EP0832187A1 - Agents de differenciation cellulaire - Google Patents

Agents de differenciation cellulaire

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Publication number
EP0832187A1
EP0832187A1 EP96916223A EP96916223A EP0832187A1 EP 0832187 A1 EP0832187 A1 EP 0832187A1 EP 96916223 A EP96916223 A EP 96916223A EP 96916223 A EP96916223 A EP 96916223A EP 0832187 A1 EP0832187 A1 EP 0832187A1
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EP
European Patent Office
Prior art keywords
serum
differentiation
cells
extract
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96916223A
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German (de)
English (en)
Inventor
Bradley Michael John Stringer
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CellFactors PLC
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CellFactors PLC
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Filing date
Publication date
Priority claimed from GBGB9510555.7A external-priority patent/GB9510555D0/en
Priority claimed from GB9522562A external-priority patent/GB2294946A/en
Priority claimed from GBGB9606373.0A external-priority patent/GB9606373D0/en
Application filed by CellFactors PLC filed Critical CellFactors PLC
Publication of EP0832187A1 publication Critical patent/EP0832187A1/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • the invention relates to agents that affect the differentiation of cells; methods including such agents and products including such agents.
  • Cellular differentiation is a process whereby cells become different from one another, they specialize so as to perform particular functions.
  • cells of the retina become specialised so that they can respond to light and convey this response to the brain
  • cells of the skeleton become specialised so that they can either manufacture bone (osteoblasts) or cartilage (chondrocytes), or alternatively resorb bone (osteoclasts).
  • osteoblast differentiation from a mesenchymal stem cell occurs either as a restriction in the multipotentiality of early precursor cells during maturation, or due to commitment to a differentiation pathway, or cell lineage, early in the mesenchymal stem cell development, but knowledge of this process is scant.
  • differentiated cells are preceded by precursor cells which at some stage become committed to a particular differentiation pathway so as to provide a fully differentiated phenotype.
  • teaching encompasses the idea of multipotentiality and thus it is thought that, depending upon the circumstances, a precursor cell can produce a number of different phenotypes. Unfortunately, the nature of the circumstances remain largely obscure.
  • immortalised cell lines and in particular immortalised human cell lines as described in our co- pending UK patent application 9522562.9.
  • These cell lines have been provided by immortalising human precursor cells, such as human bone marrow stromal cells, using retroviral transduction with a modulatable oncogene.
  • immortalising human precursor cells such as human bone marrow stromal cells
  • retroviral transduction with a modulatable oncogene This is advantageous because immortalisation is induced by a single genetic event, hence the genotype, and, consequently, the phenotype of such cell lines are clearly defined.
  • expression of the active form of the immortalising oncogene product is controllable.
  • the oncogene protein can be inactivated so allowing the cells to return to their original state of differentiation.
  • cellular differentiation does involve plasticity in that precursor cells have multipotentiality and thus can differentiate down a number of different differentiation pathways so as to provide for different cell phenotypes having regard to the nature of the pathway undertaken.
  • This multipotentiality is controlled by an external agent acting upon the precursor cells and more specifically we have found it is controlled by blood, or an extract thereof and/or neural cell extracellular milieu, or an extract thereof. This observation has significant ramifications because it clearly suggests that by controlling the environment to which a precursor cell is exposed one can control the nature of the mature phenotype.
  • human bone precursor cell lines will differentiate to the osteoblast phenotype under normal culture conditions, these conditions include exposure to dexamethasone/(OH) 2 Nitamin D 3 administration, but when exposed to blood and particularly serum, or an extract thereof, these same human bone precursor cell lines differentiate to an adipocyte phenotype.
  • This observation accords well with the fact that the volume of marrow adipose tissue is significantly increased and bone volume reduced in primary osteoporotic and osteopenic patients of all ages.
  • an increase in the volume of marrow fat occurs after the reduction of bone loss by: immobilisation, exposure to zero gravity, or ovariectomy. To date, no cell related mechanism has been identified which may account for the inverse relationship between the volume of marrow adipose tissue and bone.
  • osteoporosis or the increase in volume of marrow adipose tissue can be treated by using our human cell lines to identify agents that block the activity of blood and particularly serum, or an extract thereof, and then using such agents in vivo.
  • human neural cell precursor cell lines will differentiate to either the nerve cell phenotype under normal culture conditions, these conditions include exposure to glial cell line-derived neurotrophic factor (GDNF), but when exposed to serum, or an extract thereof, the same human neural cell precursor cell lines differentiate to a glial such as an astrocyte phenotype.
