WO2009024748A1 - Facteurs neurothropiques issus de cellules souches - Google Patents

Facteurs neurothropiques issus de cellules souches Download PDF

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WO2009024748A1
WO2009024748A1 PCT/GB2008/002719 GB2008002719W WO2009024748A1 WO 2009024748 A1 WO2009024748 A1 WO 2009024748A1 GB 2008002719 W GB2008002719 W GB 2008002719W WO 2009024748 A1 WO2009024748 A1 WO 2009024748A1
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neural
cells
stem cells
neural stem
cell
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PCT/GB2008/002719
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Stefan Przborski
Adam Croft
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Reinnervate 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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem 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
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)

Definitions

  • the invention relates to methods for promoting the differentiation of neural stem cells/neural progenitor cells that facilitate the production of differentiated neurones.
  • stem cell represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues.
  • Stem cells can be pluripotent or multipotent.
  • a pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism.
  • a multipotent cell has a restricted ability to form differentiated cells and tissues.
  • adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues.
  • a totipotent cell is a cell that has the ability to form all the cells and tissues that are found in an intact organism, including the extra-embryonic tissues (i.e. the placenta).
  • Totipotent cells comprise the very early embryo (8 cells) and have the ability to form an intact organism and are not as such considered stem cells.
  • multipotent stem cells examples include mesenchymal and neural stem cells.
  • Mesenchymal stem cells or MSCs differentiate into a variety of cell types that include osteoblasts, chondrocytes, myocytes, adipocytes and neurones. Typically MSCs are obtained from bone marrow.
  • Neural stem cells are multipotent stem cells that generate the main cell phenotypes of the nervous system. NSCs have been isolated from the brain and spinal cord. A sub-population of neural stem cells is referred to as neural progenitor cells. These have a more restricted potential to differentiate into neural tissue.
  • stem cell therapies are exploring different sources of pluripotent and multipotent stem cells and cell culture conditions to efficiently differentiate stem cells into cells and tissues suitable for use in tissue repair, in particular the replacement of damaged neurones either through trauma or disease.
  • cell growth conditions that produce cells that are functional and express typical cell markers associated with a specific differentiated cell-type.
  • Methods to derive MSCs in vitro are known in the art.
  • WO2005/1113751 describes a method for the isolation and expansion of MSCs under serum free conditions comprising culturing MSCs on a cell culture support material that includes extracellular matrix proteins.
  • WO2006/121445 describes the isolation of MSCs and their differentiation into endothelial cells.
  • the differentiated endothelial cells have utility in would healing and inflammatory disease.
  • a further application of MSCs is in the repair of cardiac muscle.
  • US2007/0003530 describes the administration of a preparation of MSCs to the heart as a liquid injectible preparation or combined with a matrix.
  • KR20040016785 describes a method for the differentiation of MSCs into neuronal cells by increasing expression of transcription factors involved on neuronal cell differentiation.
  • KR20020082239 describes the differentiation of MSCs to neuronal cells through the addition of growth factors to cell cultures. It is apparent that there exist methods to differentiate MSCs into different cell-types, in particular neurones.
  • US2007/0020608 describes a method for the generation of neural progenitor cells from pluripotent blastocyst derived stem cells.
  • WO2006/100088 a method for the derivation of neural stem cells from dental progenitor cells is disclosed that involves the culturing of dental progenitor cells as spheres that are disaggregated into single cells that re-aggregate into spheres which differentiate into neural stem cells.
  • US2006240553 and US2006263876 each describe the isolation neural stem cells from different sources; the central nervous system and neural crest respectively.
  • the present disclosure relates to a method for the differentiation of primarily neurones from neural stem cells by co-culturing with neural induced MSCs.
  • neural induced MSCs are to be defined as MSCs that express neural antigens.
  • oligodendroctyes at the expense of astrocytes (astrocytes are specialised glial cells found in the brain).
  • the primary function of oligodendrocytes is in the myelination of axons in the central nervous system.
  • the present disclosure relates to an alternative culture method that results predominantly in the formation of neurones.
  • Two populations of MSCs have been used in the disclosed method; MSCs induced to express neural antigens (nestin+, Tuj-1+, GFAP+) and neural antigen negative MSCs.
  • MSCs provide instructive signals that are able to direct the differentiation of NSC and promote axonal development in neuronal progeny. The data indicates that the nature of MSC derived signals is dependent not only on the microenvironment but on the developmental status of the MSCs.
