WO2020061627A1 - Cellules souches mésenchymateuses et produits obtenus à partir de celles-ci - Google Patents

Cellules souches mésenchymateuses et produits obtenus à partir de celles-ci Download PDF

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WO2020061627A1
WO2020061627A1 PCT/AU2019/051029 AU2019051029W WO2020061627A1 WO 2020061627 A1 WO2020061627 A1 WO 2020061627A1 AU 2019051029 W AU2019051029 W AU 2019051029W WO 2020061627 A1 WO2020061627 A1 WO 2020061627A1
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stem cell
conditioned medium
cell line
tissue
cells
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English (en)
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Kuldip Sidhu
Corey Cunningham
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CK Cell Technologies Pty Ltd
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Application filed by CK Cell Technologies Pty Ltd filed Critical CK Cell Technologies Pty Ltd
Priority to AU2019348047A priority Critical patent/AU2019348047A1/en
Publication of WO2020061627A1 publication Critical patent/WO2020061627A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N5/0664—Dental pulp stem cells, Dental follicle stem 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0212—Face masks
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • 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
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/98—Xeno-free medium and culture conditions
    • 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
    • C12N2500/99—Serum-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2502/1364—Dental pulp stem cells, dental follicle stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2502/1382—Adipose-derived stem cells [ADSC], adipose stromal 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
    • 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
    • C12N2506/1361—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 dental pulp or dental follicle stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2506/1384—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 adipose-derived stem cells [ADSC], from adipose stromal stem cells

Definitions

  • the present invention relates generally to human mesenchymal stem cell lines produced in serum- free and predominantly xenogeneic-free conditions, and to uses thereof.
  • the invention relates to human mesenchymal stem cell lines so produced, that are derived from human adipose tissue and human dental pulp tissue.
  • the invention also relates to products of the mesenchymal stem cell lines including, for example, conditioned media and exosomes comprising secreted factors and the use of such products in wound healing, tissue repair and tissue regeneration.
  • Mesenchymal stem cells are multipotent adult stem cells that have the ability to differentiate into various different cell types, including adipocytes, osteoblasts, chondrocytes, fibroblasts and myoblasts. Mesenchymal stem cells have demonstrated a range of therapeutic efficacies, for example in treating musculoskeletal injuries, cardiovascular diseases, inflammatory diseases, immune system diseases, neurological diseases, and other degenerative diseases and conditions.
  • Mesenchymal stem cells can be isolated from a range of tissues including bone marrow, cord blood and amniotic fluid, adipose tissue and dental pulp from teeth.
  • Adipose tissue has been at the forefront of stem cell research for a number of years.
  • ADSCs adipose tissue-derived stem cells
  • hDPSCs human dental pulp tissue derived stem cells
  • trophic factors released by hDPSCs in the cell culture media are able to trigger pronounced angiogenic and regenerative responses.
  • mesenchymal stem cells derived from human tissue, wherein the stem cells are isolated and cultured in serum-free and predominantly xenogeneic-free conditions and media.
  • the present invention provides an isolated human mesenchymal stem cell line deposited under Accession Number CBA20180030, wherein the stem cell line is derived from human adipose tissue.
  • the present invention provides an isolated human mesenchymal stem cell line deposited under Accession Number CBA20180031, wherein the stem cell line is derived from human dental tissue.
  • the stem cell lines of the first and second aspect are cultured and maintained in serum- free and predominantly xenogeneic-free cell culture media.
  • the present invention provides a method of producing conditioned medium, comprising: (a) culturing a stem cell line of the first or second aspect in cell culture medium for a suitable period of time for the stem cells to secrete one or more molecules into the growth medium and/or generate one or more extracellular vesicles; (b) harvesting the conditioned medium; and optionally (c) filtering the conditioned medium.
  • the method further comprises the step of lyophilizing the conditioned medium. In another embodiment, the method further comprises the step of isolating the one or more secreted molecules or one or more extracellular vesicles from the conditioned medium.
  • the present invention provides conditioned medium produced by the method of the third aspect.
  • the present invention provides one or more secreted molecules or one or more extracellular vesicles obtained by the method of the third aspect.
  • the present invention provides conditioned medium obtained from a culture of a stem cell line of the first or second aspect.
  • the present invention provides extracellular vesicles derived from a stem cell line of the first or second aspect or conditioned medium of the fourth or sixth aspect.
