FIELD OF THE INVENTION

Embodiments described here concern a method for expanding adult stem cells from whole blood, in particular but not only peripheral blood, of adult mammals, and the corresponding application in the medical field, particularly in the human or veterinary field, for the therapeutic treatment of lesions, external lesions, internal lesions, lesions of tendons, lesions of ligaments, lesions of cartilage, bone fractures, as well as therapeutic and/or preventive treatment of chronic and/or acute inflammatory pathologies, neurological and neurodegenerative pathologies, cardiac pathologies, tumorous pathologies, autoimmune pathologies, ophthalmic pathologies and pathologies of genetic origin.

Here and hereafter in the description, and as known in literature, the word “expansion” means the process to increase the number of cells, either by cell division or, as in the specific case described and claimed here, by “de differentiation” or“de-programming”, that is to say, the process by which some cells in the blood are re -transformed into stem cells following suitable in vitro treatment, as will be seen hereafter.

BACKGROUND OF THE INVENTION

In recent years, the use of stem cells in therapy has had widespread consensus, but the therapeutic results obtained have been far below expectations with the exception of stem cells obtained from blood.

In fact, many known methods for obtaining stem cells have proven to be long, laborious, expensive, with limited results and sometimes side effects.

We have embryonic and adult stem cells: the former derive from blastocysts of 8 days, the adult ones on the other hand can be obtained mainly from bone marrow, adipose tissue, muscular tissue, from peripheral blood and from the umbilical cord, etc.

The definition of stem cell is in constant evolution. For all these cells, embryonic (ES) and adult, both hematopoietic (HSC) and also mesenchymal (MSC) (Kuwana M., et al., 2003), different genetic markers have been identified, some of which are common to many cell types (Condomines M, et ah, 2006; Kang WJ, et al., 2006; Zhao Y., et al., 2003; Rabinovitch, M. et al., 1976).

On the other hand, in order to identify pluripotent stem cells (PSCs), embryonic and adult, the expression of some intracellular transcription factors is considered (Sox2, Oct3/4 and Nanog).

Initially, research was oriented toward stem cells of embryonic derivation as they are pluripotent and also qualifiable and quantifiable, therefore suitable for an experimental trial, but ethical questions and above all the contraindications due to the production of tumors caused them to be set aside. Therefore, today adult stem cells are preferred.

The adult stem cells of another individual (allogeneic) very frequently give serious rejection phenomena because they are not recognized as“self’. This mainly affects umbilical cord stem cells that are used almost exclusively as allogeneic stem cells.

Induced pluripotent stem cells produced by a process that transfers, both through viruses and also not through viruses, the pluripotency factors from embryonic stem cells to adult stem cells are cells that are not suitable for treatment, due to the contraindications similar to those found with embryonic stem cells and the high costs.

For now, in humans the use of stem cells obtained from peripheral blood through a process called“apheresis” or“leukapheresis” is accepted. The stem cells are extracted from the blood, collected, and then inoculated into patients suffering from some leukemic pathologies, immediately after a chemo or radiotherapy. These stem cells are hematopoietic, therefore they inter-react exclusively with pathologies of the blood.

In apheresis, which lasts from 6 to 8 hours, blood is drawn from the vein of an arm, or from a vein in the neck or chest and passed through a machine that removes the stem cells. The blood purified in this way returns to the patient, while the collected cells are preserved by refrigeration in liquid nitrogen (Condomines M, et al., 2006; Kang WJ, et al., 2006). This technique, as well as being painful, is also extremely stressful for the patient. It provides in vivo inoculation of growth factors to stimulate the release of stem cells from the bone marrow to the blood, and does not allow for a real discrimination and/or purification of the circulating stem cells. Today, these cells are allowed, from a regulatory point of view, in therapy to treat pathologies exclusively of the blood.

The stem cells that are being introduced onto the market today to treat different pathologies are adult stem cells and mainly mesenchymal stem cells obtained from bone marrow and fat. However, they have a number of limitations:

- invasive sampling by drilling a bone for those of the bone marrow, or surgery for those of fat;

- capacity to inter-react only with certain tissues, due to their mesenchymal derivation;

- when put into culture to obtain an adequate number for therapy, they begin to differentiate into other cell types showing membrane receptors that are always different and therefore cannot be qualified and quantified, characteristics that are indispensable for a human experimental trial.

