WO2003093487A1 - Regulation de la differenciation et de l'autorenouvellement de cellules souches par manipulation de la mapk p38 et de la voie de signalisation notch - Google Patents

Regulation de la differenciation et de l'autorenouvellement de cellules souches par manipulation de la mapk p38 et de la voie de signalisation notch Download PDF

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WO2003093487A1
WO2003093487A1 PCT/AU2003/000518 AU0300518W WO03093487A1 WO 2003093487 A1 WO2003093487 A1 WO 2003093487A1 AU 0300518 W AU0300518 W AU 0300518W WO 03093487 A1 WO03093487 A1 WO 03093487A1
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cell
differentiation
cells
mapk
agent
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Richard Hans Harvey
Thomas Yeoh
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The Victor Chang Cardiac Research Institute Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C12N5/0659—Satellite cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/42—Notch; Delta; Jagged; Serrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention relates to assays for determining cell differentiation and self renewal and uses of the assays for developing or studying treatment modalities and agents.
  • Stem cells refer to unique populations of cells in the embryo or adult, defined as clonogenic precursor cells capable of differentiation into at least one specialised cell type, as well as unlimited or prolonged self-renewal (Watt, F.M. & Hogan, B.L. Out of Eden: stem cells and their niches. Science 287, 1427-30 (2000)).
  • the traditional view of stem cells is that they exist in tissues in which there is a constant turnover of post-mitotic differentiated cells, such as the blood system, skin, gut endothelium and skeletal muscle. In addition, they are mono- or oligo-potent, differentiating only into the tissue type in which they reside. While this view has changed radically over the last few years, the principles of stem cell function have been established in some of these systems.
  • stem cells can be recognised morphologically, and their spatial relationship to non-stem cell daughters is stereotypical.
  • the two processes outlined above may be quite different, although both must certainly involve multiple feedback controls and intercellular dialogues.
  • population-based asymmetry allows for regulated responses to diverse physiological inputs.
  • the molecular mechanisms that underlie control and regulation of stem cells remain largely unknown.
  • HPC hematopoietic precursor cells
  • CNS central nervous system
  • adult stem cells are derived from the bone marrow stem cell compartment. Indeed, dispersed muscle cells contain a stem cell population which expresses HPC markers and has 10-14 fold greater hematopoietic activity than whole bone marrow (Jackson, K.A., Mi, T. & Goodell, M.A. Hematopoietic potential of stem cells isolated from murine skeletal muscle. PNAS 96, 14482-14486 (1999)).
  • skeletal muscle precursor cells can be readily isolated from explants of dorsal aorta and from limbs of met-/- and Pax3-/- mice, into which somite-derived muscle precursors fail to migrate (De Angelis, L. et al. Skeletal Myogenic Progenitors Originating from Embryonic Dorsal Aorta Coexpress Endothelial and Myogenic Markers and Contribute to Postnatal Muscle Growth and Regeneration. J. Cell. Biol. 147, 869-877 (1999)). Consistent with this model is the fact that muscle satellite cells (see below) express endothelial markers.
  • Skeletal muscle in vertebrates originates from precursor populations resident within somites, segmental mesodermal structures formed in the embryo. These cells give rise to the dermomyotome, then myotome, and form both appendicular and axial muscles. These muscles initially express high levels of MyoD and Myf-5, two members of the myogenic bHLH family of transcription factors, which also includes MRF-4 and myogenin.
  • the other well-established precursor cell for skeletal muscle is the satellite cell (Seale, P., Asakura, A. & Rudnicki, M.A. The potential of muscle stem cells. Dev Cell 1 , 333-342 (2001)).
  • Satellite cells arise late in development and account for the majority of post-natal and adult muscle growth, as well as the adaptive response of hypertrophy, and repair and regeneration after injury. Satellite cells have classically been described by their appearance as mononuclear cells located between the basal lamina and sarcolemma of myofibres. In the resting state, satellite cells are quiescent and most express distinct markers including c-met, M-cadherin, CD34, bcl2 and Pax-7. Satellite cells do not express high levels of the bHLH myogenic regulatory factors (MRFs) related to MyoD, although low-level expression of Myf5 in most (but not all) satellite cells has been detected using a Myf-5-lacZ reporter gene (Beauchamp, J. R.