  • a culture of neural induced mesenchymal stem cells or conditioned medium prepared from neural induced mesenchymal stem cells in the formation of differentiated neurones from neural stem cells or neural progenitor cells.
  • a culture of neural induced mesenchymal stem cells or conditioned medium prepared from neural induced mesenchymal stem cells in the formation neurites from differentiated neurones.
  • a culture of neural induced mesenchymal stem cells or conditioned medium prepared from neural induced mesenchymal stem cells in the promotion of neurone cell survival.
  • said mesenchymal stem cells and neural stem/ progenitor cells are mammalian.
  • said mammalian cells are mouse, rat or human.
  • said neural stem cells/ progenitor cells are brain neural stem/progenitor cells; preferably hippocampal neural stem cells or embryonic neural stem cells.
  • said mesenchymal stem cells and neural stem/ neural progenitor cells are autologous.
  • said neural induced mesenchymal stem cells express and secrete the polypeptide pigment epithelium derived factor (PEDF).
  • PEDF polypeptide pigment epithelium derived factor
  • an in vitro method for the differentiation of neurones comprising: i) providing a cell culture vessel comprising a suspension of mesenchymal stem cell neurospheres and neural stem cell neurospheres wherein the mesenchymal neurospheres and neural stem cell neurospheres are separated by a permeable membrane to allow the diffusion of cell differentiation factors; ii) culturing the cells under conditions that facilitate the differentiation of neural stem cell neurospheres into neurones.
  • Vessel is defined as any means suitable to contain the above described cell culture. Typically, examples of such a vessel is a petri dish; cell culture bottle or flask; multiwell culture dishes.
  • the suspension comprising mesenchymal stem cell neurospheres is provided in a separate vessel, but in liquid contact with the other components of the cell culture medium.
  • the separate vessel is an insert or similar means which allows the cells contained in a mesenchymal stem cell fraction to proliferate but prevents cell contact with the neural stem cell neurospheres contained in the cell culture vessel.
  • said mesenchymal stem cells and said neural stem cells are human.
  • said neural stem cells/ progenitor cells are brain neural stem/progenitor cells; preferably hippocampal neural stem cells or embryonic neural stem cells.
  • said induced mesenchymal stem cells express the neural specific gene marker Tuj-1. In a further preferred method of the invention said induced mesenchymal stem cells express the neural specific gene markers Tuj-1 and GFAP.
  • said induced mesenchymal stem cells express the neural specific gene markers Tuj-1, GFAP and nestin.
  • induced mesenchymal stem cells down regulate the expression of mesenchymal stem cell gene marker smooth muscle actin.
  • said induced mesenchymal stem cells express and secrete the polypeptide pigment epithelium derived factor (PEDF).
  • PEDF polypeptide pigment epithelium derived factor
  • said PEDF polypeptide is a mammalian PEDF.
  • mammalian PEDF polypeptide is mouse, rat or human.
  • said human PEDF polypeptide is encoded by a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 9a, 9c or 9e, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleic acid sequence in Figure 9a, 9c or 9e.
  • nucleic acid molecule encodes a polypeptide comprising an amino acid sequence as represented in Figure 9b, 9d or 9f.
  • condition medium comprises a PEDF polypeptide.
  • said neural stem cells express the neural stem cell specific gene marker Tuj-1.
  • said neural stem cells express the neural stem cell specific gene markers Tuj-1 and GFAP.
  • said neural stem cells express the neural stem cell specific gene markers Tuj-1 , GFAP and RIP.
  • the expression of neural gene markers is known in the art as a measure of differentiation. Tuj-1 and nestin are associated with neurone differentiation; GAFP is an astrocyte gene marker and RIP identifies cells with an oligodendrocyte fate.
  • a method for the formation of neurites from neurones comprising: i) forming a preparation of neurones and neural induced mesenchymal stem cells; ii) providing cell culture conditions conducive to the formation of neurites from said neurones.
  • Neurite outgrowth is an essential event in neuronal development and in the formation and remodelling of synapses and in response to damage, either as a consequence of trauma or disease.
  • a cell culture vessel comprising: i) a suspension of neural induced mesenchymal stem cells; and ii) neural stem cells/ neural progenitor cells.
