WO2017193134A1

WO2017193134A1 - Mesenchymal stem cell proliferation

Info

Publication number: WO2017193134A1
Application number: PCT/US2017/031585
Authority: WO
Inventors: Melinda SIMON; István HORNYÁK; Zsombor Lacza
Original assignee: Lacerta Technologies Inc.
Priority date: 2016-05-06
Filing date: 2017-05-08
Publication date: 2017-11-09
Also published as: EP3452500A4; EP3452500A1

Abstract

The invention relates to a use of serum fraction of platelet rich fibrin (SPRF) for increasing mesenchymal stem cell (MSC) proliferation rate wherein said differentiated MSCs maintain their potential to differentiate in several cell types. The invention also relates to SPRF for use in therapy, wherein in said therapy SPRF obtained from a donor subject used to increase proliferation rate of the patients MSCs.

Description

MESENCHYMAL STEM CELL PROLIFERATION

The invention relates to a new method for increasing proliferation rate of mesenchymal stem cells. BACKGROUND

Mesenchymal stem cells (MSCs) are defined as multipoten self -renewing, non-hematopoietic cells, which originate from the mesoderm and characterized by typical surface markers, for example ALCAM (activated leukocyte cell adhesion molecule, CD166) and STRO-1. Their multipotency permits the differentiation to bone, cartilage, reticular tissues and fat (Oreffo et al. 2006). Due to their advantageous properties MSCs have been proved to be effective as autologous cell transplantation in clinical trials in case of regeneration of periodontal tissue defects, diabetic critical limb ischemia, bone damage caused by osteonecrosis and burn-induced skin defects. However, MSCs multi-lineage potential can be lost easily, when MSCs grown in vitro on standard tissue culture plastics. Their proliferation and multilineage differentiation potential also decreases with aging or increased time in in vitro culture.

This phenomenon is the major obstacle to the clinical application of MSCs, because the patient's own stem cells cannot be harvested and expanded without phenotypical change. MSCs cultured ex vivo lose their regenerative capacity, - in case of bone- they ability to augment and promote bone formation. In addition, MSCs for human stem cell therapy are usually cultured in fetal bovine serum (FBS) supplemented medium, which provides a xenogeneic additive to the reintroduced cell population. FBS can trigger immune reactions, and due to its unpredictable lot-to-lot variability these effects are totally incidental. Therefore, for the translation of stem cells to clinical uses, would be ideal to evolve xeno-free culture conditions. Among the nowadays applied human blood separation products; platelet-rich plasma (PRP) was already proven in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. (Amable P. R. et al. Stem Cell Research & Therapy2013, 4:67, Rigotti G. et al. Aesthet Surg J. 2016; 36(3):261-70 2013) suggested a standardized PRP and the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes. Uncertainties of the process are involved. In order to have a larger overview on this field, we can also evaluate the other well-known blood separation products for their potential supplementary role. Plasma is the anticoagulated, centrifuged whole blood supernatant, which has the disadvantage of containing anticoagulant (e.g. EDTA, heparin or citrate derivatives), which affects enzymatic balance and interfere with systemic blood coagulation as well and plasma also contains fibrinogen, which is converted to fibrin just like in PRP and causes limited protein transport. Another possible candidate is serum, which is the supernatant of the coagulated whole blood. This has the same disadvantage as ACS (autologous conditioned serum) namely that during the clotting of whole blood inflammatory markers are populated in a similar manner as in a systemic inflammatory. Usually the longer the blood is coagulating, the more inflammatory cytokines are produced, which unfortunately can lead to a positive feedback loop so ultimately this can generate inflammation if injected back to the patient. In summary as a supplementing material the main criteria are to have a somewhat standardized, fibrin and/or fibrinogen and anticoagulant free autologous blood separation product, which does not induce inflammation. The only procedure, which enabled to fulfil these goals was simply centrifuging whole blood after blood drawing in a clot activating tube so platelet rich fibrin (PRF) is produced in the supernatant. Pressing out the fluid content from PRF leads to an autologous blood separation product, which does not contain fibrinogen, anticoagulants and the inflammation markers are low. After testing it as a stem cell medium supplement, the inventors have surprisingly found that use of a serum from platelet rich fibrin (SPRF) instead of PRP and FBS enhanced the proliferation rate of human mesenchymal stem cells in vitro while phenotypical changes were not observed and differentiation potential of proliferated MSCs was maintained. Moreover, culturing human subchondral bone pieces in SPRF supplemented medium cell viability was not only retained, but also significantly increased in 7-days culture without any measurable cell differentiation. The inventors revealed that predominantly mesenchymal stem cells were multiplicated in the course of the incubation time.

ABBREVIATIONS ACS (autologous conditioned serum)

DMEM (Dulbecco' s modified Eagle' s medium)

FBS (fetal bovine serum)

hMSCs (human mesenchymal stem cells)

hSBPs (human subchondral bone pieces)

PRF (platelet rich fibrin)

PRP (platelet rich plasma)

SPRF (serum from platelet rich fibrin)

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a method for use of serum fraction of platelet rich fibrin (SPRF) for increasing mesenchymal stem cell (MSC) proliferation rate in vitro, ex vivo or in vivo wherein said MSCs maintain their potential to differentiate into several cell types.

The invention relates to a use of serum fraction of platelet rich fibrin (SPRF) for increasing MSC proliferation rate in vitro, ex vivo or in vivo wherein said MSCs maintain their potential to differentiate into several cell types.

Preferably, in the method or use of the invention the MSCs are contacted or maintained in contact with

SPRF for at least 5 days, preferably for at least 8 days or at least 10 days.

Preferably, in the method or use of the invention the MSCs are contacted or maintained in contact with SPRF for until at least a time -period when osteoblast direction differentiation occurs, preferably for until at least a time-period when the expression of at least one, preferably two or at least two osteoblast specific marker gene(s) is/are increased in a medium supplemented with SPRF, preferably SPRF having the concentration range given herein, highly preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium.

Preferably expression of one or both of the following osteogenic marker genes is/are increased:

COL1 Al and ALPL, wherein preferably

- COL1A1 expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold,

- ALPL expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold, when compared to 10 % (v/v) FCS supplemented medium.

In an in vitro method or use the MSCs are maintained in culture. In an embodiment a medium for culturing mammalian cells e.g. an MSC culturing medium supplemented with SPRF is applied. MSC culturing medi- um normally comprises a carbon source eg. sugar source eg. glucose. According to the invention the medium comprises SPRF and comprises no further serum and/or no serum substitute and/or no serum derived product or supplement. In particular the medium according to the invention does not comprise fetal bovine (calf) serum (FBS or FCS) and does not comprise platelet rich plasma (PRP). In a particular embodiment the medium according to the invention does not comprise any further growth factor (only those which are present in the SPRF).

Preferably, in a method for using SPRF for selectively increasing MSC proliferation rate in vitro said differentiated MSCs maintain their potential to differentiate in several cell types. Preferably MSCs are obtained from a subject, said method comprising

- providing SPRF,

- adding SPRF to a pool of MSCs in medium,

- allowing MSCs to proliferate.

In a preferred embodiment the MSCs so proliferated are administered to a subject. In a preferred embodiment the subject is a patient in need of bone or cartilage regeneration or repair.

In a preferred embodiment the patient is treated with a condition wherein the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.

In a preferred embodiment the patient is in need of bone tissue regeneration.

Preferably the patient is suffering in osteoarthritis or osteoarthritis, preferably osteoarthritis or osteoarthritis of a joint. Preferably the patient is a mammalian or human subject.

In an ex vivo method or use the MSCs are present in or on a tissue and so cultured or maintained in culture.

In an embodiment the tissue is an explant. In an embodiment the tissue is an artificial tissue. In an embodiment the tissue is an explant, eg. a bone explant. The bone explants may be e.g. subchondral bone pieces or explants obtained by osteotomy. In an embodiment the tissue is an artificial tissue, e.g. a bone graft or a joint or cartilage graft.

