AU2022290667A1 - Use of mesenchymal stem cells and products thereof - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
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- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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- 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
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Abstract
The present invention relates to human mesenchymal stem cell lines produced in serum-free and predominantly xenogeneic-free conditions. In particular, the invention relates to exosomes derived from such human mesenchymal stem cell lines and their use in the treatment of spinal cord injury.
Description
USE OF MESENCHYMAL STEM CELLS AND PRODUCTS THEREOF FIELD OF THE INVENTION
[0001 ] The present application claims priority from Australian Provisional Patent Application No. 2021901708 (filed 8 June 2021), the contents of which are incorporated in their entirety herein.
[0002] The present invention relates generally to human mesenchymal stem cell lines produced in serum-free and predominantly xenogeneic-free conditions. In particular, the invention relates to products of such human mesenchymal stem cell lines, such as conditioned media and exosomes, and the use of such products in the treatment of spinal cord injury (SCI).
BACKGROUND OF THE INVENTION
[0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
[0004] SCI is one of the major delipidating public health issues, with an incidence of 40-80 per million people per year. Generally, young adults are involved, where the burden of permanent neurological damage is significant for patients, their caregivers, and the health system. Most of the post-traumatic degeneration of the nervous system is caused by multifactorial secondary damage including different molecular processes such as inflammation, neuronal death, ionic dysregulation, free radicals and lipid peroxidation, disconnection of normal nerve pathways, blood-brain barrier dysfunction, apoptosis, and necrosis, followed by cavitation processes and retrograde degeneration. In traumatic SCI, an early surgical decompression seems to be important in reducing secondary damage, in the range between 8 and 24 h after injury (Sohail et al. 2018, The Spine Vol 1 , 2, Update Book Company), together with spinal fixation to allow correct nursing and rehabilitation. However, such surgery does not stop the degenerative molecular processes associated with SCI.
[0005] In light of the above, there is a need for alternative therapies for the treatment of SCI.
[0006] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
SUMMARY OF THE INVENTION
[0007] It has been surprisingly found that administration of exosomes derived from mesenchymal stem cells to subjects with SCI improved sensory and motor responses.
[0008] According to one aspect, the present invention provides a method of treating SCI in a subject comprising administering to the subject exosomes derived from a human mesenchymal stem cell line, wherein the human mesenchymal stem cell line is the stem cell line deposited with CellBank Australia under Accession Number CBA20180030 or Accession Number CBA20180031.
[0009] According to another aspect, the present invention provides use of exosomes derived from a human mesenchymal stem cell line for the manufacture of a medicament for treating SCI in a subject, wherein the human mesenchymal stem cell line is the stem cell line deposited with CellBank Australia under Accession Number CBA20180030 or Accession Number CBA20180031.
[0010] According to another aspect, the present invention provides a composition comprising exosomes derived from a human mesenchymal stem cell line for use in treating SCI in a subject, wherein the human mesenchymal stem cell line is the stem cell line deposited with CellBank Australia under Accession Number CBA20180030 or Accession Number CBA20180031.
[0011] In one embodiment, the stem cell line is cultured and/or maintained in serum free and predominately xenogeneic-free media.
[0012] In another embodiment, the human mesenchymal stem cell line is the stem cell line deposited with CellBank Australia under Accession Number CBA20180030.
[0013] In another embodiment 5x106 to 1x108 exosomes are administered to the subject.
[0014] In another embodiment, about 2x107 exosomes are administered to the subject.
[0015] In another embodiment, the exosomes are administered intravenously, intranasally or intrathecally.
[0016] In another embodiment, the exosomes are administered intrathecally.
[0017] In another embodiment, the exosomes are administered monthly.
[0018] In another embodiment, the exosomes are administered every two months.
[0019] In another embodiment, the treatment improves sensory response in the subject.
[0020] In another embodiment, the treatment improves motor response in the subject.
[0021] In another embodiment, the treatment stimulates nerve regeneration at the site of the injury.
[0022] In another embodiment, the treatment at least partially restores nerve function through the injured spinal cord.
[0023] In one embodiment, the spinal cord injury may be a complete severing of the spinal cord, a partial severing of the spinal cord, or a crushing or compression injury of the spinal cord.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Embodiments of the invention are described herein, by way of example only, with reference to the accompanying drawings.
