WO2016082882A1

WO2016082882A1 - Secretomes and method for producing secretomes

Info

Publication number: WO2016082882A1
Application number: PCT/EP2014/075875
Authority: WO
Inventors: Steven James KELLNER
Original assignee: Med Cell Europe Ag
Priority date: 2014-11-27
Filing date: 2014-11-27
Publication date: 2016-06-02

Abstract

A method of producing a secretome secreted by a mesenchymal stem cell or dendritic cell comprising at least one biological property of said cell wherein the method comprises at least one period of keeping the cells at least 30 minutes under anoxic conditions. The novel anoxic production method results in a 30 fold increase in the amount of RNA in the secretome. A further advantage is the high amount of miR in the secretome after the anoxic treatment. For example miR-22 which can be used in a method of treating a cardiovascular diseases, miR-133b for neurological diseases, miR-146 for wound healing, miR-204 for pulmonary diseases. Thereby the present invention allows the person skilled in the art to produce secretome specifically for certain diseases.

Description

Secretomes and Method for producing Secretomes

Field of the invention

The present invention relates to the fields of secretomes. More particularly, the invention relates to a method of deriving secretomes from mesenchymal stem cells.

[Background

Mesenchymal stem cells, or MSCs, are multipotent stromal cells which have the potential to differentiate into a variety of mesenchymal cell types of the adipocytic, chondrocytic and osteocytic lineages, including : osteoblasts, chondrocytes, neurons, muscle cells and adipocytes. This potential has been documented in specific cells and tissues in vivo and in vitro. MSCs are distributed in mammals all over the body and are responsible for regeneration. Commonly used tissues for the isolation of MSC are bone marrow, umbilical cord, cord lining and - increasingly - adipose tissue, which has a superior amount of MSCs.

Dendritic cells, or DCs, are antigen- presenting cells (APCs) which play a critical role in the regulation of the adaptive immune response. Dendritic cells are unique APCs and have been referred to as "professional" APCs, since the principal function of DCs is to present antigens, and because only DCs have the ability to induce a primary immune response in resting naive T- lymphocytes. To perform this func- tion, DCs are capable of capturing antigens, processing them, and presenting them on the cell surface along with appropriate co- stimulation molecules. DCs also play a role in the maintenance of B-cell function and recall responses. Thus, DCs are critical in the establishment of immunological memory. The function of DCs falls broadly into three categories, each of which involve antigen presentation. The first category of DCs function is antigen presentation and activation of T-cells. The second category of DC function is not as well established, but it has been suggested that a different class of DCs exist with the function of inducing and maintaining immune tolerance. The third category of DCs, known as follicular DCs, appear to work to maintain immune memory in tandem with B-cells. DCs can be generated by transforming peripheral blood monocytes.

All cells communicate and exchange information by different ways, including the secretion of soluble factors, the cell-to-cell adhesion contact, and the intercellular exchange of organelles. The secretome of mesenchymal stem cells includes paracrine substances, exosomes and microvesicles. Over 1 50 paracrine substances, also called cytokines and chemokines, can be released by mesenchymal stem cells. Two distinct populations of vesicles with peculiar membrane structure, mechanism of production, pathophysiological relevance, and different size have been described : exosomes and microvesicles. Microvesicles and exosomes contain biomolecules, including messenger RNA and micro RNA. Exosomes and the whole secretome have been used for therapies in regenerative medicine to treat various illnesses such as osteoarthritis, cardiovascular diseases, neurological diseases, pulmonary diseases, diabetes and many more. In the known therapies success rate and duration has been very variable, mainly due to the insufficient amount of proteins and RNA used in the studies.

The secretome of both MSCs and DCs consists of various proteins, RNAs and extracellular vesicles, such as, but not limited to, exosomes and microvesicles.

