CELLS EXHIBITING NEURONAL PROGENITOR CELL CHARACTERISTICS

Description

[0001] FIELD OF THE INVENTION

[0002] The invention relates to methods of making cells exhibiting neuronal progenitor cell characteristics from mesenchymal stem cells by regulating cellular pathways in the mesenchymal stem cells that are associated with glial transdifferentiation of the mesenchymal stem cells.

[0003] BACKGROUND OF THE INVENTION

[0004] A limitation in the research and treatment of Central Nervous System (CNS) or Peripheral Nervous System (PNS) diseases is the conventional recognition that terminally differentiated neurons are significantly limited in their ability to proliferate. Accordingly, any treatment of CNS or PNS diseases that requires transplant of terminally differentiated neurons is difficult to accomplish.

[0005] One proposed approach to overcoming this difficulty has been to culture large numbers of mitotic cells exhibiting neuronal progenitor cell characteristics ("CPCs"). Such cells could theoretically differentiate In vivo into neurons that could function In the treatment of CNS and/or PNS diseases. Alternatively, CPCs might be differentiated In vitro into neurons and then transplanted into patients. However, such CPCs are rare and difficult to isolate from donors. Therefore, conventionally, researchers have attempted to obtain CPCs from treated embryonic and fetal stem cells (collectively referred to as "embryonic stem cells" hereinafter).

[0006] Embryonic stem cells, which are pluripotent cells, have been used to generate a large variety of tissue types, and could be a source of CPCs. I. Weissman, Stem cells: units of development, units of regeneration, and units in evolution (Review). Cell 100, 157-168 (2000). However, the use of embryonic stem cells raises a number of ethical concerns, and so is a disfavored source of stem cells for production of CPCs. Additionally, embryonic stem cells can be tumorigenic, which generates safety concerns as to any transplant procedure that could potentially result in the delivery of embryonic stem cells to a patient such as creation of a CPC graft from embryonic stem cells.

[0007] Some researchers have attempted to utilize other types of stem cells, such as mesenchymal stem cells in the production of CPCs. United States Patent Application 20030003090 of Prockop, et al., filed January 2, 2003, and entitled "Directed in vitro differentiation of marrow stromal cells into neural cell progenitors" discloses that the expression levels of both NSE and vimentin were increased in human mesenchymal stem cells after their incubation with 0.5 millimolar IBMX and 1 millimolar dbcAMP. The increase in NSE and vimentin mRNAs coincided with the appearance of neural cells in the cultures. However, Prockop et al. reported that there was no change in the expression level of either MAP1B or TuJ-1. Since NSE, MAP1B, and TuJ-1 are early neuron-characteristic markers, and vimentin is an early marker for glia, Prockop et al. suggested that the hMSCs transdifferentiated in vitro into some early progenitors of either neurons or glia. However, the early progenitor cells of Prockop may be undesirable for use because they seem to display a very immature neuronal phenotype whose clinical efficacy is not well understood.
WO 03/066856, discloses the production of neural stem cells from mesenchymal stem cells after transfection with the Notch intracellular domain. The neural stem cells could be further differentiated into neural cells expressing MAP-2ab and TuJ-1 after trophic factor induction.

[0008] Accordingly, there is a scarcity of conventionally available and suitable sources of CPCs for use, for example, in the research and treatment of CNS or PNS diseases. Further, there is a scarcity of methods that can be used to produce such CPCs in a suitable manner suitable for use. What are needed are methods and compositions that overcome such problems.

[0009] SUMMARY OF THE INVENTION

[0010] In an aspect, the invention relates to a method of producing cells exhibiting neuronal progenitor cell characteristics from material comprising mesenchymal stem cells, the method comprising: regulating cellular pathways in the mesenchymal stem cells that are associated with glial transdifferentiation of the mesenchymal stem cells; wherein the cellular pathways are sufficiently regulated to induce at least a portion of the mesenchymal stem cells to transdifferentiate into cells exhibiting neuronal progenitor cell characteristics; characterised in that the regulating comprises inhibition of JAK/STAT signal transduction

[0011] In another aspect, the invention relates to a method for producing cells exhibiting neuronal progenitor cell characteristics comprising: incubating mesenchymal stem cells with a JAK/STAT inhibitor in an amount sufficient to induce at least a portion of the mesenchymal stem cells to transdifferentiate into cells exhibiting neuronal progenitor cell characteristics; with the proviso that the interacting does not comprise transfection of the mesenchymal stem cells with Notch intracellular domain.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The inventor has unexpectedly and surprisingly discovered that the problems and limitations noted above can be overcome by practicing the invention disclosed herein. The present invention addresses producing CPCs from mesenchymal stem cells (MSCs) by regulating cellular pathways in MSCs that are associated with glial transdifferentiation of the MSCs. Ways to make and use the invention are disclosed herein.

[0013] Cells exhibiting neuronal progenitor cell characteristics ("CPCs") are defined as, for the purposes of this invention, being cells that are mitotic, express nestin and other cell markers specific for neural precursor/neural progenitor cells, and are derived from MSCs. CPCs can differentiate into neurons, glia, and oligodendrocytes, and precursors of any of the foregoing. CPCs can be derived from MSCs according to methods disclosed herein. In an embodiment, human CPCs are EfnB2+, CD90-, and PDGF receptor beta-. These markers may be used to separate CPCs from MSCs using FACS following glial transdifferentiation of the MSCs according to the present invention. Suitable methods of handling CPCs are known conventionally, including those methods disclosed, for example, in published United States patent application 20020012903 to Goldman et al.

[0014] Generally, according to the invention CPCs may be produced by regulating cellular pathways in MSCs that are associated with glial transdifferentiation of the MSCs, with the cellular pathways being sufficiently regulated to induce at least a portion of the MSCs to transdifferentiate into CPCs.

