differenziate in un dato tessuto o organo necessarie per mantenere e/o riparare i tessuti

4 Stem Cells: Regenerative tissues (epithelia, blood, testes) Static tissues (nervous system, liver) At the crossroad between self-renew and differentiation Differentiation Stem Cell Differentiated Cell Stem Cell

5 Stem cells Ability to self-renew Progeny committed to differentiation Asymmetric division Two sisters with different fates size, morphology gene expression pattern number of subsequent divisions

6 Two mechanisms: 1. response to extracellular signals (extrinsic mechanism) 2. cell autonomously (intrinsic mechanism) 1. response to extracellular signals From: Knoblich JA. Cell 132: , 2008

7 2. Intrinsic asymmetric division Polarized mother cell capable of segregating specific factors called determinants into one of its daughter cells to initiate a particular developmental pathway in this cell but not its sister. Three steps I II III IV From: Ahringer, Curr. Opin. Cell Biol. 15:73-81, 2003

8 Spindle orientation and slicening of a cake Vertical slice = 2 pieces of equal size and content Horizontal slice = 2 pieces of unequal size and content Spindle orthogonal to the polar axis Spindle parallel to the polar axis

9 First cell division of the C. elegans zygote ABa AB P1 ABp EMS P2 MEX-5/6 PAR-3, PAR-6,PKC3 PAR-1. PAR-2 AB P1 From: Knoblich J.A, Nature Reviews 2: 11-20

10 Proteins involved in asymmetric cell division identified in C. elegans

11 Drosophila stem cells provide examples for both the extrinsic and intrinsic mechanisms Drosophila germline stem cells (GSCs): cells surrounding the stem cell niche supply self-renewal cues and/or release stem cell maintenance short-range signals that induce the polarity necessary for stem cell asymmetric divisions. Par1 and CycA: checkpoint preventing mitotic entry until the spindle is correctly oriented Drosophila neuroblasts (NBs) NB self-renewal does not require any extrinsic factors NBs are still able to divide asymmetrically and self-renew in culture

12 Drosophila neuroblasts (NBs) NBs inherit basal-apical polarity from epithelial cells Spindle reorients Cell fate determinants localize asymmetrically Determinants segregate differentially between the daughters

13 Drosophila NBs: asymmetric division giving rise to a NB and a Ganglion Mother Cell (GMC) GMC divides once (symmetrically) to produce neurons or glial cells Embryonic NBs: Reduce in size at each division Restricted self renewed capacity Give rise to the simple larval nervou system Larval NBs: Regrow at each division Can divide hundreds of times Give rise to adult nervous system

14 Type I NBs: same mechanism of embryonic NBs Type II NBs: asymmetric division generating two Intermediate Neural Progenitors (INP) which become mature INPs. INPs: 3-5 rounds of asymmetric divisions to generate another NB and a GMC (about 5000 adult neural cells) INPs: transit amplifying population allowing type II NBs to generate more neurons than type I NBs.

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16 Summary of the key players in NB asymmetric division Two complexes accumulate at the apical cortex and induce basal localization of determinants Numb: Notch pathway repression (endocytosis of Notch receptor) Prospero: transcription factor activating pro-neural genes in GMC Brat: postranscriptional regulator Mira and Pon: adaptor proteins From: Chia et al., J Cell Biol. 180: , 2008 Disruption of cell polarity or spindle orientation: unbalance between self-renewal and differentiation

17 Asymmetric divisions of Drosophila neuroblasts Tubulin DNA Mira Cnn From: Bonaccorsi et al., Nature Cell Biol 2: 54-56

18 Setting up polarity: apical localization of Par-3 (Baz), Par-6 and apkc Spindle orientation: MT-binding protein Mud recruited by Pins and Gαi Determinants localization: Lgl inactivation by apkc phosphorylation

19 Setting up polarity: apical localization of Par-3 (Baz), Par-6 and apkc NB centrosome serves as a reference point for apical accumulation of the Par complex during interphase to establish an apicobasal axis of polarity within the cell The mechanism by which cortical polarity is oriented relative to the centrosome is still unclear.