In an embodiment an MSC culturing medium for maintaining or culturing a tissue ex vivo is a medium for culturing mammalian cells e.g. a medium for culturing MSCs supplemented with SPRF. MSC culturing medium normally comprises a carbon source eg. sugar source eg. glucose, a glutamine source and pyruvate. According to the invention the medium comprises SPRF and no further serum and/or no serum substitute and/or no serum derived product or supplement. In particular the medium according to the invention does not comprise fetal bovine (calf) serum (FBS or FCS) and does not comprise platelet rich plasma (PRP). In a particular embodiment the medium according to the invention does not comprise any further growth factor (only those which are present in the SPRF).

Preferably MSCs are obtained from a subject, said method comprising

- providing SPRF,

- adding SPRF to a pool of MSCs present in an ex vivo tissue or on an explant,

- allowing MSCs to proliferate.

In the method or use of the invention the MSCs are incubated in the presence of SPRF for at least 5 days, preferably for at least 8 days or at least 10 days.

In an embodiment the MSCs are bone marrow derived mesenchymal stem cells (BM-MSCs or bone mar- row stromal stem cells).

In an embodiment the MSCs are adipose derived mesenchymal stem cells (AD-MSC).

In an embodiment the medium is as defined above or a medium as disclosed herein.

In a preferred embodiment the tissue or explant on which MSCs so proliferated is/are administered to a subject. In a preferred embodiment the tissue or explant is a graft to be implanted into the subject.

In a preferred embodiment the subject is a subject in need of bone or cartilage regeneration or repair. Preferably the subject is suffering in osteoarthritis or osteoarthritis, preferably osteoarthritis or osteoarthritis of a joint. Preferably the subject is a mammalian or human subject.

In an embodiment the MSCs are mammalian MSCs, preferably human MSCs.

The invention relates to a method for use of serum fraction of platelet rich fibrin (SPRF) for increasing mesenchymal stem cell (MSC) proliferation rate in vitro wherein said MSCs maintain their potential to differentiate into several cell types.

The invention relates to a use of serum fraction of platelet rich fibrin (SPRF) for increasing MSC prolif- eration rate in vitro wherein said MSCs maintain their potential to differentiate into several cell types. Preferably the expression of at least one or two, preferably two or at least two osteoblast differentiation factors show increased expression after an appropriate period of time, preferably at or after 5 days culturing. Preferably the osteoblast factors are COL1 Al and ALPL.

In a preferred embodiment the MSCs are obtained from a subject. Preferably the subject is a mammalian subject, more preferably a human subject.

In an embodiment the MSCs are primary cells.

In an embodiment the MSCs are adipose derived mesenchymal stem cells (AD-MSC).

In a preferred embodiment the culture of mesenchymal stem cells is supplemented with 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.

In a preferred embodiment the culture medium used for increasing proliferation rate of mesenchymal stem cells comprises

one or more amino acid source, preferably at least glutamate source or a glutamate source only, - one or more salts, said salt being preferably selected from calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and monosodium phosphate,

one or more sugar, preferably at least glucose or glucose only and optionally or if desired one or more vitamins preferably selected from folic acid, nicotinamide, riboflavin and B12, wherein said medium comprises 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF wherein said medium comprises no FBS (FCS) and no PRP and preferably no FGF, wherein preferably said medium comprises, besides SPRF, no other serum product and/or no other serum derived product or supplement and preferably no other growth factors.

The medium may comprise further additives e.g. buffer(s), antibiotic(s), selection agent(s), preservation agent(s) etc.

In a preferred embodiment the medium is a derivative of Dulbecco' s modified Eagle's medium (DMEM) in that it is supplemented with 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.

In an embodiment the invention relates to an in vivo method or use wherein

SPRF is contacted with MSCs of a subject in vivo and

an appropriate level of SPRF is maintained for increasing mesenchymal stem cell (MSC) proliferation rate in vivo wherein said MSCs maintain their potential to differentiate into several cell types. Preferably, SPRF is administered to the subject thereby contacting said SPRF with the MSCs present in said subject.

Preferably, no other serum derived product or supplement and preferably no other growth factors are administered to the subject besides SPRF.

Preferably, in the method or use of the invention the MSCs are maintained in contact with or in the pres- ence of SPRF in vivo for at least 5 days, preferably for at least 8 days or at least 10 days.

Preferably, in the method or use of the invention the subject is in need of regeneration of cartilage and/or bone,

SPRF is administered to a site wherein it may be contacted with the bone or cartilage to be regenerated, and

MSCs present at the site of administration are contacted or maintained in contact with SPRF, and

SPRF level is maintained to proliferate MSCs for until at least a time -period when osteoblast direction differentiation occurs, preferably for until at least a time -period when the expression of at least one, preferably two or at least two osteoblast specific marker gene(s) is/are increased in a medium supplemented with SPRF, preferably SPRF having the concentration range given herein, highly preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium.

In a further preferred embodiment the MSCs present at the site of administration in the subject are MSCs propagated according to the present invention, preferably MSCs obtained from a subject, cultured and propagated in vitro and reintroduced or readministrated to said subject.

Preferably expression of one or both of the following osteogenic marker gene/s is/are increased:

COL1 Al and ALPL, wherein preferably

- ALPL expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold, when compared with 10 % (v/v) FCS supplemented medium. Preferably the subject is a mammalian subject, more preferably a human subject.

In an embodiment the MSCs are bone marrow derived mesenchymal stem cells (BM-MSCs or bone marrow stromal stem cells).

In an embodiment the MSCs are adipose derived mesenchymal stem cells (AD-MSC).

Preferably, upon culturing the MSCs in contact with SPRF expression of MSC-specific genes is maintained or MSC-specific genes remain intensely expressed. In a particular embodiment, the expression level of the following MSC-specific genes is unchanged or is increased by 1 to 150% in comparison with the same medium supplemented with the same concentration of FCS instead of SPRF or in comparison with the same medi- um supplemented with 10% of FCS instead of SPRF. Preferably, the expression level is measured by real time quantitative PCR (rt-qPCR). Preferably, the expression level is measured on or after 5 days as of starting the administration of SPRF or contacting the cells with SPRF. In particular the expression levels of the following MSC marker genes are increased: ALCAM (CD166), ITGB l, CD105, ANPEP. Thus the MSC type or features of the cells are maintained.

Preferably, the expression of hMSC-specific genes are increased after 5 days incubation in a medium supplemented with SPRF, preferably SPRF having the concentration range given above, preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium as follows:

- ALCAM expression is increased at least 1.2 fold (by 20%), preferably at least 1.4 fold or 1.6 fold, - ITGBl expression is increased at least 1.5 fold (by 50%), preferably at least 1.7 fold or 1.9 fold,

- CD105 expression is increased at least 1.05 fold (by 5%), preferably at least 1.1 fold or 1.2 fold and

- ANPEP expression is increased at least 1.1 fold (by 10%), preferably at least 1.2 fold or 1.3 fold, preferably as confirmed by real time qPCR.

Alternatively, any one of the above markers is at least not decreased.

Preferably, no adipose differentiation occurs in the MSCs when MSCs are cultured in SPRF, preferably

10% (v/v) SPRF.

In a preferred embodiment the expression level of adipogenic (adipocyte) markers FABP4, PPARG and ADIPOQ expression, that are markers of adipogenic differentiation, is not increased by more than 1.3 fold (less than by 30 %), preferably 1.2 fold (less than by 20 %), more preferably 1.1 fold (less than by 10 %) upon cultur- ing according to the invention, preferably after 5 days or further culturing, in comparison with 10 % (v/v) FCS supplementation.

However, an osteoblast direction differentiation occurs in the cells upon culturing according to the invention, preferably after 5 days or further culturing, in comparison with 10 % (v/v) FCS supplementation.

In particular, the expression of at least one preferably two osteoblast specific marker gene(s) is/are in- creased after 5 days incubation in a medium supplemented with SPRF, preferably SPRF having the concentration range given above, preferably with 10 % (v/v) SPRF (and comprising no other serum or serum derived product or supplement) when compared with 10 % (v/v) FCS supplemented medium as follows:

- ALPL expression is increased at least 5 fold (by 400%), preferably at least 6 fold or 7 fold, and prefera- bly - RUNX2 expression is at least not reduced or is increased at least 1.05 fold (by 5%), preferably at least 1.1 fold or 1.2 fold,

when compared with 10 % (v/v) FCS supplemented medium,

preferably as confirmed by real time qPCR.

