EP1501920A2 - Procede de fabrication de cellules presentant un potentiel de developpement augmente - Google Patents

Procede de fabrication de cellules presentant un potentiel de developpement augmente

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Publication number
EP1501920A2
EP1501920A2 EP03724890A EP03724890A EP1501920A2 EP 1501920 A2 EP1501920 A2 EP 1501920A2 EP 03724890 A EP03724890 A EP 03724890A EP 03724890 A EP03724890 A EP 03724890A EP 1501920 A2 EP1501920 A2 EP 1501920A2
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Prior art keywords
cells
kinase
induction
activated
hph2
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EP03724890A
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German (de)
English (en)
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Ulf R. Rapp
Bernd Neufeld
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RAPP, R. ULF, PROF. DR.
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Individual
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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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • the invention relates to a method for producing cells with increased development potential, cells obtainable with such a method and use of such cells.
  • embryonic and fetal phase an organism is formed by differentiating effector cells from stem cells. These embryonic or fetal stem cells have an increased development potential compared to somatic stem cells from adult tissues.
  • pluripotency of the embryonic or fetal stem cells since these can differentiate to all tissues, organs or cell types.
  • somatic stem cells for purposes other than in-vitro fertilization is opposed to serious ethical concerns. Therefore, a search for alternative sources for pluripotent or totipotent cells focuses on the generation of an increased development potential of somatic cells, in particular somatic stem cells from adult organisms, compared to the natural state. These research directions are encouraged by the realization that transdifferentiation of somatic stem cells is possible, for example neural ones Stem cells form blood cells, while blood stem cells can form brain and muscle cells. For this purpose, reference is made, for example, to the literature references US-6, 087, 168, US-6,093,531 and US-6,093,531.
  • Plasticity plays a particularly important role here because, with increased plasticity, those with good availability (quantity and / or easy removal), for example hematopoietic stem cells, can be used as starting cells.
  • 3pK belongs to a kinase family (MAPKAP kinases) that are activated by one or more members of the MAPK family (mitogen-activated protein kinase). 3pK is also known as MAPKAP kinase 3 (MAPK activated protein kinase 3).
  • MAPKAP kinases kinase family
  • MAPK activated protein kinase 3 MAPK activated protein kinase 3
  • the mechanisms for activating 3pK via the mitogenic kinase cascade are relatively well elucidated, for which reference is made, for example, to the literature references G. Sithanandam et al. ; Mol. Cell Biol. 16 (3), 868-876, and S. Ludwig et al. , Mol. Cell Biol. 16 (12), 6687-6697.
  • 3pK can also be via stress kinase cascades via p38 (S. Ludwig et al., Mol Cell Biol 16: 6687-6697 (1996); MM McLaughlin et al., J Biol Che 271: 8448-8492 (1996)) be activated, ie by induction of Rac (CDC42), for example by heat shock or proapoptotic substances such as TNF-alpha.
  • p38 stress kinase cascades via p38
  • p38 S. Ludwig et al., Mol Cell Biol 16: 6687-6697 (1996); MM McLaughlin et al., J Biol Che 271: 8448-8492 (1996)
  • be activated ie by induction of Rac (CDC42), for example by heat shock or proapoptotic substances such as TNF-alpha.
  • MAPKAP kinase is MK2, which is primarily activated via p38 as a cellular response to stress, for example by heat shock or cytokines such as TNF-alpha (J. Rouse et al., Cell 78: 1027-1037 (1994); K. Engel et al., J Cell Biochem 57: 321-330 (1995)).
  • 3pK and MK2 have a sequence homology of 75% at the amino acid level. Both are arranged in the non-activated state in the cell nucleus and leave it after activation or phosphorylation.
  • Other homologues are MAPKAP-Kl, RSK2, RSK3, MK4 and PRAK.
  • MAPKAP kinases such as 3pK or MK2.
  • the differential expression of homeotic genes determines the formation of different parts of the body along the longitudinal axis of an organism.
  • homeotic genes are activated in the right areas by transcription factors.
  • these factors are soon no longer active and the correct expression of homeotic genes in the course of the further development of the organism is inter alia from proteins of the so-called Polycomb-group (Pc-G) controlled.
  • Pc-G proteins keep genes that are no longer expressed after a certain developmental state suppressed. This property is also passed on to daughter cells. They form part of a cellular memory system, so they are part of an epigenetic expression control.