  • MRFs myogenic regulatory factors
  • skeletal muscle also contains a population of multipotential stem cells, which share properties with stem cells from a variety of other sources.
  • skeletal muscle contains cells that express hematopoietic stem cell markers and show blood lineage repopulation ability.
  • HPCs these cells also exclude Hoechst dye, giving rise to the so-called "side population" (SP) after FACS.
  • SP side population
  • Muscle-derived hematopoietic stem cells are hematopoietic in origin. PNAS 99, 1341-1346 (2002)). How these populations relate to satellite cells is unknown, although they may exist in a hierarchical relationship, with some or all populations showing self-renewal (Gussoni, E. et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401, 390-4. (1999); Seale, P. et al. Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777-86 (2000)). The various lines of evidence for heterogeneity in the stem cell populations within muscle, both in vitro and in vivo (Zammit, P.S. & Beauchamp, J. R.
  • the skeletal muscle satellite cell stem cell or son of stem cell? Differentiation 68, 193-204 (2001); Beauchamp, J. R., Morgan, J. E., Pagel, C. N. & Partridge, T. A. Dynamics of Myoblast Transplantation Reveal a Discrete Minority of Precursors with Stem-Cell-like Properties as the Myogenic Source. J. Cell. Biol. 144, 1113-1121 (1999); Baroffio, A. et al. Identification of self-renewing myoblasts in the progeny of single human muscle satellite cells. Differentiation 60, 47-57. (1996)), may relate to different states in this hierarchy. Pax-7 appears to be one important factor for specification of satellite cells, although not for that of the multipotential stem cell population.
  • Skeletal muscle stem cell self-renewal The ratio of satellite cells as a percentage of total myonuclei drops from 32% at birth to ⁇ 5% in rodents as a direct result of fusion to existing myocytes (Seale, P. & Rudnicki, M.A. A new look at the origin, function, and "stem-cell" status of muscle satellite cells. Dev Biol 218, 115-24. (2000)). In contrast, the number of quiescent satellite cells in adult muscle remains constant over several rounds of degeneration and regeneration, demonstrating the inherent capacity for self-renewal in this compartment. However, there is a significant progressive decline in satellite cell numbers during aging (Gibson, M.C. & Schultz, E.
  • GM high serum growth medium
  • DM low serum differentiation medium
  • a general feature of satellite cells induced to differentiate is the persistence of mononuclear cells that fail to differentiate, even after many days. This same property is seen in the established myogenic cell line C2C12, originally derived from mouse satellite cells (Yoshida, N., Yoshida, S., Koishi, K., Masuda, K. & Nabeshima, Y.
  • reserve cells Another important characteristic of reserve cells is their ability to re-express MyoD when returned to GM, and to generate both differentiated progeny and more reserve cells (see Figure 1B).
  • reserve cells meet the current definition for stem cells: they are able to self-renew and yield in their progeny both cells committed to differentiate and cells similar to the mother cells.
  • reserve cells express markers also found on the majority of satellite cells, including CD34 and Myf-5. Satellite cells also heterogeneously express bcl-2, which appears to confer apoptosis-resistance to muscle stem cells (Dominov, J.A., Dunn, J.J. & Miller, J.B.
  • Bcl-2 expression identifies an early stage of myogenesis and promotes clonal expansion of muscle cells. J Cell Biol 142, 537-44 (1998)).
  • the mechanisms regulating reserve cell generation and down-regulation of MyoD expression are unknown, although the C2C12 system appears to represent a valid model for stem cell self-renewal. Kitzmann et al. found that Myf5 and MyoD accumulate in different phases of the cell cycle, with MyoD low in GO and G1/M, suggesting that formation of reserve cells, which are MyoD-negative, may depend upon their position in, or place of exit from, the cell cycle (Kitzmann, M. et al. The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142, 1447-59. (1998)).