  • an isolated cell culture comprising: i) a suspension of neural induced mesenchymal stem cells; and ii) neural stem cells/ neural progenitor cells.
  • said neural stem cells/ progenitor cells are brain neural stem/progenitor cells; preferably hippocampal neural stem cells or embryonic neural stem cells.
  • conditioned medium in the differentiation of neural stem cells or neural progenitor cells into predominantly neurones wherein said condition medium is derived from neural induced mesenchymal stem cells.
  • said conditioned medium comprises at least PEDF.
  • kits comprising: i) mesenchymal stem cells; and ii) neural stem cells/neural progenitor cells.
  • said neural stem cells/ progenitor cells are brain neural stem/progenitor cells; preferably hippocampal neural stem cells or embryonic neural stem cells.
  • said kit includes cell culture medium and optionally an instruction manual to direct the use of the kit in the formation of neurones.
  • said mesenchymal stem cells and neural stem cells/ neural progenitor cells are autologous.
  • said neural stem cells/ progenitor cells are brain neural stem/progenitor cells; preferably hippocampal neural stem cells or embryonic neural stem cells.
  • Cell combinations according to the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example be, intravenous, intraperitoneal, intramuscular, intra-cavity, or subcutaneous.
  • the cells of the invention are administered in effective amounts.
  • An "effective amount" is that amount of the combined cell composition that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
  • a neurodegenerative disease is a condition that results, either directly or indirectly, in the deterioration of neurones which over time results in a gradual functional impairment which ultimately results in death.
  • said medicament is for the treatment of neurodegenerative disease.
  • said neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Cockanes syndrome, Creutzfeldt Jacob disease, Huntington's disease, HIV associated dementia, multiple sclerosis, spinocerebellar ataxia, or spinal muscular atrophy.
  • neurone damage is the result of trauma.
  • said trauma is spinal cord injury.
  • nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 9a, or a nucleic acid sequence that hybridizes under stringent hybridization conditions to the nucleic acid sequence in Figure 9a in the manufacture of a medicament in the repair and/or replacement of damaged neurones.
  • an effective amount of a preparation of neural induced mesenchymal stem cells that express a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 9a, or a nucleic acid sequence that hybridizes under stringent hybridization conditions to the nucleic acid sequence in Figure 9a, in the repair and/or replacement of damaged neurones.
  • said medicament is for the treatment of neurodegenerative disease.
  • neurone damage is the result of trauma.
  • said trauma is spinal cord injury.
  • a bioreactor comprising: i) a suspension of neural induced mesenchymal stem cells or conditioned medium isolated from neural induced mesenchymal stem cells; and ii) neural stem cells/progenitor cells.
  • the bioreactor is a rotary bioreactor.
  • said neural stem cells/ progenitor cells are brain neural stem/progenitor cells; preferably hippocampal neural stem cells or embryonic neural stem cells.
  • said conditioned medium comprises PEDF.
  • Bioreactors are known in the art and provide means for the large scale production of cells. For example, Chen et al (Stem Cells (2006) 24(9): 2052-2059) describes a 3D rotary bioreactor adapted for the expansion of human mesenchymal stem cells. Commercially available rotary bioreactors can be purchased from http://www.svnthecon.com.
  • said bioreactor comprises at least two separate cell culture chambers adapted to be in liquid contact with each other to facilitate the passage of differentiation factors between said cell culture chambers thereby facilitating differentiation of neural stem cells/neural progenitor cells.
  • said cell culture chambers are separated by a permeable membrane.
  • said bioreactor comprises at least one cell culture chamber in fluid contact with a further chamber wherein said further chamber comprises conditioned medium isolated from neural induced mesenchymal stem cells which is adapted to perfuse said cell culture chamber thereby facilitating the differentiation of neural stem cells/neural progenitor cells.
  • Figure 1 Generation of MSC cellular spheres positive for neural antigens following transfer to serum free media supplemented with EGF (10ng/ml) and FGF (10ng/ml).
  • Photomicrographs show the presence of small aggregations of cells, which begin to develop on the bottom of the culture flask within 3-5 days (A). Free-floating spheres were evident within 7-10 days of culture (B). Scale bars 50 ⁇ vn.
  • the open peaks indicate IgG isotype control corresponding to the antibodies in which they were generated.
  • the solid peaks indicate are counts of the cell population that is positive for the antibody indicated in each individual histogram. The number of positive cells is shown on the y-axis and the fluorescence intensity of staining on the x-axis.