In an embodiment the BAX/BCL2 ratio was elevated at least 15, preferably at least 20 or 25 fold both in case of 10 % (v/v) FCS + 1 ng/mL bFGF supplement and in case of the medium as used in the present invention, in particular when 10 % (v/v) SPRF supplementation was used, in comparison with 10 % (v/v) FCS supplementation. The invention also relates to a medium or the use of a medium as disclosed herein for increasing proliferation rate of mesenchymal stem cells (MSCs) or for culturing MSCs as disclosed herein.

Thus, the invention also relates to a use of SPRF as a cell medium supplement instead of PRP and FBS wherein said SPRF enhances the proliferation rate of human mesenchymal stem cells in vitro while phenotypi- cal changes were not observed except that the levels of osteoblast markers are increased and differentiation potential of proliferated MSCs was maintained.

Said medium comprises SPRF as a supplement and as a serum-derived product. Preferably the medium does not comprise fetal bovine serum (FBS or fetal calf serum, FCS) and does not comprise platelet rich plasma (PRP) and preferably does not comprise FGF (e.g. bFGF) and preferably does not comprise any other growth factor either.

Preferably the medium comprising SPRF does not comprise any further serum product or serum derived product or supplement and preferably does not comprise any other growth factor besides those present in SPRF.

In a preferred embodiment the culture medium comprises

one or more amino acid source, preferably at least glutamate source or a glutamate source only, one or more salts, said salt being preferably selected from calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and monosodium phosphate,

one or more sugar, preferably at least glucose or glucose only and optionally or if desired one or more vitamins preferably selected from folic acid, nicotinamide, riboflavin and B12, wherein said medium comprises 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF.

In a preferred embodiment the medium is a derivative of Dulbecco' s modified Eagle's medium (DMEM) which differs from DMEM in that it is supplemented with 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.

In an embodiment of the invention the SPRF selectively increases mesenchymal stem cell (MSC) proliferation rate which means that proliferation rate of mesenchymal stem cells (MSC) increases at a higher extent than at least one other adult stem cell type, e.g. that of hematopoietic stem cells. The invention also relates to the use of serum fraction of platelet rich fibrin (SPRF) obtained from a donor subject to test selective increase of MSC proliferation rate in vitro. Preferably, MSCs are undifferentiated cells with the potential to differentiate in several cell types.

Preferably, in the use of SPRF or in the method of the invention MSCs doesn't differentiate in vitro into adipocyte direction.

Said SPRF is a serum fraction of platelet rich fibrin prepared or is obtainable by the following method: SPRF is isolated from whole blood obtained from donors by centrifugation

- at 1000 to 4000 g, preferably 1000 to 3000 g or 1500 to 2500 g or 1000 to 2500 g or 1500 to 2000 g or more preferably 1500 to 2000 g

for 2 to 20 minutes or 3 to 15 minutes or 5 to 15 minutes or 3 to 12 minutes or e.g. 5 or 10 mins,

preferably within 4, 3, 2 or preferably within 2 or 1.5 or within 1 minute(s) from blood collection,

to obtain a fibrin clot,

wherein the fibrin clot (coagel or platelet rich fibrin) is pressed or squeezed to obtain the SPRF. Thus, SPRF is obtained as an exudate or releasate of the fibrin clot (platelet rich fibrin preferably specifically prepared as disclosed herein).

Preferably, SPRF can be collected and stored eg. frozen.

In certain embodiments, the SPRF is separated by pressing, squeezing, filtering and/or centrifuging the fibrin clot to isolate the serum fraction (fluid fraction) of platelet rich fibrin.

Preferably, upon clotting and formation of the platelet rich fibrin clot, the acellular or clear supernatant from the PRF may be isolated, or may be removed before fractionating the PRF to isolate the PRF fluid fraction. Such fluid fraction turned out to contain a high concentration of activated platelet releasate and growth factors contained therein.

In an embodiment, the SPRF may be obtained from a blood sample from a single donor subject or from multiple donor subjects and mixed together to obtain a single blood sample. Alternatively SPRF obtained from multiple donors can be mixed together.

According to a specific aspect, the SPRF is obtained from venous blood collected from a single donor. In a preferred embodiment the donor is the patient to whom, once proliferated, the MSCs are reintroduced.

Preferably, the SPRF is employed herein without exogenous anticoagulants that are commonly used in the prior art when preparing PRP, thereby an effective activation of platelets and a content of an activated platelet releasate in the isolated serum fraction is obtained according to the invention.

Highly preferably, SPRF is obtained or obtainable by the following method:

a) Whole blood obtained from a donor is immediately centrifuged at 1000 to 3000 g or 1500 to 2500 g or

1000 to 2500 g or 1500 to 2000 g or more preferably 1500 to 2000 g, for 2 to 20 minutes or 3 to 15 minutes or 5 to 15 minutes or 3 to 12 minutes or e.g. 5 or 10 mins

preferably atlOOO to 2500 g or more preferably 1500 to 2000 g, for 5 to 15 minutes

in particular for 1700 g for 10 mins;

b) the fibrin clot (platelet rich fibrin) is obtained wherein the red blood cells discarded. c) the SPRF is pressed or squeezed out of the fibrin clot,

wherein optionally or if desired the SPRF is collected and stored.

Preferably SPRF is stored frozen or lyophilized, e.g. at -20 °C. The invention also relates to the use of SPRF in stem cell therapy preferably in MSC therapy, wherein

SPRF obtained from a donor subject is used to increase proliferation rate of the patient's in vitro expanded MSCs. Preferably, the donor subject of the SPRF is the same subject as the patient to be treated with MSCs. Preferably, the donor subject of the SPRF is the same subject as the patient from whom the MSCs are obtained.

Preferably, with the use of SPRF MSCs do not differentiate in vitro. Preferably, however, osteoblast mark- ers appear on the MSCs on a given period of time e.g. 5 days culturing.

The invention also relates to the use of SPRF in stem cell therapy wherein the patient's own cells are proliferated in situ (in vivo) or in vitro or ex vivo.

Preferably, the patient is a subject in need of bone tissue regeneration. Preferably the patient is a subject in spongy bone tissue defect, osteonecrosis osteoarthrosis or osteoarthritis.

In an application the lack of differentiable MSCs in the subchondral/spongy bone can be cured/treated by stem cell therapy or stem cell transplantation.

In an application the MSC transplantation is autologous transplantation followed by ex vivo multiplication. In a preferred application the ex vivo multiplication is carried out in a medium comprising SPRF as the patient' s own blood separation product.

In a preferred application transplantation is not needed, because the proliferation of resident MSCs can be enhanced.

In a preferred embodiment the used therapy comprises the proliferation of MSCs resident in a tissue of a patient wherein the SPRF is administered to the tissue of said patient to enhance proliferation of MSCs in said tissue. Preferably in said tissue the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.

In an application the SPRF is administered to the patient to the same site as in vitro expanded own MSCs, essentially simultaneous with or after MSC transplantation.

Preferably the tissue is impaired bone tissue or cartilage tissue.

Preferably, SPRF is administered to the patient by matrix assisted transplantation.

The invention also relates to a method of treatment wherein MSCs are obtained from said patient and the patient's own cells are proliferated in vitro, wherein the MSC transplantation is autologous transplantation followed by ex vivo expansion.

The invention also relates to a method of treatment of a patient in a stem cell therapy wherein SPRF obtained from a donor subject used to increase proliferation rate of the patient's MSCs expanded ex vivo, wherein the MSCs so proliferated maintain their undifferentiated character with the potential to differentiate in several cell types. Preferably, the MSCs show increased expression of at least one or two, preferably two osteoblast markers, preferably COL1A1 and/or ALPL.

In a preferred embodiment the donor subject of blood from which the SPRF is obtained is identical with the patient. In a preferred embodiment the patient is treated with a condition wherein the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.

In a preferred embodiment the patient is in need of bone tissue regeneration.