  • GDNF glial cell line-derived neurotrophic factor
  • the invention concerns the exploitation of the multipotentiality of precursor cells in order to selectively drive differentiation along a pre-determined pathway with a view to selecting the nature of the mature phenotype.
  • a method for regulating or controlling the differentiation of precursor cells to at least one specific phenotype comprising exposing said cells to blood and particularly serum, or an extract thereof, and/or neural cell extracellular milieu, or an extract thereof, under conditions that support differentiation so as to selectively drive differentiation with a view to providing a preselected mature phenotype.
  • a method for regulating or controlling the differentiation of precursor cells to at least one specific phenotype comprising exposing said cells to an agent that blocks the activity of blood and particularly serum, or an extract thereof, and/or neural cell extracellular milieu, or an extract thereof, under conditions that support differentiation.
  • said blood and particularly serum, or an extract thereof, and/or said milieu, or an extract thereof is used to induce, what may be termed, serum directed or neural cell extracellular milieu directed differentiation, respectively, so as to provide a serum or neural cell extracellular milieu directed phenotype.
  • said agent is used to block what we have termed serum and/or neural cell extracellular milieu directed differentiation so as to provide for an alternative phenotype which may be termed a serum or nerve cell extracellular milieu independent phenotype.
  • a pharmaceutical composition comprising an effective amount of: blood and particularly serum, or an extract thereof, and/or neural cell extracellular milieu, or an extract thereof, for directing differentiation of selected precursor cells to at least one specific phenotype.
  • a pharmaceutical composition comprising an effective amount of agent that blocks the activity of blood and particularly serum, or an extract thereof, and/or neural cell extracellular milieu, or an extract thereof, so as to provide for serum or neural cell extracellular milieu independent differentiation of selected precursor cells to at least one serum or neural cell extracellular milieu independent phenotype.
  • said composition may be used in relation to bone precursor cells so as to provide for a an adipocyte phenotype; or in relation to neural precursor cells so as to provide for a glial phenotype, so safeguarding against diseases such as multiple sclerosis which is characterised by a reduction in oligodendrocytic glial cells.
  • said pharmaceutical composition may be used in relation to bone precursor cells so as to provide for an osteoblast phenotype so safeguarding against osteoporosis, osteopaenia, osteoarthritis, bone fractures, rickets and other skeletal conditions associated with a reduction in bone formation, and furthermore obesity and other conditions associated with an increase in adipose tissue and for (cardio) vascular disease whereby the build up of fat (cells) in the vascular system may lead to changes in blood flow.
  • a modification of differentiation is relevant to muscle wastage in aged individuals.
  • the fourth aspect of the invention is used in relation to neural precursor cells it provides for an increase in production of neuronal cells and thus may be used to safeguard against diseases associated with a reduction in neuronal cells such as stroke injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis or Motor Neurone Disease or Huntingtons chorea.
  • a method of preventing, mitigating or alleviating osteoporosis or obesity comprising administering to an individual to be treated a pharmaceutical composition comprising an effective amount of a blood and, particularly a serum, and/or neural cell extracellular milieu blocking agent that blocks serum and/or neural cell extracellular milieu directed differentiation so as to provide for a bone related phenotype.
  • said phenotype is an osteoblast phenotype.
  • said pharmaceutical composition is administered to a site to be treated.
  • a method for promoting or enhancing the amount of adipose tissue in an individual to be treated comprising administering to said individual a pharmaceutical composition comprising an effective amount of serum, or an extract thereof and/or neural cell extracellular milieu or an extract thereof, to induce serum and/or neural cell extracellular milieu directed differentiation of an adipose cell precursor cell so as to provide for a serum directed adipose phenotype.
  • nerve cell extracellular milieu can selectively drive differentiation so as to produce what we have termed neural cell extracellular milieu directed differentiation and a nerve cell extracellular milieu phenotype.
  • a method for preventing, mitigating or alleviating diseases associated with a reduction in neuronal cells comprising administering to an individual to be treated a pharmaceutical composition comprising an effective amount of agent that blocks serum directed differentiation and/or neural cell extracellular milieu directed differentiation and so ensures that neural cell precursor cells differentiate in a serum and/or neural cell extracellular milieu independent manner so as to provide for a neuronal cell phenotype.
  • the pharmaceutical composition is directed towards the tissue of the central nervous system.
  • the pharmaceutical composition provides for an increase in the production of neuronal cells and thus may be used to safeguard against diseases associated with a reduction in neuronal cells such as stroke injury, Alzheimer's disease, Parkinson's disease, amytrophic lateral sclerosis or Motor Neurone Disease or Huntingtons chorea.