Another known method is described by Zhao Y. et al., in the article“A human peripheral blood monocyte-derived subset acts as pluripotent stem cells” and in WO-A-2004/043990. It is a method for preparing stem cells deriving from monocytes, which includes the steps of isolating monocytes from peripheral blood, putting them into contact with a mitogenic component and subsequently effecting the culture of monocytes from peripheral blood in conditions suitable for the propagation of cells.

This method, which initially requires a step of isolating the monocyte and then an expansion step in a culture medium, is very long, about 15-20 days, to obtain a significant number of stem cells, and does not allow to obtain pluripotent stem cells.

Again within the framework of preparing stem cells from monocytes, documents WO-A-2005/046570, WO-A-2007/131200 and WO-A-03/083092 are also known. However, even the methods described in these documents, since they have to carry out a preliminary purification of the blood in order to isolate only a cell fraction, that is, the monocytes, and a subsequent expansion in order to obtain the desired stem cells, require very long times, always in the order of 15-40 days, to obtain an acceptable quantity of stem cells.

Document WO-A-2008/034370 in the name of the present Applicant is also known, which is incorporated herewith entirely as reference, concerning a method for expanding adult stem cells from blood using MCSF (Macrophage Colony Stimulating Factor). This known method provides a growth of blood adult stem cells after blood is drawn by an in vitro treatment with MCSF, in a concentration comprised between 8 nM and 15 nM, and a subsequent purification, preferably by fractionation on a Ficoll gradient. The method can also provide a growth of the purified peripheral blood stem cells by in vitro treatment with MCSF in a concentration comprised between 35 nM and 55 nM.

The effectiveness of this method is demonstrated by the presence and recognition of the CD90, CD90/34, CD34 and CD117 stem cell markers, and by the fact that the stem cells do not lose the“self’ recognition factors following division or expansion. These stem cells do not give rise to side effects such as phenomena of rejection, infection, development of teratomas once administered in the patient, they are able to differentiate in vivo and behave as pluripotent stem cells.

The authors have found that the cells thus grown by division or expanded, once injected locally or intravenously, acquire in vivo (and not in vitro as in the methods known in the prior art by means of suitable growth factors and/or chemical stimuli (Gulati R., et al., 2003; Katz RL, et ah, 2002; Okazaki T. et al 2005)), all the morphological and chemical characteristics of macrophage, lymphocyte, epithelial, endothelial, neuronal and hepatocytic cells, according to the needs and pathologies of the living organisms treated. The method is less invasive than the other methods used so far for collecting stem cells, painless (as opposed to apheresis) and economically convenient.

Finally, the possibility of easily obtaining these cells and, then, of being able to preserve them for a long time, for example refrigerated in liquid nitrogen, makes the cells obtained with the known method suitable for autologous transplants and for the treatment of many pathologies (lesions of different kinds, metabolic diseases, acute and chronic neurological and inflammatory pathologies).

Furthermore, document WO-A-2009/115522 is known in the name of the present Applicant, which is hereby incorporated entirely as a reference, and concerns a kit for collecting blood, preferably peripheral blood, for the production of pluripotent stem cells, including a container, suitable to contain the blood collected, which contains an anticoagulant and the substance MCSF (Macrophage Colony Stimulating Factor). This kit can be used within the scope of the method described in WO-A-2008/034370.

However, the present Applicant has found that the various processing and manipulation steps to which the stem cells are subjected during the execution of the method described in WO-A-2008/034370, such as the elimination of red blood cells, the purification of the stem cells with respect to other blood components, the obtaining of a larger quantity of pluripotent stem cells with respect to hematopoietic and mesenchymal stem cells, the culturing thereof, the differentiation into other cell types, can stress the adult stem cells thus obtained, leaving them alive but having less effectiveness and reduced potential energy and information capacity.