[0025] [Fig. 1] Tri-lineage differentiation potential of ADSCs. Left hand panels, HMSC-ad from ScienCell Research Laboratories (a commercially available ADSC line). Right hand panels, CKC-Endeavour 1. 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.
[0026] [Fig. 2] Quantification of adipogenic, osteogenic, and chondrogenic differentiation of HMSC-ad and CKC-Endeavour 1.
[0027] [Fig. 3] Expression of differentiation marker genes in day 7 differentiated (diff) and undifferentiated (undiff) cells derived from HMSC-ad and CKC-Endeavour 1. A = expression of adipogenic-specific marker PPARy; B = expression of osteogenic-specific marker osteocalcin; and C = expression of chondrogenic-specific marker collagen 10A1. D = expression of mesenchymal stem cell marker CD44 in undifferentiated cells. RPLP0 was used as the house-keeping control.
[0028] [Fig. 4] Tri-lineage differentiation of DPSCs. Left hand panels, differentiated cells derived from CKC-Endeavour 2. 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.
[0029] [Fig. 5] Quantification of adipogenic, osteogenic, and chondrogenic differentiation of CKC-Endeavour 2.
[0030] [Fig. 6] A = expression of mesenchymal stem cell marker CD44 in undifferentiated CKC-Endeavour 2. 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: B = expression of adipogenic-specific marker PPARy; C = expression of osteogenic-specific marker osteocalcin; and D = expression of chondrogenic-specific marker collagen 10A1. RPLP0 was used as the house-keeping control.
[0031] [Fig. 7] Flow expression of surface markers for MSCs positive for CD73, CD44, CD90 and negative for CD34, CD 45, CD105. Data shown for CKC-Endeavour 1 (A) and CKC- Endeavour 2 (B).
[0032] [Fig. 8] Levels of 105 growth factors and cytokines expressed in lyophilised conditioned media from CKC-Endeavour 1 cells (left hand bars) and CKC-Endeavour 2 cells (right hand bars) reconstituted in RO water (day 0) following storage of non-reconstituted lyophilised conditioned media powder at 4°C for 36 days.
[0033] [Fig. 9] Levels of growth factors and cytokines expressed in lyophilised conditioned media from CKC-Endeavour 1 cells (left hand bars) and CKC-Endeavour 2 cells (right hand bars) 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.
[0034] [Fig. 10] Exosome concentration and particle size distribution derived from
CKC-Endeavour 2.
[0035] [Fig. 11] Exosome concentration and particle size distribution derived from
CKC-Endeavour 1 .
[0036] [Fig. 12] Comparison of cytokine profile of CKC Endeavour-1 EXO (left hand bars) and HMSC-ad EXO (right hand bars).
[0037] [Fig. 13] Comparative analyses of protein content of secretome of CKC-Endeavour 1 and HMSC-ad.
[0038] [Fig. 14] Summary of data from treatment of SCI with exosomes derived from CKC- Endeavour 1.
DEFINITIONS
[0039] In describing and claiming the present invention, the following terminology has been used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.
[0040] The 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. By way of example, “an element” means one element or more than one element.
[0041] As the term is used herein, 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.
[0042] As used herein the term “about” can mean within 1 or more standard deviation per the practice in the art. Alternatively, “about” can mean a range of up to 20%. When particular values are provided in the specification and claims the meaning of “about” should be assumed to be within an acceptable error range for that particular value.
[0043] In the context of the invention the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, for example mammals and nonmammals, such as non-human primates, horses, cows, dogs, etc.
[0044] In the context of the present invention, the words “comprise”, “comprising” and the like are to be construed in their inclusive, as opposed to their exclusive, sense, that is in the sense of “including, but not limited to”.
[0045] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
[0046] As used herein 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.
[0047] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term ‘about’.
[0048] The recitation of a numerical range using endpoints includes all numbers subsumed.
[0049] As used herein, “spinal cord injury” means an injury in which the axons or nerve fibres of the spinal cord are interrupted, generally by mechanical forces. The spinal cord injury may be a complete severing of the spinal cord, a partial severing of the spinal cord, or a crushing or compression injury of the spinal cord. The spinal cord injury may have occurred more than three months prior to the treatment, more than three weeks prior to the treatment, or more than two weeks prior to the treatment.