[0015] A wide variety of regulating methods may be useful . These include, but are not limited to, modification of the medium and conditions in which cells are grown, if grown ex vivo; modifying the tissue environment in which the MSCs are present, if grown in vivo; or incubation of the MSCs with glial regulating agents. The precise manner of regulation does not matter, so long as glial transdifferentiation of the MASCs is effectively regulated, thus allowing differentiation of the MSCs into CPCs. Generally, the regulation of cellular pathways in MSCs that are associated with glial transdifferentiation of the MSCs takes place under conditions that are appropriate to maintain any MSCs or CPCs in a mitotic and viable state. Such conditions are known to one of skill in the art, and may be found in, for example, M. Kallos et al., Large-scale expansion of mammalian neural stem cells: a review. Med Biol Eng Comput. 2003 May;41 (3):271-82. Suitable conditions and techniques also can be found elsewhere in the literature both for cell culture and in vivo environments.

[0016] Regulation of the cellular pathways in MASCs that are associated with glial transdifferentiation of the MSCs may be accomplished by incubating the MSCs with glial regulating agents. Regulation of the cellular pathways in MSCs that are associated with glial transdifferentiation of the MSCs may be accomplished by incubating the MSCs with glial regulating agents in amounts sufficient to induce at least a portion of the MASCs to transdifferentiate into CPCs. Incubations may involve culturing MSCs in the presence of glial regulating agents with the intent that the glial regulating agents either interact with MSC cell surface receptors or are transported into the interior of the MASCs to interact with internal cellular pathways. Such transportation may be passive, such as diffusive transport, or active, such as through active transporters or a mixture of the two. In vitro incubations may be performed in a conventional manner, for instance incubating cultures of MSCs in alpha-MEM, or similar media, to which glial regulating agent(s) are added. Suitable incubation techniques may be found generally in the literature, including for example M. Kallos et al., Large-scale expansion of mammalian neural stem cells: a review. Med Biol Eng Comput. 2003 May;41(3):271-82. incubations may also take place in an in vivo environment, in which case glial regulating agents according to the invention may be administered either systemically or locally, and using conventional methods.

[0017] If the glial regulating agent is a protein or peptide, the method of incubation may be a transfection of the DNA coding for that protein or peptide into the MSCs. Transfections may be performed using commercially available transfection protocols, such as the Lipofectamine™ 2000 system available from lnvitrogen, or the Effectene™ transfection system available from Qiagen, or other conventional transfection protocols.
If the glial regulating agent is a protein or peptide, the method of incubation may be viral delivery of the glial regulating agent, using conventional viral vectors, such as Lentiviral vector systems (BLOCK-iT™ Lentiviral RNAi Expression System, Invitrogen) for stable expression and Adenoviral vector systems (BLOCK-iT™ Adenoviral RNAi Expression System, Invitrogen) for transient expression.

[0018] The incubations can take place at various times: serially, in parallel or combinations of serial and parallel incubations of the MSCs with various glial regulating agent(s).

[0019] In embodiments of the invention, there is the proviso that regulating cellular pathways in the MSCs that are associated with glial transdifferentiation of the MSCs does not comprise transfection of the MSCs with the intracellular domain of the Notch gene. In embodiments of the invention, there is the proviso that incubating the MSCs with glial regulating agents does not comprise transfection of the MSCs with the intracellular domain of the Notch gene.

[0020] Mesenchymal stem cells (MSCs) are defined as being, for the purposes of this invention, stem cells that are conventionally recognized as differentiating into several types of cells found primarily in connective tissues, including but not limited to, osteoblasts, adipocytes, chondrocytes, and myocytes. MSCs specifically exclude embryonic stem cells and fetal stem cells. MSCs may be obtained from a wide variety of animals, including but not limited to humans, and other mammals such as rats, mice, primates, pigs, cows, and sheep. MSCs may be obtained from a variety of tissues; preferred sources comprise bone marrow (marrow adherent stem cells) and cord blood. Useful sources for MSCs, and methods of obtaining them are described in Example 1 below, and elsewhere herein. In an embodiment, human MSCs useful in the practice of this invention express CD29, and CD90, but are negative for CD15, CD34, CD11b/c, CD31, CD45 and von Willebrand Factor.

[0021] In an embodiment, MSCs may be isolated from cord blood using techniques described in the literature. For instance, C. Campagnoli et al., Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. 1: Blood. 2001 Oct 15;98(8):2396-402., describes methods generally useful in obtaining fetal blood MASCs. In A. Erices et al., Mesenchymal progenitor cells in human umbilical cord blood. 1: Br J Haematol. 2000 Apr;109(1):235-42., there were described methods generally useful in obtaining MSCs from cord blood. L. Hou et al., Induction of umbilical cord blood mesenchymal stem cells into neuron-like cells in vitro. Int J Hematol. 2003 Oct;78(3):256-61, describes methods generally useful in obtaining purifying, and expanding human umbilical cord blood MSCs.

[0022] Glial regulating agents are defined as being substances that, among other characteristics, possess the characteristic of inhibiting transdifferentiation of MSCs into glial cells and promoting their transdifferentiation into CPCs. Glial regulating agents may act through a variety of different mechanisms to direct MASCs away from the glial fate. For instance, proneural basic helix-loop-helix transcription factors such as Mash 1, Math 1 and neurogenin 1 are believed to be activators of neuronal gene expression.

[0023] Proneural genes are believed to drive neuronal transdifferentiation of MSCs while inhibiting glial transdifferentiation. One mechanism by which glial transdifferentiation may be inhibited is through the regulation of STAT-mediated signal transduction. Signal transduction by STAT is believed to be triggered by phosphorylation which is believed to be catalyzed by the Janus family of tyrosine kinases (JAK). Inhibition of the JAK-STAT signal transduction therefore may regulate glial transdifferentiation pathways and promote the neuronal fate of MSCs.

[0024] Glial regulating agents used herein may comprise inhibitors or antagonists or agents that interfere with the signaling pathways for gliogenic factors. Glial regulating agents may also comprise agonists for neurogenesis, including neurogenic factors. Use of these agonists or factors may negatively control gliogenesis of MSCs in the practice of this invention. Glial regulating agents may comprise conventional forms of therapeutic molecules, including but not limited to small molecules, peptides, and whole or portions of gene products.