  • the Pc-G proteins are believed to produce a chromatin configuration that is inaccessible to transcription factors and thus inhibit the transcription of homeopathic genes.
  • the invention is based on the technical problem of specifying a method for increasing the plasticity of cells.
  • the invention teaches a method for producing cells increased development potential from cells, wherein the removed cells are cultivated in vitro and a native nuclear non-activated MAPKAP kinase of the cultivated cells is activated or an activated MAPKAP kinase is transported into the cell nucleus.
  • the term nuclear denotes a localization of the MAPKAP kinase in the cell nucleus, in particular in the chromate jelly.
  • a MAPKAP kinase can be activated if it can be phosphorylated with the result that the phosphorylated MAPKAP kinase can phosphorylate its substrates and is exported from the cell nucleus.
  • the said MAPKAP kinase can also be activated directly in vivo, the cells according to the invention then being formed directly in vivo.
  • the invention is based on the knowledge that nuclear MAPKAP kinases bind both with HPH2 and with BMI1.
  • the invention is based on the further finding that the activated, phosphorylated MAPKAP kinase in turn phosphorylates BMI1 before it is exported from the cell nucleus, which is also mobilized thereby, which ultimately makes a locus represented by the Pc-G complex accessible and transcribed.
  • This reactivates genes that are normally inactive at a certain stage of development, which leads the cells back to a status similar to that of an early embryonic stem cell, ie the plasticity of the cells used is increased.
  • the invention can in principle be used with all cell types, but is particularly suitable when using somatic stem cells. In detail, the following findings are based.
  • 3pK is a BMI1 kinase in vitro and develops a suppressive effect similar to other Pc-G proteins in an artificial repression assay. 3pK binds to putative full-length HPH2 and has the highest affinity for a 73 amino acid C-terminal fragment of HPH2, which encompasses the HDII / SEP domain. This region is part of the HPH2 dimerization domain, ie it comprises the alpha-helical HDII / SEP domain required for hetero- / homodimerization and for Bmil binding and overlaps with the domain for 3pK binding. The stronger binding to the said fragment compared to the full length can be due to a folding of full-length HPH2 over the HDII / SEP domain.
  • HPH2 The status of the phosphorylation of HPH2 is unclear and a phosphorylation of the putative full-length HPH2 by 3pK could not be observed in vitro. However, phosphorylation is not excluded either, since a mouse homolog of HPH2 (mPh.2, NCBI Accession U81491) with a long N-terminal attachment contains three potential 3pK phosphorylation sites. Since HPH1 and mouse mPhl proteins have the same length, it can be assumed that HPH2 also has the same length as mPh2.
  • 3pK is a kinase that phosphorylates BMI1. This plays an important role in that on the one hand the dissociation of Pc-G complexes and the BMI1 phosphorylation are interrelated and on the other hand hypophosphorylated BMI1 specifically in the Chromatin-associated protein fraction is kept out of the cell nucleus, while phosphorylated BMIl is not chromatin-bound. In summary, it was found that 3pK forms part of the Pc-G complex and is a BMIl kinase.
  • 3pK should therefore regulate the phosphylation-dependent Pc-G complex / chromatin interaction, namely by binding to HPH2, which provides 3pK in the Pc-G complex BMIl, with the result of its phosphorylation and release of the complex from the chromatin. Similar relationships were found in the case of MK2.
  • the invention further relates to cells obtainable by a method according to the invention and the use of cells according to the invention for the production of a pharmaceutical composition, in particular for the treatment of degenerative nerve diseases.
  • the increase in plasticity ensures colonization of the target tissue and differentiation of the introduced cells according to the invention into the desired cell type.
  • a particular aspect of the findings according to the invention of independent importance also lies in the use of a substance which promotes the expression or activation of a MAPKAP kinase, in particular 3pK, for the production of a pharmaceutical composition for the prophylaxis or treatment of cancer.
  • the tumor cells can be brought to expression of activated 3pK by genetic engineering measures.
  • the MAPKAP kinase can be 3pK, MNK1, MNK2, MSK1, MK2, MK4 or PRAK, preferably 3pK.
  • the MAPKAP kinase is preferably a wild-type MAPKAP kinase.