5 1 . Introduction to exosomes and microvesicles

Cells communicate and exchange information by different ways, including the secretion of soluble factors, the cell-to-cell adhesion contact, and the intercellular exchange of organelles through nanotubular structures'^{1 1}. Recent studies have proposed that cell-derived small circular membrane vesicles, called exosomes and i o microvesicles, represent an additional mechanism of cell-to-cell communication by transfer of membrane components as well as the cytoplasmic content ¹²·³·^Λ· ^5]. Two distinct populations of vesicles with peculiar membrane structure, mechanism of production, pathophysiological relevance, and different size have been described. Membrane fragments of 30 - 1 OO nm diameter derived from the en-

1 5 dosomal membrane compartment after fusion of secretory granules with the plasma membrane are defined as exosomes. Once released, exosomes bind the recipient cells through receptor-ligand interactions or fuse with the target cell membrane transferring membrane components, including cell receptors'²¹, and discharging the portion of cytosol segregated within their lumen into the cyto-

20 plasm of recipient cells'⁶¹. The molecular cargo content of exosomes derives from active packaging of certain nucleic acid species leading to the presence of mRNAs in exosomes that are not found in donor cells'⁷¹. Furthermore, relatively large mi- crovesicles ( 1 00 nm - 1 mm diameter) are formed from the surface membrane of activated cells in a calcium and calpain dependent manner following a disordered function of phospholipid transporters that results in the budding of altered membrane that exposes phosphatidylserine in the outer leaflet. Microvesicles and exosomes contain biomolecules, including mRNA and microRNA ( miRNA) , packaged in a random process and their release is considered an expression of a pathological process in place. Molecular transfer from microvesicles and exosomes contributes to changes in the maturation and differentiation of target cells as for example microvesicles and exosomes released by endothelial progeni- tor cells trigger neo-angiogenesis in endothelial cells'⁸¹.

2. Introduction to paracrine substances

MSCs secrete numerous growth factors and cytokines. A typical MSC secretion profile comprises growth factors, cytokines, ECM proteases, hormones, and lipid mediators. Paracrine signaling is a form of cell-cell communication in which a cell produces a signal to induce changes in nearby cells, altering the behavior or differentiation of those cells. Signaling molecules known as paracrine factors diffuse over a relatively short distance ( local action) , as opposed to endocrine factors ( hormones which travel considerably longer distances via the circulatory system) , juxtacrine interactions, and autocrine signaling. Cells that produce paracrine fac- tors secrete them into the immediate extracellular environment. Factors then travel to nearby cells in which the gradient of factor received determines the outcome. However, the exact distance that paracrine factors can travel is not certain. Although paracrine signaling elicits a diverse array of responses in the induced cells, most paracrine factors utilize a relatively streamlined set of receptors and pathways. In fact, different organs in the body - even between different species - are known to utilize a similar sets of paracrine factors in differential development. The highly conserved receptors and pathways can be organized into four major families based on similar structures: Fibroblast growth factor ( FGF) family, Hedgehog family, Wnt family, and TGF- β superfamily. Binding of a paracrine factor to its respective receptor initiates signal transduction cascades, eliciting different responses.

3. Introduction to hypoxia

Previous studies suggested that the hypoxic condition promotes self- renewal of undifferentiated mesenchymal stem cells and enhances therapeutic potential.

Hypoxic exposure activates several signal transduction pathways including hypoxia inducible factor ( H IF) , a master transcription factor that regulates the expression of hundreds of genes to promote cellular adaptation to the hypoxic condition. H I F- 1 a is a pivotal signaling molecule for hypoxia- mediated upregulation of bFGF and hepatocyte growth factor ( HGF) secretion, whereas H IF-2a is a key signaling molecule for hypoxia- mediated upregulation of VEGF secretion from MSCs. H IF- 1 a is widely expressed in almost all tissues, whereas the expression of H IF-2a is restricted to certain cell types such as vascular endothelial cells. SUMMARY OFTHE INVENTION

According to a first aspect of the present invention, a method of producing a secretome secreted by a mesenchymal stem cell comprising at least one biological 5 property of a mesenchymal stem cell or secreted by a dendritic cell comprising at least one biological property of dendritic cell is provided. According to the present invention, the cells are kept during at least one period of at least 5 minutes under anoxic conditions at an Oxygen concentration below 1 % , preferably below 0.5 % . Surprisingly the aypoxic conditions had an significant positive influence on the i o amount and quality of exosomes, microvesicles and/ or paracrine substances secreted by mesenchymal stem cells or secreted by the dendritic cells.

According to a second aspect of the present invention, a secretome or secretome vesicles or paracrine secretome substances secreted by a mesenchymal stem cell comprising at least one biological property of a mesenchymal stem cell or se- 1 5 creted by a dendritic cell comprising at least one biological property of dendritic cell is provided.