[0025] Glial regulating agents used in the invention include , JAK/STAT inhibitors, including inhibitors of STAT1 and STAT3. In certain embodiments, such JAK/STAT inhibitors may comprise RNAi for gene silencing of the JAK/STAT pathway, antisense oligonucleotides to down regulate the JAK/STAT pathway, or the small molecule JAK inhibitor 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline. Additional JAK/STAT inhibitors may be disclosed in United States Patent Application 20040209799 of George Vasios, published October 21, 2004; and United States Patent Application 20040052762 of Hua Yu et al., published March 18, 2004.

[0026] Alternative glial regulating agents include, but are not limited to, antagonists of BMP2 or 7 (bone morphogenic protein). Such antagonists may comprise whole or portions of gene products from genes expressing Noggin, Chordin, Follistatin, sonic hedgehog (SHH), or agonists of these genes.

[0027] Other alternative glial regulating agents include, but are not limited to, Hes inhibitors, including but not limited to Hes 1 and/or Hes 5 inhibitors. Such Hes inhibitors may comprise RNAi for gene silencing of Hes, or antisense oligonucleotides to down regulate Hes.

[0028] Further alternative glial regulating agents include, but are not limited to, inhibitors of Id-1. See S. Tzeng et al., Id1, Id2, and Id3 gene expression in neural cells during development. Glia. 1998 Dec;24(4):372-81. Such Id-1 inhibitors may comprise RNAi for gene silencing of Id-1, or antisense oligonucleotides to down regulate Id-1.

[0029] Other glial regulating agents include, but are not limited to, inhibitors of mammalian homologs of Drosophila glide/gcm (glial cells missing), including but not limited to Gcm1 (murine) or GCMB (human). See Y. Iwasaki et al., The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes. Development. 2003 Dec;130(24):6027-35. Epub 2003 Oct 22; and M. Kammerer et al., GCMB, a second human homolog of the fly glide/gcm gene. Cytogenet Cell Genet. 1999;84(1-2):43-7.). Such glide/gcm homolog inhibitors may comprise RNAi for gene silencing of glide/gcm homologs (such as Gcm1 (murine) or GCMB (human)), or antisense oligonucleotides to down regulate glide/gcm homologs (such as Gcm1 (murine) or GCMB (human)).

[0030] Further glial regulating agents include, but are not limited to, inhibitors of Sox9, which may be a transcription factor for oligodendrocyte lineage. See C. Stolt et al., The Sox9 transcription factor determines glial fate choice in the developing spinal cord. Genes Dev. 2003 Jul 1;17(13):1677-89.). Such Sox9 inhibitors may comprise RNAi for gene silencing of Sox9, or antisense oligonucleotides to down regulate Sox9.

[0031] Still further glial regulating agents include, but are not limited to, inhibitors of Neurogenin3, which may be a transcription factor for gliogenesis. Such Neurogenin3 inhibitors may comprise RNAi for gene silencing of Neurogenin3, or antisense oligonucleotides to down regulate Neurogenin3.

[0032] Other glial regulating agents include, but are not limited to, inhibitors of ciliary neurotrophic factor (CNTF). In certain embodiments, such CNTF inhibitors may comprise RNAi for gene silencing of CNTF, or antisense oligonucleotides to down regulate CNTF.

[0033] Glial regulating agents may also comprise whole or portions of gene products from genes expressing Wnt1, which strongly inhibits gliogenesis. See K. Tang et al., Wnt-1 promotes neuronal differentiation and inhibits gliogenesis in P19 cells. Biochem Biophys Res Commun. 2002 Apr 26;293 (1):167-73. Whole or portions of gene products from genes expressing Wnt1 may be administered by transfection or other conventional methods, such as gene therapy methods including viral vectors.

[0034] Alternatively, glial regulating agents may comprise whole or portions of gene products from genes expressing a subset of neural basic helix-loop-helix (bHLH) factors that play instructive roles during neurogenesis or are expressed in proliferating CPCs. Such glial regulating agents may comprise whole or portions of gene products from genes expressing Neurogenin1, Mash1, Math1, Math6, or NeuroD. Whole or portions of gene products from genes expressing the subset of neural basic helix-loop-helix (bHLH) factors, including but not limited to Neurogenin1, Mash1, Math1, Math6, or NeuroD, may be administered by transfection or other conventional methods, such as gene therapy methods including viral vectors.

[0035] Additionally, glial regulating agents may be administered singly or in combination. In a preferable embodiment, if a combination of glial regulating agents is used in the practice of the invention, then glial regulating agents that act on different glial regulating pathways may be selected. This may serve to enhance the overall glial regulating effect of the glial regulating agents.

[0036] For the purposes of this invention, isolating CPCs comprises isolating CPCs from non-CPC cells in a sample, such as MSCs that have not transdifferentiated into CPCs. Such isolation may comprise a single isolation or multiple isolations. If multiple isolations are to be performed, different types or techniques of isolation may be preferably used, as such different types or techniques of isolation may enhance isolation results. A wide variety of isolation methods are useful in the practice of this invention. Examples of such isolation methods include, but are not limited to flow cytometry (aka FACS sorting), magnetic separation techniques, and visual sorting. Immunocytochemistry may also be used in instances where cell viability is not critical.

[0037] FACS sorting can be performed using conventional FACS equipment and protocols with antibodies that are specific to epitopes associated with one or more characteristics of CPCs. One such epitope may be EfnB2 in the case of human CPCs. N. Ivanova et al., A stem cell molecular signature. Science 298(5593):601-4 (Oct 18, 2002). Antibodies additionally useful in the practice of the invention, although not necessarily for FACS sorting, comprise anti-CD15, anti-CD29, anti-CD34, anti-CD90, anti-CD31, anti-CD45, anti-CD11 b/c, and anti-von Willebrand factor. Cell populations FACS equipment useful in the practice of this invention include, but are not limited to, a FACScalibur™ analyzer with CeliQuest™ software (Becton Dickinson, Franklin Lakes, NJ), or FACS equipment available from Guava Technologies (Hayward, California).