  • the present inventors have determined the role of a number of cellular mechanisms in cell differentiation and self renewal which can be used to develop assays and determine the role of agents on the cellular differentiation/self renewal process. Disclosure of Invention
  • the present invention provides a method for controlling precursor cell differentiation comprising treating a precursor cell with an agent that affects activity of mitogen-activated protein kinase (MAPK) p38 or signalling of Notch in the cell such that the cell undergoes differentiation or undergoes self renewal.
  • MAPK mitogen-activated protein kinase
  • the precursor cell is a stem cell or a myogenic cell line.
  • the myogenic cell line is C2C12.
  • the MAPK p38 is p38 ⁇ or p38 ⁇ .
  • the agent is an inhibitor of MAPK p38 or an activator of MAPK p38.
  • the agent that affects activity of MAPK p38 ⁇ or MAPK p38 ⁇ is SB203580 (SB) (produced by Calbiochem) and the precursor cell is a stem cell or the myogenic cell line C2C12. It will be appreciated that other agents would also be suitable for the present invention.
  • Other possible drug inhibitors of p38 MAPK include SKF 86002 and its derivatives and HEP689 and VX-745. (Lee, J.C, Kumar, S., Griswold,
  • reagents activating or repressing the p38 pathway include: dominant-negative p38 (inhibitor), dominant- negative MKK6 (inhibits MKK6 which is the upstream activator of p38), constitutively active MKK6 (constitutively activates p38) (Raingeaud, J., Whitemarsh, A.J., Barrett, T., Derijard, B., and Davis, R.J. (1996) MKK3- and MKK6-regulated gene expressions mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Molecular and Cellular Biology 16, 1247-1255).
  • the agent that affects Notch signalling in the cell is DFK-167 (Enzyme Systems Products) and the precursor cell is a stem cell or the myogenic cell line C2C12.
  • Calbiochem produces the inhibitor MG132 and makes an inhibitor called gamma secretase inhibitor X.
  • MDL281 0 is cited in De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J.S., Shroeter, E.H., Schrijvers, V., Wolfe, M.S., Ray, W.J., Goate, A.
  • the present invention may lead to better therapies for stem cell-related diseases and loss of stem cell function during aging.
  • the present invention is directed to a cell model for studying cell differentiation and self renewal comprising a cell modified to alter its sensitivity to an agent that affects activity of MAPK p38 or Notch signalling in the cell.
  • the cell is a precursor cell, more preferably a stem cell or C2C12 cell.
  • the agent that affects activity of MAPK p38 ⁇ or p38 ⁇ is SB203580
  • SB (produced by Calbiochem) and the precursor cell is a stem cell or the myogenic cell line C2C12. It will be appreciated that other agents would also be suitable for the present invention.
  • Other possible drug inhibitors of p38 MAPK include SKF 86002 and its derivatives and HEP689 and VX-745. (Lee, J.C, Kumar, S., Griswold, D.E., Underwood, D.C., Votta, B.J., and Adams, J.L. (2000) Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology 47, 185-201).
  • reagents activating or repressing the p38 pathway include: dominant-negative p38 (inhibitor), dominant- negative MKK6 (inhibits MKK6 which is the upstream activator of p38), constitutively active MKK6 (constitutively activates p38) (Raingeaud, J., Whitemarsh, A.J., Barrett, T., Derijard, B., and Davis, R.J. (1996) MKK3- and MKK6-regulated gene expressions mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Molecular and Cellular Biology 16, 1247-1255).
  • the cell is modified to alter expression of MAPK p38 ⁇ or p38 ⁇ during pre-differentiation of the cell model during the time reserve cells are formed, or during post-differentiation of the cell model.
  • the cell may be modified to over express, constitutively express, inducibly express, or substantially not express MAPK p38 ⁇ or p38 ⁇ .
  • the cell model can be a useful tool in elucidating the cellular mechanism for cell differentiation or self renewal.
  • the cell is a modified precursor cell, more preferably a modified stem cell or a myogenic cell line, preferably C2C12.
  • the agent that affects Notch signalling in the cell is DFK-167 (Enzyme Systems Products) and the precursor cell is a stem cell or C2C12.
