WO2014053420A1 - Method for the isolation of mesenchymal stem cells from mammalian blood and use thereof - Google Patents
Method for the isolation of mesenchymal stem cells from mammalian blood and use thereof Download PDFInfo
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- WO2014053420A1 WO2014053420A1 PCT/EP2013/070257 EP2013070257W WO2014053420A1 WO 2014053420 A1 WO2014053420 A1 WO 2014053420A1 EP 2013070257 W EP2013070257 W EP 2013070257W WO 2014053420 A1 WO2014053420 A1 WO 2014053420A1
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- Prior art keywords
- stem cells
- mesenchymal stem
- composition
- blood
- cells
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Classifications
-
- 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
Definitions
- the invention relates to a method for the isolation of mesenchymal stem cells from mammalian blood , and the expansion thereof.
- the isolated stem cells can be used in regenerative therapies for joint, cartilage and tendon injuries, as well as to support and enhance the immune system.
- Stem cell therapy is a promising application in the relatively new field of regenerative medicine and surgery, including veterinary applications as such.
- Stem cells have the ability to differentiate into different cell types, can multiply massively, migrate spontaneously to damaged tissues, producing important factors for tissue repair and possess immunomodulating properties.
- Various sources of mesenchymal stem cells (MSCs) have been described in humans, horses and other mammals; mesenchymal stem cells were primarily isolated from bone marrow, adipose tissue, umbilical cord blood and umbilical cord matrix have been described (Guest et al, 2008; Hoynowski et al, 2007; Koch et al, 2009; Radcliffe et al, 2010).
- MSCs isolated from blood are a promising therapeutic tool for certain degenerative or traumatic diseases in different animal species, because of their enormous plasticity and differentiation capacity (Giovannini et al, 2008; Koerner et al, 2006; Martinello et al, 2010; Zvaifler et al , 2000).
- a disadvantage of the use of autologous MSCs (from the individual itself) is that the, time-consuming isolation is not always successful (hence therapy comes sometimes too late) and the quality of MSCs varies between the different animals.
- a solution to this is the use of allogenic MSCs from selected donors.
- WO 2008 034 740 discloses a method for the isolation and expansion of MSCs from peripheral blood of a mammal, by the addition of MCSF (macrophage colony stimulating factor) by which the cells are expanded and are then sorted.
- MCSF macrophage colony stimulating factor
- the above invention relates to a method for the isolation of mesenchymal stem cells from the blood of mammals according to claim 1.
- the invention in a second aspect relates to a composition comprising mesenchymal stem cells according to claim 11.
- the invention relates to a method for the administration of such a composition according to claim 24.
- FIGURES Figure 1 shows an example of mesenchymal stem cells isolated from the horse according to an embodiment of the present invention, wherein said stem cells are positive for vimentin, fibronectin, and Ki67.
- Figure 2 shows an example of mesenchymal stem cells isolated from human blood, in which the stem cells are positive for vimentin, fibronectin, and Ki67.
- Figure 3 is a schematic representation of the three-phase distribution of blood after centrifugation. Layer A is the plasma layer, layer B the buffy coat, layer C contains the erythrocytes among others.
- Figure 4 shows examples of compositions according to the present invention, preserved in specific sample vials, preferably stored in a container, suitable for long-term storage at a minimum of -80°C, and for administration immediately after thawing.
- Figure 5 shows the effect of mixing MSCs with different scaffolds (for example, based on hyaluronic acid or glycosaminoglycans) on the vitality of the mesenchymal stem cells.
- Figure 6 shows a graphical representation of the significance of the inner diameter of a needle used to aspirate the composition, according to the present invention, from a vial.
- Figure 7 shows the effects of various concentrations of DMSO, when freezing the compositions according to an embodiment of the present invention, on the vitality of the cells in two different methods of thawing.
- Figure 8 shows a representation of mesenchymal stem cells isolated in accordance with an embodiment of the present method.
- Figure 9 shows a representation of mesenchymal stem cells isolated in accordance with an embodiment of the present method, induced towards the formation of tenocytes.
- Figure 10 A shows undifferentiated mesenchymal stem cells according to the present invention (left picture) which are differentiated to tenocytes (right picture) with clear fiber orientation. Differentiation was confirmed by expression of collagen type I ( Figure 10 B) and Smooth Muslce Actin ( Figure 10 C).
- Figure 11 shows mesenchymal stem cells isolated according to an embodiment of the present method, in which the cells were selected by diameter.
- Figure 12 A is an ultrasound scan of a patient with tendinitis
- figure B shows the evolution after 6 months of intensive, conservative therapy.
- Figure 13 A is an ultrasound scan of a patient with tendinitis
- figure B shows the evolution, 29 days after treatment with a composition according to an embodiment of the present invention.
- Figure 14 A is an ultrasound scan of a patient with chronic desmitis
- figure B shows the evolution, 35 days after treatment with a composition according to an embodiment of the present invention.
- the invention relates to a process for the isolation of mesenchymal stem cells from the blood of mammals, as well as a composition of mesenchymal stem cells obtained according to the present invention, and a method for its administration to a subject.
- the method provides a relatively simple and rapid procedure to achieve a highly pure population of MSCs from (preferably peripheral) blood. For each lxlO 6 leucocytes only one MSG is present in the blood. Therefore, isolation and enrichment is necessary in order to come to a homogenous population.
- the resulting population of MSCs can be induced in a next step towards several specific cell types, or they can be used as such.
- the application of MSCs as such, or differentiated, are virtually unlimited. In the present invention, they will be used mainly in the treatment of lesions, as well as in the treatment of frequently occurring diseases or neurological disorders.
- Citing numerical intervals by endpoints includes all integers, fractions and/or real numbers between the endpoints, these endpoints included .
- the invention relates to a method for the isolation of mesenchymal stem cells from the blood of mammals.
- the process comprises by preference the following steps : a) the collection of one or more blood samples from donors, in a sample vial, coated with an anti-coagulant ; b) centrifuging the blood samples to obtain a 3-phase distribution, consisting of a plasma-phase, buffy coat, and erythrocytes phase; c) collecting the buffy coat and loading it on a density gradient;
- step d collecting of the blood-inter-phase obtained from the density gradient of step c) e) isolating of mesenchymal stem cells from the blood-inter-phase by centrifugation; f) seeding at least 2.5 x lOVcm 2 mesenchymal stem cells in culture and keeping them in a low glucose growth medium supplemented with dexamethasone, antibiotics and serum .
- step f) minimally 2.5 x lO'Vcm 2 cells, even more preferred between 2.5 x lOVcm 2 and 5 x 10 5 /cm 2 cells are seeded . This number is crucial to ultimately obtain a pure and viable population MSCs at an acceptable concentration .
- the density in which the cells in step f) of the present method are seeded is essential, because planting the cells too dense will lead to massive cell death during expansion and a non-homogenous population of mesenchymal stem cells.
- a too low cell concentration will result in little or no colony formation of mesenchymal stem cells, so that expansion is not or hardly possible, or it will take too much time. In both cases the viability of the cells will be negatively influenced .
- anti- coagulant it is meant a composition that can inhibit the coagulation of the blood .
- anticoagulants used in the present invention include EDTA or heparin.
- the term "buffy coat" in this invention is to be understood as the fraction of non- coagulated blood, preferably obtained by means of a density gradient centrifugation, whereby the fraction is enriched with white blood cells and platelets.
- Figure 3 shows a schematic representation of a 3-phase distribution of a blood sample obtained by means of centrifugation.
- the buffy coat is the middle phase B, located between the plasma-phase A and the erythrocyte-phase C.
- the buffy coat will be isolated from the other fractions and diluted by means of a suitable physiological buffer, such as for example, a phosphate, bicarbonate, or Tris buffer, preferably with a minimum ratio of 1 :2.
- a suitable physiological buffer such as for example, a phosphate, bicarbonate, or Tris buffer, preferably with a minimum ratio of 1 :2.
- This dilution factor is important, as lower dilution factors may lead to problems when loading the sample from step c on the density gradient, mainly due to a too heavy buffy coat fraction.
- the density gradient in step c and d of the present method is obtained by means of Percoll®. More in particular, the Percoll ® will comprise a density between the de i .08g/ml and 1.077g/ml.