  • FIG. 2 MSC soluble factors promote the differentiation of embryonic NSC/progenitor cells into distinct cell lineages.
  • progenitor cells were plated on poly-L-ornithine-coated dishes in the absence of growth factors and the presence of 0.5% FCS (control).
  • FCS 0.5% FCS
  • differentiation was initiated under identical conditions but in the presence of induced or non-induced MSCs. After 12 days, cells were fixed and immuno-stained with monoclonal antibodies directed against Tuj-1 (A, neuron), GFAP (B, astrocyte) or RIP (C, oligodendrocyte). Some cultures were dual stained for GFAP (D, green) and Tuj-1 (D, red). All cells were counterstained with Hoechst 33342 (blue). Scale bars: 50 ⁇ m.
  • Figure 3 Quantification of cellular differentiation under co-culture conditions. The number of cells positive (%) for each cell type marker was determined for each culture condition (mean + SEM; 10 fields of view) in 12d cultures. All data shown are from at least three experiments in parallel cultures with error bars representing standard error. Significant differences are indicated with an asterisk (*P ⁇ 0.05, **P ⁇ 0.01).
  • Figure 5 Figure 5.8 Effects of MSCs on the survival, proliferation and neuronal commitment of striatal NSC/progenitor cells.
  • A Proliferation of progenitor cells was accessed by BrdU incorporation. Parallel cultures were incubated with BrdU (2.5uM) on different time points for 24 hours, immediately followed by fixation and staining for BrdU.
  • FIG. 6 MSCs promote neuritogenesis of the Tuj-1 positive (neuronal) cell progeny of NSC/progenitor cells in 12-day cultures.
  • D-E Further examples of extensive neurite outgrowth in co-cultures of induced MSCs with neural progenitor cells are illustrated in D-E.
  • G-H Quantification of neurite outgrowth in control or co-culture conditions (induced or non-induced MSCs). After 12 days, cells were fixed and stained with Tuj-1 to identify neuronal progeny.
  • FIG. 7 MSCs promote the neurite outgrowth of differentiating N2a cells.
  • Undifferentiated N2a cells A were grown under standard culture conditions (DMEM+
  • Control N2a were differentiated by the removal of serum for 12 days either alone (B) or co-cultivated with induced (C) or non-induced (D) MSCs. After 12 days, cells were fixed and stained with Tuj-1 to identify neurites. Cells were then photographed and the average (E) and maximum (F) length of neurites was quantified from 10 randomly selected fields of view for each measurement. All data are mean values + SEM from 3 independent experiments in parallel cultures. Significant differences from the control group are indicated with an asterisk (*P ⁇ 0.05).
  • Figure 8 illustrates polyacrylamide gel electrophoresis of MSC conditioned medium
  • Figure 9a is the nucleic acid sequence of human PEDF;
  • Figure 9b is the amino acid sequence of human PEDF;
  • Figure 9c is the nucleic acid sequence of mouse PEDF;
  • Figure 9d is the amino acid sequence of mouse PEDF;
  • Figure 9e is the nucleic acid sequence of rat PEDF; and
  • Figure 9f is the amino acid sequence of rat PEDF.
  • Rat MSCs were isolated as previously described (Croft and Przyborski, 2006). Cells were cultured in complete culture medium (CCM; Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% fetal calf serum (FCS) (Invitrogen), 100 U/ml penicillin, 100 ug/ml streptomycin (Invitrogen) and 2 ⁇ M L-glutamine (Sigma) and 1 x nonessential amino acids (Invitrogen)) in T-75cm 2 tissue culture flasks (Nunc). Cells were grown in CCM at 37°C and 5% CO 2 until >70% confluence at which stage cultures were passed by enzymatic dissociation.
  • CCM complete culture medium
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • penicillin 100 ug/ml
  • streptomycin 100 ug
  • Rat MSCs were washed with phosphate buffered saline (PBS) and detached by incubation with 0.25% trypsin and 0.1% EDTA for 5-10 minutes at 37°C.
  • CCM was added to inactivate the trypsin.
  • the cells were centrifuged at 450 x g for 10 minutes, the medium was removed and cells were resuspended in 1-10ml of CCM.
  • the cells were counted in duplicate using a hemacytometer and then plated at a density of 10 cells / cm 2 for expansion. Passage 8 (approximately 25 population doublings) cells were used herein.