  • a method of identifying agents effective at preventing or interfering with serum directed differentiation and/or neural cell extracellular milieu directed differentiation comprising: exposing a precursor cell population to an effective amount of
  • This method of the invention may be performed in vitro or in vivo.
  • said cell population is a cell line and ideally of human origin such as a human bone marrow stromal cell line or a human neural cell line.
  • agents identified using this method of the invention would be effective at blocking serum and/or neural cell extracellular milieu directed differentiation and so when used in relation to bone precursor cells would provide for the production of a bone related phenotype such as an osteoblast phenotype, and when used in relation to neural cell precursor cells would provide for the production of neuronal cells.
  • a bone related phenotype such as an osteoblast phenotype
  • neural cell precursor cells when used in relation to neural cell precursor cells would provide for the production of neuronal cells.
  • a method of identifying agents that are effective at enhancing serum and/or neural cell extracellular milieu directed differentiation comprising: exposing a precursor cell population to an effective amount of
  • This method of the invention may be performed in vitro or in vivo.
  • said cell population is a cell line ideally of human origin such as a human bone marrow stromal cell line or a human neural cell line.
  • an agent identified by the afore method of identification.
  • Agents identified using this method of the invention when used in relation to bone precursor cells provide for enhanced differentiation to an adipose phenotype, and when used in relation to neural precursor cells provide for enhanced differentiation to a glial, including an astrocyte phenotype.
  • a method for enhancing bone formation and, at least relatively, reducing adipose formation comprising exposing a bone precursor cell population to an agent effective at blocking serum and/or neural cell extracellular milieu directed differentiation of said precursor cells to an adipose phenotype.
  • the relative balance between bone tissue and adipose tissue may be reversed by removing said agent and/or exposure to serum, or an extract thereof, and/or neural cell extracellular milieum, or an extract thereof, which promotes directed differentiation to adipose tissue.
  • This method may be performed either in-vivo or in-vitro.
  • a method for enhancing the formation of neuronal cell tissue and, at least relatively, reducing the amount of glial or more specifically astrocyte tissue comprising exposing a neural precursor cell population to an agent effective at blocking serum and/or neural cell extracellular milieu directed differentiation so that said population differentiates to a neuronal cell phenotype.
  • the balance between neuronal tissue and astrocyte or oligodendrocyte tissue may be reversed by removing said blocking agent and/or exposing said population to serum, or an extract thereof, and/or neural cell extracellular milieu, or an extract thereof; which provides for directed differentiation so as to produce a glial or more specifically an astrocyte phenotype.
  • This method may be performed either in vivo or in vitro.
  • Reference herein to blood and particularly serum, and an extract thereof, includes reference to a diverse range of serum, such as, but not limited to human, rabbit, foetal calf, new born calf, foetal horse, adult horse, goat and donkey.
  • prostaglandins and in particular prostaglandin metabolites such as prostaglandin J2 and its metabolites, and/or retinoic acid and/or to various trophic factors such as, but not limited to, ciliary neurotrophic factor CNTF, glial cell-line derived neurotrophic factor GDNF, fibroblast growth factor FGF and other molecules such as hydrocortisone, and indeed analogues and/or homologues of the aforementioned agents.
  • prostaglandins and in particular prostaglandin metabolites such as prostaglandin J2 and its metabolites, and/or retinoic acid and/or to various trophic factors such as, but not limited to, ciliary neurotrophic factor CNTF, glial cell-line derived neurotrophic factor GDNF, fibroblast growth factor FGF and other molecules such as hydrocortisone, and indeed analogues and/or homologues of the aforementioned agents.
  • Reference herein to neural cell extracellular milieu, or an extract thereof includes reference to a diverse range of such milieu, such as, but not limited to, human, rabbit, foetal calf, new born calf, foetal horse, adult horse, goat and donkey.
  • neural cell extracellular milieu includes reference to various factors found in said milieu such as but not limited to, ciliary neurotrophic factor CNTF, and/or glial cell-line derived neurotrophic factor GDNF, and/or brain derived neurotrophic factor BDNF, and/or retinoic acid and/or steroid hormones such as hydrocortisone or dexamethasone, and indeed analogues and/or homologues of the aforementioned agents.
  • factors found in said milieu such as but not limited to, ciliary neurotrophic factor CNTF, and/or glial cell-line derived neurotrophic factor GDNF, and/or brain derived neurotrophic factor BDNF, and/or retinoic acid and/or steroid hormones such as hydrocortisone or dexamethasone, and indeed analogues and/or homologues of the aforementioned agents.
  • references herein to agents effective at blocking serum and/or neural cell extracellular milieu includes reference to agents specifically blocking the activity of CNTF, retinoic acid, FGF and PG J2a .