  • Activated MAPKAP kinase can be induced in the cell nucleus in various ways. So it is possible that the cells are transformed to overexpress an activated MAPKAP kinase. Activation by induction of p38 is also possible, the induction of p38 again being possible by induction of Rac and / or Ras.
  • the activation of 3pK can be carried out in detail by induction of the mitogenic kinase cascade, in particular induction of Raf, MEK and / or ERK, for example by incubation with a serum growth factor or with TPA.
  • the activation can also be carried out by inducing a stress kinase cascade, for example i) by treating the cells with conditions inducing stress kinase, in particular a heat shock, osmolarity shock or UV, or ii) by incubation in a physiologically effective dose with at least one Stress kinase cascade inducing substance, in particular cytokines such as IL-1, TNF-alpha, anisomycin, arsenite and / or alkylating agents.
  • cytokines such as IL-1, TNF-alpha, anisomycin, arsenite and / or alkylating agents.
  • a preferred aspect of the invention is therefore the generation or induction and / or maintenance of B-raf in target cells whose development potential is to be increased.
  • the cells used are atic stem cells, for example hematopoietic or neural stem cells.
  • the cells used should be autologous, which avoids undesirable immune reactions when administered or reimplated.
  • Human PKRSPA-3pK which carries the gene under the control of the Rous Sarcoma Virus (RSV), pEBG-3pK, which expresses the gene as a GST fusion protein under the control of the human EFla promoter, and the mutation of the lysine in the area of the putative ATP binding region of the 3pK (3pK K73M) are known from the literature reference S. Ludwig et al (see above). Another mutation at 73 or derivatization can also be used, which renders 3pK inactive.
  • pcDNA3HA-3pk was generated by inserting an Nhel-Spel fragment of pPC97-3pK into pcDNA3HA.
  • pPCH-3pK K73M was obtained by inserting the Sall-Notl fragment of pPC97-3pK into pPCH (available from C. Hagemann, MSZ, Würzburg), which is a pPC97 derivative in which the LEU nutrient cassette is replaced by the TRP marker of pPC86 (PM Chevray et al., Proc Natl Acad Sei USA 89 (13), 1992, 5789-5793).
  • the BamHI-Ba HI insert of pGEXKG-3pK was ligated into the yeast two hybrid vector P ⁇ S2.1 (Clontech) to obtain pAS2.1-3pK K73M.
  • pMT2SM-HA-bmi (mouse), ppuro GAL4 DB-Bmil and ppuro GAL4 DB were obtained from Mv Lohuizen (Amsterdam).
  • PPuro GAL4 DB 3pK was cloned by cutting pGEXKG-3pK with EcoRI and subsequently inserting this fragment in ppuro GAL4 DB.
  • pBEVY-GU-Xb i was by ligation of the EcoRI fragment from pPC97-Xbmi (available from A. Otte, Amsterdam) into pBEVY-GU (available from Ch.A. Miller, New La).
  • pGAD10-HPH2 (137-432 aa), hereinafter referred to as pGAD10-HPH2 (C-295 aa), and pGAD10-HPH2 (432 aa) are available from A. Otte and described in MJ Gunster et al., Mol Cell Biol. 17 (4), 2326-2335.
  • the yeast two hybrid screens were carried out using a human heart MATCHMAKER cDNA library, which is cloned into the yeast two hybrid vector pGADIO (Clontech).
  • the kinase-active 3pK K73M was used in the single copy plasmid pPCH.
  • the yeast strain CG-1945 was manipulated according to the MATCHMAKER Library User Manual (PT1020-1, Clontech) using the sequential transformation protocol. Positive clones were identified by growth on SD / -TRP / -LEU / -HIS plates and activity determination of the lacZ reporter gene in filter assays.
  • GAL4 AD-HPH2 C-73 aa
  • GAL4 AD-HPH2 C-145 aa
  • GAL4 AD-HPH2 C -198 aa
  • GAL4 AD-HPH2 C-209 aa
  • GAL4 AD-HPH2 C-295 aa
  • GAL4 AD-HPH2 C-432 aa
  • pAS2.1-3pK K73M and pGAD10-HPH2 were co-expressed with a third yeast expression vector, either pBEVY-GU without insert or PbEVY-GU-Xbmil, and on SD / - TRP / -LEU / -URA plates are cultivated and then subjected to the liquid culture ⁇ -galactosidase assay.