  • rat MSCs The mutipotentcy of isolated rat MSCs was verified by their capacity to differentiate into mesodermal derivatives (bone and fat) in vitro and their cell surface expression of CD90, CD73, CD44 and absence of CD45 and CD11 b (data not shown). NSC/progenitor cells were isolated from the mesencephalon of day 14 rat embryos.
  • Pregnant female Wistar rats at the specified gestational age of 14 days (the day of conception was confirmed by the presence of a vaginal plug, embryonic day 0) were killed by cervical dislocation and the uteri were aseptically removed and transferred to Petri dishes containing sterile Dulbecco's phosphate buffered saline (PBS) with 30% glucose and penicillin (20 units/mL), streptomycin (20 mg/mL). E14 striata were isolated and titurated in DMEM/F12 with a sterile Pasteur pipette. The cell suspension was filtered with a 70 um-mesh and viable cells were estimated by typan blue exclusion.
  • PBS sterile Dulbecco's phosphate buffered saline
  • the cells were plated (1 x 106 cells/75-cm2 uncoated tissue culture flask (Nunc)) in a chemically defined serum-free medium DMEM/F12 including 0.6% glucose, 2mM L- glutamine, 3mM sodium bicarbonate and 5mM HEPES buffer, supplemented with N-2 (a multi-component cell culture supplement), EGF (10ng/ml, Sigma) and FGF (10ng/ml, Sigma).
  • N-2 a multi-component cell culture supplement
  • EGF 10ng/ml, Sigma
  • FGF 10ng/ml, Sigma
  • the mouse neuroblastoma N2a cell line was originally obtained from the American Tissue Culture Collection. Cells were seeded in T-25 tissue culture flasks (Gibco) plates at a density of 3 x 104 cells/cm2 and grown in DMEM (Gibco) supplemented with 2 mM
  • Rat hippocampal cells were purchased from Chemicon International http://www.chemicon.com/
  • Differentiation of neurospheres under control conditions was initiated by plating spheres on poly-L-ornithine coated tissue culture 12-well plates. Cells were transferred to serum free media (DMEMF12) devoid of growth factors but supplemented with N2 and heparin (10 units per ml). In co-culture assays, differentiation was initiated under identical conditions but in the presence of Millipore cell culture inserts in which MSCs were seeded at a density of 10,000 cells. In this system the MSCs share the same media environment as the NSC/progenitor cells and are separated only by a porous membrane through which diffusible factors can pass.
  • DEMF12 serum free media
  • N2 and heparin 10 units per ml
  • MSCs or MSC spheroids were seeded into 24mm diameter membrane cell culture inserts (Sigma) and put into six-well culture trays (Nunc).
  • Cell proliferation was accessed by addition of 1OuM BrdU (S-phase marker) (Sigma) in the culture medium for a period of 24 hours 0, 1 , 2 and 3 days after plating, immediately followed by fixation in 4% PFA and analysis of total cell numbers and BrdU incorporation.
  • 1OuM BrdU S-phase marker
  • Tuj-1 fixed cells were incubated with first anti-BrdU antibody (1 :400 rat; Accurate) for 1 hour followed by anti-Tuj-1 antibody.
  • Labelled cells were cells visualised using an inverted fluorescent microscope (model E660 Nikon) and a CCD camera (Spot RT; diagnostic instruments) with individual filter sets for each channel. Colour images were generated using Adobe photoshop (Adobe systems, mountain view, CA) The mean percentage + standard error of the mean (S. E. M) of immunofluescent cells was determined by counting 15 high power (x20 magnification) visual fields (approx 25 cells/field; 375 cells/slide) systematically across the slide, visualised under fluescence. The total number of cells was determined from Hoescht 33342 positive cells. Data was gathered from 3 independently replicated experiments carried out under identical conditions.
  • Negative cells controls were HEK 233 cells for nestin, Tuj-1 and GFAP and primary rat astrocytes for fibronectin and smooth muscle actin.
  • N2a cells or E14 progenitor cells were differentiated either alone or in the presence of induced or non-induced MSCs as described above. After 12 days cells were fixed in 4% PFA and processed for immunocytochemical staining with Tuj-1. Cells were viewed using an inverted fluorescent microscope and images acquired using digital camera. Images were taken of 10 non-overalapping visual fields (x10 magnification) for each culture condition and in 3 independent experiments in cells were cultured in parallel. The neurite lengths of every Tuj-1 + cell (10-15 cells/field approx) within each field of view was determined.