- the term blood-inter-phase is to be understood as that fraction of the blood, preferably obtained by means of a density gradient, located between the bottom fraction, mainly consisting of erythrocytes and polymorph nuclear cells, and the upper fraction, mainly consisting of plasma polymorph nuclear cells.
- the blood-interphase is the source of blood mononuclear cells (BMCs) comprising monocytes, lymphocytes, and mesenchymal stem cells.
- BMCs blood mononuclear cells
- the lymphocytes are washed away at 37 °C, while the monocytes die within 2 weeks in the absence of cytokines necessary to keep them alive. In this way, the MSCs are purified.
- the isolation of the mesenchymal stem cells from the blood- inter- phase is preferably done by means of centrifugation of the blood-inter- phase (after isolation of the inter-phase), after which the cell pellet is washed at least once with a suitable buffer, such as a phosphate buffer.
- the mesenchymal cells are kept at least 2 weeks in growth medium.
- the dexamethasone in the growth medium will caus the stem cells to retain their specific characteristics and keep/prevent them from differentiating.
- 1 % dexamethasone is used.
- mesenchymal stem cell colonies will become visible in the culture bottles.
- a subsequent step g) at least 6 x 10 3 stem cells/cm 2 are transferred to a, expansion medium containing low glucose, serum and antibiotics for the purpose of expanding the mesenchymal stem cells.
- this medium will include a maximum of 20 % serum (such as FBS or FCS). Too high serum concentrations can lead to a kind of "habituation phase" of the mesenchymal stem cells to the growth factors present in the serum, which can lead to a suboptimal division of the cells in the absence of serum. This can adversely affect the cells when they are used for regenerative purposes.
- the expansion of the mesenchymal stem cells will occur in minimal five cell passages. In this way sufficient cells can be obtained.
- the cells are split at 70 to 80 % confluency.
- the mesenchymal stem cells can be maintained up to 50 passages in culture. After this the risk of loss in vitality, senescence or mutation formation occurs.
- the cell population obtained by the method according to the present invention preferably consists of 90% mesenchymal stem cells. More preferably, it will consist of at least 95% mesenchymal stem cells, more preferably of at least 99%, most preferably 100%.
- markers specific for mesenchymal stem cells.
- markers are selected from the group consisting of vimentin, fibronectin, Ki67, or any combination thereof.
- vimentin As such the purity of the obtained cell populations can be analyzed, and the percentage of mesenchymal stem cells determined.
- Figures 1 and 2 show mesenchymal stem cells obtained according to an embodiment of the present invention, respectively isolated from horse ( Figure 1), and human blood ( Figure 2). Both stem cell populations are positive for vimentin, fibronectin and Ki67.
- the obtained mesenchymal stem cells can be induced or differentiated towards adult cells. Induction and differentiation is preferably done by the addition of specific growth factors and/or other differentiation - inducing factors to the medium of the cells. The nature of these factors will crucially depend on the differentiation and the desired adult cell type.
- the mesenchymal stem cells obtained according to the method of the present invention can differentiate into tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, or fibroblasts.
- Figures 9 and 10 show mesenchymal stem cells obtained according to the present invention, which were induced and differentiated towards tenocytes. The nature of the differentiated cells was morphologically confirmed by the observation of the typical fiber structure (Fig. 10 A), and also via the expression of specific markers such as smooth muscle actin (Figure 10 C) and collagen type I ( Figure 10 B).
- the mesenchymal stem cells are ready to use for various purposes, such as regenerative therapies.
- the stem cell population obtained or derivatives thereof (induced or differentiated cells) are frozen and stored at a temperature of at least -80 °C, optionally in liquid nitrogen containers.
- An example of a method for the freezing and storage of the mesenchymal stem cells is set forth in Example 3.
- the cells are frozen in a sample vial, the sample vial ( 1) having a pierceable septum (2) at the top (3) of thevial.
- the pierceable septum (2) consists of a flexible material such as rubber.
- the sample vial ( 1) is stored in a lockable container (4), wherein the container (4) is closable by means of a screw thread mechanism (5).
- the container (4) is closable by means of a screw thread mechanism (5).
- both the sample vials as well as the container are made of materials suitable for cryogenic application.
- Figure 4 illustrates embodiments of the sample vial and reservoir comprising a composition according to the present invention.
- Crucial step in the freezing of the mesenchymal stem cells is the composition of the cryogenic medium, in particular, the concentration of DMSO.
- DMSO prevents ice crystal formation in the medium during the freezing process, but may be toxic to the cells in high concentrations.
- the concentration of DMSO comprises up to 20%, more preferably, the DMSO concentration in the cryogenic medium comprises 10%.
- the cryogenic medium further comprises low-glucose medium such as low- glucose DMEM.
- Figure 7 shows the influence of the DMSO concentration on the vitality of the stem cells, or compositions according to the present invention during defrosting. A percentage of 10% DMSO showed the best results.
- Freezing in accordance with the method of the present invention results in a minimal conservation period of the cells at -80°C for at least 6 months.
- the stem cells according to the present invention are isolated from the blood of mammals, more preferably, from peripheral blood.
- the used blood will originate from human, cat, dog or horse, most preferably equine derived.
- Examples 1 and 2 describe the protocols according to the present invention for the isolation of mesenchymal stem cells from the blood of respectively horse and human.
- blood from a donor was used who was later also recipient of his isolated mesenchymal stem cells.
- blood is used from donors in which the donor is preferably of the same family, gender or race as the recipient of the mesenchymal stem cells isolated from the blood of donors.
- these donors will be tested on common current transmittable diseases or pathologies, in order to avoid the risk of horizontal transmission of these pathologies or diseases through the stem cells.
- the donor animals are kept in quarantine.
- EIA equine infectious anemia
- EHV-l equine rhinopneumonia
- EVA equine viral arteritis
- WNV West Nile virus
- AHS African Horse Sickness
- Dourine Terypanosoma
- piroplasmosis glanders (malleus, glanders), equine influenza A, Borreliosis (Borrelia burgdorferi, Lyme disease).
- present invention provides a composition comprising mesenchymal stem cells and/or induced or differentiated cells derived from mesenchymal stem cells isolated from the blood of mammals according to the above-mentioned method.
- the composition will comprise at least 90%, more preferably comprise at least 95% mesenchymal stem cells.
- the composition comprises at least 99% mesenchymal stem cells, more preferably 100%.
- the non- induced, non- differentiated mesenchymal stem cells in the composition express preferably vimentin, fibronectin, Ki67, or any combination thereof.
- the composition will comprise at least 90%, more preferably at least 95% induced or differentiated cells derived from mesenchymal stem cells.
- the composition comprises at least 99% induced or differentiated cells derived from mesenchymal stem cells isolated according to the method of the present invention, more preferably 100%.
- the induced or differentiated cells may differentiate towards tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, keratinocytes, neurons or fibroblasts.
- the composition is formulated for intravenous, intra-articular, intramuscular, intra-lesional administration to mammals. These modes of administration will depend heavily on the desired application of stem cells and/or their differentiated form.
- compositions based on hyaluronic acid can be used with components selected from the group consisting of platelet- rich plasma (PRP), hyaluronic acid, compositions based on hyaluronic acid, glycosaminoglycans, or compositions based on glycosaminoglycans.
- PRP platelet- rich plasma
- hyaluronic acid hyaluronic acid
- glycosaminoglycans based on glycosaminoglycans
- glycosaminoglycans can be used with components selected from the group consisting of platelet- rich plasma (PRP), hyaluronic acid, compositions based on hyaluronic acid, glycosaminoglycans, or compositions based on glycosaminoglycans.
- PRP for example, a substance rich in growth factors, stimulate the stem cells after implantation.
- both the stem cells and PRP are harvested from the same donors are for compatibility reasons.
- Carrier substances can also be used to counteract gravity: stem cells follow the law of gravity and therefore have difficulties reaching higher lesions without a carrier in which they can migrate.
- the carrier substances themselves also have beneficial effects on the pathological environment in which they contribute to the tissue repair itself and also provide a good stem cell niche to help differentiation of the cells in this area.
- hyaluronic acid, glycosaminoglycans or compositions on this basis examples include OSTENIL®, OSTENIL® +, Adant® and Adequan® (see Figure 5).