In the veterinary field there are different techniques and apparatuses to produce stem cells, in particular to concentrate stem cells from fat, from bone marrow and to obtain growth factors.

However, a first obstacle for stem cells is the difficulty of harvesting; in fact, as stated, to obtain them from bone marrow it is necessary to drill a bone or penetrate the sternum, and to obtain them from fat a real surgical operation with stitches can be provided, or, even if this intervention were not required, it would always constitute an invasive technique.

There is then the logistical complexity: sending the sample and receiving the stem cells, keeping them all alive and stable.

Other obstacles are the manual skills, the costs and the preparation times for all the adult stem cells used so far.

These difficulties, combined with poor clinical results, have almost completely made stem cells disappear from veterinary clinical practice, and only a few veterinarians continue to use them for mostly experimental purposes.

The cell preparation described in WO-A-2008/034370 and reported in various scientific publications manages to qualify and quantify the stem cells obtained, confirming their pluripotency characteristics. It is also much easier than the techniques used so far, both because the sampling is simple - a few milliliters of blood - and also because the cells do not need to be cultured.

However, this system can also be improved to arrive at the use of stem cells in human clinical practice, to overcome obstacles linked to the shipment of the sample to the laboratory, the subsequent de-programming with MCSF and the re sending to the facilities where the therapeutic treatment takes place. Another limitation can be the complete purification of the stem cells obtained from blood with the method described in WO-A-2008/034370 which allows greater safety for a possible allogeneic inoculation, the safety of which however still needs to be proven. In fact, purifications and processes carried out to eliminate red blood cells and the passage of cells in a sorter (for qualification) create considerable stress on the stem cells obtained, causing them to lose part of their healing capacity. Therefore, there is strong need to reduce to a minimum the manipulation of the blood to obtain effective stem cells and thus achieve better results.

Another limitation of all therapies with stem cells obtained with known methods, including that which is described in WO-A-2008/034370, is that they require the presence of specialized laboratories.

Furthermore, document WO-A-2008/036374 is known, which describes methods and compositions for transplanting stem cells in patients who have not previously been immunosuppressed.

The following scientific articles are also known:

- Spaas J.H., Gambacurta A., Polettini M. Broeckx S. et al.,“Purification and expansion of stem cells from equine peripheral blood, with clinical applications”, vol. 80, no. 2, pages 129-135 retrieved from the Internet: URL: http: //hdl.handle.net/1854/LU- 1215157;

- G. E: Garber et ah,“The use of ozone-treated blood in the therapy of HIV infection and immune disease: a pilot study of safety and efficacy” AIDS, 1 January 1991, pages 981-984, retrieved from the Internet: URL: http: //graphics.tx.ovid.com/ovftpdfs/FPDDNCFBHADJCP00/fs047/ovft/ live/gv039/0 0002030/00002030- 199108000-00009.pdf;

- Larini et ah,“Effects of ozone on isolated peripheral blood mononuclear cells”, in vitro Toxicology, Elsevier Science, GB, vol. 19, no. 1, 1 February 2005, pages 55-61.

The international application WO-A-2015/170291 in the name of the present Applicant is also known, in which a method for expanding adult stem cells from blood is described, which comprises growth and de-programming of the adult blood stem cells of a drawn blood sample, by in vitro treatment of the blood sample with MCSF and ozonization of the blood sample.

There is therefore the need to perfect a method for expanding adult stem cells from whole blood which can overcome at least one of the disadvantages of the state of the art.

Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

Unless otherwise defined, all the technical and scientific terms used here and below have the same meaning commonly understood by a person of ordinary experience in the field of technology to which the present invention belongs.

Although methods and materials similar or equivalent to those described here can be used in the practice or in the verification tests of the present invention, the methods and materials are described below by way of example. In the event of a conflict, the present application prevails, including the definitions. The materials, methods and examples are of a purely illustrative nature and should not be understood in a limiting manner.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claims, while the dependent claims describe other characteristics of the invention or variants to the main inventive idea.