  • Neurites exhibited by differentiating neuronal progeny from cultures of E14 neural progenitor cells or serum deprived N2a cells, that were immunopositive for TUJ1 were analysed using ImageJ 1.33 software, a public domain JAVA image processing program (NIH, USA). Pixel scale was set to microns according to image magnification. JPEG files obtained from light or fluorescent microscopy were opened in ImageJ and neurite lengths were measured by tracing along neurites with the freehand line tool then measuring length using the measurement tool. Statistical analysis
  • MSCs have been shown to express neural proteins in culture and following transplantation into the rodent brain (Tondreau et al., 2004; Deng et al., 2006). Although this expression can occur spontaneously in culture and is present at low levels in MSCs maintained under standard serum culture conditions, expression can be induced to increased levels through the culture of these cells under defined conditions (Hermann et al., 2006). Two populations of MSCs have been detected following transplantation into the intact and injured brain, those cells, which express markers of neural cell lineages, and those cells, which are negative for these markers.
  • MSCs previously maintained under standard culture conditions and expanded until passage 8 were subsequently transferred to serum free media (DMEM F12) supplemented with N2, EGF (10ng/ml) and FGF (10ng/ml) for 7 days in low attachment culture dishes. Under these conditions, small aggregations of cells were evident within 4-5 days of culture ( Figure 1a). By 7 days these aggregations form free-floating cellular spheres ( Figure 1b), which can be harvested and sub-cultured.
  • DMEM F12 serum free media
  • N2 serum free media
  • EGF (10ng/ml) and FGF (10ng/ml) FGF (10ng/ml
  • MSC soluble factors influence the cell fate determination of differentiating embryonic NSC/progenitor cells.
  • NS/progenitor cells isolated from day 14 embryos. These cells were propagated as neurosphere structures in non-adherent culture conditions according to established procedures (Tropepe et al., 1997). Under these conditions the cells retain their multi-potent stem cell characteristics. First, they give rise to all three principal neural cell types, as defined by cell type specific markers for neurons (Tuj-1 +), Oligodenrocytes (RIP+) and astrocytes (GFAP+) in vitro ( Figure 2). Second, undifferentiated cells are positive for nestin, an immature cell marker (97.8 + 2.2) ( Figure 4) and but negative for markers of differentiated cell types (data not shown).
  • NSC/progenitor cells were cultured either alone (control) or co-cultivated in the presence of MSCs (induced or non-induced) under standard differentiation conditions for a period 12 days.
  • Cell phenotype specific markers as described for control conditions
  • Quantification of lineage commitment was carried out by image analysis ( Figure 3). In the presence of non-induced MSCs, 6.3 + 0.8 progenitor cells differentiate into neurons, 5.5 + 0.6 into oligodenrocytes and 68.8 + 1.8 into astrocytes.
  • induced MSCs promote neurogenic differentiation. This increase in neuronal differentiation occurs in the absence of a significant reduction in the number of RIP+ or GFAP+ cells. This indicates that the presence of induced MSCs promotes an increase in the total number of progenitor cells that differentiate compared to control conditions and that this additional differentiation is predominately neuronal.
  • NSCs at the expense of neurogenesis and had no effect on the number of oligodendrocytes.
  • MSCs provide instructive signals that regulate the cell fate determination of NSC/progenitor cells in culture
  • MSCs have previously been shown to promote cell survival of neuronal progeny both in culture and in vivo.
  • cell death in control cultures was significantly higher than 3 and 6 day intervals but in co-cultures of progenitor cells and induced MSCs this increase in cell death was not observed.
  • Soluble factors released by MSCs promote the neurite outgrowth of differentiating neuronal progeny of NSC/progenitor cells
  • MSCs have been reported to promote axonal and neurite outgrowth of neuronal cell populations. As reconstruction of neural circuitry will be vital to promote recovery from CNS injury we investigated whether the soluble interactions of MSCs were extended to include effects on the differentiated progeny of progenitor cells. NSC/progenitor cells were first differentiated under control conditions to establish a baseline to compare the effects of MSC co-culture. Differentiated cultures were fixed and labelled with Tuj-1 at the specified time point in order to identify neuronal progeny.