  • Calbiochem produces the inhibitor MG132 and makes an inhibitor called gamma secretase inhibitor X.
  • MDL28170 is cited in De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J.S., Shroeter, E.H., Schrijvers, V., Wolfe, M.S., Ray, W.J., Goate, A. and Kopan, R.
  • Presenilin-1 and -2 are Molecular Targets for Gamma-Secretase Inhibitors. Journal of Biological Chemistry 275, 34086-34091.
  • the cell is modified to alter Notch signalling during pre- differentiation of the cell model during the time reserve cells are formed, or during post- differentiation of the cell model.
  • the cell may be modified to over express, constitutively express, inducibly express, or substantially not express components that play a role in Notch signalling of the cell.
  • the cell model can be a useful tool in elucidating the cellular mechanism for cell differentiation or self renewal.
  • the present invention is directed to a method for assaying an agent for a capability of influencing cell differentiation or self renewal, the method comprising:
  • stem cell The capacity for tissue regeneration via stem cells is an integral part of animal physiology. Furthermore, the clinical potential of stem cell therapies seems enormous, and the recent focus on adult stem cells avoids the sensitive ethical issues surrounding possible human therapies based on embryo-derived stem cells. Self-renewal is a key hallmark of all stem cells. This work is significant in that it seeks to define the molecular basis of stem cell self-renewal, seen as a dynamic equilibrium, in a muscle cell model. We believe that this information may be applicable to many stem cell systems. Stem cell implantation to effect regeneration in disease, and manipulation of the self-renewal capacity of endogenous stem cells, may be equally valid therapeutic goals. Our studies are innovative in that we have discovered a key player in muscle stem cell self-renewal and have established a cell culture system that may be valuable in screening for new stem cell markers, and for new ways to manipulate the self-renewal equilibrium.
  • Figure 1 is a schematic showing model for role of p38 during myogenisis (panel A) and model for reserve cells (panel B).
  • Figure 2 shows the results of SB treatment on a number of cell types and cell markers.
  • Figure 3 shows MLC p38 DR clone: (A) Differentiation at day 4 shows a complex mix of multinucleate myotubes (M - some outlined) and reserve cells (R) and; (B) differentiation at day 4 in the presence of SB 20 mM shows virtual "clearing" of reserve cells ( * ) with fusion into massive myosacs (F).
  • Figure 4 shows ld(1-3) mRNA expression is down-regulated during differentiation.
  • Figure 5 shows Western blot analyses of Id1 and Id2 protein expression.
  • A Id1 and Id2 protein expression in C2C12 myoblasts and during differentiation ⁇ SB 10 and 20 ⁇ M; ⁇ -tubulin present as loading control.
  • Figure 6 shows Western blotting analyses of the expression of Id1 and Id2 and cell cycle-related proteins p21 , cyclinDI and PCNA, relative to tubulin control, in C2C12 cells after 3 days of differentiation in low serum in the presence and absence of SB and after fractionated into mononuclear cells (m) and myotubes (t).
  • Figure 7 shows inhibition of p38 with SB increases the number of thin and elongated myocytes and induces a loss of mononuclear cells in high density cultures of primary mouse myoblasts.
  • the MAP kinases comprise several families of intermediate signalling kinases which mediate a diverse range of cellular responses(Kyriakis, J.M. & Avruch, J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81 , 807-69. (2001)).
  • p38 is one of the two stress-induced MAPKs, and has four isoforms ( ⁇ , ⁇ , ⁇ and ⁇ ), each the product of a different gene. They share 60-70% amino acid identity but vary in pattern of expression.
  • p38 ⁇ and ⁇ isoforms are sensitive to the specific inhibitor SB203580 (SB), which interacts with Thr- 106 in the hinge region of the ATP binding pocket (Wilson, K.P. et al. The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase. Chem Biol 4, 423- 31. (1997)).
  • SB specific inhibitor SB203580
  • Thr- 106 in the hinge region of the ATP binding pocket
  • p38 is classically activated by extracellular stresses such as- inflammation, UV irradiation and ischaemia/reperfusion, and by growth factors and cytokines such as FGF, IGF-1, IL1 and TNF- ⁇ .