Preferably, in the treatment subchondral and/or spongy bone is treated in a stem cell therapy or stem cell transplantation by MSCs proliferated using said SPRF.

Preferably said therapy comprises the proliferation of MSCs resident in a tissue of a patient wherein the SPRF is administered to the tissue of said patient to enhance proliferation of MSCs in said tissue, and

wherein the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis, osteoarthrosis or osteoarthritis.

Preferably the tissue is impaired bone tissue or cartilage tissue.

In a further preferred embodiment in said treatment administration of SPRF is applied together with a treatment as defined in any of claims 2 to 8.

In an embodiment SPRF is administered to the patient in a matrix.

DEFINITIONS

The term "subject" as used herein shall refer to a warm-blooded mammalian, particularly a human being. In particular, the medical use of the invention or the respective method of treatment applies to a subject in need of administration of a pool of MSCs. The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. The term "treatment" is thus meant to include both prophylactic and therapeutic treatment, in particular a treatment including the step of proliferation of MSCs in vitro.

"Platelet rich plasma" (PRP) is a blood fraction prepared by separating the red blood cell fraction from a venous blood sample, removing the red blood cell fraction and, if appropriate, the buffy coat, obtaining thereby a platelet poor plasma fraction (PPP), separating - preferably by centrifugation - a platelet rich fraction from the PPP or pelleting platelets, and recovering the platelets in a platelet rich plasma (PRP) fraction, optionally by resus- pending the pelleted platelets in an appropriate medium, optionally in PPP.

Platelet rich fibrin is clotting spontaneously during its preparation by centrifuging a blood sample, preferably accelerated upon contact with negatively charged surfaces and without adding exogenous coagulation activators.

The "serum fraction of platelet rich fibrin", hereinafter also referred to as SPRF (serum of platelet rich fibrin) is a serum fraction as defined or described in WO2014126970A2. Preferably SPRF, as used herein is isolated from whole blood obtained from donors by centrifugation to obtain a fibrin clot and wherein the fibrin clot (platelet rich fibrin) is pressed or squeezed to obtain the SPRF as an exudates or releasate of the fibrin clot. Preferably, SPRF said centrifugation to obtain the fibrin clot is carried out at 1000 to 4000 g, preferably 1000 to 3000 g or 1500 to 2500 g or 1000 to 2500 g or 1500 to 2000 g or more preferably 1500 to 2000 g for 2 to 20 minutes or 3 to 15 minutes or 5 to 15 minutes or 3 to 12 minutes or e.g. 5 or 10 mins, within 4, 3, 2 or preferably within 2 or 1.5 or within 1 minute(s) from blood collction, and SPRF can be collected and stored eg. frozen.

Specifically, the coagel is separated by pressing, squeezing, filtering and/or centrifuging the coagel to isolate the serum fraction containing the fluid fraction of platelet rich fibrin.

Preferably, the SPRF may be obtained from a blood sample from a single donor or from multiple donors and mixed together to obtain a single blood sample or SPRF. According to a specific aspect, the SPRF is obtained from venous blood collected from a single donor. In a preferred embodiment the donor is the patient to whom, once proliferated, the MSCs are reintroduced.

"Stem cells" are undifferentiated or partially differentiated cells with a strong potential to differentiate into several or multiple differentiated cell types and which are also capable of a limited number of cell division to maintain themselves. Thus, stem cells have a limited capability to proliferate and a high potential to differentiate.

"Adult stem cells" ("somatic stem cells" or "tissue stem cells") are partially differentiated stem cells capable of proliferation, self -renewal, production of a large number of differentiated functional progeny, and are capable of regenerating tissue after injury and having a flexibility in the use of these options

Without limitation, adult stem cells are e.g.:

Hematopoietic stem cells,

Mammary stem cells,

Intestinal stem cells,

Mesenchymal stem cells,

Endothelial stem cells,

Neural stem cells,

Olfactory adult stem cells,

Neural crest stem cells,

Testicular cells.

"Mesenchymal stem cells" (MSC) are stem cells of stromal origin and/or localization which have the potential to differentiate in several cell types, and are

- adherent,

- capable of differentiation into mesenchymal tissue, preferably bone, cartilage or adipose tissue in vitro, and preferably are

- CD105, CD73 and CD90 positive, do not carry surface markers of blood progenitor cells or heamatopoietic stem cells, and preferably are CD45, CD34, CD14, CDl lb, CD79a es CD19 negative.

"Cell therapy" is the transplantation of human or animal cells to a patient to replace or repair damaged tissue. "MSC therapy" is a cell therapy wherein MSCs are administered to a patient having an impaired tissue and wherein said MSCs are differentiated into cells of said tissue or tissue-specific cells or tissue-resident cells in the patient.

"Osteoarthritis" is a degenerative disease characterized by erosion of articular cartilage, which becomes soft, frayed, and thinned with eburnation of subchondral bone and outgrowths of marginal osteophytes; pain and loss of function result; mainly affects weight-bearing joints. Osteoarthritis is also called degenerative joint disease, or osteoarthrosis. Osteoarthrosis may be considered as a chronic noninflammatory bone disease variant and also may be a synonym for osteoarthritis. "Spongy bone" is the tissue that makes up the interior of bones; compact bone is the tissue that forms the surface of bones. In long bones, spongy bone forms the interior of the epiphyses.

"Osteonecrosis" is bone death in particular caused by poor blood supply.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic comparison of exemplary isolation of platelet rich plasma (PRP) and serum from platelet rich fibrin (SPRF).

Figure 2. Time-course effect of serum supplements on hMSCs. Subconfluent hMSCs were cultured in DMEM in the absence of supplement (□, col. 1 at day 2 and 5), 10% (v/v) of FCS (ϋ, col. 2 at day 2 and 5) or 10% (v/v) FCS + 1 ng/mL bFGF (¾ col. 3 at day 2 and 5), or 10% (v/v) PRP (■, col. 4 at day 2 and 5) OR 10% (v/v) SPRF (^, col. 5 at day 2 and 5). Results are presented as means of triplicate sapmples in three experiments + SD. p < 0,0001 ***.

Figure 3. Cell morphology of hMSCs using phase contrast microscopy (magnification x 10). 3.A: 10% (v/v) of FCS (upper row, left); 3.B: 10% (v/v) FCS + 1 ng/mL bFGF (upper row, right), 3.C: 10% (v/v) PRP; (lower row, left) 3.D: 10% (v/v) SPRF (lower row, right).

Figure 4. Cell immunophenotypes of hMSCs cultured in differently supplemented media. Mesenchymal stem cells were cultured in (4.A) 10% (v/v) of FCS (1) or (4.B) 10% (v/v) FCS + 1 ng/mL bFGF (¾, or (4.C) 10% (v/v) PRP (■) or (4.D) 10% (v/v) SPRF (SS) and stained with specific antibodies. In the first three FACS diagrams (/l, 12 and /3 figures) representative images of expression of hMSC markers CD90, CD105, CD73, respectively, are shown. On the fourth FACS diagrams (/4 figures) representative images of expression of hematopoietic markers CD34, CD l ib, CD 19 and CD45 is shown. The fluoro- chromes applied were, respectively, FITC (fluorescein isothiocyanate), Cy5 cyanine dye, APC (Allo- phycocyanin) and PE (Phycoerythrin).

Figure 5 Gene expression analysis of differently supplemented hMSCs cultures - On this figure hMSC, adipocyte, osteoblast and apoptotic marker levels are shown in hMSC cultures after 5 days culturing. Mesenchymal stem cells were cultured in 10% (v/v) of FCS (85; background color, control) or (5.B)

10% (v/v) FCS + 1 ng/mL bFGF (SS; col. 1), or (5.C) 10% (v/v) PRP (■; col. 2) or (5.D) 10% (v/v) SPRF (^; col. 3). On the Y axis the relative protein expression level ratio is presented, the base of comparison is the level in 10% FCS cultures ( i.

Data are presented as fold change values to the expression of hMSCs cultured in 10 (v/v) % FCS- supplemented medium that was considered as the standard growing medium.