- Figure 5 shows a schematic representation of the effect of dilution of the composition with hyaluronic acid or glycosaminoglycan components on the vitality of the composition according to the present invention.
- Arthramid® or R- Gel® are toxic and it must be avoided to use them in combination with MSCs.
- the cells from the composition are isolated from the blood of man, cat, dog or horse.
- composition according to the present invention has very broad applicability.
- composition is suitable for the following purposes:
- trauma selected from the group comprising skin traumas, trauma of cartilage, tendon traumas, traumas of the ligaments, traumas of the bones, traumas of the mucus membranes, cysts or fractures
- acute or chronic inflammatory disease states selected from the group of laminitis, periostitis, gastritis, osteoarthritis, inflammation caused by viral, bacterial, parasitic or mycotic agents in mammals, and/or
- hypersensitivity reactions such as insect hypersensitivity (summer eczema for example), drug hypersensitivity, hypersensitivity to dust and other types of hypersensitivity and/or;
- a method for administering a composition according to the present invention to a subject may include the following steps: a) thawing a sample bottle comprising the composition, frozen at at least -80°C, whereby thawing is carried out at a temperature between 20°C and 37°C, preferably between 25°C and 37°C, and in a time span of maximal 20 minutes, more preferably maximal 5 minutes; b) aspirating the sample from the sample vial by means of a needle with an inner diameter of at least 0.3mm, preferably at least 0.35mm; c) optional mixing of the composition with components selected from the group of platelet- rich plasma (PRP), hyaluronic acid or glycosaminoglycans; d) administrating the composition or mixture thereof to a
- the cell diameter of the injection needle is crucial in this respect, in order to avoid damage to the cells.
- Figure 6 shows the essential effect of the inner diameter of the needle used for aspiration of the composition.
- a 23G needle (inner diameter 0 : 33 mm) had a significantly more positive effect on the vitality of the cells present in the composition than a 25G needle (inner diameter 0: 26 mm).
- Defrosting the composition can be accomplished by thawing in a hot water bath or in the palm of one's hand or by any other method within the temperature limits.
- the composition is administered within 2 minutes after thawing, in order to safeguard the vitality of the composition.
- Example 1 Example Protocol for the isolation of mesenchymal stem cells from the blood of orses
- Figure 1 shows mesenchymal stem cells isolated according to the protocol of Example 1 , wherein the cells are positive for the markers vimentin, fibronectin, and Ki67.
- Example 2 Example Protocol for the isolation of mesenchymal stem cells from the blood of humans, cats or dogs
- Example 1 can be used without problems in blood samples from other mammals, such as human, dog or cat.
- Figure 2 shows mesenchymal stem cells are isolated from human blood, positive for the markers vimentin, fibronectin, and Ki67.
- Example 3 Example protocol for cryo-preservation
- h Remove the supernatant; i . Resuspend the cells in an appropriate quantity of medium ; j Add 1ml cells/cryotube; k. Place the cryotubes overnight in a plastic container with isopropanol (KT) at -80°C.
- KT isopropanol
- Example 4 Example of composition of media used
- compositions of the present invention may be marketed as follows a. a composition comprising non-differentiated mesenchymal stem cells isolated from mammals, preferably for use in orthopedic lesions and pathologies such as osteoarthritis, cartilage damage, cyst structures (see Figure 8). Preferably, such composition is administered intra-articularly. b. a composition comprising mesenchymal stem cells induced towards chondrocytes, preferably for use in cases of severe cartilage injuries. c. a composition comprising mesenchymal stem cells induced towards tenocytes. Preferably, such composition is used in applications of tendon lesions, lesions of the ligaments and other tendopathies (see figure 9 and 10). d.
- composition comprising non-differentiated mesenchymal stem cells, with a diameter of less than 40 ⁇ , preferably for use in treatment of Musco-skeletal pathologies, endocrine pathologies or for regional perfusion (see Figure 11).
- the composition is preferably administered intravenously.
- Example 6 Treatment of tendinitis in horses with a composition comprising mesenchymal stem cells induced towards tenocytes
- Ten horses with severe tendinitis of the superficial digital flexor tendon and 15 horses with chronic desmitis of the musculus interosseous maxims were treated with a composition according to the present invention, including mesenchymal stem cells are induced (pre-differentiation) towards tenocytes in combination with PRP as a carrier substance.
- Figure 12 A is an example of an ultrasound scan of a patient with tendinitis, which was treated using conventional conservative therapy. Recovery was only observed after 6 months of therapy ( Figure 12 B).
- Figure 13 A is an example of an ultrasound scan of a patient with tendinitis, treated with a composition of tenocytes according to the present invention.
- Figure 13 B shows the evolution, 29 days after treatment. A remarkable improvement was visible.
- Figure 14 A is an ultrasound scan of a patient with chronic desmitis, figure B shows the evolution, 35 days after treatment with a composition according to an embodiment of the present invention. Again, a remarkable improvement was observed in a very short time. Moreover, local calcification of tendons before treatment also disappeared after treatment.
- Example 7 Infected wound after abdominal colic
- a patient was suffering from lameness as a result of a lesion to the collateral ligament of the fetlock : less than 2 months after the local injection of a composition according to the invention, the horse was able to walk again normal and no relapse had been noticed.
- a 4-year-old gelding with a difficult healing of the hoof bone fracture was, after 10 months of conservative treatment, iocaiiy treated with a composition according to the present invention. Fuii recovery occurred after 4 months.
- a 19-year old horse with Cushing's syndrome was treated with 2 consecutive treatments of the composition described above (with 2 month interval). After 2 cycles, the symptoms were improved remarkably. Two more horses were treated in the same way with the same result.
- Example 13 Vascular reconstruction
- Example 14 Hypersensitivity reaction A horse with diffuse noduies (bumps) on the body caused by a hypersensitivity reaction has been treated by the composition described, resulting in the disappearance of the bumps the following day. This result lasted for 2 months.
- Carrade DD Carrade DD, Affolter VK, Outerbridge CA, Watson JL, Galuppo LD, Buerchler S, Kumar V, Walker NJ, Borjesson DL, 2011a.
- Intradermal injections of equine allogenic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions. Cytotherapy 13, 1180- 1192.
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Abstract
The present invention relates to a method for the isolation of mesenchymal stem cells from the blood of mammals, comprising the isolation of mesenchymal stem cells from the blood-interphase of blood samples and preserving said cells in a low glucose growth medium supplemented with antibiotics, serum and dexamethasone for preventing the differentiation of the cells. In a further aspect, present invention provides a composition comprising the mesenchymal stem cells obtained according to the method of the current invention as well as a method for the administration of this composition to a subject.
Description
METHOD FOR THE ISOLATION OF MESENCHYMAL STEM CELLS FROM MAMMALIAN BLOOD AND USE THEREOF
TECHNICAL FIELD
The invention relates to a method for the isolation of mesenchymal stem cells from mammalian blood , and the expansion thereof. The isolated stem cells can be used in regenerative therapies for joint, cartilage and tendon injuries, as well as to support and enhance the immune system.
STATE OF THE ART
Stem cell therapy is a promising application in the relatively new field of regenerative medicine and surgery, including veterinary applications as such. Stem cells have the ability to differentiate into different cell types, can multiply massively, migrate spontaneously to damaged tissues, producing important factors for tissue repair and possess immunomodulating properties. Various sources of mesenchymal stem cells (MSCs) have been described in humans, horses and other mammals; mesenchymal stem cells were primarily isolated from bone marrow, adipose tissue, umbilical cord blood and umbilical cord matrix have been described (Guest et al, 2008; Hoynowski et al, 2007; Koch et al, 2009; Radcliffe et al, 2010). However, a disadvantage is that the sampling to isolate mesenchymal stem cells from bone marrow or fat is highly invasive and there is a low degree of similarity between the various clinical trials (seen in trials in horses, among others). Therefore, there is a need for another, more standardized and better source of mesenchymal stem cells. Blood appears to be an optimal source of these MSCs. Blood is not only a non-invasive and painless source, but also simple and safe to collect and, consequently, easily accessible. Furthermore, MSCs isolated from blood are a promising therapeutic tool for certain degenerative or traumatic diseases in different animal species, because of their enormous plasticity and differentiation capacity (Giovannini et al, 2008; Koerner et al, 2006; Martinello et al, 2010; Zvaifler et al , 2000). A disadvantage of the use of autologous MSCs (from the individual itself) is that the, time-consuming isolation is not always successful (hence therapy comes sometimes too late) and the quality of MSCs varies between the different animals. A solution to this is the use of allogenic MSCs from selected donors. The safe use of allogenic MSCs is already reported for humans (Fang et al, 2007; Ringden et al , 2006) and horses (Carrade et al, 2011a ; Carrade et al, 2011b).
