WO2010136752A1 - Milieu de différenciation neurale - Google Patents

Milieu de différenciation neurale Download PDF

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WO2010136752A1
WO2010136752A1 PCT/GB2010/001028 GB2010001028W WO2010136752A1 WO 2010136752 A1 WO2010136752 A1 WO 2010136752A1 GB 2010001028 W GB2010001028 W GB 2010001028W WO 2010136752 A1 WO2010136752 A1 WO 2010136752A1
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cells
stem cells
cell culture
neural
bone marrow
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PCT/GB2010/001028
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English (en)
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Hassan Tawhid Hassan
John Alan Goodacre
Xiao Qun Zhai
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University Of The West Of Scotland
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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
    • C12N5/0619—Neurons
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/086—Coculture with; Conditioned medium produced by cells of the nervous system glial 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)

Definitions

  • the invention relates to the preparation of a serum free differentiation medium comprising cell free human astrocyte conditioned medium; a cell culture and method for the differentiation of neural stem cells/neural progenitor cells into differentiated neurones; and their use in the repair of diseased or damage neural tissue and in drug screening.
  • eukaryotic cells for example some mammalian cells has become a routine procedure and cell culture conditions which allow certain cells to proliferate are well defined.
  • cell culture of mammalian cells requires a sterile vessel, usually manufactured from plastics and defined growth medium.
  • serum and feeder cells that provide essential growth factors for the maintenance of the cultivated cell.
  • 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, haematopoietic 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 (NSCs) 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 but nevertheless are considered stem cells.
  • CD133 also called prominin 1
  • prominin 1 is a pentaspan membrane protein first identified as a marker of haematopoietic and neural stem cells. It addition to CD133 as a marker of stem cells [see pancreatic and liver stem cells in WO/03/026584; see renal stem cells in WO2007/027905; its expression is also associated with tumour initiating cancer stem cells in a number of human cancers, for example prostate cancer [see WO2005/089043] and pituitary adenoma [see WO2008/024832].
  • An example of a neural stem cell marker is nestin. Nestin is an intermediate filament protein transiently expressed by neural stem/progentitor cells. It is rarely expressed by adult neurones and is therefore a reliable marker of neural stem/progenitor cells. The co-expression of CD133 and nestin is known in the art.
  • CD133 and nestin in bone marrow derived mesenchymal cells predisposes these cells to differentiation into neural cells.
  • the proportion of differentiated neurones derived from this sub-population is not high.
  • Human astrocytes are star shaped glial cells found in the brain and spinal cord. Astrocytes are known to perform various functions in the maintenance and function of the nervous system. For example, they provide nutrients and growth factors to neurones, they secrete and sequester neural transmitters, regulate ion concentration in the brain, enhance the myelinating function of oligodendrocytes and repair of damaged neural tissue to name but a few.
  • 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.
  • simple cell culture conditions could be established which did not require the addition of xenobiotic materials such as fetal bovine serum or murine feeder cells since their use increases the likelihood of adventious infectious agents (e.g. viruses and prions, in particular for bovine products, and murine viruses for mouse feeder cells) infecting mammalian cells grown in culture.
  • This disclosure relates to the preparation of a serum free differentiation medium comprising conditioned astrocyte medium and the identification of cell growth conditions and cell growth medium that enhances the formation of differentiated neurones derived from bone marrow neural stem cells that co-express CD133 and nestin.
  • differentiated neurones have utility in the repair of damaged or diseased neural tissue and also in testing agents which may enhance or inhibit neural cell differentiation.
  • This disclosure relates to conditioned medium of primary adult human astrocytes that induces neural differentiation of adult human bone marrow stem cells and has the unique feature of using adult human and not mouse/rat embryonic source in serum-free culture conditions.
  • the present disclosure of conditioned medium of primary adult human astrocytes refers to the serum-free culture supernatant of these primary cultured adult human astrocytes. We disclose the ability of this culture supernatant to induce the neural differentiation of easily accessible (CD133 positive) adult human bone marrow stem cells into neural cells in serum-free cultures.