[0050] The terms “parenteral administration” and “administered parenterally” are art- recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid, intraspinal, epidural, intracerebral and intrasternal injection or infusion.
[0051] As used herein, the term “treating” or “treatment” includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize a subject's condition. As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether
partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
[0052] The terms "promoting" and "inducing", and variations thereof such as "promotion" and "inducement", as used herein 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. Thus, 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.
[0053] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
[0054] As used herein, the term “sensory response” means sensitivity to pinpricking and touch.
[0055] As used herein, the term “motor response” means movement of limbs or body parts through motor muscle response.
[0056] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term ‘about’.
[0057] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
PREFERRED EMBODIMENT OF THE INVENTION
[0058] Although the invention has been described with reference to certain embodiments detailed herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be obvious to those skilled in the art and the invention is intended to cover all such modifications and equivalents.
[0059] 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 xenogeneic infectious agents.
[0060] In particular, provided herein are mesenchymal stem cell lines produced under serum-free and xenogeneic-free conditions, derived from human adipose tissue and from human dental tissue. In one aspect, there is provided 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. In another aspect, there is provided 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.
[0061] In particular embodiments, the medium in which the stem cells of the invention are cultured does not comprise serum. In embodiments, 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) or StemMACS™ MSC Expansion Media Kit XF. The antibiotic may be, for example, penicillin-streptomycin. The basal medium may further comprise an artificial serum replacement.
[0062] Also provided is 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. In alternate embodiments 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. Those skilled in the art will appreciate that 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, in particular exosomes.
[0063] Exosomes may be purified and/or enriched from conditioned media by methods such as magnetic particles, filtration, dialysis, ultracentrifugation or chromatography, or by commercially-available kits such as ExoQuick™ (Systems Biosciences, CA, USA) and qEV exosome isolation kit (Izon)
[0064] In one aspect 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. In certain embodiments, the conditioned medium (media) is processed, producing concentrated, processed conditioned medium. For example, the conditioned medium may be filtered by ultra-filtration to produce a processed conditioned medium, typically with an increased concentration of secreted factors.
[0065] Depending on the application to which it is to be put, 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.
[0066] 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 10°C, about 12°C, about 14°C, about 16°C, about 18°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.
[0067] Also provided herein are pharmaceutical 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.
[0068] 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.
[0069] 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. By way of non-limiting example only, the cells, conditioned media, extracellular vesicles and compositions of the invention may find application in nerve regeneration. Such applications may involve, for example, the injection or 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.
[0070] The present invention is further described by the following non-limiting examples.
EXAMPLES
EXAMPLE 1 - ISOLATION AND CHARACTERISATION OF AN ADIPOSE TISSUE- DERIVED HUMAN MESENCHYMAL STEM CELL LINE
[0071] The lipoaspirate (100mL) collected from a patient with informed consent undergoing liposuction surgery was transported in sterile culture tubes to the laboratory on ice (4°C). Upon receipt, the separated fat layer (30mL) was aspirated and washed three times with equal volumes of PBS containing 1% antibiotic-antimycotic in a closed Class III isolator (Biospherix).
Following washes, the fat was subjected to enzymatic digestion (1.5mg/mL collagenase) at 37°C for 30 minutes. After digestion, the enzyme was deactivated, and the slurry of fat tissue centrifuged to obtain the stromal vascular fraction (SVF) in the pellet. The SVF was then resuspended in serum-free and predominantly xenogeneic-free media (StemMACS™ MSC Expansion Media Kit XF). The final pellet obtained after centrifugation is then seeded into cell culture flasks.
[0072] 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. The continued propagation of the derived ADSCs for 3-5 population doublings, their survival of freezing-thawing cycles and their differentiation potentials into adipogenic, osteogenic, and chondrogenic lineages (see below) indicated the formation of a new ADSC line.
[0073] This cell line was designated CKC-Endeavour 1 , and was deposited on 13 June 2018 with CellBank Australia (214 Hawkesbury Rd, Westmead NSW 2145, Australia) acting as International Depositary Authority for the purposes of deposits pursuant to the Budapest Treaty. The deposit was accorded Accession Number CBA20180030.
[0074] 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. Briefly, the cells were subjected to adipogenic, osteogenic, and chondrogenic differentiation conditions over a period of 7-14 days using respective differentiation inducing media [Fig. 1] Over this duration, evident morphological changes started appearing in ADSCs even in the first 2-3 days indicating their commitment to the differentiation pathway.