  • a method of identifying an extract that is effective at providing serum directed differentiation comprising fractionating blood and particularly serum and then exposing a precursor cell population to an effective amount of at least one fraction of said serum for an effective amount of time under conditions that support differentiation and then examining said population to determine whether and/ or to what degree serum directed differentiation has taken place.
  • This method may be performed in vitro or in vivo.
  • a further aspect of the invention comprises an extract identified by the afore method.
  • a method of identifying a neural cell extracellular milieu extract that is effective at providing neural cell extracellular milieu directed differentiation comprising fractionating said milieu and then exposing a precursor cell population to an effective amount of at least one fraction of said milieu for an effective amount of time under conditions that support differentiation and examining said population to determine whether and/or to what degree neural cell extracellular milieu directed differentiation has taken place.
  • This method may be performed in vitro or in vivo.
  • a further aspect of the invention comprises an extract identified by the afore method.
  • fractionation may be undertaken having regard to size, thus differing molecular weight components of serum may be separated and tested as per the above methods. Identification may also be pursued by ionic separation or indeed by any method known to those skilled in the art, as described in, but not limited to, the following references:
  • agents that provide for either of the above types of directed differentiation it will be possible to identify, using the above techniques, further agents that block this effect and so identify antagonists to agents that are effective at providing directed differentiation.
  • Reference herein to precursor cells includes reference to cells which have not yet developed to their mature phenotype and ideally includes reference to cells which are in a very early stage of development and thus ideally have not begun the differentiation process and so are characterised by a lack of or shortage of tissue specific markers.
  • Reference herein to conditions that support differentiation will of course vary having regard to the nature of the cell population under consideration but in any event these conditions will be known by those skilled in the art. Thus, for example, for human bone precursor cells conditions which support differentiation would include exposure to dexamethasone/(OH) 2 Nitamin D 3 .
  • Reference herein to differentiation includes transdifferentiation where a mature cell type de-differentiates to what is considered a precursor cell type before going through a further process of differentiation to give a fully mature cell type. Alternatively transdifferentiation may occur without the said de-differentiation step.
  • a method for diagnosing the existence of, susceptibility to, or predisposition towards serum directed differentiation of a precursor cell population so as to provide a serum directed phenotype comprising obtaining a test sample of blood and particularly serum from an individual to be tested and exposing precursor cells to said test sample under conditions that support differentiation and then observing the nature of the differentiated phenotype of said precursor cells.
  • a method for diagnosing the existence of, susceptibility to, or predisposition towards serum directed differentiation of a precursor cell population so as to provide a serum directed phenotype comprising obtaining a test sample of precursor cells from an individual to be tested and exposing said precursor cells to blood and particularly serum, or an extract thereof, under conditions that support differentiation and then observing the nature of the differentiated phenotype of said precursor cells.
  • a method for diagnosing the existence of, susceptibility to, or predisposition towards neural cell extracellular milieu directed differentiation of a precursor cell population so as to provide a neural cell extracellular milieu directed phenotype comprising obtaining a test sample of neural cell extracellular milieu from an individual to be tested and exposing precursor cells to said test sample under conditions that support differentiation and then observing the nature of the differentiated phenotype of said precursor cells.
  • a method for diagnosing the existence of, susceptibility to, or predisposition towards neural cell extracellular milieu directed differentiation of a precursor cell population so as to provide a neural cell extracellular milieu directed phenotype comprising obtaining a test sample of precursor cells from an individual to be tested and exposing said precursor cells to nerve cell extracellular milieu, or an extract thereof, under conditions that support differentiation and then observing the nature of the differentiated phenotype of said precursor cells.
  • said precursor cells comprise a human cell line such as a bone marrow stromal cell line or a human neural cell line, ideally, as herein described.
  • a human cell line such as a bone marrow stromal cell line or a human neural cell line, ideally, as herein described.
  • adipo/osteoprogenitor precursor cells express what, up to now, was thought to be a marker for the mature adipocyte phenotype, ie a receptor known as peroxisome profilerator- activated receptor ⁇ (PPAR ⁇ ).
  • PPAR ⁇ peroxisome profilerator- activated receptor ⁇
  • serum and/or neural cell extracellular milieu includes reference to ligands that bind this receptor; and reference herein to agents that block serum and/or neural cell extracellular milieu directed differentiation includes reference to antagonists for these ligands and/or agents which affect the functioning of said receptor.
  • Figure 1 Fluorescence immunolocalisation of a temperature sensitive form of the simian virus-40 derived large tumour antigen expressed in immortalised human marrow stromal cell clone 7.