  • the extracellular vesicles may comprise, for example, microparticles or exosomes.
  • the present invention provides a secretome derived from a stem cell line of the first or second aspect.
  • the present invention provides a pharmaceutical or cosmetic composition
  • a pharmaceutical or cosmetic composition comprising a stem cell line of the first or second aspect, conditioned medium of the fourth or sixth aspect, one or more secreted molecules of the fifth aspect, one or more extracellular vesicles of the fifth or seventh aspect or a secretome of the eighth aspect, optionally in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the present invention provides a method for treating or preventing a disease or condition comprising administering to a subject in need thereof a stem cell line of the first or second aspect, conditioned medium of the fourth or sixth aspect, one or more secreted molecules of the fifth aspect, one or more extracellular vesicles of the fifth or seventh aspect, a secretome of the eighth aspect or a pharmaceutical composition of the ninth aspect.
  • the present invention provides method for aiding, inducing or promoting wound healing, tissue repair, tissue generation or tissue regeneration, comprising administering to a subject, or to cells or tissue derived therefrom, an effective amount of a stem cell line of the first or second aspect, conditioned medium of the fourth or sixth aspect, one or more secreted molecules of the fifth aspect, one or more extracellular vesicles of the fifth or seventh aspect, a secretome of the eighth aspect or a pharmaceutical composition of the ninth aspect.
  • stem cell lines of the first and second aspects are also provided.
  • conditioned medium of the third aspect or extracellular vesicles or a secretome of the fourth aspect in the manufacture of medicaments for therapeutic or cosmetic application.
  • the present invention provides a method of preserving one or more growth factors and/or cytokines secreted by a stem cell line according to the first or second aspect, comprising obtaining conditioned medium from the culture of the stem cell line and lyophilizing the conditioned medium.
  • the lyophilised conditioned medium may be stored at, for example, 4°C or at a temperature up to about room temperature.
  • the lyophilised conditioned medium may be stored, for example, for a period of up to about one month, two months, three months, four months, five months or six months.
  • the lyophilised conditioned medium may be mixed with a solid or liquid carrier or vehicle, for example a cream.
  • FIG. 1 Tri-lineage differentiation potential of ADSCs. Left hand panels, commercially available ADSC line. Right hand panels, ADSC line of the present invention. Top row, presence of lipid globules stained with Oil Red O in adipogenic-differentiated ADSCs (day 7). Middle row, presence of mineralized nodules in osteoblast-differentiated ADSCs (day 8) stained by Alizarin Red S stain. Bottom row, presence of carboxylated and sulphated proteoglycans in chondrocyte-differentiated ADSCs (day 11) stained by Alcian blue.
  • FIG. 1 Quantification of adipogenic, osteogenic, and chondrogenic differentiation of a commercially available ADSC line (left hand columns) and the ADSC line of the present invention (right hand columns).
  • FIG. 3 Expression of differentiation marker genes in day 7 differentiated (diff) and undifferentiated (undiff) cells derived from a commercially available ADSC line (Cell line) and the ADSC line of the present invention (In-house).
  • A expression of adipogenic- specific marker PPARy
  • B expression of osteogenic-specific marker osteocalcin
  • C expression of chondrogenic- specific marker collagen 10A1.
  • Expression of mesenchymal stem cell marker CD44 in undifferentiated cells is shown in D. RPLPO was used as the house keeping control.
  • FIG. 1 Tri-lineage differentiation of DPSCs.
  • Left hand panels differentiated cells derived from the DPSC line of the present invention.
  • Right hand panels undifferentiated DPSC line.
  • Top row presence of lipid globules stained with Oil Red O in adipogenic- differentiated DPSCs (day 9).
  • Middle row presence of mineralized matrix in osteoblast- differentiated DPSCs (day 21) stained by Alizarin Red S stain.
  • Bottom row presence of carboxylated and sulphated proteoglycans in chondrocyte-differentiated DPSCs (day 10) stained by Alcian blue.
  • FIG. 1 Expression of mesenchymal stem cell marker CD44 in undifferentiated DPSC cells of the present invention.
  • B-D Expression of differentiation marker genes in day 7 differentiated (diff) and undifferentiated (undiff) cells derived from the DPSC line of the present invention: A, expression of adipogenic-specific marker PPARy; B, expression of osteogenic-specific marker osteocalcin; and C, expression of chondrogenic - specific marker collagen 10A1.