  • the human embryonic kidney cell line HEK293 which was used for immune complex kinase assays and coimmune precipitation experiments, was added to Dulbecco's modified Eagle's Medium (DMEM), supplemented with 10% (v / v) FCS (at 56 ° C for 30 min. Heat inactivated) 37 ° C cultivated in humidified air with 6% C02.
  • DMEM Dulbecco's modified Eagle's Medium
  • FCS at 56 ° C for 30 min. Heat inactivated
  • the human osteosarcoma cell line U20S GAL4-TKluc which is stably transfected with a TK-luciferase reporter plasmid with five GAL4 binding sites above the TK promoter and was used for the repression assay, was cultivated in DMEM with 10% FCS (v / v).
  • Polyclonal rabbit antiserum against bacterially expressed 3pK was obtained according to G. Sithananda (see above).
  • Anti-glutathione S-transferase (GST) antisera were obtained from rabbits which were immunized with bacterially expressed and purified GST. 1: 750 dilutions of both sera were used for immunoblots.
  • the monoclonal anti-HA tags (12CA5) were used in a concentration of 1 ⁇ g / ml for Western blots.
  • HEK293 and U-20S For the transfection of both cell lines HEK293 and U-20S, 5 x 10 A 5 cells were sown in a 10 cm dish and cultured for 24 hours in DMEM with 10% FCS before the transfection. The transfections were carried out using the calcium phosphate coprecipitation method using 5-10 ⁇ g DNA for HEK293 or 15 ⁇ g DNA for U-20S according to a modified Stratagene protocol (according to S. Ludwig et al., See above). HEK293 cells were starved 48 hours before transfection in DMEM with 0.3% FCS. To activate 3pK, HEK293 cells with 0.5 M sodium metaarsenite were used for 30 min. before the Harvest or with 20% FCS in combination with 100 ng tetradecanoylphorbol acetate (TPA) per ml 60 min. stimulated before harvest.
  • TPA tetradecanoylphorbol acetate
  • Transfected HEK293 cells were in Tritonlysis buffer (TLB, 20 mM Tris (pH 7.4), 50 mM sodium ⁇ -glycerophosphate, 20 mM sodium pyrophosphate, 137 mM NaCl, 10% (v / v) glycerol, 1% ( v / v) Triton X-100, 2 mM EDTA, 1 mM Pefabloc, 1 mM sodium orthovanadate, 5 M benzamidine, 5 mg per ml apritinin, 5 mg per ml leupeptin) on ice for 10 min. lysed. Cell solids were removed by centrifugation at 15,000 rpm for 10 min. away.
  • TLB Tritonlysis buffer
  • 50 mM sodium ⁇ -glycerophosphate 20 mM sodium pyrophosphate
  • 137 mM NaCl 10% (v / v) glycerol
  • In-complex complex kinase assay with 3pK Separately immunoprecipitated GST-3pK and HA-BMIl were combined and twice with a high NaCl concentration (25 mM Tris (pH 8), 1 M NaCl, 10% (v / v) glycerol, 0.1% SDS, 0.5% Sodium deoxycholate, 1% NP40, 2 mM EDTA (pH 8), 1 mM Pefabloc, 1 mM sodium orthova nadate, 5 mM benzamidine, 5 mg per ml aproptin, 5 mg per ml leupeptin) and specific kinase buffer (10 mM MgCl2, 25 mM ⁇ -glycerophosphate, 25 HEPES (pH 7.5), 5 mM benzamidine, 0.5 mM dithiothreitol, 1 mM sodium vanadate).
  • specific kinase buffer 10 mM MgCl2,
  • the kinase assays were carried out in the same buffers, supplemented with 5 mCi [gamma32-P] -ATP, 0.1 mM ATP, at 30 ° C. and the reaction after 30 min. ended by adding Laemmli buffer and 3 min. at 100 ° C. After gel electrophoresis and blotting on nitrocellulose membrane, the BMIl phosphorylation was analyzed using an X-ray film (Amersham).
  • this medium was replaced by DMEM with 10% FCS and 10 ⁇ g / l puromycin, in which the cells were cultured for a further 38-62 h for removal of all non-transfected cells.
  • Cells transfected with the non-puromycin-resistant vector pKRSPA served as a control for the complete elimination.