  • the culture vessel utilised is a sterile tissue culture flask coated with PLL (Poly-L-Lysine) used in culturing astrocytes.
  • the PLL coating has the property of changing the electrostatic conditions of the plastic surface of the culture flasks to ensure the maximum 96% required rapid attachment and fibre outgrowth of astrocytes in cultures.
  • An aspect of the present disclosure is to provide a reliable method for production of a large amount of conditioned medium of accessible primary adult human astrocytes in a simple way using serum-free cultures containing no non-human compounds. A feature of this is the ability to induce the neural differentiation of easily accessible adult human bone marrow stem cells whilst maintaining their proliferation potential in serum-free cultures containing no non-human compounds.
  • a method for the preparation of a conditioned minimal medium comprising: i) forming a preparation comprising astrocytes in a cell culture vessel which is coated with a non-proteinacous based cell culture support, astrocyte growth factor and supplemental cell culture medium additives; ii) culturing said astrocytes in said cell culture vessel; and optionally iii) separating and storing said conditioned medium from the astrocytes contained in the cell culture vessel.
  • a method for the preparation of a conditioned minimal medium comprising: i) forming a preparation comprising adult human astrocytes in a cell culture vessel which is coated with a poly-l-lysine cell culture support, astrocyte growth factor and supplemental cell culture medium additives but in the absence of serum; ii) culturing said astrocytes in said cell culture vessel; and optionally iii) separating and storing said conditioned medium from the astrocytes in the cell culture vessel.
  • said astrocytes are primate cells.
  • said astrocytes are human cells.
  • conditioned medium obtained or obtainable by the method according to the invention.
  • a cell culture vessel comprising conditioned medium according to the invention.
  • Cell culture vessel is defined as any means suitable to contain the above described cell culture.
  • an example of such a vessel is a petri dish; cell culture bottle or flask or multiwell culture dishes or well insert or rotary bioreactor.
  • Multiwell culture dishes are multiwell microtitre plates with formats such as 6, 12, 48, 96 and 384 wells which are typically used for compatibility with automated loading and robotic handling systems.
  • high throughput screens use homogeneous mixtures of agents with an indicator compound that is either converted or modified resulting in the production of a signal. The signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, substrate/agent and indicator compound. This will have utility in the analysis of differentiation.
  • said vessel is selected from the group consisting of: a petri dish, a multi-well cell culture vessel, a spinner flask, a rotary bioreactor.
  • Bioreactors are known in the art and provide means for the large scale production of cells.
  • Chen et al Stetem Cells (2006) 24(9): 2052-2059
  • a 3D rotary bioreactor adapted for the expansion of human mesenchymal stem cells which can be adapted for the large scale production of astrocyte conditioned medium and differentiated neurones
  • a cell culture container comprising conditioned cell culture medium according to the invention.
  • Container is defined as any sealable bottle or the like suitable for the storage of conditioned medium for transport or storage prior to use.
  • the container is adapted to prevent photo damage to the conditioned medium and is suitable for freezing and maintaining sterility of the conditioned medium contained therein.
  • conditioned human astrocyte medium formed by the method according to the invention in the formation of differentiated neurones from neural stem cells and/or neural progenitor cells; preferably CD133 + adult human bone marrow stem cells.
  • a method to differentiate neural stem cells in a minimal medium comprising: i) forming a preparation comprising neural stem cells and/or neural progenitor cells, cell free human astrocyte conditioned medium and supplementary cell culture medium additives; ii) culturing said neural stem cells and/or neural progenitor cells, in conditions that differentiate and maintain said cells into differentiated neurones.
  • a method to differentiate CD133 * adult human bone marrow stem cells in a minimal medium comprising: i) forming a preparation comprising CD133 + adult human bone marrow stem cells, cell free astrocyte conditioned medium and supplementary cell culture medium additives; ii) culturing said CD133 + adult human bone marrow stem cells in conditions that differentiate and maintain said cells into differentiated neurones.