[0038] Alternatively, isolation may be performed using magnetic separation techniques, such as the BioMag™ protocols and reagents, available in kit form from Qiagen. Immunocytochemistry may be another separation technique useful in the practice of this invention; useful Immunocytochemical methods are described in M. Dezawa et al., Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur, J. Neurosci. 14, 1771-1776 (2001). Immunocytochemical inspections may be made under a confocal laser scanning microscope, such as the Radians 2000 (Bio-Rad, Hertfordshire, UK). Conventional visual cell sorting techniques may be used in the practice of this invention.

[0039] Neurons are defined as, for the purposes of this invention, being any of the impulse-conducting cells that constitute the brain, spinal column, and nerves, consisting of a nucleated cell body with one or more dendrites and a single axon. Biochemically, neurons are characterized by reaction with antibodies for neurofilament-M, beta3-tubulin, and TuJ-1. These reactions may be used to isolate neurons or cells exhibiting one or more characteristics of neurons using techniques such as FACS sorting. Neural cells are also characterized by secreting neurotransmitters, neurotransmitter synthetases or neurotransmitter-related proteins, for example neuropeptide Y and substance P.

[0040] Neurotrophic agents are defined as being, for the purposes of this invention, substances that, among other characteristics, possess the characteristic of causing or promoting the differentiation of CPCs into neurons or cells that exhibit one or more characteristics of neurons. Neurotrophic agents useful in the practice of this invention comprise but are not limited to basic-fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), and forskolin (FSK). Neurotrophic agents may be combined with the CPCs of the present invention using cell handling techniques known in the art. Preferred methods may be found generally in PCT/JP03/01260 of Dezawa et al. In a preferred embodiment, bFGF, CNTF and FSK are combined with CPCs in cell culture in amounts effective to cause or promote the differentiation of CPCs into neurons or cells that exhibit one or more characteristics of neurons.

[0041] Glial cells are defined as, for the purposes of this invention, being any of the cells that make up the network of branched cells and fibers that support the tissue of the central nervous system. Glial cells include, but are not limited to astrocytes, Schwann cells, oligodendrocytes, and microglia.

[0042] Genes are defined as, for the purposes of this invention, being a set of connected transcripts, wherein a transcript is a set of exons produced via transcription followed (optionally) by pre-mRNA splicing. Gene products are defined as, for the purposes of this invention, being proteins translated from genes. Portions of genes are defined as, for the purposes of this invention, being a subset of a gene. Portions of gene products are defined as, for the purposes of this invention, being a subset of a gene product.

[0043] Patient means an animal, typically a mammal, and more typically, a human, that is the subject of medical observation or study.

[0044] CPCs produced according to the invention may be administered to patients through a variety of methods, including but not limited to infusion through an injection cannula, needle or shunt, or by implantation within a carrier, e.g., a biodegradable capsule, but other routes of administration, are also within the scope of the invention. Inventive routes of administration comprise local and systemic routes. Local administration may preferably include administration to targeted portions of the CNS or PNS, and preferably includes intraparenchymal routes. Systematic routes of administration comprise parenteral routes, with intravenous (i.v.), or intra-arterial (such as through internal or external carotid arteries) administration being preferred routes of systemic administration. Systemic administration techniques can be adapted from techniques used to administer precursor cells generally, such as those disclosed in D Lu et al., Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury. J Neurotrauma. 2001 Aug;18(8):813-9.

[0045] Amounts of CPCs administered to a patient may be determined clinically, using conventional dose ranging techniques, and clinical assessments of a particular patient's disease.

EXAMPLES

Materials and Methods:

[0046] MSCs: Rat MSCs (Wistar strain) are isolated and cultured as described in M. Dezawa et al., Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur. J. Neurosci. 14, 1771-1776 (2001). As for human MSCs, commercially purchased MSCs (PT-2501, BioWhittaker. Walkersville, MD) and MSCs obtained from healthy donors are used. Cells may be maintained in alpha-MEM (Sigma, M-4526) with 10% fetal bovine serum (FBS).

[0047] In the case of obtaining MSCs from healthy donors, an initial step is to obtain bone marrow aspirate from healthy donors using conventional aspiration techniques. The cell aspirate is then transferred into a 50 ml tube. 13 ml Histopaque is then carefully underlayed, using a 10 ml pipette. The tube is then centrifuged @ 2000rpm for 20 minutes. Cells at the interphase are then harvested. PBS is then added (at least 3x the volume of the interphase) and the mixture centrifuged @ 1200 rpm. The cells are washed twice more with PBS. The cell pellet is then resuspended in DMEM +10% FCS, and the cells counted. 5x10^6 cells are replated per T-75 tissue culture flask, and incubated for 3 days. On day 4, the non-adherent cells are removed and the flask washed three times with medium. The adherent cells are allowed to grow in the flask. When the cells reach 20-30% confluence, the content of 2-3 flasks are pooled and re-plated in one T-75 flask. When the cells in this pooled reach confluence, the cells are trypsinized using 0.05% trypsin and 0.02% EDTA. The cells are then washed and counted. The cells are then resuspended in Sigma alpha MEM +10% FBS (M-4526). In experiments where lipofection is to be used, it is important to insure that the medium contains no I-glu. Glutamine is not added. The cells are expanded for 2-4 weeks and are frozen in early passages.

[0048] Cell surface markers in rat and human MSCs are analyzed with fluorescence activated cell analysis (FACS). In an embodiment, the MSCs express CD29, and CD90, but are negative for CD34, CD31, CD45, CD11 b/c, and von Willebrand Factor consistent with M. Pittenger et al., Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147 (1999); and J. Kohyama et al., Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent. Differentiation 68, 235-244 (2001) (Fig. 1A). The same result is obtained by immunocytochemistry. Adipogenic, chondrogenic and osteogenic differentiation of both rat and human MSCs are confirmed according to the method described by M. Pittenger et al., Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147.