20 Spindle orientation Bazooka (one of the Par complex members) links apical polarity to spindle orientation by anchoring Inscuteable (Insc) Insc connect the Par complex and the Gαi/Pins (Rap)/Mud complex Mud binds this complex to astral microtubules and directs spindle orientation wild type mud mutant 12-13% defective orientation increased number of NBs

21 asterless mutants

22 Determinants localization The apical complexes induce asymmetric localization of cell fate determinants Brain tumor (Brat), Prospero (Pros) and Numb to the opposite (basal) side of the cell Polar phosphorylation is the underlying mechanism for asymmetric protein segregation during mitosis.

23 Role of mitotic kinases in asymmetric divisions Par complex in interphase: apkc, Par6 and Lgl At mitosis Par6 phosphorylation by Aurora A triggers rearrangement of the Par complex apkc more active and phosphorylates Lgl that dissociates from the cortex and localizes to the cytoplasm Dissociation of Lgl allows Baz to interact with apkc/par6 Novel substrate specificity for the complex Baz recruits Numb Polo phosphorylates Pon Pon directs Numb basal localization apkc directly phosphorylates Numb, leading to its release from the apical NB cortex and its asymmetric distribution

24 Prospero: transcription factor activating pro-neural genes and repressing proliferation genes in GMC Mira prevents Pros from regulating transcription in the NB by tethering it to the basal cortex during mitosis In GMC Mira is degraded and Pros enters the nucleus to prevent the expression of cell cycle genes contributing to terminal differentiation Numb: Notch pathway repression (endocytosis of Notch receptor) Brat: postranscriptional regulator

25 Asymmetric divisions in the mammalian brain Mouse early embryo (E9.0): symmetric divisions to expand progenitor pool E11.0: neurogenesis starts and progenitors become radial glial (RG) cells that give rise to neurons Resembling Drosophila Type I NB divisions

26 Many molecules regulating asymmetric divisions in flies are conserved in mammals Mouse Insc Insc, Pins and Mud homologues: spindle orientation Baz, Par-6 and apkc homologues: apical localization and polarity axis Prospero and Numb homologues: not functioning as determinants

27 Drosophila NBs as a model system for the study of tumor growth

28 For a tumor to establish itself it must integrate several hallmarks of cancer, which include the ability to promote sustained proliferative signaling while concomitantly evading apoptotic signals. For this to occur, cells must acquire activating mutations in oncogenes and deactivating mutations in tumor suppressor genes. It is widely thought that once a tumor has exhausted its site of origin, a tumor must undergo further changes to allow invasion and metastasis for colonization of new sites (Hanahan and Weinberg, 2011) Hanahan, D., Weinberg, R.A., Hallmarks of cancer: the next generation. Cell 144, 646e674

29 Mutant brains transplanted into a host Gonzalez C: Spindle orientation, asymmetric division and tumour suppression in Drosophila stem cells. Nat Rev Genet 2007, 8:

30 Two weeks after transplantation: no growth of wild type pieces 100 fold growth from pins, numb, pros or mira mutant pieces

31 Neuroblastomas resulting from uncontrolled division of NBs and largely composed of undifferentiated cell types Possible mechanism: NBs producing two daughter NBs thus altering balance between differentiated vs undifferentiated cells

32 Only centrosome mutants that also affect asymmetry induce tumor growth after transplantation Castellanos E, Dominguez P, Gonzalez C. (2008). Centrosome dysfunction in Drosophila neural stem cells causes tumors that are not due to genome instability. Curr Biol 18:

33 Neoplastic transformation triggered by perturbing functions that mediate asymmetric stem cell division Common etiology: perturbation of NB polarity, impairment of cell-fate detemination and alteration of cell homeostasis If impaired segregation of determinats can cause hyperproliferation of larval NBs in Drosophila it may similarly affect tissue stem cells in other species Stem cells as tumor founder cells?