Figure 6. Culture of human subchondral bone chips - Following 5 days incubation cells in SPRF shows alike if not better viability increase as cells in FCS medium. Blood serum free medium did not induce proliferation of cells. Serum-free medium: (O), 10% (v/v) of FCS (Δ),10% (v/v) SPRF (O).

Figure 7 Histological analysis of hSBPs Culturing hSBPs in 10 % (v/v) SPRF supplemented medium for 5 days preserved bone marrow integrity as hematoxylin and eosin -stained sections (A) and Masson's tri- chrome sections (B) show. However, 10 % (v/v) FCS supplementation for 5 days appeared less effective therein. That means, SPRF revealed higher level of hMSC accumulation (C) compared to FCS (G), and preserved better the local vasculature (D,H).

Figure 8. Gene expression analysis of human subchondral bone chips - Relative gene expression level on Y axis is shown compared to the values measured right after the bone chip explantation. . Serum-free me- dium: (O), 10% (v/v) of FCS (Δ),10% (v/v) SPRF (O).

A: demonstrates that hMSC markers did not change in average.

A/1: ENG A/2: ITGB1 A/3: ANPEP A/4: ALCAM

B: indicates that the hematopoetic cells were not induced, however they could be present in bone chips. B/l: CD34 B/2: CD14 B/3: PTPRC

C: shows that adipocites were not induced in PSRF medium.

C/l: PPARg C/2: FABP4 C/3: ADPOQ

D: the increase of the expression level of osteoblastic genes is demonstrated.

D/l: COL1A1 D/2: P4HA2 D/3: ALPL D/4: RUNX2

DETAILED DESCRIPTION

One of the most important functions of MSCs is natural tissue repair, which is mainly the result of the wide distribution and multipotent differentiation in the human body. Clinical and preclinical models already proved this reparative effect and the critical role of MSCs in injury healing was strongly suggested as well. MSCs are believed to be responsible for replacing cells that are lost in diseases or pathological conditions. Due to these functions the approach of supplementing stem cells to enhance tissue regeneration and treat degenerative diseases were also successfully tested and were shown to be effective. MSCs are also responsible for therapeutic effects in the musculoskeletal system, and were found to be effective in periodontal tissue and bone damage caused by e.g. osteonecrosis and has been successfully applied in cartilage and long bone repair. Besides supplementing MSCs, which were harvested from the patient and either injected or cultured and injected back to the patient, there may be an alternative solution as well. The distribution of stem cells may alternatively be redistributed using our method, which basically enables selective proliferation of the available stem cells, which means that the proliferative effect can be localized and thus a selective tissue repairing treatment can be realized. In order to overcome the uncertainty, which is posed by the circulating excess stem cell concentration, we focus on local therapeutic effect, which means that musculoskeletal and degenerative bone and joint diseases are the main therapeutical targets. This solves the majority of the circulation problems as the circulation of these parts of the body is limited thus the effect of the enhanced proliferation of the stem cells is concentrated on local tissue repair. In our case these tissues are mainly musculoskeletal tissues, more specifically bone and joint tissue.

Another important aspect of our application is that the MSCs preserve their stem cell character during the first 5 days of culturing as no differentiation occurs into adipocyte direction with the use of our SPRF culture supplement except the increase in osteoblast factors after 5 days of culture or further culturing. This enables the advantage of not interfering with the MSCs, thus the MSCs will only differentiate as an effect of the surrounding cells at site of the treatment. This gives the opportunity that the stem cells will differentiate in a manner that accelerates the regeneration of the treated tissue.

In case of diseases of the bone and cartilage this osteogenic differentiation indicates a further unforeseen advantage as it shows that SPRF in a surprising manner prepares the cells for osteogenic differentiation whereas no other direction of differentiation is observed. This means that SPRF and MSCs proliferated thereby are particularly suitable for treatment of diseases of the bone, particular osteoarthrosis and osteoarthritis. Platelet rich plasma (PRP)

In the prior art before the invention PRP was suggested for treatments with similar concept in case of bone and joint diseases in particular osteoarthritis.

A 2015 Meta-analysis reviewed 551 studies on PRP for osteoarthritic knee and found that only nine were worth considering and concluded that with respect to short term outcomes, PRP was not more efficacious than placebo in total WOMAC score but was more efficacious than hyaluronic acid (HA) on that measure; it was no different than placebo or HA with regard to adverse events. [Kanchanatawan, W; Arirachakaran, A; Chaijenkij, K; Prasathaporn, N; Boonard, M; Piyapittayanun, P; Kongtharvonskul, J (May 2016). "Short-term outcomes of platelet-rich plasma injection for treatment of osteoarthritis of the knee.". Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA. 24 (5): 1665-77.].

Results varied in this regard and the prior art was no unambiguous as to the usefulness of PRP treatment.

Since 2004, proponents of PRP therapy have argued that negative clinical results are associated with poor-quality PRP produced by inadequate single spin devices.

In the present invention SPRF showed surprisingly consequently better results than PRP and similar to or even better than the gold standard cell medium supplement FBS plus growth factors. However, FBS obviously is not appropriate for medicine and is not advisable in cell cultures in transplantation applications.

Source of MSCs

While MSCs are available from various sources it appears the present invention is not limited to BM derived MSCs and also for example adipogen derived MSC-s could be applied. Literature opinions vary in as- sessing the capabilities of MSCs of various sources, an advantage of the present invention may be that due to culturing as disclosed herein multiple sources may become useful and available.

Culturing MSCs

In the present invention MSCs are obtained from a subject and said MSCs are cultured by an in vitro method, as disclosed in the Brief Description of the Invention.

In the invention usual MSC culturing conditions can be applied, for example a DMEM basal medium with sugar source like high glucose, glutamate source like GlutaMax™ Supplement, pyruvate and antibiotics or selective agents like penicillin/streptomycin and 1% amphotericin. Instead of FBS regularly used in media like DMEM, SPRF and only SPRF are to be uses. In a particular embodiment even no further growth factors are to be used.

Various methods for the culturing of MSCs are well known and such methods may be considered for use in the present invention wherein using SPRF they may be developed further [Panagiota A. Sotiropoulou et al. Stem Cells Volume 24, Issue 2 February 2006 Pages 462^-71 Characterization of the Optimal Culture Conditions for Clinical Scale Production of Human Mesenchymal Stem Cells] .

SPRF as a medium supplement/additive

Pressing out the fluid content from PRF leads to an autologous blood separation product, which does not contain fibrinogen, anticoagulants and the inflammation markers are low. After testing it as a stem cell medium supplement, the inventors have surprisingly found that use of a serum from platelet rich fibrin (SPRF) instead of PRP and FBS enhanced the proliferation rate of human mesenchymal stem cells in vitro while phenotypical changes were not observed and differentiation potential of proliferated MSCs was maintained. Moreover, culturing hu- man subchondral bone pieces in SPRF supplemented medium cell viability was not only retained, but also signifi- cantly increased in 7-days culture without any measurable cell differentiation. The inventors revealed that predominantly mesenchymal stem cells were multiplicated in the course of the incubation time.

Treatment with MSCs

Osteoarthritis (OA) is one of the most prevalent joint diseases with prominent symptoms affecting the daily life of millions of middle aged and elderly people. Despite this, there are no successful medical interventions that can prevent the progressive destruction of OA joints.

Administration of SPRF in Osteoarthritis

Administration of SPRF is conveniently carried out by injection at the site of impaired bone or chondrocyte tissue. In order to maintain an appropriate level so as to maintain contact with MSCs multiple injection can be applied. For example, injection can be added regularly, e.g. every day or in every 2 days or 1, 2 or 3 times a week.

Another possibility to maintain the level of SPRF may be e.g. matrix assisted administration.

EXAMPLES

Cell and tissue cultures

All tissue culture procedures were carried out in a sterile laminar flow tissue culture hood. Cells and ex vivo explant cultures were maintained in an incubator at 37 °C and 5% CO2 and 95% of humidity.

hMSC proliferation assay

Cells were seeded in standard growing medium into 5 parallel wells of a 96-well plate (2000 cells/well). Cell-free wells were used as background control. 48 hours after the start of the incubation standard growing medium was refreshed only in a group (same medium type was kept), in the others the 10 % (v/v) FCS supplement was changed for 10% (v/v) FCS + bFGF, or 10% (v/v) platelet rich plasma (PRP), or 10% (v/v) serum from platelet rich fibrin (SPRF). PRP-supplemented medium contained 2 U/mL heparine (Clexane, Sanofi Aventis, Paris, France). As negative control, serum-free medium was used. Following 2 and 5 days incubation cell viability assay was performed.