WO 2008 034 740 discloses a method for the isolation and expansion of MSCs from peripheral blood of a mammal, by the addition of MCSF (macrophage colony stimulating factor) by which the cells are expanded and are then sorted. However, this method does not lead to a completely homogeneous population of MSCs, but to a mixture of hematopoietic, mesenchymal and pluripotent stem cells.
There is thus a need for a process to isolate mesenchymal stem cells from blood in which a homogeneous population of MSCs is obtained, and for compositions comprising these homogeneous population of MSCs, to use in regenerative applications. Therefore, a rapid and simple process to obtain a MSG composition of high quality is necessary. It is well known that treatment with stem cells have the highest success rate when it is administered immediately after the inflammation phase caused by the damage (before infiltration of fibroblasts, and scar tissue formation) due to the ideal environment for cell growth at that time (Richardson et al, 2007).
With such a pure composition of MSCs, the theoretical applications are virtually unlimited and very accessible (especially when using allogenic MSCs). SUMMARY OF THE INVENTION
The above invention relates to a method for the isolation of mesenchymal stem cells from the blood of mammals according to claim 1.
In a second aspect the invention relates to a composition comprising mesenchymal stem cells according to claim 11.
In a final aspect, the invention relates to a method for the administration of such a composition according to claim 24.
DESCRIPTION OF THE FIGURES Figure 1 shows an example of mesenchymal stem cells isolated from the horse according to an embodiment of the present invention, wherein said stem cells are positive for vimentin, fibronectin, and Ki67.
Figure 2 shows an example of mesenchymal stem cells isolated from human blood, in which the stem cells are positive for vimentin, fibronectin, and Ki67. Figure 3 is a schematic representation of the three-phase distribution of blood after centrifugation. Layer A is the plasma layer, layer B the buffy coat, layer C contains the erythrocytes among others.
Figure 4 shows examples of compositions according to the present invention, preserved in specific sample vials, preferably stored in a container, suitable for long-term storage at a minimum of -80°C, and for administration immediately after thawing.
Figure 5 shows the effect of mixing MSCs with different scaffolds (for example, based on hyaluronic acid or glycosaminoglycans) on the vitality of the mesenchymal stem cells.
Figure 6 shows a graphical representation of the significance of the inner diameter of a needle used to aspirate the composition, according to the present invention, from a vial.
Figure 7 shows the effects of various concentrations of DMSO, when freezing the compositions according to an embodiment of the present invention, on the vitality of the cells in two different methods of thawing.
Figure 8 shows a representation of mesenchymal stem cells isolated in accordance with an embodiment of the present method.
Figure 9 shows a representation of mesenchymal stem cells isolated in accordance with an embodiment of the present method, induced towards the formation of tenocytes.
Figure 10 A shows undifferentiated mesenchymal stem cells according to the present invention (left picture) which are differentiated to tenocytes (right picture) with clear fiber orientation. Differentiation was confirmed by expression of collagen type I (Figure 10 B) and Smooth Muslce Actin (Figure 10 C).
Figure 11 shows mesenchymal stem cells isolated according to an embodiment of the present method, in which the cells were selected by diameter.
Figure 12 A is an ultrasound scan of a patient with tendinitis, figure B shows the evolution after 6 months of intensive, conservative therapy.
Figure 13 A is an ultrasound scan of a patient with tendinitis, figure B shows the evolution, 29 days after treatment with a composition according to an embodiment of the present invention.
Figure 14 A is an ultrasound scan of a patient with chronic desmitis, figure B shows the evolution, 35 days after treatment with a composition according to an embodiment of the present invention.
Figure 15 shows statistical results of patients (suffering from tendinitis or desmitis) treated with compositions according to embodiments of the present
invention. Score 0 indicates 0% improvement 6 weeks after treatment and score 5 indicates 100% improvement (1 =20%, 2=40%, 3=60% and 4=80%).
DETAILED DESCRIPTION
The invention relates to a process for the isolation of mesenchymal stem cells from the blood of mammals, as well as a composition of mesenchymal stem cells obtained according to the present invention, and a method for its administration to a subject.
The method provides a relatively simple and rapid procedure to achieve a highly pure population of MSCs from (preferably peripheral) blood. For each lxlO6 leucocytes only one MSG is present in the blood. Therefore, isolation and enrichment is necessary in order to come to a homogenous population. The resulting population of MSCs can be induced in a next step towards several specific cell types, or they can be used as such. The application of MSCs as such, or differentiated, are virtually unlimited. In the present invention, they will be used mainly in the treatment of lesions, as well as in the treatment of frequently occurring diseases or neurological disorders.
Unless defined otherwise, all terms used in the description of the invention, including technical and scientific terms, have the meaning as they are generally understood by a professional in the technical field of the invention. For a better evaluation of the description of the invention, the following terms are explained explicitly.
"A ", "an" and " the " refer in this document to both the singular and the plural, unless the context clearly implies otherwise. For example, " a segment " means one or more than one segment.
When "about" or "round" are used in this document additional to a measurable quantity, a parameter, a time period or time, etc., variations of +/- 20 % or less, preferably +/-10% or or less, more preferably +/-5% or less , still more preferably +/-1% or less , and even more preferably +/-0.1 % or less than the value cited and, to the extent that such variations can apply in the described invention. It should, however, be understood to mean that the value of the quantity in which the term "about" or "round " is used, itself is not disclosed specifically.
The terms "comprise", "comprising", "air", "include", "including", "law claiming", "content", "holding" are synonyms and are inclusive or open terms that indicate the presence of what follows and which do not preclude the presence or prevent
other components, features, elements, members, steps, as known from, or described in the prior art.
Citing numerical intervals by endpoints includes all integers, fractions and/or real numbers between the endpoints, these endpoints included .
In a first aspect, the invention relates to a method for the isolation of mesenchymal stem cells from the blood of mammals. In particular, the process comprises by preference the following steps : a) the collection of one or more blood samples from donors, in a sample vial, coated with an anti-coagulant ; b) centrifuging the blood samples to obtain a 3-phase distribution, consisting of a plasma-phase, buffy coat, and erythrocytes phase; c) collecting the buffy coat and loading it on a density gradient;
d ) collecting of the blood-inter-phase obtained from the density gradient of step c) e) isolating of mesenchymal stem cells from the blood-inter-phase by centrifugation; f) seeding at least 2.5 x lOVcm2 mesenchymal stem cells in culture and keeping them in a low glucose growth medium supplemented with dexamethasone, antibiotics and serum . Preferably, in step f) minimally 2.5 x lO'Vcm2 cells, even more preferred between 2.5 x lOVcm2 and 5 x 105/cm2 cells are seeded . This number is crucial to ultimately obtain a pure and viable population MSCs at an acceptable concentration . The density in which the cells in step f) of the present method are seeded, is essential, because planting the cells too dense will lead to massive cell death during expansion and a non-homogenous population of mesenchymal stem cells. A too low cell concentration, however, will result in little or no colony formation of mesenchymal stem cells, so that expansion is not or hardly possible, or it will take too much time. In both cases the viability of the cells will be negatively influenced .
By the term anti- coagulant, it is meant a composition that can inhibit the coagulation of the blood . Examples of anticoagulants used in the present invention include EDTA or heparin.
The term "buffy coat" in this invention, is to be understood as the fraction of non- coagulated blood, preferably obtained by means of a density gradient centrifugation, whereby the fraction is enriched with white blood cells and platelets. Figure 3 shows a schematic representation of a 3-phase distribution of a blood sample obtained by means of centrifugation. The buffy coat is the middle phase B, located between the plasma-phase A and the erythrocyte-phase C.