[0075] At Day 7, the presence of lipid globules within the ADSCs were observed by staining with Oil Red O stain that selectively targets neutral lipids (triglycerides) within the cells [Fig. 1 , top row]. This provides evidence of differentiation of the ADSCs into adipocytes. This adipogenic differentiation was quantified using isopropanol elution [Fig. 2]
[0076] At Day 8, the presence of mineralised nodules was observed throughout the culture surface, confirmed by staining with Alizarin Red S stain that selectively targets calcium deposits in these deposited mineralised nodules [Fig. 1 , middle row]. This provides evidence
of differentiation of the ADSCs into osteoblasts. This osteogenic differentiation was quantified using the known acetic acid/ammonium hydroxide-based method [Fig. 2]
[0077] At Day 11 , the presence of proteoglycan-rich extracellular matrix was observed throughout the culture surface, confirmed by specific staining with Alcian blue stain that selectively targets carboxylated and sulphated proteoglycan-rich extracellular matrix [Fig. 1 , bottom row]. This provides evidence of differentiation of the ADSCs into chondrocytes. This chondrogenic differentiation was quantified using the known guanidine hydrochloride-based method [Fig. 2]
[0078] On Day 7 of differentiation, cells derived from both HMSC-ad from ScienCell Research Laboratories (a commercially available ADSC line) and the Endeavour 1 cell line were analyzed for the expression of specific differentiation markers by RT-PCR. The absence or relatively low expression of these differentiation markers in respective undifferentiated ADSCs are also shown. RPLP0 was used as the house-keeping control in all experiments.
[0079] 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 HMSC-ad and the Endeavour 1 cell line [Fig. 3A-C] Moreover, CD44, a key mesenchymal stem cell marker, is shown to be expressed in both HMSC-ad and the Endeavour 1 cell line [Fig. 3D]
EXAMPLE 2 - ISOLATION AND CHARACTERISATION OF A DENTAL PULP-DERIVED HUMAN MESENCHYMAL STEM CELL LINE
[0080] Third molarteeth from healthy individuals aged between 18-25 years, were extracted by a dentist and transferred to the laboratory in 10 ml of sterile phosphate buffer solution (PBS). Each tooth was transferred into a petri dish labelled with the patient identifier and with informed consent. Dental pulp tissue was retrieved from each tooth using a tweezer and the spoon excavator and sliced into three to four pieces using a disposable scalpel. The excavated dental pulp tissue was transferred into a fresh 6 well plate and washed with PBS. Serum-free and predominantly xenogeneic-free media was then added to each sample (StemMACS™ MSC Expansion Media Kit XF). Cell growth was observed within 3-5 days of culture.
[0081] Passaging and expansion of the cells, and their differentiation potential to adipocytes, chondroblasts and adipocytes (see below) indicated the formation of a new dental pulp tissue-derived mesenchymal stem cell (DPSC) line. This cell line was designated CKC- Endeavour 2, and was deposited on 21 June 2018 with CellBank Australia (214 Hawkesbury Rd, Westmead NSW 2145, Australia) acting as International Depositary Authority for the
purposes of deposits pursuant to the Budapest Treaty. The deposit was accorded Accession Number CBA20180031.
[0082] Early passages of the DPSCs between P-1 and P-4 were cryopreserved and stored as primary stock. DPSCs were used to test the multilineage differentiation potential before cryopreserving. Cells were seeded at a density of 22,500 cells per well on a 6 well plate. Approximately 70% confluence was obtained in 2-4 days. The cells were subjected to adipogenic, osteogenic, and chondrogenic differentiation conditions over a period of 7-14 days using respective differentiation inducing media [Fig. 4]
[0083] At Day 9, the presence of lipid globules within the DPSCs were observed by staining with Oil Red O stain that selectively targets neutral lipids (triglycerides) within the cells [Fig. 4, top row]. This provides evidence of differentiation of the DPSCs into adipocytes. This adipogenic differentiation was quantified using isopropanol elution [Fig. 5]
[0084] At Day 10, the presence of mineralised nodules was observed throughout the culture surface, confirmed by staining with Alizarin Red S stain that selectively targets calcium deposits in the mineralized matrix [Fig. 4, middle row]. This provides evidence of differentiation of the DPSCs into osteoblasts. This osteogenic differentiation was quantified using the known acetic acid/ammonium hydroxide-based method [Fig. 5]
[0085] At Day 21 , the presence of proteoglycan-rich extracellular matrix was observed in the cell micromasses, confirmed by specific staining with Alcian blue stain that selectively targets carboxylated and sulphated proteoglycan-rich extracellular matrix [Fig. 4, bottom row]. This provides evidence of differentiation of the DPSCs into chondrocytes. This chondrogenic differentiation was quantified using the known guanidine hydrochloride-based method [Fig. 5]
[0086] On Day 7 of differentiation, cells derived from both HMSC-ad cell line and the CKC- Endeavour 1 cell line were analysed for the expression of specific differentiation markers by RT-PCR. RPLP0 was used as the house-keeping control.