  • p38 There are numerous downstream substrates identified for p38 including protein kinases such as MAPK-activated protein kinase 2 (MAPKAP-K2) and p38-activated kinase (PRAK), and transcription factors ATF-2 and MEF2A/C.
  • protein kinases such as MAPK-activated protein kinase 2 (MAPKAP-K2) and p38-activated kinase (PRAK), and transcription factors ATF-2 and MEF2A/C.
  • MAPKAP-2 MAPK-activated protein kinase 2
  • PRAK p38-activated kinase
  • p38 activates the critical myogenic transcription factors MyoD and MEF2C, and is essential for activation of PI3 kinase pathway, also necessary for myogenic differentiation (Cuenda, A. & Cohen, P. Stress-activated protein kinase-2/p38 and a rapamycin-sensitive pathway are required for C2C12 myogenesis. J Biol Chem 274, 4341-6. (1999)).
  • the knockout of p38 ⁇ dies at mid-gestation due to erythropoietic and placental defects (Tamura, K. et al. Requirement for p38 ⁇ in
  • This finding may relate to the observed heterogeneity in reserve cell self-renewal capacity observed in human primary muscle cultures, and to the fact that not all satellite cells express the above marker set. Early identification of the reserve cell will enable us to address the dynamics of their formation, and potentially to purify them by FACS for microarray and proteomics applications.
  • Notch is an evolutionarily conserved signalling system used by metazoans to control cell fate decisions during development (Artavanis-Tsakona, S., Rand, M., and Lake, R.J. (1999). Notch Signalling: Cell Fate Control and Signal Integration in Development. Science 284, 770-776).
  • the Notch protein is a single-pass transmembrane receptor activated by its membrane-tethered ligands Delta and Jagged.
  • the transcription factor Suppressor of Hairless (Su(H); CBF1/RJBk in mammals) appears to be the main downstream effector of Notch signalling and transcription factors of the Enhancer of split (E(spl)) family are their primary transcriptional targets.
  • ⁇ - secretase is a membrane-associated protein complex containing presenilin and nicastrin, which proteolytically cleaves Notch, liberating its intracellular domain and allowing it to passage to the nucleus.
  • the activity of this complex is essential for activation of Notch signalling by its ligands.
  • Notch signalling system controls an extraordinarily broad spectrum of cell fates and developmental processes and has been implicated in self-renewal of neural stem cells (Hitoshi, S., Alexson, T., Tropepe, V., Donviel, D., Elia, A.J., Nye, J.S., Conlon, R.A., Mak, T.W., Bernstein, A., and van der Kooy, D. (2002) Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes and Development 16, 846-858).
  • Notch has also been suggested to be important in skeletal myogenesis in that over-expression of a constitutively activate form of Notch inhibits myogenic differentiation in C2C12 myogenic cells in vitro (Kopan, R., Nye, J.S., and Weintraub, H. (1994).
  • the intracellular domain of mouse Notch a constitutively activated represser of myogenesis directed at the basic helix-loop-helix region of MyoD.
  • Notch signalling inhibits muscle cell differentiation through a CBF1- independent pathway.
  • Notch signalling was involved in reserve cell formation.
  • Using a Notch dependent luciferase reporter gene we observed that endogenous Notch activity was high in myoblasts, but fell progressively during their differentiation in low- serum medium. This suggests that Notch may play a role in maintaining the undifferentiated state in C2C12 cells.
  • the ⁇ -secretase inhibitor DFK-167 (Enzyme Systems Products), has been previously used to inhibit Notch cleavage and thus activation of the pathway dependent on Notch ligands (Wolfe, M. S., Citron, M., Diehl, T.S., Xia, W., Donkor, I. O., and Selkoe, D. J. (1998).
  • a substrate-based difluoro ketone selectively inhibits Alzheimer's gamma-secretase activity. J Med Chem 41, 6-9; De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J.S.,
  • DFK-167 appeared to induce precocious fusion, potentially through conversion of reserve cells to fusion-competent myocytes.