  • the remaining cells were then cultured for 6 hours in DMEM with 10% FCS, after which the cells were examined for luciferase and ⁇ -gal activity.
  • U20S were harvested in 100 ml lysis buffer (50 mM Na-2 (N-morpholino) ethanesulfonic acid, pH 7.8, 50 mM Tris HC1, pH 7.8, 10 mM dithiothreitol, 2% Triton X-100).
  • the untreated cell lysates were clarified by centrifugation and 50 ml of the pre-clarified cell extract was added to 50 ml luciferase assay buffer (125 mM Na-2 (N-morpholino) ethanesulfonic acid, pH 7.8, 125 mM Tris HCl, pH 7.8, 25 mM magnesium acetate, 2 mg / ml ATP) added.
  • FIG. 1 shows as a result that 3pK binds in vivo in the yeast two-hybrid system to the C-terminal part, including the homology domain II (HDII), of HPH2.
  • Figure 1A is a schematic representation of the 3pK interacting HPH2 clones of different lengths. Interestingly, both the domain required for homo- or heterodimerization with HPH2 or HPH1 (104 C-terminal amino acids) and the binding domain for BMIl (295 C-terminal amino acids) overlap with the interaction domain for 3pK binding.
  • the minimal 3pK binding domain of HPH2 contains a 67 amino acid long homology domain II (called HDII, SAM or SEP), which is identical or similar to corresponding domains of other polyhomeotic proteins (eg Mph2, Mphl / Rae-28 or HPH1).
  • HDII long homology domain II
  • SAM SAM
  • SEP 67 amino acid long homology domain II
  • the strongest interaction 3pK / HPH2 was determined with the smallest HPH2 fragment, as can be seen from the X-Gal test (FIG. 1B) and the quantitative two-hybrid test with ONPG as the substrate (FIG. IC).
  • Y190 yeast was cotransformed with pAS2.1 / 3pK K73M and the specified pGAD10 / HPH2 constructs and cultivated on SD / -TRP / -Leu plates.
  • the colonies obtained were streaked on filters on SD / -TRP / -LEU / -HIS and cultured for 12-24 at 30 ° C before the ß-Gal assay was performed.
  • a liquid culture ß-galactosidase assay with ONPG as substrate was carried out to quantify the binding affinities.
  • the activity for 3pK-HPH2 (C-73 aa) was set to 100% for comparison purposes.
  • FIG. 2 shows that 3pK interacts with HPH2 in vivo, even in the case of mammalian cells.
  • 3pK should be part of the Polycomb complex, which contains HPH2.
  • Coimmune precipitation experiments were carried out to analyze whether 3pK is present in a Pc-G together with HPH2.
  • HEK293 cells were co-transfected with GST tagged HPH2 (C-73 aa) and 3pK wt.
  • polyclonal anti-GST serum was used for the immunoprecipitation and immunoblotting of GST-HPH2 (C-73 aa) (FIG. 2A, lane 5).
  • the bands at 55 kDa are the result of a cross reaction with heavy chains of the antibodies in the samples and have nothing to do with the above relationships.
  • the procedure was as follows. HEK 293 cells were transfected with the plasmids indicated. 3pK was expressed either untagged by an RSV promoter (FIG. 2A) or as an HA-tagged version by a CMV promoter (FIG. 2B). The proteins were immunoprecipitated, as indicated, with either anti-3pK (A), anti-GST (A, B) or anti-HA (12CA5) (B) antibodies, washed twice in TLB buffer, separated on SDS-PAGE gels ( 10%) and blotted in nitrocellulose filters. Immunoblots were sampled with the appropriate antiserum and detected with ECL.
  • FIG. 3 shows results that demonstrate an in vivo interaction between 3pK and BMIl.
  • the detailed procedure was as follows. The cells were transfected with HA-BMI1 or pEBG-3pK DNA. Immunoprecipitation was performed with either anti-GST or anti-HA (12CA5) antibodies, followed by two washes with TLB buffer. The proteins were then on separated on SDS-PAGE gels (10%) and blotted in nitrocellulose filters. Immunoblots were sampled with the appropriate antiserum and detected with ECL. The bands at 55 kDa have the above cause.
  • OHMI1 is specifically coprecipitated with GST-3pK (lane 1) and vice versa (lane 3). Lanes 2 and 4 show no interactions with HA-BMIl or GST alone.