[0049] FACS analysis. Cells at a final concentration of 1 x 10^7/ml are incubated with 1 mg of a monoclonal antibody in phosphate buffered saline (PBS). Incubations may be performed in the presence of 10 mg of mouse immunoglobulin to prevent nonspecific antibody binding. In rat MSCs, mouse anti-CD34 (Santa Cruz Antibodies) and hamster anti-CD29 (PharMingen, San Diego, CA) may be labeled with FITC, and controls may be incubated either with FITC-labeled anti-mouse or hamster IgG. Mouse anti-CD54 and CD11b/c may be all purchased from PharMingen. Mouse anti- von Willebrand factor and other antibodies needed in the practice of this invention may be obtained commercially. Controls may include cells stained either with non-immune mouse serum. If these antibodies are conjugated to FITC, the cells may be subsequently incubated with 1mg of FITC-conjugated anti-mouse IgG. In human MSCs, phycoerythrin labeled mouse anti-CD34, CD29, CD54, CD11b/c and von Willebrand factor may be used, and controls may include cells stained with phycoerythrin labeled and-mouse IgG. Data may be acquired and analyzed on a FACScalibur with CellQuest software (Becton Dickinson, Franklin Lakes, NJ).

[0050] Immunocytochemistry. The general procedure is described in M. Dezawa et al., Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur. J. Neurosci. 14, 1771-1776 (2001). After the fixation of cells with 4% paraformaldehyde in phosphate-buffered saline (PBS), they are incubated with primary antibodies for overnight at 4 Deg. C. Antibodies to nestin may be purchased commercially from PharMingen. Cells may be then incubated with secondary antibodies to Alexa Fluor 488 or 546 conjugated anti-mouse IgG, IgM, or rabbit IgG (Molecular Probes, Eugene, OR) for 1 hour at room temperature, and TOTO-3 iodide (Molecular Probes) counter staining may be performed. Inspections may be made under a confocal laser scanning microscope (Radians 2000, Blo-Rad, Hertfordshire, UK).

Example 1:

[0051] Human MSCs (PT-2501, BloWhiftaker, Walkersville, MD) were allowed to grow in culture in alpha-MEM containing 10% FBS generally according to E. Sudbeck et al., Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin. Cancer Res. 5, 1569-1582 (1999). The MSCs were incubated with 40 ug/ml 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P131, Calbiochem, San Diego, CA) for two days. The WHI -P131 was washed off after 2 days.

Example 2:

[0052] Human MSCs, prepared according to the Materials and Methods section, are allowed to grow in culture in alpha-MEM containing 10% FBS generally according to E. Sudbeck et al., Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin. Cancer Res. 5, 1569-1582 (1999). Once the culture has reached 90% confluence, several RNAs, designed using the BLOCK-iT™ RNAi Designer (Invitrogen) are incubated with the culture for a period of time sufficient to silence Sox9 expression, using BLOCK-iT™ protocols available from Invitrogen. Resulting CPCs are isolated from untransdifferentiated MSC's by sequential selection using magnetic beads coated with appropriate antibodies such as anti-EfnB2 (positive selection for CPCs), anti-CD90 (negative selection for CPCs), and anti-PDGF receptor beta (negative selection for CPCs). The antibodies and coated beads may be obtained from commercial suppliers. The cells in PBS are incubated with coated beads for 1 hr. @ room temperature. The cell-bound beads are removed using a magnet. The CPCs are washed free of the antibody and re-suspended In alpha-MEM containing 10% FBS and allowed to proliferate.

Example 3:

[0053] Human MSCs, prepared according to the Materials and Methods section, are allowed to grow in culture in alpha-MEM containing 10% FBS generally according to E. Sudbeck et al., Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-indudng antileukemic agents. Clin. Cancer Res. 5, 1569-1582 (1999). Antisense oligomers to Hes 1 are generated according to techniques disclosed in any one of H. Moulton et al., Peptide-assisted delivery of steric-blocking antisense oligomers. Curr Opin Mol Ther. 2003 Apr;5(2):123-32; C. Stein et al., Antisense oligonucleotides as therapeutic agents-is the bullet really magical? Science. 1993 Aug 20;261(5124):1004-12; or C. Helena, The anti-gene strategy: control of gene expression by triplex-forming-oligonucleotides. Anticancer Drug Des. 1991 Dec;6(6):569-84. Once the MSC culture reaches 90% confluence, the Hes-1 antisense oligomers are incubated with the MSCs for a period sufficient to downregulate Hes-1 expression, according to techniques disclosed in any of the three references cited in this example. Resulting CPCs are isolated from untransdifferentiated MSC's by sequential selection using magnetic beads coated with appropriate antibodies such as anti-EfnB2 (positive selection for CPCs), anti-CD90 (negative selection for CPCs), and anti-PDGF receptor beta (negative selection for CPCs). The antibodies and coated beads may be obtained from commercial suppliers. The cells in PBS are incubated with coated beads for 1 hr. @ room temperature. The cell-bound beads are removed using a magnet. The CPCs are washed free of the antibody and re-suspended in alpha-MEM containing 10% FBS and allowed to proliferate.