In accordance with the above purposes, a method for expanding adult stem cells from blood, which overcomes the limitations of the state of the art and eliminates the defects present there, comprises:

- making available a sample of whole blood;

- obtaining Platelet Rich Plasma (PRP) from a first portion of the sample of whole blood, and causing a lysis of platelets contained in the PRP, obtaining a PRP -based blood derivative product containing growth factors that include autologous MCSF (Macrophage Colony Stimulating Factor);

- an in vitro treatment in which the PRP -based blood derivative product containing growth factors which include MCSF is put into contact with the remaining part of the sample of whole blood, triggering growth and de- programming of adult blood stem cells present in the sample of whole blood.

Advantageously, the present invention allows, therefore, to obtain pluripotent adult stem cells from the blood sample, without manipulating the blood sample, without adding, or only in a limited manner, components that have an origin that is external or extraneous to the organism and without risk of possible side effects, because for the growth and deprogramming, that is, the expansion, of the adult blood stem cells present in the blood sample, the PRP-based blood derivative product is used, which contains autologous MCSF, advantageously derived from the same blood sample.

Applicant has found that the quantity of autologous MCSF present in the growth factors which are released by the lysis of the platelets contained in the PRP obtained according to the method described here, allows growth and de programming of the adult stem cells present in the sample of whole blood subjected to the treatment in vitro. In this way, it is possible to largely, if not completely, replace MCSF of a different derivation from the autologous one that is used in the state of the art. In this way, the subsequent use of the blood sample thus treated, in which the expanded stem cells are present, is comparable in practice with an autologous blood transfusion, thereby overcoming risks and contraindications due to possible side effects which can be attributed to a growth factor for example of bacterial origin, and therefore extraneous to the organism, as well as overcoming many bureaucratic and regulatory limitations that could hinder the application of treatment to patients.

Advantageously, therefore, the sample of whole blood is a sample of autologous blood of a patient.

According to possible embodiments, the lysis of the platelets releases the growth factors, among which MCSF is advantageously included, contained in the platelets of the PRP.

According to possible embodiments, the lysis of the platelets is caused by centrifuging the first portion of the sample of whole blood.

According to possible embodiments, the method uses the plasma obtained by the centrifugation, in which, as a result of the lysis of the platelets, the growth factors contained in the platelets, among which MCSF is advantageously included, have been released. According to further embodiments, it is possible to add the blood sample, for the purposes of growth and de-programming of the adult blood stem cells present in the blood sample, not only to the autologous PRP-based blood derivative product which includes autologous MCSF, but also to MCSF of a different derivation, typically obtained from bacterial cells. This can be advantageous, as the quantity of MCSF natively contained in the autologous PRP-based blood derivative product can vary from individual to individual, and therefore could, in some cases, not be sufficient to allow the growth and de-deprogramming of the adult stem cells present. However, thanks to the present invention, it is possible to add a smaller quantity of the MCSF of extraneous origin to the organism, compared to the known technique, thanks to the presence of the autologous MCSF present in the autologous growth factors contained in the PRP-based blood derivative autologous product derived from the same blood sample. In other words, the use of autologous MCSF contained in the PRP-based blood derivative product obtained as described above according to the present method, decreases the quantity of MCSF of extraneous origin, in particular bacterial, and therefore extraneous to the organism, necessary for growth and de-programming of adult stem cells present in the blood sample being treated.

According to further embodiments, the method provides that an anticoagulant is present in the sample of whole blood that is made available.

According to further embodiments, the method provides the ozonization of the blood sample before the division into portions, or of the blood sample while it is subjected, or after it has been subjected, to the in vitro treatment, or a combination of these possibilities.

Advantageously, the ozonization has a sterilizing effect of possible contaminations exogenous and/or endogenous, of the blood sample being treated. In particular, if a pathogen is present in a blood sample that is subjected to treatment with the PRP-based blood derivative product that includes autologous MCSF, the introduction/inoculation, in a biological tissue to be treated, of the blood sample treated according to the method described here could create problems due to the presence of the pathogen. Instead, thanks to the effect of complete sterilization of the ozone, there is a sterilization of the blood sample and the introduction/inoculation becomes much safer. Additionally, the ozonization has an advantageous catalytic effect on the growth and de programming, that is, the expansion, of the adult stem cells present in the blood sample.