  • FIG. 4 shows that 3pK BMIl phosphorylates.
  • HEK 293 cells were transfected with HA-BMIl or pEBG-3pK DNA.
  • the kinase was stimulated with arsenite or serum / TPA for 60 min. activated.
  • the substrate and kinase were immunoprecipitated with anti-HA (12CA5) or anti-GST antibodies. Immune complexes were combined, washed twice under stringent conditions with RIPA buffer containing 1 mM NaCl and subjected to an in vitro kinase reaction in the presence of [gamma32P] ATP. Lanes 2 and 3 of the upper panel show that a strong phosphorylation occurs when 3pK is activated.
  • pBEVY-GU In addition to the transformation of pAS2.1 / 3pK K73M and pGAD10 / HPH2 (C-73 aa), either pBEVY-GU or pBEVY-GU / XBMIl were co-transformed.
  • XBMI1 Xenopus BMIl
  • Xenopus BMIl is 90% identical and 95% similar to human and mouse BMIlDa since an ONPG ß-galactosidase assay is 6 orders of magnitude less sensitive than with X-Gal, only the (C-73 aa) fragment was used.
  • Each bar represents 5 parallel experiments and the data shown are representative of 5 independent assays. It can be seen that XBMI1 reduces the bond strength from 100% to 44%.
  • FIG. 6A shows a schematic representation of the stably integrated luciferase reporter construct.
  • FIG. 6B shows the fusion proteins used with the GAL4 DNA-binding domain (Gal4 DB).
  • GAL4 DB GAL4 DNA-binding domain
  • Gal4 DB-BMI1 Expression of Gal4 DB-BMI1 was used as a positive control and exactly four-fold inhibition was used, as described by Alkema et al. reported (see above) found. The same result was also found for Gal4 DB-3pK, which shows that non-activated and Pc-G associated 3pK is able to recruit Pc-G proteins such as BMIl or HPH2 to the chromatin-rearranged promoter, where an inhibitory complex is formed ,

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Abstract

L'invention concerne un procédé de fabrication de cellules présentant un potentiel de développement augmenté, à partir de cellules ayant été prélevées sur un organisme. Selon ledit procédé, les cellules prélevées sont cultivées in vitro, une MAPKAP kinase non activée, nucléique, endogène des cellules cultivées est activée, ou une MAPKAP kinase activée est introduite dans le noyau cellulaire des cellules.
EP03724890A 2002-05-07 2003-05-07 Procede de fabrication de cellules presentant un potentiel de developpement augmente Withdrawn EP1501920A2 (fr)

Applications Claiming Priority (3)

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DE10220480A DE10220480C1 (de) 2002-05-07 2002-05-07 Verfahren zur Herstellung von Zellen mit erhöhtem Entwicklungspotential
DE10220480 2002-05-07
PCT/DE2003/001516 WO2003095633A2 (fr) 2002-05-07 2003-05-07 Procede de fabrication de cellules presentant un potentiel de developpement augmente

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WO (1) WO2003095633A2 (fr)

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US6218136B1 (en) * 1996-03-12 2001-04-17 Smithkline Beecham Corporation Methods of the identification of pharmaceutically active compounds
US7122360B1 (en) * 1998-06-24 2006-10-17 University Of Dundee Polypeptides, polynucleotides and uses thereof
US6087168A (en) * 1999-01-20 2000-07-11 Cedars Sinai Medical Center Conversion of non-neuronal cells into neurons: transdifferentiation of epidermal cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BEN-LEVY R. ET AL.: "Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2", CURRENT BIOLOGY, vol. 8, no. 19, 1998, pages 1049 - 1057, XP002453911, DOI: doi:10.1016/S0960-9822(98)70442-7 *
ENGEL K. ET AL.: "Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation", EMBO JOURNAL, vol. 17, no. 12, 1998, pages 3363 - 3371, XP002120627, DOI: doi:10.1093/emboj/17.12.3363 *
NEININGER A. ET AL.: "FRET-based detection of different conformations of MK2", EMBO REPORTS, vol. 2, no. 8, 2001, pages 703 - 708 *

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WO2003095633A3 (fr) 2004-03-04
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AU2003227513A1 (en) 2003-11-11
DE10220480C1 (de) 2003-09-11

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