According to a third aspect of the present invention, a pharmaceutical composition comprising a secretome or secretome vesicles or paracrine substances produced according to the present invention together with a pharmaceutically ac- 20 ceptable carrier, excipient or diluent are provided.

According to a fourth aspect of the present invention, there is provided such a pharmaceutical composition for use in a method of treating a disease. According to a fifth aspect the present invention provides the use of such a secretome or secretome vesicle or paracrine substance in a method of autogenous medical treatment of a disease or autogenous cosmetic treatment in an individual (which acted as donor for the mesenchymal stem cell or the dendritic cell used for 5 producing the secretome or secretome vesicle or paracrine substance) .

The disease may be selected from the group consisting of: osteoarthritis, cardiovascular diseases, lung diseases, liver diseases, neurodegenerative diseases including multiple sclerosis, Parkinson, Alzheimer and Graft-versus- host-disease (GVHD) or Crohn's disease. i o The secretome or secretome vesicle or paracrine secretome substance may be used to aid wound healing, scar reduction or cartilage- or bone-formation.

"Oxic conditions" refers in the present description to ambient atmospheric oxygen tension of approximately 5-21 % 0₂ (volume:volume; 1 Ό 1 3 hPa ; 50 Ό00 - 21 O'OOO parts per million Oxygen ;) .

1 5 "Hypoxic conditions" refers to oxygen tensions between approximately 1 -5 % 0₂ ( 1 O 'OOO - 50 Ό00 ppm Oxygen) .

"Anoxic conditions" refers to oxygen tensions below approximately 1 % 0₂ ( 1 O 'OOO ppm Oxygen) , preferably below 0.5 % 0₂ (5 Ό00 ppm Oxygen) . The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities and the general knowledge of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example [1 3], [1 4] and [1 5] .

BRI EF DESCRI PTION OFTHE FIGU RES

Figure 1 shows in a bar graph the semiquantitatively measured ( ocular density

( OD) measurement in photospectrometer) amounts of surface markers CD 63 , 81 and 9 via RAP/ PAN technology compared with standard hypoxic conditions and the total indicating a 30 fold increase;

Figure 2 shows a comparison between free RNA in secretome produced by 1 0 x

1 0⁶ MSCs under standard hypoxia and 1 x 1 0⁶ MSCs with PAN technology according to Example 1 ;

Figure 3 shows a comparison of free RNA content of secretome produced under standard hypoxia and the PAN system according to Example 1 ;

Figure 4 shows the amount of cytokines measured (ocular density ( OD) measurement in photospectrometer) after secretome production ;

Figure 5 shows the carbondioxide/ temperature/ oxygen curves during a 1 20 minute anoxic period ( x-axis: time in minutes; y-axis: temperature in degrees celcius, C0₂ in % , 0₂ in % ) ; Figure 5a shows the single carbondioxide curve according to Figure 5 ; Figure 5 b shows the single temperature curve according to Figure 5 ; and Figure 5c shows the oxygen curve according to Figure 5.

Figure 6 shows an oxygen/ carbondioxide curve according to manufacturer of

AnaeroGEN compact AnaerobJar.

Detailed description of the invention

Preferably MSCs are thawed and cultivated and DCSs are thawed in cell culture flasks with a special cell medium ( nutrient) . According to a first preferred method (called RAP system by the applicant) the cells are then detached and washed three times in warm phosphate buffered saline ( PBS) or other saline solutions.

According to a second preferred method the cells are washed directly in the cell culture flasks without detachment (called PAN system) .

After washing according to either one of the above mentioned systems ( RAP or PAN) 2 to 1 5 ml of warm PBS is added and the cells are kept for at least one period of at least 5 minutes under anoxic conditions before leaving the cells at oxic conditions at room temperature or at 37°C for a total of between 5 and 24 hours. The duration of the period under anoxic conditions lies preferably in a range of between 5- 1 20 minutes. According to further preferred embodiments of the present invention the cells are exposed to more than one period under anoxic conditions, preferably the cells are exposed to 2 to 4 periods of anoxic conditions each followed by a subsequent period of keeping the cells under oxic conditions. Each period under anoxic conditions followed by a period under oxic conditions is called a cycle. Durnig the anoxic cycle the Oxygen concentration is below 1 % , preferably below 0.5 % .