In particular, the buffy coat will be isolated from the other fractions and diluted by means of a suitable physiological buffer, such as for example, a phosphate, bicarbonate, or Tris buffer, preferably with a minimum ratio of 1 :2. This dilution factor is important, as lower dilution factors may lead to problems when loading the sample from step c on the density gradient, mainly due to a too heavy buffy coat fraction.
Preferably, the density gradient in step c and d of the present method is obtained by means of Percoll®. More in particular, the Percoll ® will comprise a density between the de i .08g/ml and 1.077g/ml.
For the purpose of the present invention, the term blood-inter-phaseis to be understood as that fraction of the blood, preferably obtained by means of a density gradient, located between the bottom fraction, mainly consisting of erythrocytes and polymorph nuclear cells, and the upper fraction, mainly consisting of plasma polymorph nuclear cells. The blood-interphase is the source of blood mononuclear cells (BMCs) comprising monocytes, lymphocytes, and mesenchymal stem cells.
In the present invention, the lymphocytes are washed away at 37 °C, while the monocytes die within 2 weeks in the absence of cytokines necessary to keep them alive. In this way, the MSCs are purified.
In a further aspect, the isolation of the mesenchymal stem cells from the blood- inter- phase is preferably done by means of centrifugation of the blood-inter- phase (after isolation of the inter-phase), after which the cell pellet is washed at least once with a suitable buffer, such as a phosphate buffer.
In particular, the mesenchymal cells are kept at least 2 weeks in growth medium. Surprisingly, the dexamethasone in the growth medium will caus the stem cells to retain their specific characteristics and keep/prevent them from differentiating. Preferably, 1 % dexamethasone is used.
Following a minimum period of 2 weeks ( 14 days), preferably 3 weeks (21 days) mesenchymal stem cell colonies will become visible in the culture bottles.
In a subsequent step g) at least 6 x 103 stem cells/cm2 are transferred to a, expansion medium containing low glucose, serum and antibiotics for the purpose of expanding the mesenchymal stem cells.
In particular, this medium will include a maximum of 20 % serum (such as FBS or FCS). Too high serum concentrations can lead to a kind of "habituation phase" of the mesenchymal stem cells to the growth factors present in the serum, which can lead to a suboptimal division of the cells in the absence of serum. This can adversely affect the cells when they are used for regenerative purposes. Preferably, the expansion of the mesenchymal stem cells will occur in minimal five cell passages. In this way sufficient cells can be obtained. Preferably, the cells are split at 70 to 80 % confluency. The mesenchymal stem cells can be maintained up to 50 passages in culture. After this the risk of loss in vitality, senescence or mutation formation occurs.
The cell population obtained by the method according to the present invention, preferably consists of 90% mesenchymal stem cells. More preferably, it will consist of at least 95% mesenchymal stem cells, more preferably of at least 99%, most preferably 100%.
The nature of the cells obtained through this method can be ascertained by means of markers, specific for mesenchymal stem cells. Preferably, markers are selected from the group consisting of vimentin, fibronectin, Ki67, or any combination thereof. As such the purity of the obtained cell populations can be analyzed, and the percentage of mesenchymal stem cells determined. Figures 1 and 2 show mesenchymal stem cells obtained according to an embodiment of the present invention, respectively isolated from horse (Figure 1), and human blood (Figure 2). Both stem cell populations are positive for vimentin, fibronectin and Ki67.
If desired, the obtained mesenchymal stem cells can be induced or differentiated towards adult cells. Induction and differentiation is preferably done by the addition of specific growth factors and/or other differentiation - inducing factors to the medium of the cells. The nature of these factors will crucially depend on the differentiation and the desired adult cell type. In particular, the mesenchymal stem cells obtained according to the method of the present invention can differentiate into tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, or fibroblasts. Figures 9 and 10 show mesenchymal stem cells obtained according to the present invention, which were induced and differentiated towards tenocytes. The nature of the differentiated cells was morphologically confirmed by the
observation of the typical fiber structure (Fig. 10 A), and also via the expression of specific markers such as smooth muscle actin (Figure 10 C) and collagen type I (Figure 10 B).
After the expansion stage the mesenchymal stem cells are ready to use for various purposes, such as regenerative therapies. However, preferably, the stem cell population obtained or derivatives thereof (induced or differentiated cells) are frozen and stored at a temperature of at least -80 °C, optionally in liquid nitrogen containers. An example of a method for the freezing and storage of the mesenchymal stem cells is set forth in Example 3. Preferably, the cells are frozen in a sample vial, the sample vial ( 1) having a pierceable septum (2) at the top (3) of thevial. The pierceable septum (2) consists of a flexible material such as rubber. The sample vial ( 1) is stored in a lockable container (4), wherein the container (4) is closable by means of a screw thread mechanism (5). Preferably, both the sample vials as well as the container are made of materials suitable for cryogenic application. Figure 4 illustrates embodiments of the sample vial and reservoir comprising a composition according to the present invention.
Crucial step in the freezing of the mesenchymal stem cells is the composition of the cryogenic medium, in particular, the concentration of DMSO. DMSO prevents ice crystal formation in the medium during the freezing process, but may be toxic to the cells in high concentrations. In a preferred form, the concentration of DMSO comprises up to 20%, more preferably, the DMSO concentration in the cryogenic medium comprises 10%. The cryogenic medium further comprises low-glucose medium such as low- glucose DMEM. Figure 7 shows the influence of the DMSO concentration on the vitality of the stem cells, or compositions according to the present invention during defrosting. A percentage of 10% DMSO showed the best results.
Freezing in accordance with the method of the present invention results in a minimal conservation period of the cells at -80°C for at least 6 months.
Preferably, the stem cells according to the present invention are isolated from the blood of mammals, more preferably, from peripheral blood. By preferencethe used blood will originate from human, cat, dog or horse, most preferably equine derived. Examples 1 and 2 describe the protocols according to the present invention for the isolation of mesenchymal stem cells from the blood of respectively horse and human.
In a possible embodiment, blood from a donor was used who was later also recipient of his isolated mesenchymal stem cells. In another embodiment, blood is used from donors in which the donor is preferably of the same family, gender or race as the recipient of the mesenchymal stem cells isolated from the blood of donors.
In particular, these donors will be tested on common current transmittable diseases or pathologies, in order to avoid the risk of horizontal transmission of these pathologies or diseases through the stem cells. Preferably, the donor animals are kept in quarantine.
When using donor horses they can be, for example tested for the following pathologies: equine infectious anemia (EIA), equine rhinopneumonia (EHV-l, EHV- 4), equine viral arteritis (EVA), West Nile virus (WNV), African Horse Sickness (AHS), Dourine (Trypanosoma), piroplasmosis, glanders (malleus, glanders), equine influenza A, Borreliosis (Borrelia burgdorferi, Lyme disease).
In a second aspect present invention provides a composition comprising mesenchymal stem cells and/or induced or differentiated cells derived from mesenchymal stem cells isolated from the blood of mammals according to the above-mentioned method.
In a preferred form, the composition will comprise at least 90%, more preferably comprise at least 95% mesenchymal stem cells. Preferably, the composition comprises at least 99% mesenchymal stem cells, more preferably 100%. The non- induced, non- differentiated mesenchymal stem cells in the composition express preferably vimentin, fibronectin, Ki67, or any combination thereof. In another preferred form, the composition will comprise at least 90%, more preferably at least 95% induced or differentiated cells derived from mesenchymal stem cells. Preferably, the composition comprises at least 99% induced or differentiated cells derived from mesenchymal stem cells isolated according to the method of the present invention, more preferably 100%. Preferably, the induced or differentiated cells may differentiate towards tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, keratinocytes, neurons or fibroblasts.
In particular, the composition is formulated for intravenous, intra-articular, intramuscular, intra-lesional administration to mammals. These modes of administration will depend heavily on the desired application of stem cells and/or their differentiated form.
In one embodiment, especially when said composition is used for joint or tendon pathologies, can be used with components selected from the group consisting of platelet- rich plasma (PRP), hyaluronic acid, compositions based on hyaluronic acid, glycosaminoglycans, or compositions based on glycosaminoglycans. Mixing of the composition with such carrier substances may in some cases be desirable to increase the effectiveness of the composition or create a synergistic effect. PRP, for example, a substance rich in growth factors, stimulate the stem cells after implantation. Preferably, both the stem cells and PRP are harvested from the same donors are for compatibility reasons. Carrier substances can also be used to counteract gravity: stem cells follow the law of gravity and therefore have difficulties reaching higher lesions without a carrier in which they can migrate. In addition, the carrier substances themselves also have beneficial effects on the pathological environment in which they contribute to the tissue repair itself and also provide a good stem cell niche to help differentiation of the cells in this area.