[0087] The adipogenic marker PPARy showed weak expression in differentiated cells [Fig. 6B], while stronger expression of the osteogenic differentiation marker osteocalcin and the chondrogenic differentiation marker collagen 10A1 was observed [Fig.6C&D] Undifferentiated DPSCs expressed the CD44 marker specific to mesenchymal cells [Fig. 6A] Flow expression of surface markers for MSCs was positive for CD73, CD44, CD90 and negative for CD34, CD 45, CD105 in both adipose-derived MSCs [Fig. 7A] and dental-derived MSCs [Fig. 7B]
EXAMPLE 3 - CONDITIONED MEDIA OF CKC-ENDEAVOUR 1 AND CKC- ENDEAVOUR 2
[0088] Following the successful generation of the CKC-Endeavour 1 and CKC-Endeavour 2 mesenchymal stem cell lines as described in Examples 1 and 2, the inventors optimised conditions to harvest the secreted factors from these cells by collecting conditioned media. Specifically, the inventors investigated: the expression of the secretome profile of conditioned media derived from CKC-Endeavour 1 and CKC-Endeavour 2 following lyophilisation and storage of lyophilised conditioned media powder at 4°C for over a month; the stability of expression of secretome components following storage of the lyophilised conditioned media powders at room temperature for 28 days; and the stability of expression of the secretome components after mixing lyophilised conditioned media from with a proprietary cream and storing at 4°C for 1-3 months.
[0089] Lyophilisation of frozen conditioned media was performed overnight, independently for CKC-Endeavour 1 and CKC-Endeavour 2 cells. It was carried out in 15 mL tubes overnight containing 10 mL 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 was as follows:
• Samples (<100 mg) of lyophilised conditioned media from each of CKC-Endeavour 1 and CKC-Endeavour 2 were taken after 36 days storage at 4°C and reconstituted in RO water (1 mL) before secretome profile analysis.
• Samples (<100 mg) of lyophilised conditioned media from each of CKC-Endeavour 1 and CKC-Endeavour 2 were taken after 43 days storage at 4°C, stored at room temperature in a plastic container containing a silica gel pack for a further 28 days, and reconstituted in RO water (1 mL) before secretome profile analysis.
[0090] As shown in Figures 8 and 9, levels of 105 growth factors and cytokines (secretome) expressed in the conditioned media from both CKC-Endeavour 1 and CKC-Endeavour 2 was retained following prolonged storage at 4°C and at room temperature, demonstrating stability of the secreted factors.
EXAMPLE 4 - EXOSOMES FROM CKC-ENDEAVOUR 1 AND CKC-ENDEAVOUR 2
[0091] Exosomes or extracellular vesicles were derived using CKC-Endeavour 1 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 Fig. 10, the concentration of exosome particles derived from
CKC-Endeavour 2 conditioned media was 1.07 x 108 ± 2.11 x 107 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).
[0092] Fig. 11 is a representative image of an analysed exosome sample derived from CKC- Endeavour 1 conditioned media (see Example 2). Here, 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 108 ± 1 .7 x 107 particles/mL.
EXAMPLE 5 - COMPARISON OF CKC-ENDEAVOUR 1 EXOSOMES WITH COMMERCIALLY-AVAILABLE EXOSOMES
[0093] ADSC exosomes have been studied extensively and their cargo include various macromolecules that are very similar to their source of origin and they perform a significant role in cell-cell communication. These macromolecules include a variety of proteins, enzymes, transcription factors, lipids, extracellular matrix proteins, receptors, and nucleic acids, and can be found both on the inside and outside of the exosomal surface.