  • said neural stem cells express nestin.
  • neural stem cells express CD133.
  • said neural stem cells express both nestin and CD133.
  • said astrocyte conditioned medium and said neural stem cells and/or neural progentor cells are autologous; preferably autologous
  • a method to screen for an agent wherein said agent affects the, proliferation, neural differentiation or function of a neural stem cell/neural progenitor cell comprising the steps of: i) providing a cell culture according to the invention; ii) adding at least one agent to be tested; and iii) monitoring the activity of the agent with respect to the proliferation, differentiation or function of said cells.
  • said screening method includes the steps of: collating the activity data in (iii) above; converting the collated data into a data analysable form; and optionally providing an output for the analysed data.
  • high throughput screens use homogeneous mixtures of agents with an indicator compound which is either converted or modified resulting in the production of a signal.
  • the signal is measured by suitable means (for example detection of fluorescence emission, optical density, or radioactivity) followed by integration of the signals from each well containing the cells, agent and indicator compound.
  • a method for the identification of genes associated with neural stem cell/neural progenitor cell differentiation comprising the steps of: i) providing a cell culture according to the invention; ii) extracting nucleic acid from cells in said cell culture; iii) contacting said extracted nucleic acid with a nucleic acid array; and iv) detecting a signal which indicates the binding of said nucleic acid to a binding partner on said nucleic acid array.
  • said method includes the additional steps of: i) collating the signal(s) generated by the binding of said nucleic acid to said binding partner; ii) converting the collated signal(s) into a data analysable form; and optionally; iii) providing an output for the analysed data.
  • Methods used in the identification of cell differentiation markers include immunogenic based techniques (e.g. using the cells as complex immunogens to develop antisera to for example cell surface markers and the like) nucleic acid based techniques (e.g. differential screening using cDNA from differentiated and differentiating cells).
  • immunogenic based techniques e.g. using the cells as complex immunogens to develop antisera to for example cell surface markers and the like
  • nucleic acid based techniques e.g. differential screening using cDNA from differentiated and differentiating cells.
  • a kit comprising: i) human astrocyte conditioned medium according to the invention; and ii) neural stem cells and/or neural progenitor cells.
  • said kit includes cell culture medium and optionally an instruction manual to direct the use of the kit in the formation of neurones.
  • Figure 3-17 IF staining of adult human BM CD133+ stem cells with anti-CD133-2 antibody after 7 days in SS (A-F) and in SF (a-f) neural differentiation induction cultures (x 1000 magnification);
  • Figure 3-18 IF staining of adult human BM CD133+ cells with anti-CD133-2 antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);
  • Table 3-6 Percentages of day-14 culture BM CD133+ cells that remained positive to CD133-2 in SF neural differentiation induction culture (figures represent the mean ⁇ SD of three determinations); Figure 3-25 IF staining of adult human BM CD133+ cells with anti-Nestin antibody after 7 days in SS (A-E) and SF (a-e) neural differentiation induction cultures (x1000 magnification);
  • Figure 3-19 IF staining of adult human BM CD133+ cells with anti-nestin antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);
  • Table 3-7 Percentage of day-14 culture BM CD133+ cells that were positive to Nestin in SF neural differentiation induction cultures (figures represent the mean ⁇ SD of three determinations);
  • Figure 3-26 IF staining of adult human BM CD133+ cells with anti-GFAP antibody after 7 days in SS (A-F) and SF (a-f) neural differentiation induction cultures (x 1000 magnification);
  • Figure 3-20 IF staining of adult human BM CD133+ cells with anti-GFAP antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);
  • Table 3-8 Percentage of day-14 culture BM CD133+ cells that were positive to GFAP in SF neural differentiation induction cultures (figures represent the mean ⁇ SD of three determinations);
  • Figure 3-27 IF staining of adult human BM CD133+ cells with anti-NF-h antibody after 7 days in SS (A-F) and SF (a-f) neural differentiation induction cultures (x 1000 magnification);
  • Figure 3-21 IF staining of adult human BM CD133+ cells with anti-NF-h antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);
  • FIG. 3-23 ICC staining of BM CD133+ cells with anti human-NF-h antibody after 14 days cultured in SF neural differentiation induction cultures (x1000 magnification);
  • Table 3-9 Percentage of day-14 culture BM CD133+ cells that were positive to NF-h in SF neural differentiation induction cultures (figures represent the mean ⁇ SD of three determinations); Figure 3-22 IF staining of adult human BM CD133+ cells with anti-NSE antibody after 14 days in SF neural differentiation induction cultures (x1000 magnification);
  • Table 3-10 Percentage of day-14 culture BM CD133+ cells that were positive to NSE in SF neural differentiation induction cultures (figures represent the mean ⁇ SD of three determinations).