Example 4:

[0054] Wnt-1 expression plasmids are generated according to M. Sen et al., Regulation of fibronectin and metalloproteinase expression by Wnt signaling in rheumatoid arthritis synoviocytes. Arthritis Rheum. 2002 Nov;46(11):2867-77. Human MSCs, prepared according to the Materials and Methods section, are allowed to grow in culture in alpha-MEM containing 10% FBS generally according to E. Sudbeck et al., Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin. Cancer Res. 5, 1569-1582 (1999). Once the culture reaches 90% confluence, the MSCs are incubated with the Wnt-1 expression plasmids for two days at 37 deg C and 5% CO2 using the Lipofectamine™ 2000 reagent and protocols available from Invitrogen. After the two days of incubation, the culture is selected for transfected cells using conventional selection techniques for a period of 10 days. Resulting CPCs are isolated from untransdifferentiated MSC's by sequential selection using magnetic beads coated with appropriate antibodies such as anti-EfnB2 (positive selection for CPCs), anti-CD90 (negative selection for CPCs), and anti-PDGF receptor beta (negative selection for CPCs). The antibodies and coated beads may be obtained from commercial suppliers. The cells in PBS are incubated with coated beads for 1 hr. @ room temperature. The cell-bound beads are removed using a magnet. The CPCs are washed free of the antibody and re-suspended in alpha-MEM containing 10% FBS and allowed to proliferate.

Example 5:

[0055] The cells produced according to Example 1 were placed in Minimum Essential Medium Alpha Eagle Modification (M4526, Sigma Co.) containing 20% fetal bovine serum (14-501 F, Lot #61-1012, BioWhittaker Co.). 5 microM of forskolin (344273, Calbiochem, La Jolla, CA), 10 ng/ml of recombinant human basic fibroblast growth factor (100-18B, Peprotech EC, Ltd., London, UK) and 10 ng/ml of ciliary neurotrophic factor (557-NT, R&D Systems, Minneapolis, MN) were added. The culture was grown for 3 days, at which point cells exhibiting neuronal characteristics were recognized, with the result of 29.46+/-3.0% of MAP-2ab-positive cells. MAP-2ab was analyzed for using Western blotting, with cell lysates prepared from incubated cells, and 50 ug of lysate proteins electrophorased on 5% and 10% SDS-polyacrylamide gel. Antigens to MAP-2 (1:500, Chemicon) were detected using alkaline phosphatase.

Example 6:

[0056] The cells exhibiting neuronal characteristics of Example 5 are harvested, and grown to 90% confluence in culture in alpha-MEM containing 10% FBS generally according to E. Sudbeck et al., Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin. Cancer Res. 5, 1569-1582 (1999). Next, 5 mM of forskolin (344273, Calbiochem), 10 ng/ml of basic fibroblast growth factor (100-18B, Peprotech EC, Ltd.) and 50 ng/ml of ciliary neurotrophic factor (557-NT, R&D Systems) are added to the cell culture.

[0057] The cells are grown for ten days in the presence of the neurotrophic agents, and then are analyzed for the characteristic morphology of neural cells and for positive reaction for antibodies against MAP-2 (MAB364, Chemicon), neurofilament (814342, Boehringer Manheim) and nestin (BMS4353, Bioproducts)

PUBLICATIONS

[0058] Bishop, A.E., Buttery, L.D. & Polak, J.M. Embryonic stem cells (Review). J. Pathol. 197, 424-429 (2002).

[0059] Weissman, I.L. Stem cells: units of development, units of regeneration, and units in evolution (Review). Cell 100, 157-168 (2000).

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[0061] Dezawa, M. et al. Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur. J. Neurosci. 14, 1771-1776 (2001).

[0062] Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41-49 (2002).

[0063] Eglitis, M.A., Dawson, D., Park, K.W. & Mouradian, M.M. Targeting of marrow-derived astrocytes to the ischemic brain. Neuroreport 10, 1289-1292 (1999).

[0064] Kopen, G.C., Prockop, D.J. & Phinney, D.G. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc. Natl. Acad. Sci. U S A 96, 10711-10716 (1999).

[0065] Terada, N. et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416, 542-545 (2002).

[0066] Wagers, A.J., Sherwood, R.I., Christensen, J.L. & Weissman, I.L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297, 2256-2259 (2002).

[0067] Woodbury, D., Schwarz, E.J., Prockop, D.J. & Black, I.B. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364-370 (2000).

[0068] Kohyama, J. et al. Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent. Differentiation 68, 235-244 (2001).

[0069] Sanchez-Ramos, JR. Neural cells derived from adult bone marrow and umbilical cord blood. J. Neurosci. Res. 69, 880-893 (2002).

[0070] Lundkvist, J. & Lendahl, U. Notch and the birth of glial cells (Review). Trends Neurosci. 24, 492-494 (2001).

[0071] Morrison, S.J. et al. Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 101, 499-510 (2000).

[0072] Gaiano, N., Nye, J.S. & Fishell, G. Radial glial identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26, 395-404 (2000).

[0073] Nye, J.S., Kopan, R. & Axel, R. An activated Notch regulates neurogenesis and myogenesis but not gliogenesis in mammalian cells. Development 120, 2421-2430 (1994).

[0074] Yamamoto, N. et al. Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor. J. Biol. Chem. 276, 45031-45040 (2001).

[0075] Shibata, T. et al. Glutamate transporter GLAST is expressed in the radial glia-astrocyte lineage of developing mouse spinal cord. J. Neurosci. 17, 9212-9219 (1997).

[0076] Yamasaki, M. et al. 3-Phosphoglycerate dehydrogenase, a key enzyme for I-serine biosynthesis, is preferentially expressed in the radial glia/astrocyte lineage and olfactory ensheathing glia in the mouse brain. J. Neurosci. 21, 7691-7704 (2001).

[0077] Gregg, C.T., Chojnacki, A.K. & Weiss, S. Radial glial cells as neuronal precursors: the next generation? J. Neurosci. Res. 69, 708-713 (2002).

[0078] Roy, N.S. et al. In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat. Med. 6, 271-277 (2000).

[0079] Ip, N.Y. The neurotrophins and neuropoietic cytokines: two families of growth factors acting on neural and hematopoietic cells (Review). Ann. N. Y. Acad. Sci. 840, 97-106 (1998).

[0080] Grosse, G. et al. Expression of Kv1 potassium channels in mouse hippocampal primary cultures: development and activity-dependent regulation. J. Neurosci. 20, 1869-1882 (2000).

[0081] Morrison, S.J. Neuronal differentiation: proneural genes inhibit gliogenesis (Review). Curr. Biol. 11, R349-351 (2001).