Human mesenchymal stem cell (hMSC) 2D culture

Human mesenchymal stem cells (hMSCs) purchased from LONZA were seeded at 5000 cells/cm² in normal T-75 tissue culture flasks and maintained in standard growing medium: Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX™ Supplement, pyruvate (Gibco, Paisley, Scotland), supplemented with 10 % (v/v) fetal calf serum (FCS, Gibco, Paisley, Scotland), fibroblast growth factor (bFGF) 1 ng/ml (Sigma- Aldrich, St. Louis, USA), 2% Penicillin/Streptomycin (Sigma- Aldrich, St. Louis, USA) and 1% Amphotericin (Sigma- Aldrich, St. Louis, USA). Cell culture medium was refreshed twice a week.

Cell culture conditions for hMSCs

Four different media were used in the course of the experiments with hMSCs. (1) Basal medium: DMEM, high glucose, GlutaMax™ Supplement, pyruvate containing 10% FBS, 2% penicillin/streptomycin and 1% amphotericin. (2) Serum-free medium: DMEM, high glucose, GlutaMax™ Supplement, pyruvate (Gibco, Paisley, Scotland), containing 2% penicillin/streptomycin and 1% penicillin/streptomycin. (3) SPRF-medium: DMEM, high glucose, GlutaMax™ Supplement, pyruvate containing 10% SPRF, 2% penicillin/streptomycin and 1% amphotericin. (4) PRP-medium: DMEM, high glucose, GlutaMax™ Supplement, pyruvate containing 10% PRP, 2% penicillin/streptomycin and 1% amphotericin. Media were changed every 48 hours (support for 2 days injection). Cells were incubated in normal cell culture conditions (5% CO₂, humidified atmosphere, 37 °C). 2000-3000 cells/well were seeded in 5 parallel wells for all sample type and for all time point. Percentage ratios are given in respect of the total volume.

Isolation and culture of human subchondral bone pieces (bone explants; hSBEs)

hSBEs, 2 mm in diameter were harvested from patients undergoing total hip replacement surgery at the Orthopedic Clinics of Semmelweis University (Budapest, Hungary). All procedures were performed with permission of hungarian Ethical Committee. The donors had osteoarthritis, otherwise they were diagnosed not to have cancer, or any infectious or autoimmune disease. Only tissue that would have otherwise been discarded was used.

In an embodiment femoral heads (those would have been otherwise discarded) are sawn off that results in intense cell death at the sawn surface due to friction based heat shock. Therefore, bone explanted from the body was cut in half with a bone chisel and hSBEs were picked from the cut surface with a small chisel.

Explants were delivered to the laboratory in the same medium that was used for hMSC culture.

All further experiments were started following 48 hours of preincubation in the medium described above.

In an embodiment tissue cultures were maintained in basal medium (DMEM containing 10% FBS and 1% penicillin/streptomycin) at 37 °C and 5% CO₂ in a humidified atmosphere. After 2 days incubation samples were used for experiments with special media or harvested for R A.

Viability test for cell culture and bone explants

Cell viability of hMSCs and hSBEs was determined using Cell Proliferation Kit II (XTT; Roche, Mannheim, Germany) according to the manufacturer's instructions. Absorbance were measured after 4 hours incubation in the staining solution using a PowerWave Microplate Spectrophotometer (BioTek, Winooski, VT, USA) at 480 nm with a reference wavelength at 650 nm. In case of hSBEs, bone pieces were removed from the labeling mixture right before absorbance measurement. Results were normalized with the weight of the chips considering that the bone size is proportional to the number of active cells.

On Figure 2. growth for the hMSC cells grown with FCS+bFGF or with FCS, only SPRF or PRP and without serum on plastic surface are shown. On the first day 2000-3000 cells were seeded pro well into a 96-well plate. Viability of cells was measured by XTT viability test. The viability on the 5^th day in case of cells supplemented with FCS + bFGF showed OD₄₅o=l.428+0.064 absorbance values, only FCS supplemented OD₄₅o=l.306+0.069, only SPRF supplemented

only PRP supplemented OD450 =0.954+0.075. The values are the average of five parallel measurements. It can be seen that the medium supplemented with SPRF provided the best result in terms of cell viability.

Tissue culture conditions for hSBPs

Three different media were used in the course of the experiments with hSBPs (1) Basal medium: DMEM containing 10% FBS and 1% penicillin/streptomycin. (2) Serum-free medium: DMEM containing 1% penicillin/streptomycin. (3) SPRF-medium: DMEM containing 10% SPRF and 1% penicillin/streptomycin. Media were changed every 48 hours. Tissue cultures were incubated in normal cell culture conditions (5% CO₂, humidified atmosphere, 37 °C).

Cell viability test of hMSCs and hSBPs

XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium)) assay (Roche, Cell Proliferation Kit II) was performed to measure the viability of cells. XTT Labeling Mixture was added to the culture medium and bones or cell monolayers in 0,3 mg/ml final concentration on a 96-well plate and the plate was placed back into the incubator for 4 hours. Bone pieces were removed and absorbance was measured on 450 nm on a plate reader. In case of hSBPs absorbance values were normalized to the dry weight of the bone pieces.

RNA extraction and reverse transcription

Total RNA was isolated from 5 bone pieces with TRIzol Reagent (Ambion) following homogenization with liquid nitrogen in a mortar. 500 ul of TRIzol reagent was added to the homogenized tissue. Following 5 min incubation on room temperature centrifugation was carried out (12000g, 1 minute, room temperature) to remove particulate debris from homogenized samples. The supernatant was transferred into a new tube and RNA was purified with Direct-zol™ RNA MiniPrep Kit (Zymo Research). RNA was eluted in 40 ul diethylpyrocarbonate-treated (DEPC) water. Measurement of RNA yield was performed by agarose gel electrophoresis and using a NanoDrop 1000A Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The purity of RNA was accepted, when values A260 >2.0 and A260 >2.0 were measured. Reverse transcription was performed using the first- strand cDNA synthesis kit as instructed by the manufacturer (ReadyScript™, Sigma Aldrich), i.e. reverse transcription to synthesize first strand cDNA was carried out for 30 min at 42 °C, primed with an oligo (dT) primer bearing a T7 promoter.

Quantitative PCR

Real-time quantitative PCR was performed using ABI for quantifying the expression of activated leukocyte cell adhesion molecule (ALCAM/CD166: Hs00977641_ml). Values were calculated using the comparative threshold cycle (C_t) method and normalized to GAPDH (Hs02758991_gl) expression. Values were expressed as the mean +SD. Experiments were performed at least three times. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey-Kramer Multiple Comparison post-test.

Assessment of MSC proliferation on bone explants

hSBPs were incubated in basal medium two days long. On the second day their viability showed 48.267+15.626 (n=3). On this day medium was changed for fresh basal medium, serum-free medium or SPRF- medium. Results are shown on Figure 6. Viability values on the 4th day were 92.997+17.025 (n=19), 117.357+19.383 (n=24) and 187.527+18.814 (n=24) in serum-free medium, basal medium and SPRF-medium, respectively. Viability values on the 7th day were 77.711+21.734 (n=7), 199.02+27.367 (n=15) and 224.212+28.023 (n=15) in serum-free medium, basal medium and SPRF-medium, respectively. Statistically significant differences at p < 0.05 were determined by one-way analysis of variance (ANOVA).

Quantitative reverse transcription -polymerase chain reaction analysis was used to evaluate the expression of the hMSC associated gene CD166 (ALCAM, Figure 9). Compared to the second day expression of CD166 (AL- CAM) molecule expression was 2,2-times higher in case of basal medium, and 2,1-times higher in case of SPRF- medium. All expression values are normalized to the expression of GAPDH. Our SPRF supplemented medium was as effective as the one, which was supplemented with specific stem cell media (FBS supplemented basal me- dium), however SPRF is from human autologous origin. All expression values are normalized to the expression of GAPDH.