Examples of hyaluronic acid, glycosaminoglycans or compositions on this basis include OSTENIL®, OSTENIL® +, Adant® and Adequan® (see Figure 5). Figure 5 shows a schematic representation of the effect of dilution of the composition with hyaluronic acid or glycosaminoglycan components on the vitality of the composition according to the present invention. Arthramid® or R- Gel® are toxic and it must be avoided to use them in combination with MSCs.
Preferably, the cells from the composition are isolated from the blood of man, cat, dog or horse.
The composition according to the present invention has very broad applicability. In particular, the composition is suitable for the following purposes:
- treatment of trauma selected from the group comprising skin traumas, trauma of cartilage, tendon traumas, traumas of the ligaments, traumas of the bones, traumas of the mucus membranes, cysts or fractures
- treatment of neurological and neurodegenerative diseases selected from the
group of Cushing's syndrome, respiratory paralysis or paresis of the extremities, and/or
- treatment of acute or chronic inflammatory disease states selected from the group of laminitis, periostitis, gastritis, osteoarthritis, inflammation caused by viral, bacterial, parasitic or mycotic agents in mammals, and/or
- treatment of hypersensitivity reactions such as insect hypersensitivity (summer eczema for example), drug hypersensitivity, hypersensitivity to dust and other types of hypersensitivity and/or;
- treatment of gastric dilatation and torsion complexes, and/or
- treatment of infertility in mares and precocity in foals. In particular, comprises the current invention each application whereby a subject benefits from administering the composition to said subject. The subject may include a horse, cat, dog or human. More particularly, a method for administering a composition according to the present invention to a subject, may include the following steps: a) thawing a sample bottle comprising the composition, frozen at at least -80°C, whereby thawing is carried out at a temperature between 20°C and 37°C, preferably between 25°C and 37°C, and in a time span of maximal 20 minutes, more preferably maximal 5 minutes; b) aspirating the sample from the sample vial by means of a needle with an inner diameter of at least 0.3mm, preferably at least 0.35mm; c) optional mixing of the composition with components selected from the group of platelet- rich plasma (PRP), hyaluronic acid or glycosaminoglycans; d) administrating the composition or mixture thereof to a subject by intravenous, intra-articular, intramuscular, intra-lesional injection. The cell diameter of the injection needle is crucial in this respect, in order to avoid damage to the cells. Figure 6 shows the essential effect of the inner diameter of the needle used for aspiration of the composition. A 23G needle (inner diameter
0 : 33 mm) had a significantly more positive effect on the vitality of the cells present in the composition than a 25G needle (inner diameter 0: 26 mm). Defrosting the composition can be accomplished by thawing in a hot water bath or in the palm of one's hand or by any other method within the temperature limits.
Preferably, the composition is administered within 2 minutes after thawing, in order to safeguard the vitality of the composition.
In what follows, the invention is described on the basis of non-limiting examples which illustrate the invention, and are not intended to limit the scope of the invention.
EXAMPLES
Example 1 : Example Protocol for the isolation of mesenchymal stem cells from the blood of orses
- Take five samples peripheral blood of horses and collect it in EDTA tubes (5 x 10 ml)
Transport the blood at 4 °C
- Centrifuge the tubes for 20 minutes at room temperature ( 10 accel and decel
10)
- Collect the buffy coat into a sterile 15 ml tube and dilute the cells 1 :2 with PBS at room temperature
- Transfer the solution in the same amount of Percoll (between 1 : 08 and 1.77 g/ml) at room temperature
- Centrifuge for 15 minutes at room temperature (without brake: 10 accel and decel 2)
Collect the interphase
- Wash the interphase 3x with PBS by centrifugation for 8 minutes at room temperature
- Resuspend the obtained pellet in growth medium with dexamethasone and count the cells
- Seed 20 to 40 x 106 BMCs per T75 flask (seed 4 flasks)
Change the growth medium 2 times per week
Trypsinize the cells at 70-80% confluency
- Seed 0.5 x iO6 cells per T75 flask of the first passage in expansion medium
- Split cells at 70-80% confluence until passage 5 (P5)
Figure 1 shows mesenchymal stem cells isolated according to the protocol of Example 1 , wherein the cells are positive for the markers vimentin, fibronectin, and Ki67.
Example 2 : Example Protocol for the isolation of mesenchymal stem cells from the blood of humans, cats or dogs
The protocol of Example 1 can be used without problems in blood samples from other mammals, such as human, dog or cat. Figure 2 shows mesenchymal stem cells are isolated from human blood, positive for the markers vimentin, fibronectin, and Ki67.
Example 3 : Example protocol for cryo-preservation
Trypsin 0.25% -EDTA 0.02% [50ml] :
• 10% Trypsin (stock solution = 2.5%) [5ml] 1% Versene (EDTA) [500μΙ]
• Dilute with PBS lx [44.5ml] (or commercially available Tryp-EDTA 0.25%) a. Remove the media b. Add 0.25% trypsin-EDTA to the cells; c. Incubate for up to 10 minutes at 37°C (check that all cells came loose); d . Add the same amount of warm medium (37°C, containing FBS) or pure FBS in order to block the trypsin activity; e. Centrifuge the solution at 300G for 8 minutes at room temperature ( 10 accel and decel 10) ; f. Remove the supernatant; g . Add 10ml lx PBS and resuspend the cells, to wash away the remaining medium : centrifuge at 300G for 8 minutes at room temperature ( 10 accel & decal 10);
h . Remove the supernatant; i . Resuspend the cells in an appropriate quantity of medium ;
j Add 1ml cells/cryotube; k. Place the cryotubes overnight in a plastic container with isopropanol (KT) at -80°C.
I. Transfer the labeled tubes in a box at -80°C. - General principle: [ lml/cryotube]
• MSCs + DMEM low glucose [0.9ml]
• + 10% DMSO [ΙΟΟμΙ]
Example 4 : Example of composition of media used
- Growth Medium [50ml] :
• Low glucose DMEM [39ml] 30% FBS [10ml]
• 1% low dexamethasone (stock = 10 9 M) [500μΙ]
• 1% Antibiotics - antifungals (penicillin/streptomycin/amphotericin B) [500μΙ]
Expansion medium [500ml] :
• Low glucose DMEM [395ml] 20% FBS [ 100ml]
• 1% Antibiotics - antimycotics [5ml]
Example 5
Compositions of the present invention may be marketed as follows a. a composition comprising non-differentiated mesenchymal stem cells isolated from mammals, preferably for use in orthopedic lesions and pathologies such as osteoarthritis, cartilage damage, cyst structures (see Figure 8). Preferably, such composition is administered intra-articularly. b. a composition comprising mesenchymal stem cells induced towards chondrocytes, preferably for use in cases of severe cartilage injuries.
c. a composition comprising mesenchymal stem cells induced towards tenocytes. Preferably, such composition is used in applications of tendon lesions, lesions of the ligaments and other tendopathies (see figure 9 and 10). d. a composition comprising non-differentiated mesenchymal stem cells, with a diameter of less than 40 μιη, preferably for use in treatment of Musco-skeletal pathologies, endocrine pathologies or for regional perfusion (see Figure 11). The composition is preferably administered intravenously. e. A composition similar to the composition disclosed in step d, whereby the cells are stimulated so that an increased capacity for migration to pathological sites in the body.
Example 6 : Treatment of tendinitis in horses with a composition comprising mesenchymal stem cells induced towards tenocytes
Ten horses with severe tendinitis of the superficial digital flexor tendon and 15 horses with chronic desmitis of the musculus interosseous medius were treated with a composition according to the present invention, including mesenchymal stem cells are induced (pre-differentiation) towards tenocytes in combination with PRP as a carrier substance.
Administration of the composition occurred intra-lesional under ultrasound guidance into the injured tendon. 80% of the treated horses showed improvement after only 6 weeks, where normally only after at least 6 months of intensive, conventional conservative therapy similar results are obtained.