[0082] Sun, Y. et al. Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 104, 365-376 (2001).

[0083] Ishibashi, M. et al. Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system. EMBO J. 13, 1799-1805 (1994).

[0084] Furukawa, T. et al. rax, Hes1, and notch1 promote the formation of Muller glia by postnatal retinal progenitor cells. Neuron 26, 383-394 (2000).

[0085] Kahn, M.A. et al. Ciliary neurotrophic factor activates JAK/Stat signal transduction cascade and induces transcriptional expression of glial fibrillary acidic protein in glial cells. J. Neurochem. 68, 1413-1423 (1997).

[0086] Seidel, H.M., Lamb, P. & Rosen, J. Pharmaceutical intervention in the JAK/STAT signaling pathway (Review). Oncogene 19, 2645-2656 (2000).

[0087] Burdon, T., Smith, A. & Savatier, P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 12, 432-438 (2002).

[0088] Nakashima, K. et al. BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis. Proc. Natl. Acad. Sci. U S A. 98, 5868-5873 (2001).

[0089] Stork, P.J. & Schmitt, J.M. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 12, 258-266 (2002).

[0090] Neufeld, B. et al. Serine/Threonine kinases 3pK and MAPK-activated protein kinase 2 interact with the basic helix-loop-helix transcription factor E47 and repress its transcriptional activity. J. Biol. Chem. 275, 20239-20242 (2000).

[0091] Shimazaki, T., Shingo, T. & Weiss, S. The ciliary neurotrophic factor/leukemia inhibitory factor/gp130 receptor complex operates in the maintenance of mammalian forebrain neural stem cells. J. Neurosci. 21, 7642-7653 (2001).

[0092] Kurooka, H., Kuroda, K. & Honjo, T. Roles of the ankyrin repeats and C-terminal region of the mouse notch1 intracellular region. Nucleic Acids Res. 26, 5448-5455 (1998).

[0093] Franklin, J.L. et al. Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1. Curr. Biol. 9, 1448-1457 (1999).

[0094] Akerud, P. et al. Differential effects of glial cell line-derived neurotrophic factor and neurturin on developing and adult substantia nigra dopaminergic neurons. J. Neurochem. 73, 70-78 (1999)

[0095] Sakurada, K., Ohshima-Sakurada, M., Palmer, T.D. & Gage, F.H. Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development 126, 4017-4026 (1999).

[0096] Kim, J.H. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418, 50-56 (2002).

[0097] Song, H.J., Stevens, C.F. & Gage, F.H. Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat. Neurosci. 5, 438-445 (2002).

[0098] Weinmaster, G., Roberts, V.J. & Lemke, G. A homolog of Drosophila Notch expressed during mammalian development. Development 113, 199-205 (1991).

[0099] Peyton, M. et al. BETA3, a novel helix-loop-helix protein, can act as a negative regulator of BETA2 and MyoD-responsive genes. Mol. Cell Biol. 16, 626-33 (1996).

[0100] Rozovsky, 1. et al. Estradiol (E2) enhances neurite outgrowth by repressing glial fibrillary acidic protein expression and reorganizing laminin. Endocrinology 143, 636-646 (2002).

[0101] Sudbeck, E.A. et al. Structure-based design of specific inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin. Cancer Res. 5, 1569-1582 (1999).

[0102] Seta, Y., Toyono, T., Takeda, S. & Toyoshima, K. Expression of Mash1 in basal cells of rat circumvallate taste buds is dependent upon gustatory innervation. FEBS Lett. 444, 43-46 (1999).

[0103] Schwaiger, F.W. et al. Peripheral but not central axotomy induces changes in Janus kinases (JAK) and signal transducers and activators of transcription (STAT). Eur. J. Neurosci. 12, 1165-1176 (2000).

[0104] Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31-40 (2000).

[0105] Tanaka, H. et al. Role of serotonergic neurons in L-DOPA-derived extracellular dopamine in the striatum of 6-OHDA-lesioned rats. Neuroreport 10, 631-634 (1999).

Claims

1. A method of producing cells exhibiting neuronal progenitor cell characteristics from material comprising mesenchymal stem cells, the method comprising: regulating cellular pathways in the mesenchymal stem cells that are associated with glial transdifferentiation of the mesenchymal stem cells; wherein the cellular pathways are sufficiently regulated to induce at least a portion of the mesenchymal stem cells to transdifferentiate into cells exhibiting neuronal progenitor cell characteristics;
characterised in that the regulating comprises inhibition of JAK/STAT signal transduction and does not comprise transfection of the mesenchymal stem cells with Notch intracellular domain.

2. A method of claim 1, wherein the regulating comprises incubation of a JAK/STAT inhibitor with the mesenchymal stem cells.

3. A method for producing cells exhibiting neuronal progenitor cell characteristics comprising:

incubating mesenchymal stem cells with a JAK/STAT inhibitor in an amount sufficient to induce at least a portion of the mesenchymal stem cells to transdifferentiate into cells exhibiting neuronal progenitor cell characteristics.;

characterised in that the method does not comprise transfection of the mesenchymal stem cells with Notch intracellular domain.

4. A method of any one of claims 1 to 3, wherein the mesenchymal stem cells are selected from the group consisting of human mesenchymal stem cells, rat mesenchymal stem cells, mouse mesenchymal stem cells, primate mesenchymal stem cells, pig mesenchymal stem cells, cow mesenchymal stem cells, and sheep mesenchymal stem cells.

5. A method of any one of claims 2 to 4, wherein the incubation comprises transfection of the JAK/STAT inhibitor into the mesenchymal stem cells.

6. A method of any one of the preceding claims, wherein the mesenchymal stem cells are derived from cord blood.

7. A method of any one of the preceding claims, wherein the mesenchymal stem cells are derived from bone marrow.

8. A method of any one of claims 2 to 7, wherein the JAK/STAT inhibitor is selected from:

RNAi for gene silencing of the JAK/STAT pathway;

antisense oligonucleotides to down-regulate the JAK/STAT pathway;

inhibitors of STAT1 and STAT3;

the small molecule JAK inhibitor 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline.