  • SB203580 and DFK-167 were used together, we observed a greater degree of reserve cell clearing compared with use of either drug alone.
  • Microarray studies cDNA microarray represents a powerful technology allowing mRNA expression to be examined on a very large scale. This technology has been made possible and manageable through advances in robotics, miniaturisation and bioinformatics. The resulting data generated represent a snapshot of the transcriptional state of the cell and current algorithms can cluster genes according to expression behaviour that may be involved in the same biochemical pathway.
  • Microarray technology has been applied to various aspects of biology and there have been several reports examining different aspects of myogenesis through microarray. These studies have examined the changes in gene expression during the different phases of myogenesis including proliferation of myoblasts and in differentiating and differentiated myocytes. Two of these studies utilised the C2C12 cell line as the model for myogenesis whilst the other one examined the effects of enforced MyoD expression into the mouse fibroblast cell line, C3H10T1/2.
  • RNA from each time-point was labelled with Cy5 then co-hybridised with a Cy3-labelled reference pool of C2C12 mRNA that comprised approximately equal proportions from 0, 24, 48 and 72 hours post-differentiation. The labelled RNA was then co-hybridised to the NIA/NIH 15K mouse clone set.
  • Ids The Id family of proteins (Ids) are important positive regulators of cellular proliferation and inhibitors of differentiation in various tissues. Ids contain a HLH domain which can heterodimerise with and sequester E-proteins from other bHLH transcription factors to negatively regulate differentiation, including MRFs in skeletal muscle. In addition, Id proteins lack the basic domain necessary for DNA binding. Furthermore, Id2, one of four Id isoforms in mammals, has been shown to exert an additional unique effect on cellular proliferation as it can bind pRb and induce release from E2F proteins. Id4 may also be able to target pRb but not Id1 or Id3. Id genes are widely expressed in various undifferentiated and proliferating cells during development but are down- regulated in differentiating cells.
  • Id genes have been previously shown to play important roles in myogenesis. Id3 is co-expressed with myogenin in somites and developing muscles during mouse embryogenesis. In myogenic cultures, mRNAs for lds1-3 are expressed in proliferating myoblasts and down-regulated during differentiation. In addition, over-expression of Id1, Id2 and Id3 in myoblasts in vitro negatively regulates myogenic differentiation. Recently, Id3 has been demonstrated to be under direct transcriptional regulation of MyoD in undifferentiated myoblasts. Our 15K NIA/NIH array chips contained mRNA of Id1, Id2 and Id3; Id4 was not represented although this isoform appears not to be expressed in skeletal muscle.
  • the data are expressed as the mean fold change of mRNA expression from baseline (myoblasts) to time post-differentiation (6, 10, 24, 48 and 72 hours). The results are the mean ( ⁇ SEM) from the two independent experiments. Consistent with previous data, we found that Id1, Id2 and Id3 expression was highest in proliferating C2C12 myoblasts and was rapidly down-regulated from the onset of differentiation, indicating that expression of these genes were sensitive to the effects of serum withdrawal ( Figure 4). Inhibition of p38 with SB further suppressed expression of Id1 and Id2 but not Id3, with the greatest difference in expression seen for Id2.
  • Id1 protein expression closely paralleled the mRNA expression results (Figure 5A). Expression was highest in myoblasts and was rapidly down-regulated by day 1 post-differentiation, and remained expressed at similar levels thereafter. SB induced a further reduction in expression in a dose-dependent manner at all time points post-differentiation.
  • Id2 protein expression was also very similar to the results from the microarray experiments. Id2 expression was highest in myoblasts and was down-regulated by day 1 post-differentiation ( Figure 5A). Furthermore, inhibition of p38 with SB resulted in a greater reduction in Id2 expression with each day of differentiation. After three days of differentiation, there was virtually no detectable Id2 expression in the SB-treated cultures. These data raised the possibility that Id2 expression may be restricted to reserve cells since SB induces a diminishment of this cell population. Alternatively, Id2 expression may be dependent on the formation of multinucleate myotubes which are absent in the presence of SB.