Figure 12 A is an example of an ultrasound scan of a patient with tendinitis, which was treated using conventional conservative therapy. Recovery was only observed after 6 months of therapy (Figure 12 B).
Figure 13 A is an example of an ultrasound scan of a patient with tendinitis, treated with a composition of tenocytes according to the present invention. Figure 13 B shows the evolution, 29 days after treatment. A remarkable improvement was visible.
Figure 14 A is an ultrasound scan of a patient with chronic desmitis, figure B shows the evolution, 35 days after treatment with a composition according to an embodiment of the present invention. Again, a remarkable improvement was observed in a very short time. Moreover, local calcification of tendons before treatment also disappeared after treatment.
Figure 15 shows statistical results of patients (tendinitis or desmitis) treated with compositions according to embodiments of the present invention. Score of 0 indicates 0% improvement again after 6 weeks of treatment with Tendo-Cell® in combination with PRP and score 5 100% improvement ( 1 =20%, 2=40%, 3 = 60%, and 4=80%). From this figure it can be seen that having independent veterinarians score 20 of the 25 horses (80%) had a score of 4 or more given 6 weeks after the treatment.
Example 7 : Infected wound after abdominal colic
After a colic operation in an Arabian stallion, the stitches became infected at the level of the linea alba which is at the level of the abdomen. The patient developed an infected skin wound. After 3 months of conservative therapy, the wound still didn't cure and the owners decided to treat with mesenchymal stem cells from the blood of another donor horse. About 4 weeks later, the wound was considered completely cured by the attending veterinarian.
Example 8 : Tendon Sheath Problems and Lesions of the ligaments
Three horses with a swollen tendon sheath, already present for 6 months, were treated with the composition described and as a result complete recovery was observed after 2 months.
A patient was suffering from lameness as a result of a lesion to the collateral ligament of the fetlock : less than 2 months after the local injection of a composition according to the invention, the horse was able to walk again normal and no relapse had been noticed.
Example 9 : Fractures
A 4-year-old gelding with a difficult healing of the hoof bone fracture was, after 10
months of conservative treatment, iocaiiy treated with a composition according to the present invention. Fuii recovery occurred after 4 months.
Example 10 : Mucous membranes
Improvement of a horse with severely inflamed ulcers (polyfolliculaire lymphangitis) in the throat region after 4 weeks of treatment. After 6 weeks, the lesions were completely healed.
Example 11 : Cushinq's syndrome
A 19-year old horse with Cushing's syndrome was treated with 2 consecutive treatments of the composition described above (with 2 month interval). After 2 cycles, the symptoms were improved remarkably. Two more horses were treated in the same way with the same result.
Example 12 : Head Shaking
This is a neurological pathology of the central nervous system which leads to continuous shaking of the head. A horse that suffered already for 4 months was treated once. Already in the third week after treatment the symptoms disappeared.
Example 13 : Vascular reconstruction
In a horse affected by laminitis (detachment of the lamelles in the foot, with a consequent destruction of the peripheral vasculature in the foot) the pain disappeared after nearly 24 hours after a dose with a composition according to the current invention was administered. However, we noticed that this horse relapsed after 3 weeks.
Example 14: Hypersensitivity reaction
A horse with diffuse noduies (bumps) on the body caused by a hypersensitivity reaction has been treated by the composition described, resulting in the disappearance of the bumps the following day. This result lasted for 2 months.
Another horse with sweet itch (hypersensitivity caused by culicoides mosquitos) rubbed during the first two months of summer his full mane and tail bald. By a single treatment with the component described this horse was spared of further itchiness during the rest of the summer with full recovery of mane and tail.
References
Carrade DD, Affolter VK, Outerbridge CA, Watson JL, Galuppo LD, Buerchler S, Kumar V, Walker NJ, Borjesson DL, 2011a. Intradermal injections of equine allogenic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions. Cytotherapy 13, 1180- 1192. Carrade DD, Owens SD, Galuppo LD, Vidal MA, Ferraro GL, Librach F, Buerchler S, Friedman MS, Walker NJ, Borjesson DL, 2011b. Clinicopathologic findings following intra-articular injection of autologous and allogenic placentally derived equine mesenchymal stem cells in horses. Cytotherapy 13, 419-430. Fang BJ, Song YP, Lin QD, Zhang YL, Cao Y, Zhao RCH, Ma YF, 2007. Human adipose tissue-derived mesenchymal stromal cells as salvage therapy for treatment of severe refractory acute graft- vs. -host disease in two children. Pediatric Transplantation 11, 814-817. Giovannini S, Brehm W, Mainil-Varlet P, Nesic D, 2008. Multilineage differentiation potential of equine blood-derived fibroblast-like cells. Differentiation; research in biological diversity 76, 118-129.
Guest DJ, Ousey JC, Smith MRW, 2008. Defining the expression of marker genes in equine mesenchymal stromal cells. Stem Cells and Cloning : Advances and Applications 1, 1-9.
Hoynowski SM, Fry MM, Gardner BM, Leming MT, Tucker JR, Black L, Sand T, Mitchell KE, 2007. Characterization and differentiation of equine umbilical cord- derived matrix cells. Biochemical and Biophysical Research Communications 362, 347-353.
Koch TG, Thomsen PD, Betts DH, 2009. Improved isolation protocol for equine cord blood-derived mesenchymal stromal cells. Cytotherapy 11, 443-447.
Koerner J, Nesic D, Romero JD, Brehm W, Mainil-Varlet P, Grogan SP, 2006. Equine peripheral blood-derived progenitors in comparison to bone marrow- derived mesenchymal stem cells. Stem cells 24, 1613-1619. Martinello T, Bronzini I, Maccatrozzo L, Iacopetti I, Sampaolesi M, Mascarello F, Patruno M, 2010. Cryopreservation does not affect the stem characteristics of multipotent cells isolated from equine peripheral blood. Tissue engineering. Part C, Methods 16, 771-781. Radcliffe CH, Flaminio MJ, Fortier LA, 2010. Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations. Stem cells and development 19, 269-282.
Richardson LE, Dudhia J, Clegg PD, Smith R, 2007. Stem cells in veterinary medicine— attempts at regenerating equine tendon after injury. Trends in biotechnology 25, 409-416.
Ringden O, Uzunel M, Rasmusson I, Remberger M, Sundberg B, Lonnies H, Marschall HU, DIugosz A, Szakos A, Hassan Z, Omazic B, Aschan J, Barkholt L, Le Blanc K, 2006. Mesenchymal stem cells for treatment of therapy-resistant graft- versus-host disease. Transplantation 81, 1390-1397.
Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, Maini RN, 2000. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Research 2, 477-488.
Claims
1. A method for the isolation of mesenchymal stem cells from the blood of mammals, comprising the isolation of mesenchymal stem cells from the blood- interphase of blood samples and preserving said cells in a low glucose growth medium supplemented with antibiotics, serum and dexamethasone for preventing the differentiation of the cells. :
2. Method according to claim 1, comprising the steps of:
a) collecting one or more blood samples from donors, in a sample bottle, coated with an anti-coagulant;
b) centrifuging said blood samples to obtain a 3-phase distribution phase consisting of plasma-phase, buffy coat, and an erythrocyte-phase;
c) collecting the buffy coat and loading it on a density gradient;
d) collecting the blood-inter-phase obtained from the density gradient of step c ) e) isolating of mesenchymal stem cells from the blood-inter-phase by centrifugation;
f) seeding at least 2.5 x lOVcm2 mesenchymal stem cells in growth medium.
3. Method according to claim 2, characterized in that step d) is preceded by a step c'), wherein the buffy coat from step c) is at least 1 : 2 is diluted in a physiological buffer.
4. Method according to any of the preceding claims, characterized in that the mesenchymal stem cells are kept at least 2 weeks in growth medium.
5. Method according to any one of the preceding claims, characterized in that in a step g) 6xl03 cells/cm2 are transferred to an expansion medium comprising a low glucose medium, serum and antibiotics for the purpose of expanding the mesenchymal stem cells.
6. Method according to claim 5, characterized in that the expansion medium comprises up to 20% FBS or FCS.
7. Method according to claim 6, characterized in that the expansion of the mesenchymal stem cells take place in at least five cell passages.