Isolation of SPRF

a) Whole blood obtained from donors was immediately centrifuged at 1700 g for 10 mins at RT in BD Vacutainer® Z.

b) The fibrin clot from the tube was gently removed with a tweezer and placed onto a sterile Petri-dish and the red blood cells at the bottom of the fibrin clot were cut with a sharp scissor and discarded, c) The lysate was squeezed out of the fibrin clot, collected and stored at -20 °C.

Isolation of PRP

a) Whole blood obtained from donors was centrifuged at 320 g for 12 mins at RT in the BD Vacutainer® 6 ml K2E (EDTA).

b) Three layers had been formed in the collection tube. The bottom layer containing the red blood cells, middle layer containing the buffy coat and the top layer containing the platelet-poor plasma (PPP). c) PPP was aspirated along the middle layer and transferred into a 15 ml Falcon tube and centrifuged at 1700 g for 10 mins.

d) The pellet was resuspended into a corresponding volume to the isolated SPRF in the supernatant. Stored at -20 °C.

Mesenchymal stem cell proliferation assay and cell morphology

To examine cell proliferation in presence of different serum derivatives, subconfluent hMSC cultures were incubated for 2 or 5 days in serum-free DMEM (SF) or in DMEM supplemented either with FCS, FCS + bFGF, PRP or SPRF, 10 % (v/v) each. Viability of the samples was measured with XTT assay on the 1^st, 2^nd and 5^th day of the experiment.

SF, FCS and FCS + bFGF had no mitogenic effect after 2 days incubation. In the presence of PRP and SPRF viability of cells was elevated 7.18-fold and 9.57-fold, respectively. This significant proliferation could be due to the human origin of the applied supplement.

In SF medium viability was not significantly higher as on day 2. In the period between the 2^nd and 5^th day FCS + bFGF caused an intense (14.18-times) cell number elevation. FCS and PRP reached similar viability to FCS + bFGF for the 5^th day, 11.83-fold and 15.53-fold, respectively. Clearly the strongest effect had SPRF in the medium, where the cell viability 20.1-fold higher was compared to the zero day. This effect is very remarkable considering that the official culture medium and a treatment with a growth factor were less effective (Fig. 2).

The morphology of the cells was not visibly altered by the various treatments, all preserved the typical hMSC morphology (Fig. 3).

Flow cytometry analysis of hMSC cultured in different serum supplements

Immunophenotyping analysis was used to characterize that hMSCs cultured 5 days long in differently supplemented media preserved their hMSC-characteristic. hMSCs cultured in 10% (v/v) FCS or 10% (v/v) FCS + 1 ng/mL bFGF, or 10% (v/v) PRP or 10% (v/v) SPRF were positive for CD90-FITC, CD105-PerCP-Cy5.5 and CD73-APC in more than 93.94%. While CD90-FITC and CD73-APC expression was above 99% in case of the PRP-supplemented samples (Fig. 4.A/1 and /3). CD105-PerCP-Cy5.5 level decreased by 13.99% and two cell populations were appeared. This thought to be the result of an unknown differentiation process in connection with PRP (Fig. 4.A/2).

No expression of hematopoietic markers (CD34/CDl lb/CD19/CD45/HLA-DR-PE) was detected in any of the experiments with 10 % (v/v) FCS-, 10 % (v/v) PRP- and 10 % (v/v) SPRF-supplemented samples (Fig 5.B), showing that no hematopoietic differentiation occurs; however 10 % (v/v) FCS + 1 ng/mL bFGF supplement caused the formation of a positive subpopulation for those markers (16.2%) (Fig.4.B). This finding suggests that not hematopoietic stem cells (HSCs) but MSCs are proliferated according to the invention. Gene expression analysis of differently supplemented hMSCs cultures

The result of this analysis is shown on Figure 6.

Panel A shows that mesenchymal stem cell marker levels remained compared to standard culture medium (10 v/v% FCS). On the Y axis the protein expression level ratio is presented, the base of comparison is the level in 10% FCS cultures.

MSCs may differentiate in either to osteoblasts or adipocytes, consequently both cell type markers have been checked to complete the study. On panels B and C it is shown that PPARGG and ADIPOQ expression level did not show any change in contrast with osteoblast markers ALPL and COL1A1 which have been significantly increased in case of SPRF supplementation.

Panel D: Apoptotic genes' expression were analyzed as well: BCL2 is antiapoptotic, while BAX is an apoptotic protein. BAX/BCL2 ratio is in equilibrium under normal physiological conditions (taken as 100% in FCS). Increase is demonstrable if cells undergo apoptotic process due to inner or under external effects. FCS+BFGF and SPRF supplementation results in an approximate 80-90%, while PRP cultured cells show a tremendous increase.

hMSC-specific genes stayed intensely expressed in culture of mesenchymal stem cells supplemented with 10 % (v/v) SPRF

The expression of hMSC-specific genes was confirmed by real time qPCR after 5 days incubation in the media indicated above. ALCAM (CD166), ITGB l, CD105 and ANPEP expression showed significant increase in the 10 % (v/v) SPRF supplemented samples when compared with 10 % (v/v) FCS supplemented group 1.68- fold, 2.03-fold, 1.29-fold and 1.37-fold, respectively. While ALCAM (CD166), ITGBl, CD105 and ANPEP expressions were almost unchanged after culturing in 10 % (v/v) FCS + 1 ng/mL bFGF (0.95-fold, 1.35-fold, 0.98-fold and 1.29-fold, respectively), the expression of the same markers decreased in case of the 10 % (v/v) PRP culturing at CD105 0.87-fold and ALCAM (CD166) 0.55-fold. Increase was found when 10 % (v/v) PRP culturing in case of ITGBl (1.67-fold) and ANPEP (2.19-fold) expression.

Adipose differentiation was not observed but osteoblastic gene expression was elevated when hMSCs were cultured in 10 % (v/v) SPRF

Since hMSCs are the common progenitor cells of adipocytes and osteoblasts, adipocyte- specific and os- teoblast-specific gene expression was investigated in the variously supplemented hMSC cultures by RT-qPCR. FABP4, PPARG and ADIPOQ expression, that are markers of adipogenic differentiation were not elevated when 10 % (v/v) FCS supplementation was changed for 10 % (v/v) FCS + 1 ng/mL bFGF, 10 % (v/v) PRP or 10 % (v/v) SPRF, i.e. the expression level stayed unvaried compared to standard culturing method (Fig.3B). COL1 Al, ALPL and RUNX expression was slightly changed in cultures supplemented with 10 % (v/v) FCS + 1 ng/mL bFGF, 0.78-fold, 1.3-fold and 1.49-fold respectively. 10 % (v/v) PRP had almost the same effect 1.43- fold change in COL1A1 expression, 3.11-fold change in ALPL expression and RUNX2 expression decreased 0.47-fold compared to the control group.

10 % (v/v) SPRF supplement showed a significant increase in osteblastic gene expression. COL1A1 mRNA level elevated 8.38-fold, ALPL level 8.28-fold and RUNX2 level 1.31-fold compared to control (Fig.5C).

BAX/BCL2 ratio was highly increased when hMSC culture supplemented with 10 % (v/v) PRP BAX/BCL2 ratio was elevated 29.97-fold in case of 10 % (v/v) FCS + 1 ng/mL bFGF supplement, 31.99-fold when 10 % (v/v) SPRF supplementation was used.

Histological analysis of hSBPs

Bone explants were fixed in 4% formalin solution. The samples were dehydrated in an ascending alcohol series at room temperature and infiltrated and embedded in a resin specifically developed for mineralized tissues (Technovit 9100 Kulzer). Infiltrated explants were placed in specific molds filled with polymerization mixture. 4 m-sections were cut using Leica RM2255 sawing microtome and stretched on slides. For hematoxylin-eosin staining the sections were immersed into hematoxylin solution and washed with 1% eosin solution. For Mas- son's trichrome staining sections were immersed in hematoxylin solution containing picric acid. After washing, Fuchsin Ponceau staining was performed, and unspecific parts were washed with with 1% phosphomolybdic acide solution.