  • Figure 3 Cellular alkaline phosphatase activity in immortalised human marrow stromal cell clone 7 cells in response to 7 days dexamethasone (10 "7 M) treatment. Error bars relate to the standard error of the mean of 6 samples per point. Statistical analysis was performed by student's t-test.
  • Cells were treated with a range of concentrations of 1,25 (OH) 2 D 3 in the presence of vitamin K for 4 days.
  • the media was then removed and osteocalcin measured by radio immunoassay.
  • Osteocalcin levels were normalised to nanogrammes of osteocalcin per 10000 cells. Results are expressed as the mean +/- standard error of the mean of 4 samples per point. Student's t-test was used for statistical analysis.
  • Figure 4 demonstrates the effects of two agonists on cAMP levels in immortalised human marrow stromal cell clone 7.
  • Cells were pre-treated with ImM IBMX for 5 minutes, and then with agonist for 20 minutes.
  • cAMP levels were quantified by ELISA, and the amounts of cAMP for each treatment compared to the control (IBMX only). Results are expressed as the mean +/- standard error of the mean of 4 samples. The statistical analysis was performed by student's t-test.
  • This figure demonstrates the effects of dexamethasone (10-7M) on the mineralisation of immortalised human marrow stromal cell clone 7.
  • Cells were incubated for 28 days in medium containing 10% foetal bovine serum along with ⁇ -glycerophosphate at 39° C, the oncogene's non-permissive temperature.
  • Cells treated with dexamethasone were shown to mineralise by the von-Kossa staining procedure. Light microscopic analysis shows some nodule formation in the cultures but also evidence of mineralisation elsewhere. The mineral deposits are clearly localised to the extracellular matrix.
  • Figure 7a shows that Type I ⁇ l collagen expression (lane 2) is lost (lane 3) after one weeks treatment with NRS (123 bp ladder marker, lane 1).
  • Figure 7b induces the expression of an early marker of adipogenesis, lipoprotein lipase (lane 2), which is not present (lane 3) in the absence of NRS (123 bp ladder marker, lane 1).
  • Figure 9 Demonstrates the effects of pre and post-menopausal serum on adipogenesis of clone 7 immortalised human bone marrow stromal cells.
  • Costar tissue culture plastics were used for cell culture (High Wycombe, UK).
  • Minimum essential medium - alpha (oc-MEM), Dulbecco's modified eagles medium (DMEM), HAM'S F12, foetal bovine serum (FCS), glutamrne, penicillin/streptomycin, G418 (geneticin), Trizol reagent and Taq DNA polymerase were purchased from Life Technologies (Paisley, UK).
  • Ascorbic acid vitamin K, PTH fragment 1-34, PGE 2 , forskolin, dexamethasone, polybrene, alkaline phosphatase, histochemical detection kit, para-nitrophenyl phosphate, para-nitrophenyl standard, oil-red o, trypan blue, dimethyl sulfoxide (DMSO), 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), percoll and anti-mouse IgC FITC conjugated antibody were purchased from Sigma Chemical Co. (Dorset, UK). Immu-mount was purchased from Life Sciences International (UK) Ltd. (Basingstoke, UK).
  • the SV40 T Antigen-specific monoclonal antibody was purchased from Cambridge Bioscience (Cambridge, UK) and the human osteocalcin RIA kit was purchased from Nichols Institute (Ca., USA).
  • the cAMP assay kit was purchased from Amersham (Buckinghamshire, UK) and moloney leukaemia virus reverse transcriptase (M-MLV RT) and dNTPs were purchased from Promega (Southampton, UK).
  • PCR primers for interleukins 1 ⁇ , 1 ⁇ , 3, 4, 6, and 8, GM-CSF and TNF ⁇ were generous gifts from Dr. W. Wishart (Sandoz Pharma, Basle, Switzerland) and collagen type 1 PCR primers were generous gifts from Dr. K. A.
  • Fresh human rib was obtained from a 68 year old female patient undergoing thoracic surgery, with no prior history of bone disease. The rib was immediately washed twice with sterile phosphate buffered saline (PBS) and all adherent tissue scraped off. The rib was then split into two halves exposing the marrow cavity, and the trabecular bone minced. Bone marrow cells were removed from the minced trabecular bone by flushing with ⁇ -MEM using a 10ml syringe fitted with a 19-gauge needle.
  • PBS sterile phosphate buffered saline
  • the resulting cell suspension was transferred to a universal container and allowed to stand undisturbed for 10 minutes, after which time fat deposits that had floated to the top were scraped away.