  • MSCs promote neuritogenesis in primary neurons derived from NSC/progenitor cells we co-cultiuivated MSCs (induced and non-induced) in the presence of NSC/progenitor from the onset differentiation.
  • Tuj-1 was determined under each culture condition following image analysis of cells within 10 random fields of view in 3 independent experiments. The average neurite length of Tuj-1 + cells co-cultivated in the presence of non-induced MSCs was 38.63 +
  • N2a cells differentiate exclusively into neuronal progeny upon withdrawal of serum from the culture. Under these conditions modest levels of sprouting and neurite extension were observed by 12 days serum deprivation ( Figure 6a, e,).
  • Croft AP Przyborski SA. (2004) Generation of neuroprogenitor-like cells from adult mammalian bone marrow stromal cells in vitro. Stem Cells Dev. Aug;13(4):409-20.
  • Croft AP Przyborski SA. Formation of neurons by non-neural adult stem cells: potential mechanism implicates an artifact of growth in culture. Stem Ceils. 2006 Aug;24(8):1841-51.
  • Kirino T Nakafuku M. (2002) Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell. 23;110(4):429-41.
  • Notchi and Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells to an astroglial fate.Neuron. 29(1):45-55.
  • Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration.
  • Fibroblast growth factor 2 is required for maintaining the neural stem cell pool in the mouse brain subventricular zone. Dev Neurosci. 26(2-4): 181 -96.

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Abstract

L'invention décrit un procédé pour favoriser la différenciation de cellules souches neurales comportant la co-culture de cellules souches mésenchymateuses induites et de cellules souches/cellules progénitrices neurales dans un système de culture cellulaire qui facilite la production de neurones différenciés.
PCT/GB2008/002719 2007-08-17 2008-08-08 Facteurs neurothropiques issus de cellules souches WO2009024748A1 (fr)

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GBGB0716066.6A GB0716066D0 (en) 2007-08-17 2007-08-17 Stem cell derived neurotrophic factors
GB0716066.6 2007-08-17

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2012123712A1 (fr) 2011-03-17 2012-09-20 University College Cardiff Consultants Limited Essai de criblage cellulaire
CN113122536A (zh) * 2021-03-31 2021-07-16 南通大学 一种促进神经干细胞向神经元分化的长链非编码rna及其筛选方法

Non-Patent Citations (5)

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Title
CRIGLER ET AL: "Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis", EXPERIMENTAL NEUROLOGY, ACADEMIC PRESS, NEW YORK, NY, US, vol. 198, no. 1, 1 March 2006 (2006-03-01), pages 54 - 64, XP005291454, ISSN: 0014-4886 *
LIANHUA BAI ET AL: "Human Mesenchymal Stem Cells Signals Regulate Neural Stem Cell Fate", NEUROCHEMICAL RESEARCH, KLUWER ACADEMIC PUBLISHERS-PLENUM PUBLISHERS, NE, vol. 32, no. 2, 27 December 2006 (2006-12-27), pages 353 - 362, XP019483230, ISSN: 1573-6903 *
RAMIREZ-CASTILLEJO CARMEN ET AL: "Pigment epithelium-derived factor is a niche signal for neural stem cell renewal", NATURE NEUROSCIENCE, vol. 9, no. 3, March 2006 (2006-03-01), pages 331 - 339, XP002508591, ISSN: 1097-6256 *
RIVERA FRANCISCO T ET AL: "Mesenchymal stem cells instruct oligodendrogenic fate decision on adult neural stem cells", STEM CELLS (MIAMISBURG), vol. 24, no. 10, October 2006 (2006-10-01), pages 2209 - 2219, XP002508592, ISSN: 1066-5099 *
WISLET-GENDEBIEN SABINE ET AL: "Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4", BMC NEUROSCIENCE, BIOMED CENTRAL, LONDON, GB, vol. 5, no. 1, 15 September 2004 (2004-09-15), pages 33, XP021002866, ISSN: 1471-2202 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012123712A1 (fr) 2011-03-17 2012-09-20 University College Cardiff Consultants Limited Essai de criblage cellulaire
CN113122536A (zh) * 2021-03-31 2021-07-16 南通大学 一种促进神经干细胞向神经元分化的长链非编码rna及其筛选方法
CN113122536B (zh) * 2021-03-31 2023-06-20 南通大学 一种促进神经干细胞向神经元分化的长链非编码rna及其筛选方法

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