We have found that culturing hSBPs in 10 % (v/v) SPRF supplemented medium for 5 days preserved bone marrow integrity as hematoxylin and eosin-stained sections (Figure 7, A) and Masson's trichrome sections (B) show, in a surprisingly good state. However, 10 % (v/v) FCS supplementation for 5 days appeared less effective and gave an inferior result in this regard. That means, SPRF revealed higher level of hMSC accumula- tion (C) compared to FCS (G), and preserved better the local vasculature (D,H).

Gene expression analysis of human subchondral bone chips

Culturing MSC on bone chips and rt-qPCR analysis were carried out as described above.

On Figure 81 relative gene expression level on Y axis is shown compared to the values measured right after the bone chip explantation. As previously, three media were applied: serum- free medium: (O), 10% (v/v) of FCS (Δ),10% (v/v) SPRF (O).

It has been found that hMSC markers did not change in average (Figure 8. A A/1: ENG A/2: ITGBl A/3: ANPEP A/4: ALCAM). Figure 8/B indicates that the hematopoetic cells were not induced, however they could be present in bone chips. This indicates the selectivity of the MSC proliferation method (B/l: CD34 B/2: CD14 B/3: PTPRC). Furthermore, Figure 8/C shows that adipocites were not induced in PSRF medium, thus, similarly to the MSC- cultures, adipocytes differentiation does not occur on explants either (C/l: PPARg C/2: FABP4 C/3: ADPOQ).

However, surprisingly, while MSC character of the cell is maintained, an osteoblast direction differentiation can be observed on the 5^th day of culturing. We monitored the expression of the following osteoblast marker genes: D/l: COL1A1 D/2: P4HA2 D/3: ALPL D/4: RUNX2, among which COL1A1 and to a lesser extent ALPL seem to be upregulated.

INDUSTRIAL APPLICABILITY

The present invention is applicable both in research and medicine among others to improve proliferation of MSCs with maintaining their proliferation potential preferably for the purpose of bone regeneration or for using them in MSC cell therapy.

Claims

1. A method for use of serum fraction of platelet rich fibrin (SPRF) for selectively increasing MSC proliferation rate in vitro, in vivo or ex vivo wherein said differentiated MSCs maintain their potential to differentiate in several cell types, preferably MSCs are obtained from a subject, said method comprising

i. providing SPRF,

ii. adding SPRF to a pool of MSCs,

iii- allowing MSCs to proliferate,

for at least 5 days.

2. The method according to claim 1, wherein upon proliferation of MSCs expression of one or both of the following osteogenic marker gene/s is/are increased: COL1A1 and ALPL.

3. The method according to claim 1 or 2, wherein said method is an in vitro method and in step ii. the SPRF is added to a pool of MSCs is a culture medium, said medium comprises 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.

4. The method according to claim 3 wherein the MSCs so propagated are (for) reintroduction to a patient is a subject in need of bone tissue regeneration or the patient is a subject suffering in spongy bone tissue defect, osteonecrosis osteoarthrosis or osteoarthritis.

5. The method according to claim 1 or 2, wherein said method is an ex vivo method, wherein in step ii SPRF is added to a pool of MSCs on a tissue or explant in a medium

wherein said medium comprises 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12%

(v/v) or about 10 % (v/v) SPRF and comprises no other serum derived product or supplement and preferably no other growth factors.

6. The method according to claim 5, wherein the ex vivo tissue is a bone or cartilage graft and said graft is reintroduced into a patient in need thereof, wherein said patient is a subject in need of bone tissue regenera- tion or the patient is a subject suffering in spongy bone tissue defect, osteonecrosis osteoarthrosis or osteoarthritis.

7. The method according to claim 1 or 2, wherein said method is an in vivo method, wherein

SPRF is contacted with MSCs of a subject in vivo and

an appropriate level of SPRF is maintained for increasing mesenchymal stem cell (MSC) prolifera- tion rate in vivo wherein said MSCs maintain their potential to differentiate into several cell types and wherein upon proliferation of MSCs expression of one or both of the following osteogenic marker gene/s is/are increased: COL1 Al and ALPL, and wherein

no other serum derived product or supplement and preferably no other growth factors are administered to the subject besides SPRF, and

the MSCs are maintained in contact with or in the presence of SPRF in vivo for at least 5 days.

8. The method according to claim 7, wherein SPRF is contacted with MSCs of a subject in vivo by administering SPRF to a site of said subject wherein it may be contacted with the bone or cartilage to be regenerated, and MSCs present at the site of administration are contacted or maintained in contact with SPRF for at least 5 days.

9. The method according to any of the previous claims wherein

- preferably within 4, 3, 2 or preferably within 2 or 1.5 or within 1 minute(s) from blood collection,

to obtain a fibrin clot,

wherein the fibrin clot (coagel or platelet rich fibrin) is pressed or squeezed to obtain the SPRF.

10. The use of SPRF as a cell medium supplement instead of PRP and FBS wherein said SPRF enhances the proliferation rate of human mesenchymal stem cells in vitro, ex vivo or in vivo, while maintaining their potential to differentiate in several cell types, wherein upon proliferation of MSCs expression of one or both of the following osteogenic marker gene/s is/are increased: COL1 Al and ALPL, and wherein

said medium comprises SPRF, preferably 2-20% (v/v), preferably 5-15% (v/v), highly preferably with 8 to 12% (v/v) or about 10 % (v/v) SPRF, as a supplement and

the medium does not comprise fetal bovine serum (FBS or fetal calf serum, FCS) and does not comprise platelet rich plasma (PRP) and preferably does not comprise FGF (e.g. bFGF) and preferably does not comprise any other growth factor either.

11. SPRF for use in therapy, preferably in stem cell therapy, wherein in said therapy SPRF obtained from a donor subject is used to increase proliferation rate of the patient's MSCs expanded in vitro, ex vivo or in vivo, wherein the MSCs so proliferated maintain their undifferentiated character with the potential to differentiate in several cell types,

wherein proliferation of MSCs is carried out for at least 5 days.

12. The SPRF for use according to claim 11 wherein upon proliferation of MSCs expression of one or both of the following osteogenic marker gene/s is/are increased: COL1 Al and ALPL.

13. The SPRF for use according to claim 12 wherein MSCs are obtained from said patient and the patient's own cells are proliferated in vitro, wherein the MSC transplantation is autologous transplantation.

14. The SPRF for use according to claim 11 to 13 wherein

said SPRF is a serum fraction of platelet rich fibrin prepared or is obtainable by a method according to claim 9, wherein the donor of blood is identical with the patient.

15. The SPRF for use according to any of claims 11 to 14 wherein the patient is treated with a condition wherein the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis or osteoarthritis, preferably where- in the patient is in need of bone tissue regeneration.

16. The SPRF for use according to claim 15 wherein in the treatment subchondral and/or spongy bone is treated in a stem cell therapy or stem cell transplantation by MSCs proliferated using said SPRF.

17. The SPRF for use in a therapy comprising the proliferation of MSCs resident in a tissue of a patient wherein the SPRF is directly administered to the tissue of said patient to enhance proliferation of MSCs in said tissue.

18. The SPRF for use according to claim 17, wherein the level of differentiable MSCs is low, preferably pathogenically low, preferably said condition being selected from impaired bone tissue, spongy bone tissue defect, osteonecrosis or osteoarthritis, preferably wherein the tissue is impaired bone tissue and/or cartilage tissue.

19. The SPRF for use according to any of claims 17 to 18 wherein in said treatment administration of SPRF is applied together with a treatment as defined in any of claims 11 to 16.

20. The SPRF for use according to any of claims 11 to 19 wherein SPRF is administered to the patient by

- matrix assisted transplantation,

- multiple injections,

- implantation of a graft carrying SPRF,

to provide SPRF at the site of administration and maintain a level of SPRF for at least 5 days sufficient to increase MSC proliferation rate in vivo wherein said differentiated MSCs maintain their potential to differentiate in several cell types, and

wherein upon proliferation of MSCs expression of one or both of the following osteogenic marker genes is/are increased: COL1A1 and ALPL.