  • RPLPO was used as the house-keeping control.
  • Figure 7 Schematic diagram of production of conditioned media as described in Example 4.
  • FIG. 8 Levels of 105 growth factors and cytokines expressed in lyophilised conditioned media from CKC-Endeavour-l cells (left hand columns for each growth factor/cytokine) and CKC -Endeavour-2 cells (right hand columns for each growth factor/cytokine) reconstituted in RO water (day 0) following storage of non-reconstituted lyophilised conditioned media powder at 4°C for 36 days.
  • FIG. 9 Levels of 105 growth factors and cytokines expressed in lyophilised conditioned media from CKC-Endeavour-l cells (left hand columns for each growth factor/cytokine) and CKC -Endeavour-2 cells (right hand columns for each growth factor/cytokine) reconstituted in RO water (day 28) following storage of non-reconstituted lyophilised conditioned media powder at 4°C for 43 days and subsequent storage of lyophilised conditioned media powder at room temperature for 28 days.
  • FIG. 10 Levels of 105 growth factors and cytokines expressed in lyophilised conditioned media from CKC-Endeavour-l cells (left hand columns for each growth factor/cytokine) and CKC -Endeavour-2 cells (right hand columns for each growth factor/cytokine).
  • the lyophilised conditioned media powders were mixed with a proprietary cream and stored at 4°C for 1 month before protein extraction and reconstitution in RO water for analysis of growth factors and cytokines.
  • Figure 11 Exosome concentration and particle size distribution derived from CKC-Endeavour-2 (A) and from CKC-Endeavour-l (B).
  • Figure 12 In vitro scratch assay of scratch gap width in HDF01 cells following exposure to using liquid conditioned media (CM Liquid), exosomes (EXO) or lyophilised conditioned media (6.6 mg CM, 10 mg CM and 20 mg CM) derived from CKC-Endeavour- 2 (A) and CKC-Endeavour-l (B) at Day 0 (left hand columns) and Day 1 (right hand columns).
  • CM Liquid liquid conditioned media
  • EXO exosomes
  • lyophilised conditioned media 6.6 mg CM, 10 mg CM and 20 mg CM
  • articles“a” and“an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • “an element” means one element or more than one element.
  • a“mesenchymal stem cell” refers to an undifferentiated multipotent cell that has a self-replicating ability and the potential for differentiation into various cell types including, but not limited to, adipocytes, chondrocytes, osteocytes, myoblasts, fibroblasts and stromal cells. Typically, in the context of the present specification the mesenchymal stem cells are human stem cells.
  • treating and“treatment” refer to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
  • treatment refers not only to treatment designed to cure or remove symptoms in an individual, but also to ongoing therapy designed to control and suppress the occurrence of symptoms. Treatment may be for a defined period of time, or provided on an ongoing basis depending on the particular circumstances of any given individual.
  • prevention does not necessarily mean that the subject will not eventually contract a particular condition or disease. Rather, “prevention” encompasses reducing the severity of, or delaying the onset of, a particular condition or disease.
  • methods of the present invention involve“treating” the condition in terms of reducing or eliminating the occurrence of a highly undesirable and irreversible outcome of the progression of the condition but may not of itself prevent the initial occurrence of the condition. Accordingly, treatment and prevention include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • promotion and inducement do not necessarily imply the complete promotion or inducement of the specified event, activity or function (for example wound healing, tissue repair or tissue regeneration). Rather, the promotion or inducement may be to an extent, and/or for a time, sufficient to produce the desired effect.
  • the promotion or inducement of wound healing, tissue repair or tissue regeneration by products or compositions of the present invention may be direct or indirect, may be variable in magnitude and/or may be temporal in nature.
  • the term "effective amount” includes within its meaning a non-toxic but sufficient amount or dose of a product or composition as disclosed herein to provide the desired effect.
  • the exact amount or dose required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount”. However, for any given case, an appropriate "effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • the term“subject” as used herein refers to mammals and includes humans, primates, livestock animals (e.g.
  • the mammal is human or a laboratory test ani al. Even more preferably, the mammal is a human.