[0094] There are only isolated reports about the presence of various growth factors and cytokines in ADSC exosomes that are directly relevant to wound and bone healing such as MCP-1 , MCP-3 and SDF-1 , IL-6, FGF-2, and PDGF-BB. Although VEGF is present in the soluble part of the secretome, it is also reported in MSC-EVs at similar levels to those found in the rest of the BM-MSCs secretome. VEGF acts as an effective growth factor for bone regeneration by enhancing the migration of endogenous vascular endothelial and stem cells. VEGF also improves the cellular activity of osteoblasts, overall promoting bone regeneration. These functions reflect the close relationship between osteogenesis and vascularization and the important role of cell-to-cell communication between vascular endothelial cells and osteoblasts. VEGF is overexpressed in CKC-Endeavour 1 as compared to exosomes isolated from commercially sourced cell line. Proteomic analyses have provided further information on the protein contents of EVs in the last few years such as the presence angiopoietin-related protein 2 (ANGPTL2), fibronectin 1 , IGF-I and TGF -1 , which are abundantly expressed in CKC-Endeavour 1 as opposed to the commercial one.
[0095] A comprehensive protein array analyses of 105 different growth factors and cytokines was conducted for CKC-Endeavour 1 exosomes and exosomes derived from HMSC- ad from ScienCell Res Lab (commercially available exosomes) [Fig. 12]
[0096] Importantly, Angiopoietin-1 (Ang1), a vascular anti-inflammatory growth factor implicated in endothelial function and blood vessel remodelling, is highly expressed selectively in CKC-Endeavour 1. Moreover, elevated levels of pro-inflammatory cytokines like Complement component C5a, Complement Factor D, Chitinase Factor- 3 like 1 , C-reactive protein, Criptol , Cystatin C and ENA-78, all of which play crucial roles in complement activation and neutrophil recruitment were also observed. Higher expression of DKK-1 , FGF- 19, HGF, GRO-a, M-CSF, MIG, and FLT3 ligand that are crucial in regulating the delicate balance between differentiation and proliferation of progenitor stem cells we also observed for CKC-Endeavour 1. GCSF, GDF-15 and GM-CSF that regulate injury response in a broad range of tissues are also highly expressed. Abundant expression of the growth hormone, IGFBP-2 and IGFBP-3 was observed in CKC-Endeavour 1 , indicating a role in metabolic regulation. Excellent expression profiles of the chemokines like ICAM-1 , IP10, LIF, MCP-1 and MCP-3 were observed. Elevated levels of IFN-gamma secreted by CKC-ENDEAVOUR 1 is indicative of roles in regulation of adaptive immunity while MIF and Myeloperoxidase are crucial for innate immunity. IL-6, that exhibits an anti-inflammatory profile in models of acute inflammation, is higherwhereas IL-8, the pro-inflammatory cytokine is lowly expressed in CKC- Endeavour 1. Other cytokines that exhibit elevated levels are IL-11 , 12,13, 15 and 17A, IL-19, IL-23, 24,27, 31 , 31 , 33, and IL-34. MIP-1 alpha/MIP-1 B and MIP-3 alpha are higher in CKC- Endeavour 1 compared to the ScienCell HMSC-ad exosomes, implicating roles in, wound healing, and effector immune response maintenance. Other highly expressed cytokines include MMP-9 (tissue remodelling), Lipocalin (renal regeneration), PDGF-AA and PDGF- AB/BB (bone remodelling). TNF-a which is one of the most important pro-inflammatory cytokine providing resistance to infections and cancer, is highly expressed in CKC-Endeavour 1. Lastly, CKC-Endeavour 1 highly express TIM-3, an immune checkpoint protein which has recently been identified as a promising target for immunotherapy.
[0097] To summarize, CKC-Endeavour 1 secretes a variety of pro and anti-inflammatory cytokines, chemokines and growth factors that are vital for immunomodulation, tissue remodelling, cell proliferation and metabolic regulation, and this reflects in the findings that the total protein content secreted by CKC-Endeavour 1 cells is higher than that secreted by HMSC- ad [Fig. 13]
[0098] CKC-Endeavour 1 and exosomes derived thereof show great promise for therapeutic applications as they outperform commercially-available cell lines, such as HMSC-ad from ScienCell Res Lab.