  • the marrow derived cells were transferred to a centrifuge tube and spun at lOOxg for 5 minutes to pellet the cells. The media and fat deposits were again removed and the cell pellet resuspended in 5ml of fresh media. The resuspended cells were loaded onto a 70% percoll gradient which was spun at 460g for 15 minutes. Following centrifugation the top 25% of the gradient volume, containing the low density osteoblast precursor cells, was removed (to be finished: Haynesworth et al 1992).
  • the bone marrow stromal cells were then centrifuged at lOOxg for 5 minutes and the cell pellet resuspended in an appropriate volume of medium containing an amphotropically packaged retroviral shuttle vector comprising ts-SN40-T plus selection marker, and polybrene to a final concentration of 8 ⁇ g/ml.
  • the cells and media were incubated for 2 hours after which the cell suspension was again centrifuged and the cell pellet resuspended in an appropriate volume of normal medium.
  • Cells were seeded into T75 tissue culture flasks at a final density of 4xl0 7 cells per flask. The flasks were incubated at 33° C and the cells given 3 days to adhere to the tissue culture treated plastic. Once the majority of cells had adhered to the flask, non- adherent cells were washed away and the media replaced with normal media containing G418 at a final concentration of 400 ⁇ g/ml.
  • a G418 resistant replicating cell clone was selected and expanded at the permissive temperature (33° C) in ⁇ -MEM culture medium containing: IX glutamine, IX non-essential amino acids, pen/strep, lOO ⁇ g/ml G418 and 8% heat inactivated FCS (basal medium). Cells were routinely passaged twice per week, ensuring that the cells were not allowed to reach confluence. All experiments were carried out in the same basal medium unless otherwise stated.
  • Cells were seeded in 24-well tissue culture plates at 2500, 5000 or 10000 cells/cm 2 and incubated at the oncogene's permissive temperature (33° C). The number of cells per cm 2 was determined at regular intervals, by using a haemocytometer to count viable cells as shown by toluidine blue exclusion, until confluence was reached.
  • Immunostaining for large-T antigen was performed after fixing subconfluent cells with methanol and acetone (1:1 ratio) at -20° C for 20 minutes.
  • the primary IgC mouse monoclonal antibody against S V40-T was added at a 1 :20 dilution in PBS and incubated at 37° C for 1 hour.
  • Control cells were incubated in PBS alone.
  • the cells were rinsed twice with PBS and a secondary antibody (goat anti-mouse IgC FITC conjugated) was added to the cells at a 1:20 dilution in PBS and incubated at 37° C for a further 60 minutes. Excess antibody was then washed away with several washes with PBS and the cells overlayed with immu-mount prior to fluorescence microscopy.
  • the medium of postconfluent cells was supplemented with 50 ⁇ g/ml ascorbic acid and 10 '5 M ⁇ -glycerophosphate, in the presence or absence of 10 "7 M dexamethasone, at the non-permissive temperature (39° C) for 28 days.
  • Cells were washed in PBS and fixed in 10% buffered formol saline for 10 minutes at room temperature. Cells were then stained by a modification of the von Kossa method (Cook 1974). Briefly cells were treated with silver nitrate and exposed to ultraviolet light for 1 hour. Excess silver nitrate was washed away with distilled water and the cells treated with 5% sodium thiosulfate for 5 minutes at room temperature. The cells were subsequently washed twice with distilled water and allowed to air dry.
  • Cells were seeded at a density of 10,000 cells per cm 2 and allowed to reach confluence, at the permissive temperature (33° C) before treatment with either dexamethasone, l,25-(OH) 2 D 3 or vehicle. Cells were incubated at the non- permissive temperature (39° C) and culture media, along with the appropriate agent, replenished every three days. The media was removed and stored at -80° C prior to being used for osteocalcin measurement. Osteocalcin was measured using an immunoradiometric assay kit (Nicholl's Institute), which detects both intact osteocalcin and its large N-terminal mid- fragment.
  • Alkaline phosphatase was measured in the cell layer by a modification of the method initially described by Bessey et al (1946). Briefly, cell ly sates were prepared by washing the cells several times in PBS, scraping the cell layer into 200 ⁇ l of ice-cold 0.1 % Triton-X 100 and subjecting the samples to mild sonication. Aliquots of the samples were then incubated with pre-warmed substrate solution (0.1M Diethanolamine, ImM MgCl 2 and 2mM p-NPP pH 10.5) for 1 hour and the reaction stopped by the addition of 0.1M NaOH. Samples were quantified spectrophotometrically at 410nM and the final p-NP concentration estimated using a standard curve of known p-NP standards.