  • the present inventors have successfully produced mesenchymal stem cells from different human tissue, employing serum-free and mostly xenogeneic-free conditions and defined media, thereby providing clinically relevant stem cells, and products such as conditioned media and exosomes derived therefrom, with therapeutic potential, for example in aiding, promoting or inducing wound healing, tissue repair, tissue generation and tissue regeneration.
  • the prior art use of exogenous and xeno factors introduces unacceptable risks of tumorigenicity or transmission of xenogenic infectious agents.
  • mesenchymal stem cell lines produced under serum-free and xenogeneic-free conditions, derived from human adipose tissue and from human dental tissue.
  • an isolated human mesenchymal stem cell line deposited pursuant to the Budapest Treaty with CellBank Australia under Accession Number CBA20180030, wherein the stem cell line is derived from human adipose tissue.
  • an isolated human mesenchymal stem cell line deposited pursuant to the Budapest Treaty with CellBank Australia under Accession Number CBA20180031, wherein the stem cell line is derived from human dental tissue.
  • the medium in which the stem cells of the invention are cultured does not comprise serum.
  • the medium comprises a basal medium and may further comprise one or more of L- glutamine, one or more non-essential amino acids, and an antibiotic.
  • the basal medium may be, for example, Dulbecco's Modified Eagle's Medium (DMEM).
  • the antibiotic may be, for example, penicillin- streptomycin.
  • the basal medium may further comprise an artificial serum replacement.
  • conditioned media obtained from a culture of the human mesenchymal stem cells disclosed herein (i.e. media conditioned by the culture of the mesenchymal stem cells).
  • Such conditioned media typically comprises molecules and/or extracellular vesicles such as exosomes, microvesicles, microparticles and the like that are produced or secreted by the mesenchymal stem cells.
  • Conditioned media may be generated by culturing the human mesenchymal stem cells disclosed herein in any suitable basal medium, for a predetermined length of time. Suitable basal media will be well known to those skilled in the art. In an exemplary embodiment, the cells are cultured for 24 hours.
  • the cells may be cultured, for example, for about 12 hours, about 36 hours, about 48 hours, about 72 hours or about 96 hours or more.
  • the length of time of culture will typically be sufficient for the stem cells to secrete one or more molecules into the growth medium and/or generate one or more extracellular vesicles.
  • the present invention is directed to conditioned medium comprising biological factors secreted by the stem cells of the invention.
  • the conditioned medium is obtained by culturing the stem cells in media and separating the resulting media, which contains stem cells and their secreted stem cell products (molecules and extracellular vesicles) into conditioned medium that contains biological factors and is typically substantially free or free of stem cells.
  • Secreted molecules that may be in the conditioned medium or extracellular vesicles include, for example, hormones and other growth factors, cytokines, extracellular matrix proteins, antibodies, and chemokines.
  • the conditioned medium (media) is processed, producing concentrated, processed conditioned medium.
  • the conditioned medium may be filtered by ultra-filtration to produce a processed conditioned medium, typically with an increased concentration of secreted factors.
  • the conditioned media may be subject to one or more treatment steps such as UV treatment or filter sterilization.
  • the conditioned media may also be concentrated by any suitable means, for example by dialysis or ultrafiltration.
  • the molecules and/or extracellular vesicles produced or secreted by the mesenchymal stem cells may also be isolated and purified from the conditioned medium by methods known to those skilled in the art.
  • Conditioned media, extracellular vesicles such as exosomes and secreted molecules expressed in conditioned media or extracellular vesicles may be referred to herein as“products” of the mesenchymal stem cells of the invention.
  • the conditioned media may be stored in liquid form or may be lyophilised. Lyophilised conditioned media may be reconstituted prior to use, for example in water. Lyophilised conditioned media may be stored at a temperature from about 4°C to about room or ambient temperature for a period of days or months prior to use. The storage temperature of the conditioned media may be, for example, about 4°C, about 6°C, about 8°C, about l0°C, about l2°C, about l4°C, about l6°C, about l8°C, about 20°C, about 22°C, about 24°C, or about 25°C.
  • the conditioned media may be stored, for example, for up to about seven days, up to about 14 days, up to about 21 days, up to about 28 days, up to about one month, up to about six weeks, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months or more.
  • the conditioned media or extracellular vesicles may be mixed with one or more solid or liquid carriers or vehicles prior to or following storage.
  • the carrier or vehicle may be, for example, a cream, ointment, lotion, gel, spray or powder.