  • BM CD133+ stem cells cultured in both SF and SS neural differentiation induction cultures were harvested from chamber slides and IF stained with antibodies against stem cell markers: CD133-2 and nestin as well as the glial and neural markers: GFAP and NF-h respectively ( Figure 3-17).
  • the bone marrow CD133+ stem cells after 7 days in both SF and SS neural differentiation induction culture did not show significant morphological change or any neural differentiation comparing to Day 0 as they remained GFAP and NF-h negative in all culture conditions. Cell bodies were not elongated and did not show any neuron like projection.
  • staining of Day 14-culture BM CD133+ cells in both SS and SF neural differentiation induction cultures with anti-CD133-2, -nestin, -GFAP and -NF-h antibodies were counted under fluorescent microscope.
  • the percentage of these bone marrow cells that expressed stem cell markers: CD133-2 and nestin remained > 94.2 ⁇ 4.2% ( Figure 3- 17) and ⁇ 86.9 ⁇ 0.8% ( Figure 3-25) respectively.
  • BM CD133+ cells cultured only in SF neural differentiation induction cultures were harvested and were IF and ICC stained with antibodies against stem cell markers: CD133-2 and nestin as well as the glial and neural markers: GFAP, NSE and NF-h.
  • the IF staining results are shown in Figure 3-18 to Figure 3-22 and ICC staining results are shown in Figure 3-23 and Figure 3-24.
  • longer incubation time of two weeks had dramatic effect in inducing neural differentiation of BM CD133+cells.
  • Figure 3-20, 3-21 , and 3-22 show the significant cell morphological changes comparing to after 7 days in SS and SF neural differentiation induction cultures (Figure 3-17, 3-25, 3-26, and 3-27) and the increasing expression of glial and neural markers in the neuron like cells. Also, ICC staining of day14-culture BM CD133+ cells ( Figure 3-23, 3-24) showed similar dramatic morphological changes comparing to Day-7-culture BM CD133+ cells.
  • CD133+ cells After 14 days in SF neural differentiation induction culture, the majority of the remaining adult human bone marrow CD133+ cells show typical undifferentiated features, eg. big cell body, high N/C ratio etc. Most bone marrow CD133+ cells are bigger than CD133 negative (CD133-) cells as shown in Figure 3-18.
  • Cell "a” cultured in cytokine cocktail supplemented neural A basal medium (NAM) in the presence of ACM represents a typical dim stained CD133+ cell, which maintained relative large size, but had lost most of CD133 antigen comparing to cell "b", which was cultured in [NAM+ACM (1 :1)] and kept high level of CD133.
  • CD133 expression in bone marrow cells had not dropped from 97% on day 0, as it remained 96.5 ⁇ 0.14% in NAM only culture and only marginally non- significantly dropped to 91.0 ⁇ 4.9% in NAM supplemented with RA and cytokine cocktail culture. Meanwhile, in NAM + ACM treated culture, the CD133 expression significantly dropped after 14 days to 82.6 ⁇ 1.1% (Table 3-6).