  • pre-warmed substrate solution 0.1M Diethanolamine, ImM MgCl 2 and 2mM p-NPP pH 10.5
  • Cells were seeded and cultured in media, containing 10% FCS, at the permissive temperature until approximately 60% confluent. The media was then replaced with media containing 10% rabbit serum, and the cells cultured at the non-permissive temperature (39° C) for 3 days. Following culture the cells were washed twice with PBS and then fixed with 10% buffered formol saline. Cells were stained for lipid content using the oil-red-0 method as described by Cook (1974).
  • RNA samples were seeded into flasks at 10,000 cells per cm 2 and cultured at the permissive temperature until confluent. Media was removed and the cells washed twice with PBS, to which Trizol reagent was added (1ml per 10cm 2 ) and the RNA extracted as recommended by the manufacturers.
  • First strand cDNA was synthesised from 5 ⁇ g of RNA with 0.2 ⁇ g of oligo(dT) primer in the presence of dNTP mix (0,5mM of each) by MMLV-RT (100 units) in a final volume of 20 ⁇ l.
  • RNA and primer were incubated together at 70° C for 10 minutes and then flashed cooled on ice before adding the rest of the components, and the reaction incubated at 37° C for 1 hour. Samples were then heat treated at 95 °C for 5 minutes to inactivate the enzyme.
  • cDNA was amplified in a lOO ⁇ l reaction, using the "hot-start" method described by D'Aquilla et al (1991), containing dNTP mix (0,2mM of each), specific primers (0.5.mM of each), Taq DNA polymerase (2.5 units), 20mM Tris-HCI (pH 8.4), 50mM KCI and 1.5mM MgCl 2 . Primers and amplification (30 cycles) conditions used for the PCR reactions are listed in table 1.
  • rt-PCR reverse transcriptase polymerase chain reaction
  • Neural cells from the 12 - 13 day embryonic rat raphe nuclei were immortalized by transduction with a retrovirally-packaged, temperature- sensitive oncogene (ts)SV40T (Stringer et al., 1994). After isolation and expansion of clones derived from single precursor cells at the permissive temperature of the oncogene, they were allowed to differentiate by raising the temperature to its non-permissive value. Under such basal conditions, cells from several clones exhibited similar neuronal characteristics, including neurofilament and neurone-specific enolase (NSE) positivity. In addition, markers of a serotoninergic phenotype were manifest.
  • ts temperature- sensitive oncogene
  • NSE neurone-specific enolase
  • Neural precursor cells from the 8-week embryonic human cortex were immortalized as described above, and expanded from single cells to flask level. Cells were plated onto fibronectin-coated 24- well plates, held at 33° C for 3 - 4 days until they were about 70% confluent. Drugs were added either prior to raising the temperature to 39° C, or at the time of the temperature shift, and after a further 7 - 10 days the cultures were then fixed and immunostained. Specific factors and drugs exerted a marked effect on the resulting phenotype of the cortical cell lines.
  • glial cell line-derived neurotrophic factor caused some cells to exhibit neuronal characteristics such as neurone-specific enolase immunoreactivity and a phase-bright morphology.
  • CNTF ciliary neurotrophic factor
  • GFAP staining became apparent in some flat, phase-dark astrocyte-like cells. In the absence of either of these two factors, no hallmarks of either glial of neuronal phenotypes were demonstrable.
  • weak GFAP immunostaining was present in some cells under basal incubation conditions, but upon the removal of fibroblast growth factor (FGF) from the stock medium and the addition of dexamethasone, a large proportion of the cells developed a neurone-like morphology, and intense NSE-like immunoreactivity. FGF prevented this dexamethasone effect.
  • FGF fibroblast growth factor
  • Immunocytochemistry demonstrated the expression of SV40 large T-antigen in all cells within the immortalised clone (Fig. 1), the protein being localised to the cell nucleus, as expected. No staining was seen with the control cells which were not treated with the primary antibody. Furthermore, polyacrylamide gel electrophoresis and western blotting identified the T antigen in these cells, without the need for antigen precipitation and selection prior to blotting, giving an expected Mr for full length T-antigen of around 96Kd (data not shown).
  • the immortalised clone was examined for both alkaline phosphatase activity (Fig. 3a) and osteocalcin protein expression (Fig. 3b), in order to determine whether the cells constitutively or inducibly express these two proteins, which are characteristic of the osteoblast phenotype.
  • alkaline phosphatase expression At the permissive temperature alkaline phosphatase expression is seen, and its activity can be increased by treatment with dexamethasone at a dose of 10 '7 M.
  • the increase in activity due to dexamethasone is significantly retarded by the presence of active oncogene product, with activities at the permissive temperature of 0.848+/-0.21 ⁇ Moles p-NP/mg protein/hour and at the non- permissive temperature of 7.441 +/- 1.97 ⁇ Moles p-NP/mg protein/hour.