5 It is clearly visible from Figure 5 , that it takes several minutes in the experimental setup as described herein to reach the anoxic conditions, i.e. an oxygen tension below 1 % . Accordingly the cells are exposed to normoxic conditions and then hypoxic conditions before the system reaches anoxic conditions. In the experimental setup used by the inventor, it takes about 25 Minutes to reach anoxic i o conditions.

The secretome is then harvested for example by simple aspiration of PBS. The secretome is then preferably centrifuged to discard cells and afterwards microfil- trated to include or exclude various components of the secretome.

Compared to previously known systems exposing MSC cells to hypoxic conditions 1 5 the novel production method results in a 30 fold increase in the amount of RNA in the secretome.

A further advantage is the high amount of miR in the secretome after the anoxic treatment. For example miR-22 which can be used in a method of treating a cardiovascular diseases, miR- 1 33 b for neurological diseases, miR- 1 46 for wound 20 healing, miR-204 for pulmonary diseases. Thereby the present invention allows the person skilled in the art to produce secretome specifically for certain diseases. In particular clinical grade secretome for therapies in regenerative medicine for a host of diseases such as osteoarthritis, cardiovascular diseases, lung diseases, liver diseases, neurodegenerative diseases including multiple sclerosis, Parkinson, Alzheimer, GVHD, Crohn's disease and many more.

5 All of the above said is not only true for secretome, but also for secretome vesicles, in particular for exosomes and/ or microvesicles, and for paracrine secretome substances. The person skilled in the art knows how to purify, extract etc. said vesicles and substances from the whole secretome for example by utilizing the teachings from [1 6] and [1 7] which are incorporated herein by reference. o

[Example 1 (PAN System)

According to a first example secretome is produced by the following steps:

1 .1 . Fill a precoated T75 culture flask with 1 5 ml of SM P alpha minus stem cell medium and transfer into incubator for 30- 1 20 minutes. Then spray culture5 flask with 70% I PA and transfer to laminar air flow.

1 .2. Thaw a cryopreserved vial with approximately 1 mio MSC by removing it from the liquid nitrogen storage tank and transfer it immediately into the warm water bath preheated to 37-40 °C for 1 -4 minutes. Remove vial from bath immediately after last bit of solid ice has been thawed 1 .3. Spray vial with 70% alcohol and transfer to laminar air flow. Pipette MSCs with 2 ml serological pipette into the ready culture flask and swirl gently and transfer to incubator (5% C0₂ and 37 °C)

1 .4. Pipette 1 5 ml of SM P alpha minus stem cell medium into a 50 ml centrifuge tube and transfer to water bath for at least 30 minutes.

1 .5. Change stem cell media 2-8 hours after transfer of vial into cell culture flask. Verify that at least 90% of the cells have settled to the plastic floor.

1 .6. Leave culture flask in the incubator for 1 to 3 days until a cell confluence of 75% -90% has been achieved.

1 .7. a. Transfer flask to laminar air flow bench after spraying with 70% I PA and remove supernatant. Wash Flask with warm Dulbecco's PBS or similar three times with 1 0 ml each carefully. Cover the cells with 1 0 ml ( Kommentar SK: kann auch 5 ml oder 1 5 ml sein) prewarmed Ringer's lactate or other saline solutions such as PBS.

1 .8. a. Prepare the AnaerobJar Oxoid system by spraying with 70% I PA and leaving to dry in the laminar air flow.

1 .9. a. Place the culture flask into the lying Anaerobjar, insert the oxygen capture sachet and close the lid of the jar. Leave for 1 hour in the laminar air flow bench at room temperature, then open the jar carefully and place flask in the C0₂ incubator or at room temperature in the bench for 4 hours. ( Kommentar SK: Ver- sion Raumtemperatur und Version Inkubator moglich; Anoxie Varianten: 2 Stun- den am Stuck und 2-4x 1 Stunde mit einer Stunde Pause im normalen 0₂ U mge- bung ; Dauer der Produktion 5 Stunden, 1 2 und 24 Stunden)

1 .7.b.- 1 .9. b. Instead of the anaerob jars a C0₂/ 0₂ incubator or an alternative device for culturing the cells under anoxic conditions in an enclosed volume can be used ( e.g. an Hypoxia Incubator Chamber produced by STEMCELL Technologies SARL, 40 Rue des Berges, Miniparc Polytec, Batiment Sirocco, 38000 Grenoble, France) or other newly designed anoxia systems.