  • the delivery vehicle may comprise a biocompatible, optionally biodegradable, material such as a polymeric material.
  • the polymeric material may be a gelatinous protein mixture resembling the complex extracellular environment found in many tissues, such as MatrigelTM.
  • the conditioned media (optionally lyophilised) or extracellular vesicles may be impregnated (embedded) in a delivery vehicle and incorporated into, for example a therapeutic bandage such as a gauze bandage.
  • a therapeutic bandage such as a gauze bandage.
  • lyophilised conditioned media may be reconstituted, either by addition of moisture (e.g., water, saline, PBS) or as a result of contact with wound moisture.
  • compositions comprising the stem cells of the invention, conditioned media obtained from culture of the stem cells, or one or more extracellular vesicles derived from the conditioned media. Also provided are uses of stem cells of the invention and compositions comprising the same, in therapeutic methods.
  • Conditioned media derived from the culture of mesenchymal stem cells disclosed herein, and molecules and extracellular vesicles produced or secreted by the mesenchymal stem cells may also be used in the treatment or prevention of disease. They may be used to supplement the activity of, or in place of, the mesenchymal stem cells.
  • the stem cells of the invention can be employed without modification. However, to improve the efficiency of therapy, they may be transplanted as compositions combined with one or more additional agents.
  • the preparation of compositions may comprise, for example the addition of one or more substances that improve the proliferation rate of the cells, enhance the differentiation of the cells in a desired cell lineage, improve the viability of cells in vivo , prolong the lifetime of donor cells, suppress the immunoreaction or inflammation, or improve the migration of donor cells in host tissues.
  • the cells may be engineered to incorporate a gene(s) capable of producing one or more of these effects.
  • the mesenchymal stem cells may be expanded via in vitro culture prior to use, however it is also possible to use the mesenchymal stem cells without the in vitro culture expansion.
  • suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and may include a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable styrene, aminoethyl styrene, aminoethyl sulfate, aminoethyl sulfate, aminotyl, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol sulfate, aminoethyl sulfate, aminoethyl sulfate, aminoethyl sulfate,
  • Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 -butylene glyco
  • non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.
  • Compositions may be prepared as described in, for example, US 8,465,733, the disclosure of which is incorporated herein.
  • compositions may be administered in the form of a cream, ointment, liniment, gel, paste, lotion, solution, spray, powder or the like.
  • compositions may prepared so as to contain liposomes, micelles, and/or microspheres.
  • Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil.
  • Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase.
  • the oil phase may be comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol.
  • the aqueous phase may exceed the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.
  • Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives.
  • the specific ointment base to be used is one that will provide for optimum drug delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like.
  • Suitable ointment bases are typically grouped into four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases.
  • Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum.
  • Emulsifiable ointment bases contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.
  • Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.
  • Water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight.
  • the composition may be in the form of a water-based gel wherein the gel includes at least one gelling agent, a solubilising agent and water.
  • Gelling agents that may be used in the compositions of the invention include, but are not limited to: algal extracts, gums, polysaccharides, starches, pectins, hydrolysed proteins, cellulose derivatives and polymers comprising pendant carboxylic acid groups, or esters thereof, polymers comprising pendant anhydrides of dicarboxylic acid groups and block co polymers, including poloxomers, based on ethylene oxide and/or propylene oxide.
  • the scope of the present invention is not limited in any way by the potential applications, including therapeutic applications, of the stem cells disclosed herein, conditioned media or extracellular vesicles derived therefrom, or of compositions comprising these stem cells, conditioned media or extracellular vesicles.
  • the cells, conditioned media, extracellular vesicles and compositions of the invention may find application in the treatment or prevention of musculoskeletal injuries, cardiovascular diseases, inflammatory diseases, immune system diseases, neurological diseases, and other degenerative diseases and conditions.
  • Such applications may involve, for example, the injection or transplantation (autologous or heterologous transplantation) of the stem cells or conditioned medium into a subject, or compositions comprising the stem cells or conditioned medium, or the in vitro regeneration of tissue using the stem cells or conditioned medium, or compositions comprising the stem cells or conditioned medium.
  • An aspect of the invention provides a method for aiding, promoting or inducing wound healing, tissue repair, tissue generation or tissue regeneration, comprising administering to a subject, or to cells or tissue derived therefrom, an effective amount of one or more stem cells of a stem cell line of the invention, or conditioned media, one or more secreted molecules, one or more extracellular vesicles, secretome or pharmaceutical composition as disclosed herein.