8. Method according to any of the preceding claims, characterized in that the mesenchymal stem cells can be stored in a solution of low- glucose medium and 10% dimethyl sulfoxide for at least 6 months at a maximum temperature of - 80°C.
9. Method according to any of the preceding claims, characterized in that the mesenchymal stem cells are isolated from the blood of human, cat, dog or horse.
10. Method according to claim 9, characterized in that the mesenchymal stem cells are isolated from the blood of horses.
11. Composition comprising mesenchymal stem cells and/or induced or differentiated cells derived from said mesenchymal stem cells, whereby said mesenchymal cells are isolated from the blood of mammals according to any one of the claims 1 to 10.
12. Composition according to claim 11 , characterized in that the composition contains at least 90%, preferably at least 95% mesenchymal stem cells, wherein the mesenchymal stem cells show positive expression of vimentin, fibronectin, Ki67 or a combination.
13. Composition according to claim 11, characterized in that the composition contains at least 90%, preferably at least 95%-induced or differentiated cells derived from mesenchymal stem cells.
14. Composition according to claims 11 to 13, characterized in that the composition is formulated for intravenous, intra-articular, intramuscular, intra- lesional administration to mammals.
15. Composition according to any of the claims 11 to 14, characterized in that the mesenchymal stem cells are differentiated towards tenocytes, chondrocytes, osteocytes, myocytes, adipocytes, keratinocytes, neurons or fibroblasts are.
16. Composition according to any of the preceding claims, characterized in that the composition is mixed with components selected from the group consisting of platelet- rich plasma (PRP), hyaluronic acid or glycosaminoglycans.
17. Composition according to any of the preceding claims , characterized in that the mesenchymal stem cells are isolated from the blood of human, cat, dog or horse.
18. Composition according to any of the claims 11 to 17 for treatment of a trauma selected from the group of skin trauma, cartilage trauma, tendon traumas, traumas of ligaments, traumas of bones, traumas of mucus membranes, cysts or fractures.
19. Composition according to any of the claims 11 to 17, for treatment of neurological and neurodegenerative diseases is selected from the group of Cushing's syndrome, difficulty of breathing or paresis of the extremities.
20. Composition according to any of the claims 11 to 17, for the treatment of acute or chronic inflammatory disease states selected from the group of laminitis, periostitis, gastritis, osteoarthritis, inflammation caused by viral, bacterial, parasitic or mycotic agents in mammals.
21. Composition according to any of the claims 11 to 17, for the treatment of hypersensitivity reactions.
22. Composition according to any of the claim 11 to 17, for the treatment of stomach dilatation and torsion-complexes.
23. Composition according to any one of the claims 11 to 17, for the treatment of infertility in mares or precocity in foals.
24. A method for administering a composition according to any one of the claims 11 to 23 to a subject, wherein the subject is a horse, cat, dog or human being, comprising the steps of:
a) thawing a sample bottle comprising the composition, frozen at at least -80°C, at which the thawing is carried out at a temperature between 20°C and 37°C, preferably between 25°C and 37°C, and in a time span of maximal 20 minutes, more preferably maximal 5 minutes; b) aspirating the sample from the sample vial by means of a needle with an inner diameter of at least 0.3mm, preferably at least 0.35mm;
c) optionally mixing of the composition with components selected from the group consisting of platelet-rich plasma (PRP), hyaluronic acid or glycosaminoglycans; d) administering the composition or mixture thereof to a subject by intravenous, intra-articular, intramuscular or intra-lesional injection.
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| BEBE2012/0656 | 2012-10-01 | ||
| BE2012/0656A BE1020480A5 (en) | 2012-10-01 | 2012-10-01 | METHOD FOR INSULATION OF MESENCHYMAL STEM CELLS FROM BIRD BLOOD AND USE THEREOF |
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| PCT/EP2013/070247 WO2014053418A2 (en) | 2012-10-01 | 2013-09-27 | Method for obtaining mesenchymal stem cells and use thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3666298A1 (en) | 2018-12-13 | 2020-06-17 | Global Stem Cell Technology | A collagen formulation suitable for injection |
| WO2021058758A1 (en) | 2019-09-27 | 2021-04-01 | Global Stem Cell Technology Nv | Cell composition comprising radiolabled mesenchymal stem cells, use thereof and method for preparing radiolabeled mesenchymal stem cells |
| EP3911339A4 (en) * | 2019-07-30 | 2022-06-15 | T.C. Erciyes Universitesi | Platelet rich-fibrin derived mesenchymal stem cells |
| WO2022155213A1 (en) * | 2021-01-12 | 2022-07-21 | Qcdx Llc | Detection and analysis of circulating tumor cells |
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| WO2024133886A1 (en) | 2022-12-23 | 2024-06-27 | Boehringer Ingelheim Veterinary Medicine Belgium | Primed mesenchymal stem cells for use in the treatment of chronic kidney disease |
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| EP3412768A1 (en) * | 2013-12-06 | 2018-12-12 | Global Stem Cell Technology | Method and composition for inducing chondrogenesis or tenogenesis in mesenchymal stem cells |
| CN111733128A (en) * | 2020-05-14 | 2020-10-02 | 厚朴生物科技（苏州）有限公司 | Human adipose-derived mesenchymal stem cells preparation method and in vitro differentiation ability identification method |
| CN112116555B (en) * | 2020-08-12 | 2024-03-26 | 清华大学深圳国际研究生院 | Method for detecting physiological functions of mesenchymal stem cells and application thereof |
| CN112402364B (en) * | 2020-10-23 | 2023-08-04 | 中科细胞科技(广州)有限公司 | Umbilical cord mesenchymal stem cell-platelet-rich plasma-containing composite repair gel for injection |
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| EP3666298A1 (en) | 2018-12-13 | 2020-06-17 | Global Stem Cell Technology | A collagen formulation suitable for injection |
| EP3911339A4 (en) * | 2019-07-30 | 2022-06-15 | T.C. Erciyes Universitesi | Platelet rich-fibrin derived mesenchymal stem cells |
| WO2021058758A1 (en) | 2019-09-27 | 2021-04-01 | Global Stem Cell Technology Nv | Cell composition comprising radiolabled mesenchymal stem cells, use thereof and method for preparing radiolabeled mesenchymal stem cells |
| WO2022155213A1 (en) * | 2021-01-12 | 2022-07-21 | Qcdx Llc | Detection and analysis of circulating tumor cells |
| WO2023280832A1 (en) | 2021-07-08 | 2023-01-12 | Boehringer Ingelheim Veterinary Medicine Belgium | Mesenchymal stem cells for use in the treatment of osteoarthritis in animals |
| WO2023280835A1 (en) | 2021-07-08 | 2023-01-12 | Boehringer Ingelheim Veterinary Medicine Belgium | Mesenchymal stem cells for use in the treatment of chronic gingivostomatitis |
| WO2023280836A1 (en) | 2021-07-08 | 2023-01-12 | Boehringer Ingelheim Veterinary Medicine Belgium | Mesenchymal stem cells for use in the treatment of chronic kidney disease |
| WO2023280834A1 (en) | 2021-07-08 | 2023-01-12 | Boehringer Ingelheim Veterinary Medicine Belgium | Mesenchymal stem cells for use in the treatment of atopic dermatitis |
| US20230022259A1 (en) * | 2021-07-08 | 2023-01-26 | Boehringer Ingelheim Veterinary Medicine Belgium | Mesenchymal stem cells for use in the treatment of osteoarthritis in animals |
| US12329782B2 (en) | 2021-07-08 | 2025-06-17 | Boehringer Ingelheim Veterinary Medicine Belgium | Mesenchymal stem cells for use in the treatment of atopic dermatitis |
| WO2024033462A1 (en) | 2022-08-11 | 2024-02-15 | Boehringer Ingelheim Vetmedica Gmbh | Mesenchymal stem cells for use in the treatment of insect-bite hypersensitivity in equines |
| WO2024133886A1 (en) | 2022-12-23 | 2024-06-27 | Boehringer Ingelheim Veterinary Medicine Belgium | Primed mesenchymal stem cells for use in the treatment of chronic kidney disease |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2014053418A2 (en) | 2014-04-10 |
| WO2014053418A9 (en) | 2014-07-17 |
| BE1020480A5 (en) | 2013-11-05 |
| WO2014053418A3 (en) | 2014-05-30 |
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