  • Osteocalcin synthesis was not detected in cells cultured with basal medium for up to 3 weeks, but when 1,25 (OH) 2 vitamin D 3 was added to the medium along with vitamin k, osteocalcin was detected by the second week of culture.
  • the osteocalcin response to 1,254 (OH) 2 vitamin D 3 was dose dependent with the lowest dose of 10 "n M stimulating the production of 0.41+/-0.17 ng of osteocalcin per 10 4 cells, and the top dose of 10 "7 resulting in 6.37+/-0.55 ng of osteocalcin per 10 4 cells.
  • exposure to dexamethasone did not lease to the synthesis of measurable quantities of osteocalcin.
  • cAMP responses to forskolin, PGEj and PTH were studied to investigate whether these known bone agonists would induce an elevation in cAMP levels. It is evident that all three agonists have a stimulatory effect on cAMP levels (Fig. 4). The best response, as expected, was seen with forskolin at a dose of 10 "5 M, which increased levels over 2-fold.
  • Clone 7 cells cultured in the presence of 10% normal rabbit serum for 3 days, showed a dramatic change in cell lipid content, as demonstrated by oil- red O staining. Untreated cells showed no detectable lipid content. However, when treated with rabbit serum, the proportion of cells showing positive staining increased to 100% (Fig. 6a, b). In contrast ts-SV40-T immortalised osteoblast-like cells derived from adult human trabecular bone chips could not be induced to differentiate down an adipocytic pathway (data not shown).
  • Fig. 7a Treatment of the immortalised stromal cells with normal rabbit serum for one week at 39° C also induced the expression of lipoprotein lipase, an early marker of the adipocytic lineage (Fig. 7a). Furthermore, type I collagen expression, a marker of the osteoblastic cell lineage, was lost after incubation with 10% normal rabbit serum for 7 days (Fig. 7b).
  • osteoporosis which is characterised by an increase in marrow adipose tissue and a reduction in bone volume, tends to occur in post- menopausal females and that our results suggest that serum, or an extract thereof, provides for adipogenic switching we exposed our clone 7 immortalised human bone marrow stromal cells to rabbit sera, which we know contains an agent responsible for directing differentiations so as to provide adipocytes, pre-menopausal human female serum, which we speculate contains relatively little or no such agent, and post-menopausal human female serum which we speculate may contain such an agent.
  • the results are shown in figures 8 and 9.
  • adipo/osteoprogenitor precursors differentiate down a pathway which involves the peroxisome profilerator-activated receptor ⁇ PPAR ⁇ .
  • the role of this receptor in differentiation is remarkable given that, conventionally, this receptor is thought to be a marker of a differentiated and thus fully mature phenotype. Accordingly, our data suggests that agents which activate/deactivate PPAR ⁇ will have a role to play in the differentiation of these precursor cells.
  • bone precursor cells can be made to differentiate to produce either bone cells or adipocytes
  • nerve precursor cells can be made to differentiate to produce either neuronal cells or astrocytes.
  • Our results indicate that the choice of pathway to follow is governed at least by soluble factors present in serum and/or nerve cell extracellular milieu.
  • low molecular weight soluble factors are involved and that furthermore these molecular weight soluble factors are likely to be carried by large molecular weight carrier proteins.
  • differentiation along alternative pathways becomes increasingly difficult to reverse as the differentiation process progresses until there is an apparent complete loss of ability to adopt to anything other than the committed pathway.

Abstract

Cette invention concerne la pluripotentialité de cellules précurseurs et plus spécifiquement l'exploitation de cette pluripotentialité à l'aide de procédés et de systèmes qui permettent de produire sélectivement la différenciation le long de la voie prédéterminée afin de produire un phénotype différencié mature présélectionné.
EP96916223A 1995-05-24 1996-05-22 Agents de differenciation cellulaire Withdrawn EP0832187A1 (fr)

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GBGB9510555.7A GB9510555D0 (en) 1995-05-24 1995-05-24 Human cell lines
GB9510555 1995-05-24
GB9522562 1995-11-03
GB9522562A GB2294946A (en) 1994-11-08 1995-11-03 Preparation of human cell-lines of fully-differentiated cells of specific tissue type
GBGB9606373.0A GB9606373D0 (en) 1996-03-26 1996-03-26 Differentiation agents
GB9606373 1996-03-26
PCT/GB1996/001232 WO1996037601A1 (fr) 1995-05-24 1996-05-22 Agents de differenciation cellulaire

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CA2218817A1 (fr) 1996-11-28
CZ365097A3 (cs) 1998-02-18
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