  • An amount effective to aid, promote or induce wound healing, tissue repair, tissue generation or tissue regeneration is an amount that decreases (reduces) the amount of time for healing, repair, generation or regeneration of the tissue relative to the time it would take for the healing, repair, generation or regeneration in the absence of the stem cells, conditioned medium, secreted molecule(s), extracellular vesicle(s), secretome(s) or pharmaceutical composition.
  • the wound may be a chronic, acute or surgical wound.
  • wounds that are amenable to treatment in accordance with embodiments of the invention include incisions, scars, surgical wounds, infected wounds, wounds associated with a disease state such as ulcers and decubitus ulcers, burns and other forms of injured tissue and tissue trauma.
  • Embodiments of the invention relate to the treatment of surgical wounds and to the promotion of post-operative recovery.
  • the surgical wound amenable to treatment may be any wound resulting from surgery or induced in the course of surgery.
  • Embodiments of the invention provide products comprising stem cells, conditioned media, secreted molecules, extracellular vesicles, secretomes or pharmaceutical compositions as disclosed herein for use as cosmetics and anti-aging treatments, for example in the form of creams, lotions, ointments and gels.
  • An exemplary embodiment provides a peel-off face mask comprising conditioned media of the present invention.
  • methods for improving the appearance of skin including for reducing wrinkles and lines on the skin, by applying a product, such as a cream, ointment, lotion, gel or skin or face mask comprising stem cells, conditioned media, secreted molecules, extracellular vesicles, secretomes or pharmaceutical compositions as disclosed herein.
  • Also provided herein are methods of treating injured tissue in a subject including administering to the subject any one of the stem cells, conditioned media, secreted molecules, extracellular vesicles, secretomes or pharmaceutical compositions disclosed herein in an amount effective to treat the injured tissue in the subject.
  • the injured tissue may be, for example, burned tissue/skin, tissue/skin damaged from grafting (e.g., when undamaged skin is taken from one part of the body to function as donor skin for another part of the body), a ruptured tendon or broken bone, a decubitis ulcer or other skin ulceration.
  • An amount effective to treat an injured tissue is an amount that decreases (reduces) the amount of time for healing of the tissue relative to the time it would take for the injured tissue to heal in the absence of the stem cells, conditioned medium, secreted molecule(s), extracellular vesicle(s), secretome(s) or pharmaceutical composition.
  • Example 1 Isolation and characterisation of an adipose tissue-derived human
  • SVF stromal vascular fraction
  • the SVF was then resuspended in cell culture media comprising basal media (Stemmacs MSC expansion media, Miltenyi Biotec) + 10% FBS + supplement + 1% antibiotic-antimycotic followed by sequential straining using lOOpm and 40pm strainers to remove the undigested pieces of tissue. The final pellet obtained after centrifugation is then seeded into cell culture flasks.
  • basal media Stemmacs MSC expansion media, Miltenyi Biotec
  • FBS + supplement + 1% antibiotic-antimycotic followed by sequential straining using lOOpm and 40pm strainers to remove the undigested pieces of tissue.
  • the final pellet obtained after centrifugation is then seeded into cell culture flasks.
  • the SVF contains a heterogeneous population of mesenchymal stem cells as well as other hematopoietic cells. These hematopoietic cells do not attach to the cell culture surface and remain in suspension, and hence are washed off with subsequent media changes.
  • the adherent population of cells contain the adipose tissue-derived human mesenchymal stem cells (ADSCs) which possess high proliferative ability and evident colony-forming ability. These are selectively expanded with subsequent passages.
  • ADSCs adipose tissue-derived human mesenchymal stem cells
  • This cell line was designated CKC-Endeavour-l, and was deposited on 13 June 2018 with CellBank Australia acting as International Depositary Authority for the purposes of deposits pursuant to the Budapest Treaty. The deposit was accorded Accession Number CBA20180030.
  • ADSCs Following expansion of the ADSCs, a significant portion of the cells were cryostored in liquid nitrogen to create a‘Master stock’ for future use. The remaining cells were used to investigate multipotency using tri-lineage differentiation experiments. The cells were subjected to adipogenic, osteogenic, and chondrogenic differentiation conditions over a period of 7-14 days using respective differentiation inducing media. Adipogenic, osteogenic and chondrogenic differentiation was assessed quantitatively after staining with Oil Red O, Alizarin Red S and Alcian Blue, respectively.