1 .1 0. Remove the saline under sterile conditions under the laminar air flow and centrifuge at 300 G for 3 minutes to remove detached stem cells. If only Exo- somes are needed for the therapy, filtrate the saline afterwards through a 0.45 μιη syringe filter to remove microvesicles. Microvesicles can be retrieved per reverse washing of the filter.

1 .1 1 . Transfer saline into a glass vial for immediate application or store at - 800 °C in a RNAase free plastic centrifuge tube.

1 .1 2. Add 1 5 ml of SM P alpha minus stem cell medium to the culture flask and return it to the incubator.

1 .1 3. Leave in incubator, with media changes every 2-3 days if necessary until cells have regrown to 80% confluence before repeating the harvesting procedure or freezing them according the standard procedures in liquid nitrogen. Material und Devices

1 Reagents and Solvents

( 1 ) SM P alpha minus

(2) PBS Dulbecco or similar w/ o Ca/ Mg

(3) Ringer's lactate or other saline approved for use in humans

2 Devices

( 1 ) C02 Incubator / C02/ 02 incubator

(2) Laminar flow bench

(3) Centrifuge

(4) Water bath 35 + 4°C

(5) Pipetting aid and serological pipettes

(6) Inverted Microscope Bresser

(7) Anaerob Jar or similar system

(8) Precoated culture flasks or self coated flasks

(9) Centrifuge tubes, RNAase free

[Example 2 (RAP System)

According to a second example secretome is produced by the following steps:

2.1 . Fill a precoated T75 culture flask with 1 5 ml of SM P alpha minus stem cell medium and transfer into incubator for 30- 1 20 minutes. Then spray culture flask with 70% I PA and transfer to laminar air flow. 2.2. Thaw a cryopreserved vial with approximately 1 mio MSC by removing it from the liquid nitrogen storage tank and transfer it immediately into the warm water bath preheated to 37-40 °C for 1 -4 minutes. Remove vial from bath immediately after last bit of solid ice has been thawed

2.3. Spray vial with 70% alcohol and transfer to laminar air flow. Pipette MSCs with 2 ml serological pipette into the ready culture flask and swirl gently and transfer to incubator (5% C0₂ and 37°C)

2.4. Pipette 1 5 ml of SM P alpha minus stem cell medium into a 50 ml centrifuge tube and transfer to water bath for at least 30 minutes.

2.5. Change stem cell media 2-8 hours after transfer of vial into cell culture flask. Verify that at least 90% of the cells have settled to the plastic floor.

2.6. Leave culture flask in the incubator for 1 to 3 days until a cell conflu- ency of 75% -90% has been achieved.

2.7. Subcultivate the MSCs to 3 T75 and then again to 9 T75 flasks to achieve around 8 mio cells. Repeate expansion process accordingly to achieve higher cell amounts.

2.8. Detach stem cells when the required amount of cells and confluence has been achieved. This can be done by scraping or enzymatical detachment. 2.9. Centrifuge the detached cells and wash thrice with 1 0 ml warm Dul- becco's PBS or similar saline solution by resuspending the cells in new PBS and discarding the supernatant after centrifugation.

2.1 0. Resuspend the cells in 2-20 ml Ringer's lactate, Dulbecco's PBS or similar saline solution.

2.1 1 .a. Prepare the AnaerobJar Oxoid system by spraying with 70% isopropa- nol ( I PA) and leaving to dry in the laminar air flow.

2.1 2. a. Place the Centrifuge tube with the MSC suspension into the standing Anaerobjar, insert the oxygen capture sachet and close the lid of the jar. Leave for 1 hour in the laminar air flow bench at room temperature, then open the jar carefully and place tube in the C0₂ incubator or at room temperature in the bench for 4 hours.

2.1 1 . b.-2.1 2.b. As an alternative to the anaerob jars a C0₂/ 0₂ incubator or an alternative device for culturing the cells under anoxic conditions in an enclosed volume can be used (e.g. an Hypoxia Incubator Chamber produced by STEMCELL Technologies SARL, 40 Rue des Berges, Miniparc Polytec, Batiment Sirocco, 38000 Grenoble, France) or other newly designed anoxia systems.