Ansprüche

1. Verfahren zur Herstellung von Zellen, die Eigenschaften neuraler Progenitorzellen aufweisen, aus Material, das mesenchymale Stammzellen umfasst, wobei das Verfahren Folgendes umfasst: das Regulieren zellulärer Signalwege in den mesenchymalen Stammzellen, die mit glialer Transdifferenzierung der mesenchymalen Stammzellen zusammenhängen, worin die zellulären Signalwege hinlänglich reguliert werden, um zumindest einen Teil der mesenchymalen Stammzellen dazu zu bringen, zu Zellen zu transdifferenzieren, die Eigenschaften neuraler Progenitorzellen aufweisen;
dadurch gekennzeichnet, dass das Regulieren die Hemmung von JAK/STAT-Signaltransduktion umfasst und dass es nicht die Transfektion der mesenchymalen Stammzellen mit der intrazellulären Notch-Domäne umfasst.

2. Verfahren nach Anspruch 1, worin das Regulieren die Inkubation eines JAK/STAT-Inhibitors mit den mesenchymalen Stammzellen umfasst.

3. Verfahren zur Herstellung von Zellen, die Eigenschaften neuraler Progenitorzellen aufweisen, umfassend:

das Inkubieren mesenchymaler Stammzellen mit einem JAK/STAT-Inhibitor in einer Menge, die ausreicht, um zumindest einen Teil der mesenchymalen Stammzellen dazu zu bringen, zu Zellen zu transdifferenzieren, die Eigenschaften neuraler Progenitorzellen aufweisen;

dadurch gekennzeichnet, dass das Verfahren nicht die Transfektion der mesenchymalen Stammzellen mit der intrazellulären Notch-Domäne umfasst.

4. Verfahren nach einem der Ansprüche 1 bis 3, worin die mesenchymalen Stammzellen aus der aus mesenchymalen Stammzellen von Menschen, mesenchymalen Stammzellen von Ratten, mesenchymalen Stammzellen von Mäusen, mesenchymalen Stammzellen von Primaten, mesenchymalen Stammzellen von Schweinen, mesenchymalen Stammzellen von Kühen und mesenchymalen Stammzellen von Schafen bestehenden Gruppe ausgewählt sind.

5. Verfahren nach einem der Ansprüche 2 bis 4, worin die Inkubation die Transfektion des JAK/STAT-Inhibitors in die mesenchymalen Stammzellen umfasst.

6. Verfahren nach einem der vorangegangenen Ansprüche, worin die mesenchymalen Stammzellen aus Nabelschnurblut stammen.

7. Verfahren nach einem der vorangegangenen Ansprüche, worin die mesenchymalen Stammzellen aus Knochenmark stammen.

8. Verfahren nach einem der Ansprüche 2 bis 7, worin der JAK/STAT-Inhibitor aus
RNAi zum Verstummen eines Gens des JAK/STAT-Signalwegs; Antisense-Oligonucleotiden zum Herabregulieren des JAK/STAT-Signalwegs; Inhibitoren von STAT1 und STAT3;
dem Kleinmolekül-JAK-Inhibitor 4-(4'-Hydroxyphenyl)amino-6,7-dimethoxychinazolin
ausgewählt ist.

Revendications

1. Méthode de production de cellules présentant des caractéristiques de cellules neuronales progénitrices à partir d'un matériau comprenant des cellules souches mésenchymales, la méthode comprenant:

réguler des chemins cellulaires dans les cellules souches mésenchymales qui sont associés à une trans-différentiation gliale des cellules souches mésenchymales; où les chemins cellulaires sont suffisamment régulés pour induire au moins une portion des cellules souches mésenchymales pour la trans-différentiation dans des cellules présentant des caractéristiques de cellules neuronales progénitrices;

caractérisée en ce que la régulation comprend l'inhibition de la transduction du signal JAK/STAT et ne comprend pas la transfection des cellules souches mésenchymales avec le domaine intracellulaire du Notch.

2. Méthode selon la revendication 1, où la régulation comprend l'incubation d'un inhibiteur de JAK/STAT avec des cellules souches mésenchymales.

3. Méthode de production de cellules présentant des caractéristiques de cellules neuronales progénitrices comprenant:

incuber des cellules souches mésenchymales avec un inhibiteur de JAK/STAT en une quantité suffisante pour induire au moins une portion des cellules souches mésenchymales pour la trans-différentiation dans des cellules présentant des caractéristiques de cellules neuronales progénitrices;

caractérisée en ce que la méthode ne comprend pas la transfection des cellules souches mésenchymales avec le domaine intracellulaire du Notch.

4. Méthode selon l'une quelconque des revendications 1 à 3, où les cellules souches mésenchymales sont sélectionnées dans le groupe consistant en cellules souches mésenchymales humaines, cellules souches mésenchymales du mésenchymales du porc, cellules souches mésenchymales de la vache et cellules souches mésenchymales des moutons.

5. Méthode selon l'une quelconque des revendications 2 à 4, où l'incubation comprend la transfection de l'inhibiteur de JAK/STAT dans les cellules souches mésenchymales.

6. Méthode selon l'une quelconque des revendications précédentes, où les cellules souches mésenchymales sont dérivées du sang de cordon ombilical.

7. Méthode selon l'une quelconque des revendications précédentes, où les cellules souches mésenchymales sont dérivées de la moelle osseuse.

8. Méthode selon l'une quelconque des revendications 2 à 7, où l'inhibiteur de JAK/STAT est sélectionné parmi:

ARNi pour le silençage des gènes du chemin JAK/STAT;

des oligonucléotides anti-sens pour régler vers le bas le chemin JAK/STAT;

des inhibiteurs de STAT1 et STAT3;

l'inhibiteur de JAK petite molécule 4-(4'-hydroxyphényl)amino-6,7-diméthoxyquinazoline.