  • ADSCs undergoing differentiation at Day 11 were fixed with 4% paraformaldehyde and rinsed with PBS. Following this, the cells were stained with lmL of Alcian Blue stain per well overnight at room temperature and rinsed with PBS before observing under the microscope. For quantification, lmL of 6M guanidine HC1 solution was added to each well and incubated at room temperature for 2 hours. The absorbance of the solution was measured at 650nm using a spectrophotometer
  • PCR cycling was: denaturation at 95°C for 5 mins; 40 cycles of 95°C for 30 sec, 60°C for 30 sec (50°C in the case of PPARy) and 72°C for 1 min; and a final extension step for 7 mins at 72°C. Table 1
  • the adipogenic-specific marker PPARy, osteogenic-specific marker osteocalcin and the chondrogenic-specific marker collagen 10A1 are each expressed in Day 7 differentiated cells derived from the commercially available ADSC cell line and the Endeavourl cell line ( Figure 3A-C). The absence or relatively low expression of these differentiation markers in respective undifferentiated ADSCs are also shown. Moreover, CD44 is shown to be expressed in both the commercially available ADSC cell line and the Endeavourl cell line ( Figure 3D).
  • Example 2 Isolation and characterisation of a dental pulp-derived human
  • DPSC dental pulp tissue-derived mesenchymal stem cell
  • the quality control of stem cells lines is paramount for their use in biomedical engineering and therapeutic applications.
  • the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) has proposed minimal criteria to define human MSCs. They can be identified by adherence to plastic, positive expression of cell surface markers such as CD105, CD73 and CD90, and lack of expression of hematopoietic markers such as CD45, CD34 and other surface molecules including CD 14 or CDl lb, CD79alpha or CD19 and HLA-DR. MSCs must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. Based on ISCT guidelines, the inventors determined the cell surface marker expression of C KC -Endeavour- 1 and CKC-Endeavour-2.
  • Cell surface antigen expression was analysed by flow cytometry (Attune NXT), using MSC-specific monoclonal antibodies including CD44-Allophycocyanin (APC), CD90- R-phycoerythrin (R-PE), CD45-APC, CD-73 fluorescein isothiocyanate (FITC), CD105- Super Bright 436, and CD34-R-PE.
  • APC CD44-Allophycocyanin
  • R-PE CD90- R-phycoerythrin
  • FITC CD-73 fluorescein isothiocyanate
  • CD105- Super Bright 436 CD34-R-PE
  • the array membranes were blocked with array buffer 6 at room temperature for 1 hour on a shaker. After blocking, l.5mL samples containing 775uL of ADSC-CM was mixed with 725uL array buffer 6 and added to the membranes for an overnight incubation at 4°C on a shaker. On the next day, the membranes were washed three times with IX wash buffer for 10 minutes on a shaker followed by the addition of the l.5mL detection antibody cocktail on the membranes. An incubation of lhr at room temperature on the shaker was performed followed by 3 washes with IX wash buffer for 10 minutes each.
  • Lyophilisation of frozen conditioned media was performed overnight, independently for CKC-Endeavour-l and CKC-Endeavour-2 cells. It was carried out in 15 mL tubes overnight containing lOmL conditioned media per tube, and the lyophilised samples were stored at 4°C for subsequent antibody array assays (RDSARY022B, R&D Systems). Sample collection and treatment is shown schematically in Figure 7. Briefly, three separate samples of lyophilised conditioned media from each of CKC-Endeavour-l and CKC-Endeavour-2 were taken and treated as follows:
  • Exosomes or extracellular vesicles were derived using CKC-Endeavour-l and CKC- Endeavour-2 conditioned media (see Example 2) using the qEV exosome isolation kit (Izon, according to manufacturer’s instructions). Exosomes isolated from conditioned media using the Izon columns were stored at -80°C and used for size and yield measurement using the Nanosight NS300. As shown in Figure 11 A, the concentration of exosome particles derived from CKC-Endeavour-2 conditioned media was 1.07 x 10 8 ⁇ 2.11 x 10 7 particles/mL.
  • Extracellular vesicles derived from CKC- Endeavour-2 conditioned media displayed a size range of 25 nm to 135 nm (70% of particles), 255 nm to 315 nm (20% of particles) and 588 nm to 765 nm (10% of particles).