  • Id2 may be restricted to the reserve cell population
  • myotube and mononuclear cell compartments from a well- differentiated C2C12 culture, and examined each population for Id2 expression.
  • Markers of differentiation and proliferation were analysed in mononuclear cells (m) and multinucleate myotubes (t) following 3 days of differentiation in the absence or presence of SB.
  • Mononuclear cells were enriched for markers of proliferation (PCNA [proliferating cell nuclear antigen] and cyclin D1), and the anti-differentiation proteins Id1 and Id2, reflecting the relatively uncommitted phenotype of this cell type.
  • Myotubes were enriched for the early marker of muscle differentiation, p21.
  • cells were trypsinised and plated on 24 well plates coated with matrigel. Half of the cells were maintained in a proliferation medium containing 20% FBS (no bFGF) and the other half of the cells were switched to a differentiation medium containing 2% horse serum. Each set of cells was grown in the absence or presence of SB (20 ⁇ M) which was added at the onset of differentiation and replenished daily thereafter.
  • p38 appears to be necessary for the creation and/or maintenance of the reserve cell population, and required for terminal fusion of differentiated myocytes.
  • C2C12 model of skeletal myogenesis represents a valid template for studying the process of self-renewal in at least one class of muscle stem cells.
  • a myoblast could undergo symmetric and/or asymmetric cell divisions.
  • the common progeny could either be MyoD-positive myoblasts, or MyoD-negative reserve cells.
  • myoblasts may divide asymmetrically, giving rise to one reserve cell and one cell that is more committed.
  • proliferation and differentiation Our model recognises that conditions of high or low serum will increase or decrease the probability of a particular form of division occurring, rather than inducing an all-or-none phenomenon.
  • This invention relates to how skeletal muscles and other tissues renew their stem cell populations in normal physiology and disease.
  • the benefit of this work lies in its potential value to cell-based or drug therapies for degenerative diseases of the aged, such as stroke, ischaemic heart disease, and skeletal atrophy, as well as myopathies, burns, blood disorders and any other condition in which tissue regeneration via stem cells occurs.
  • the invention sheds light on the molecular basis of stem cell self-renewal in a muscle satellite cell culture system (C2C12), and in particular the role of the mitogen- activated protein kinase (MAPK) p38 and Notch in this process.
  • C2C12 muscle satellite cell culture system
  • MAPK mitogen- activated protein kinase
  • p38 and Notch are important components of the regulatory machinery that sets the proportion of self-renewing (reserve) and differentiating cells in this system. This may occur as a dynamic equilibrium, capable of modulation by physiological inputs.
  • important aspects are effects of modifying expression of p38 and Notch at the early stages of C2C12 differentiation; links between C2C12 reserve cells and in vivo muscle stem cells; and links between reserve cells and other stem cell systems.

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Abstract

L'invention concerne des procédés permettant de réguler la différenciation d'une cellule précurseur par traitement de ladite cellule précurseur avec un agent qui affecte l'activité de la protéine kinase activée par le mitogène (MAPK) p38 ou de la voie de signalisation Notch dans la cellule de façon que celle-ci subisse une différenciation ou un autorenouvellement. L'invention concerne également des modèles de cellules permettant d'étudier la différenciation et l'autorenouvellement de cellules.