  • day-14 culture BM CD133+ cells treated with NAM culture supplemented with RA and or Cytokine cocktail in the presence of ACM had shown significant drop in CD133 expression to 72.9 ⁇ 3.0% - 75.5 ⁇ 2.0% (P ⁇ 0.01) (Table 3-6).
  • condition 4 > condition 6 (C6) > condition 5 (C5) > condition 2 (C2) > condition 3 (C3).
  • Figure 3-25 shows the variable extent of Nestin expression levels in the BM CD133+ cells after 7 days in both SF and SS culture conditions.
  • Nestin negative day-14 culture bone marrow CD133+ cells were detected in cultures supplemented with various combination of ACM, RA as well as cytokine cocktail. Most nestin positive cells remained large cell bodies, especially those cultured in NAM only medium. interestedly, cells that lost neural stem/progenitor antigen nestin with relative big size were detected in cultures that contained RA, cytokine cocktail and in the absence or presence of ACM ("a" and "b" in Figure 3-19).
  • NAM did not affect the expression of neuron stem/progenitor cell antigen nestin in day-14 culture BM CD133+ cells, however the addition of ACM, RA and cytokine cocktail contributed to the lost of sternness of these BM CD133+ cells.
  • All the tested neural differentiation induction cultures that contained ACM had significantly induced reduction of nestin, this suggests the crucial role of ACM in inducing nestin reduction in BM CD133+ stem cells in SF cultures.
  • the effect of different SF neural differentiation induction cultures in reducing neuron stem/progenitor cell antigen nestin in day-14 culture BM CD133+ cells are in the following order: C4 > C2 > C5 > C6 > C3.
  • BM CD133+ cells showed the dramatic morphological changes of some of the BM CD133+ cells treated in cultures that contained NAM and various combinations of ACM, RA and cytokine cocktail as shown in Figure 3-20.
  • ACM is able to maintain >7% glial differentiation (P ⁇ 0.05%), whereas, RA is weaker in inducing glial differentiation (6.0 ⁇ 0.3%) in the presence of ACM (P ⁇ 0.05%).
  • RA is weaker in inducing glial differentiation (6.0 ⁇ 0.3%) in the presence of ACM (P ⁇ 0.05%).
  • P>0.05 the number of cells that contain both RA and cytokine cocktail.
  • ACM only is the most potent inducing agent for glial differentiation, this suggests the normal astrocytes produced cytokines play crucial role in inducing glial differentiation of BM CD133+ cells in SF cultures.
  • the interaction between ACM 1 cytokine cocktail, and RA does not specifically contribute to the induction of glial differentiation in BM CD133+ cells in SF conditions.
  • the total percentage of induced neural/glial differentiation of CD133+ marrow cells reached the highest level of 21.1-25.5% (P ⁇ 0.01) in culture that contained NAM, ACM and cytokine cocktail followd by 23.0-23.9% (P ⁇ 0.01) in NAM containing ACM, RA and cytokine cocktail treated culture and 20.1-22.4% (P ⁇ 0.01) in culture that contained NAM, ACM and RA.
  • the ACM alone has induced 18.9-22.8% (P ⁇ 0.01) neural/glial differentiation of CD133+ marrow cells, whereas in the absence of ACM, basal medium supplemented with RA and cytokine cocktail failed to induce significant neural/glial differentiation (only 8.5-8.7%) on day 14 (P>0.05).

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Abstract

La présente invention concerne la préparation d'un milieu de différenciation sans sérum comprenant un milieu conditionné par des astrocytes humains acellulaires, ainsi qu'une culture cellulaire et un procédé pour la différenciation des cellules souches neurales/cellules progénitrices neurales en neurones différenciés et leur utilisation dans la réparation de tissu neural malade ou lésé et dans le criblage de médicaments.
PCT/GB2010/001028 2009-05-26 2010-05-26 Milieu de différenciation neurale WO2010136752A1 (fr)

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