  • Figure 11B is a representative image of an analysed exosome sample derived from CKC-Endeavour-l conditioned media.
  • the particles within the size range of 75-155 nm peak represent the exosome fraction and account for about 80% of the extracellular vesicles.
  • a small peak at about 415 nm indicates the possible presence of extracellular vesicles other than exosomes (about 20% of the total yield).
  • the total yield ( ⁇ standard error) of this exosome fraction was 3.57 x 10 8 ⁇ 1.7 x 10 7 particles/mL.
  • the inventors then investigated the ability of conditioned media and exosomes derived from the culture of CKC-Endeavour-l and CKC-Endeavour-2 to promote wound healing in an in vitro scratch assay using human dermal fibroblasts (HDF01).
  • liquid conditioned media, exosomes and lyophilised conditioned media derived from both CKC-Endeavour-l and CKC-Endeavour-2 reduced the gap width of the scratch compared to controls, demonstrating the regenerative capacity of these mesenchymal stem cell-derived products and their application in wound healing.
  • Example 7 Exemplary peel-off face mask containing lyophilized conditioned media
  • a peel off mask is designed to adhere to human skin for delivering growth factors and cytokines present in lyophilized conditioned media derived from CKC -Endeavour- 1 and/or CKC-Endeavour-2 to aid in skin regeneration and repair and to provide a moisturizing effect.
  • An exemplary face mask comprises, per 100 g:
  • All ingredients are pulverised using a high speed blender to form a homogenous blend. 2 g of the resultant powder is mixed in 12 ml RO water to develop a homogeneous paste. The mask is applied immediately on the skin before gelation and can be used for between 15 mins and 4 hours. The mask has been demonstrated to stick to human skin under laboratory conditions and can be peeled off softly.

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Abstract

L'invention concerne des cellules souches mésenchymateuses dérivées de tissu adipeux et de pulpe dentaire, en particulier des cellules souches Mésenchymateuses déposées sous les numéros d'accès CBA2018K Et CBA201837. L'invention concerne également des procédés de production et de culture desdites cellules souches. L'invention concerne également des produits dérivés desdites cellules souches comprenant des milieux conditionnés et des exosomes comprenant des facteurs sécrétés, et l'utilisation thérapeutique et cosmétique de tels produits dans la cicatrisation des plaies, la réparation tissulaire et la régénération tissulaire.
PCT/AU2019/051029 2018-09-26 2019-09-25 Cellules souches mésenchymateuses et produits obtenus à partir de celles-ci WO2020061627A1 (fr)

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Publication number Priority date Publication date Assignee Title
IT202000017746A1 (it) * 2020-07-22 2022-01-22 ALGO BIOTECHNOLOGIES srl Composizione farmaceutica comprendente mezzo condizionato da secretoma di cellule mesenchimali del cavo orale
CN114073714A (zh) * 2020-08-11 2022-02-22 北京大学口腔医学院 干细胞外泌体制剂及其应用
IT202100005438A1 (it) * 2021-03-09 2022-09-09 Pharmaexceed S R L Scaffold per la rigenerazione ossea e relativo metodo di fabbricazione
IT202100005441A1 (it) * 2021-03-09 2022-09-09 Pharmaexceed S R L Scaffold per la rigenerazione tissutale, in particolare per la rigenerazione ossea, e relativo metodo di fabbricazione
WO2022189993A1 (fr) * 2021-03-09 2022-09-15 Pharmaexceed S.R.L. Échafaudage destiné à la régénération osseuse et son procédé de fabrication
WO2022189997A1 (fr) * 2021-03-09 2022-09-15 Pharmaexceed S.R.L. Échafaudage pour la régénération tissulaire, en particulier pour la régénération osseuse, et son procédé de fabrication
WO2022256865A1 (fr) * 2021-06-08 2022-12-15 Kuldip Sidhu Utilisation de cellules souches mésenchymateuses et produits associés
CN113769103A (zh) * 2021-10-11 2021-12-10 中山大学 一种治疗糖尿病皮肤溃疡的间充质干细胞制剂及其制备方法
CN113769103B (zh) * 2021-10-11 2023-12-05 中山大学 一种治疗糖尿病皮肤溃疡的间充质干细胞制剂及其制备方法

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