2.1 3. Remove the saline under sterile conditions under the laminar air flow and centrifuge at 300 G for 3 minutes to remove detached stem cells. If only Exo- somes are needed for the therapy filtrate the saline afterwards through a 0.45 μιη syringe filter to remove microvesicles. Microvesicles can be retrieved per reverse washing of the filter.

2.1 4. Transfer saline into a glass vial for immediate application or store at - 800 °C in a RNAase free plastic centrifuge tube.

2.1 5. Add 1 5 ml of SM P alpha minus stem cell medium to the culture flask and return it to the incubator.

2.1 6. Leave in incubator, with media changes every 2-3 days if necessary until cells have regrown to 80% confluence before repeating the harvesting procedure or freezing them according the standard procedures in liquid nitrogen.

Material und Devices

Reagents and Solvents

( 1 ) SM P alpha minus

(2) PBS Dulbecco or similar without Ca/ Mg

(3) Ringer's lactate or other saline approved for use in humans

(4) Cell Dissociation Reagent such as Accutase®

Devices used in Example 2 are identical to devices according to example 1 .

According to a further embodiment, step 2.1 2a is performed with a 2 hour exposure in the AnaerobJar under anoxic conditions followed by a 1 hour break at nor- moxic conditions. This cycle is repeated 2-4 times. Duration of the production is for example 5 hours, 1 2 and 24 hours.

Ther person skilled in the art can, based on the technical information provided above and utilizing his technical knowledge, upscale the production methods according to the examples provided above without inventive activity.

References

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Claims

Patent Claims

1 . A method of producing a secretome secreted by a mesenchymal stem cell comprising at least one biological property of a mesenchymal stem cell or secreted by a dendritic cell comprising at least one biological property of a dendritic cell wherein the method comprises at least one period of keeping the cells at least 5 minutes under anoxic conditions.

2. The method according to claim 1 , characterized in that the period of keeping the cells under anoxic conditions is followed by a period of keeping the cells under oxic conditions.

3. The method according to claim 2 , characterized in that a cycle comprising a period of keeping the cells under anoxic conditions and a subsequent period of keeping the cells under oxic conditions is performed more than one time, preferably 2 to 4 times.

4. The method according to any of the preceeding claims, characterized in that the period of keeping the cells under anoxic conditions lasts between 5 to 1 20 minutes.

5. The method according to any of the preceeding claims, characterized in that the Oxygen concentration during the anoxic step is below 1 % , preferably below 0.5 % .

6. The method according to any of the preceeding claims, characterized in that exosomes, microvesicles and/ or paracrine secretome substances are isolated separately or in groups from the secretome.

7. The method according to any of the preceeding claims, wherein the cells are kept under anoxic conditions in an AnaerobJar, a C0₂/ 0₂ incubator or another device for culturing the cells under anoxic conditions.

8. A secretome produced by a method according to any of the preceeding claims comprising exosomes, microvesicles and/ or paracrine secretome substances which comprise at least one biological property of a mesenchymal stem cell or a dendritic cell.

9. A secretome according to claim 8 , characterized in that it is a clinical grade secretome for therapies, preferably in regenerative medicine for osteoarthritis, cardiovascular diseases, Type 1 and Type 2 Diabetes, lung diseases, liver diseases, neurodegenerative diseases including multiple sclerosis, Parkinson, Alzheimer and GVHD or Crohn's disease.

10. An exosome, microvesicle and/ or paracrine secretome substance isolated from a secretome according to claim 8 or 9.

1 1 . A pharmaceutical composition comprising a secretome or secretome vesicles or paracrine substances produced according to any of claims 8 to 1 0 together with a pharmaceutically acceptable carrier, excipient or diluent.

12. A pharmaceutical composition according to claim 1 1 for use in a method of treating a disease.

13. A pharmaceutical composition according to claim 1 2 , wherein the disease is selscted from the group of osteoarthritis, cardiovascular diseases, Type 1

5 and Type 2 Diabetes, lung diseases, liver diseases, neurodegenerative diseases including multiple sclerosis, Parkinson, Alzheimer, Graft- versus- host- disease (GVHD) or Crohn's disease.

14. The use of a secretome or a secretome vesicle or a paracrine secretome substance or a combination thereof in a method of autogenous medical treat- o ment of a disease or autogenous cosmetic treatment in an individual.

15. The use of a secretome or a secretome vesicle or a paracrine secretome substance or a combination thereof in aiding wound healing, scar reduction, cartilage- or bone-formation.