PCT/AU2003/000518 2002-05-02 2003-05-02 Regulation de la differenciation et de l'autorenouvellement de cellules souches par manipulation de la mapk p38 et de la voie de signalisation notch WO2003093487A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003221625A AU2003221625A1 (en) 2002-05-02 2003-05-02 Control of stem cell differentiation and self renewal by manipulation of p38 or notch

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPS2094A AUPS209402A0 (en) 2002-05-02 2002-05-02 Cell differentiation and self renewal
AUPS2094 2002-05-02

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WO2003093487A1 true WO2003093487A1 (fr) 2003-11-13

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2004053144A2 (fr) * 2002-12-10 2004-06-24 Ottawa Health Research Institute Modulation de la differenciation de cellules souches par modulation de l'activite de la caspase-3
GB2444686B (en) * 2005-09-12 2010-08-25 Es Cell Int Pte Ltd Differentiation of pluripotent stem cells using p38 MAPK inhibitors or prostaglandins
US20120276064A1 (en) * 2011-04-05 2012-11-01 Blau Helen M Methods and compositions for rejuvenation and expansion of stem cells
US8883504B2 (en) 2003-06-27 2014-11-11 Asahi Kasei Kabushiki Kaisha Cell differentiation suppressing agent, method of culturing cells using the same, culture solution, and cultured cell line
CN114015647A (zh) * 2021-12-07 2022-02-08 中国农业科学院饲料研究所 一种鸡胚胸肌成肌细胞的分离培养方法
US11963964B2 (en) 2011-06-16 2024-04-23 President And Fellows Of Harvard College Small molecules for mouse satellite cell proliferation

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WO2002059285A1 (fr) * 2000-10-27 2002-08-01 Fred Hutchinson Cancer Research Center Methodes permettant d'immortaliser des cellules
WO2002077204A2 (fr) * 2001-03-23 2002-10-03 Axordia Limited Cellule embryonnaire
WO2002094842A2 (fr) * 2001-05-24 2002-11-28 University Of Western Ontario Regulation de la myogenese par modulation de l'activite de la proteine-kinase p38 associee aux membranes

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WO2002059285A1 (fr) * 2000-10-27 2002-08-01 Fred Hutchinson Cancer Research Center Methodes permettant d'immortaliser des cellules
WO2002077204A2 (fr) * 2001-03-23 2002-10-03 Axordia Limited Cellule embryonnaire
WO2002094842A2 (fr) * 2001-05-24 2002-11-28 University Of Western Ontario Regulation de la myogenese par modulation de l'activite de la proteine-kinase p38 associee aux membranes

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CONBOY I.M., RANDO T.A.: "The regulation of notch signalling controls satellite cell activation and cell fate determination in postnatal myogenesis", DEVELOPMENTAL CELL, vol. 3, no. 3, September 2002 (2002-09-01), pages 397 - 409, XP055182008, DOI: doi:10.1016/S1534-5807(02)00254-X *
KURODA K. ET AL.: "Delta-induced notch signalling mediated by RBP-J inhibits MyoD expression and myogenesis", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 11, 1999, pages 7238 - 7244 *
ZETSER A. ET AL.: "p38 Mitogen-activated protein kinase pathway promotes skeletal muscle differentiation: participation of the MEF2C transcription factor", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 8, 1999, pages 5193 - 5200, XP002251466, DOI: doi:10.1074/jbc.274.8.5193 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053144A2 (fr) * 2002-12-10 2004-06-24 Ottawa Health Research Institute Modulation de la differenciation de cellules souches par modulation de l'activite de la caspase-3
WO2004053144A3 (fr) * 2002-12-10 2004-08-12 Ottawa Health Research Inst Modulation de la differenciation de cellules souches par modulation de l'activite de la caspase-3
US8883504B2 (en) 2003-06-27 2014-11-11 Asahi Kasei Kabushiki Kaisha Cell differentiation suppressing agent, method of culturing cells using the same, culture solution, and cultured cell line
GB2444686B (en) * 2005-09-12 2010-08-25 Es Cell Int Pte Ltd Differentiation of pluripotent stem cells using p38 MAPK inhibitors or prostaglandins
AU2006292021B2 (en) * 2005-09-12 2012-05-31 Es Cell International Pte Ltd. Cardiomyocyte production
US9422521B2 (en) 2005-09-12 2016-08-23 Es Cell International Pte Ltd. Differentiation of pluripotent stem cells with a kinase inhibitor or PGI2
US20120276064A1 (en) * 2011-04-05 2012-11-01 Blau Helen M Methods and compositions for rejuvenation and expansion of stem cells
US11963964B2 (en) 2011-06-16 2024-04-23 President And Fellows Of Harvard College Small molecules for mouse satellite cell proliferation
CN114015647A (zh) * 2021-12-07 2022-02-08 中国农业科学院饲料研究所 一种鸡胚胸肌成肌细胞的分离培养方法

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