EXAMPLE 6 - USE OF CKC-ENDEAVOUR 1 DERIVED CONDITIONED MEDIA AND EXOSOMES IN TREATMENT OF SCI
[0099] The primary objective of the study was to test the safety and to mitigate pain due to SCI using MSC-conditioned media-derived exosome by intrathecal injections based upon the following clinical assessments before (baseline) and after 6 months:
• Pain scores [numerical rating scale (NRS) or visual analog scale (VAS)];
• Oswestry Disability Index (ODI); and
• X-ray/MRI of lower spine.
[00100] The secondary objectives were volunteers’ self-assessments questionnaire on the products’ sensory aspects, efficacy and safety; as well as safety assessment based on adverse events occurred during the study.
[00101] 12 volunteers for exosomes group with SCI at different levels were recruited for this study. The base-line parameters were compared before and after six months of treatments with exosomes.
[00102] 12 volunteers aged 35 to 60 years with SCI and unresponsive to conservative treatments were recruited. Exclusion criteria were spine surgery, disc herniation discogenic pathology, radiculitis .facet joint arthropathy, spinal stenosis, post-surgery syndrome, lumber facet joint pain, sacroiliac joint pain.
[00103] Volunteers were recruited after hospital ethics approval and with written informed consent not involving the surgeon caring for the patients.
[00104] A lumber puncture needle was inserted intrathecally and 2x107 exosomes from CKC- Endeavour 1 conditioned media (prepared as described in Example 4) was injected every two months. Patients were evaluated as per protocol after 3, 6, 9 months of injection. Out of twelve patients, six (6) had thoracic spine injury and six (6) had lumber spine injury.
[00105] Three months after the first injection no adverse event was observed in any of the patients, sensory nerve response (sensitivity to pinpricking or touch) was observed in 5/12 patients (41 .66%) and no motor response (movement of limbs or body parts) was observed (0%) - see Table 1.
[00106] Six months after the first injection (total 3 injections), no adverse event observed, sensory nerve response was observed in 7/12 patients (58%) and motor response was observed in 3/12 patients (25%) - see Table 2.
[00107] Nine months after first injection (total 4 injections), no adverse events were recorded, sensory response was observed in 9/12 patients (75%) and motor response was observed in 2/12 pateitns (16.6%) - see Table 3.
[00108] The results showed progressive increase in sensory response and a non-statistically significant response for motor recovery [Fig. 14] No adverse event observed in any of the patients and no side effects like hypotension, infection, abscess, and worsened symptoms were observed.
TABLE 1
TABLE 2
TABLE 3
Claims (14)
1. A method of treating spinal cord injury in a subject comprising administering to the subject exosomes derived from a human mesenchymal stem cell line, wherein the human mesenchymal stem cell line is the stem cell line deposited with CellBank Australia under Accession Number CBA20180030 or Accession Number CBA20180031.
2. The method according to claim 1 , wherein the the stem cell line is cultured and/or maintained in serum free and predominately xenogeneic-free media.
3. The method according to claim 1 or claim 2, wherein about 2x107 exosomes are administered to the subject.
4. The method according to any one of claims 1 to 3, wherein the exosomes are administered intrathecally.
5. The method according to any one of claims 1 to 4, wherein the exosomes are administered every two months.
6. The method according to any one of claims 1 to 5, wherein the treatment improves sensory response in the subject.
7. The method according to any one of claims 1 to 6, wherein the treatment improves motor response in the subject.
8. Use of exosomes derived from a human mesenchymal stem cell line for the manufacture of a medicament for treating spinal cord injury, wherein the human mesenchymal stem cell line is the stem cell line deposited with CellBank Australia under Accession Number CBA20180030 or Accession Number CBA20180031.
9. The method according to claim 8, wherein the the stem cell line is cultured and/or maintained in serum free and predominately xenogeneic-free media.
10. The method according to claim 8 or claim 9, wherein about 2x107 exosomes are to be administered to the subject.
11 . The method according to any one of claims 8 to 10, wherein the exosomes are to be administered intrathecally.
12. The method according to any one of claims 8 to 11 , wherein the exosomes are to be administered every two months.
13. The method according to any one of claims 8 to 12, wherein sensory response is improved in the subject.
14. The method according to any one of claims 8 to 13, wherein motor response is improved in the subject.
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