According to possible embodiments, the method provides that the ozonization of the blood sample is carried out before the sample of whole blood is treated in vitro with the PRP-based blood derivative product which includes autologous MCSF. In this way, the growth and de-programming of adult blood stem cells is carried out in the already ozonized blood sample.

According to other possible embodiments, the method provides that the ozonization of the blood sample is carried out during the in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF. In particular, treatment with the PRP-based blood derivative product which includes autologous MCSF can be carried out on the blood sample during ozonization.

According to further possible embodiments, the method provides that the ozonization of the blood sample is carried out after the in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF. In particular, the treatment with the PRP-based blood derivative product which includes autologous MCSF can be carried out on the blood sample before it is ozonized.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides that the ozonization provides the blood sample with a mixture of 0 ₂ -0 ₃ .

According to possible embodiments, which can be combined with all the embodiments described here, the method provides a blood/0 ₂ -0 ₃ mixture volumetric ratio equal to 1 : 1.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides a quantity of 0 ₂ -0 ₃ mixture in the blood sample greater than, or equal to, about 1 mcg/1.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides that the quantity of 0 ₂ -0 ₃ mixture in the blood sample can be selected in a range from about 1 mcg/ml to about 42 mcg/ml.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides to add an anticoagulant to the blood sample.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides to use a kit for collecting blood which includes at least a container able to contain at least the blood drawn, containing at least the PRP-based blood derivative autologous which includes autologous MCSF.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides that the quantity of blood sample made available and treated is comprised between 10 ml and 1000 ml, in particular from 10 ml to 500 ml, more particularly from 15 ml to 250 ml, even more particularly from 20 ml to 150 ml. A possible example of the sample is 100 ml.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides a growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product, containing autologous MCSF, comprised between 4 hours and 120 hours, in particular between 24 hours and 96 hours, more particularly between 24 hours and 72 hours, even more particularly between 36 hours and 72 hours.

Embodiments described here also concern a blood sample containing adult stem cells which is obtainable with a method according to the present description.

According to possible embodiments, the blood sample is provided for use in therapeutic and/or preventive treatment of pathologies, for example by autologous introduction/inoculation, locally or intravenously.

According to possible embodiments, the blood sample is provided for use in therapeutic treatment, including therapy for lesions, external lesions, internal lesions, tendon lesions, ligament lesions, cartilage lesions, bone fractures, the therapy and/or the prevention of chronic and/or acute inflammatory pathologies, neurological and neurodegenerative pathologies, cardiac pathologies, tumorous pathologies, autoimmune pathologies, ophthalmic pathologies and genetic pathologies.

According to possible embodiments, a blood sample is provided for use in a treatment which provides intravenous or intra-arterial or local administration (for example subcutaneous, intramuscular or intra-tissue) of the blood sample treated with the PRP-based blood derivative product which includes autologous and ozonized MCSF.

According to other possible embodiments, a blood sample is provided for use in a treatment which provides intravenous or intra-arterial or local administration (for example subcutaneous, intramuscular or intra-tissue) of the blood sample treated with the PRP-based blood derivative product which includes autologous MCSF and the systemic ozonization of the patient.

Embodiments described here also concern a kit including at least a container containing a blood sample containing adult stem cells obtainable with a method according to the present description.

These and other aspects, characteristics and advantages of the present disclosure will be better understood with reference to the following description and attached claims.

The various aspects and characteristics described in the present description can be applied individually where possible. These individual aspects, for example aspects and characteristics described in the attached dependent claims, can be the object of divisional applications.

It is understood that any aspect or characteristic that is discovered, during the patenting process, to be already known, shall not be claimed and shall be the object of a disclaimer.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

We will now refer in detail to the various embodiments of the present invention. Each example is supplied by way of illustration of the invention and shall not be understood as a limitation thereof. For example, the characteristics shown or described insomuch as they are part of one embodiment can be adopted on, or in association with, other embodiments to produce another embodiment. It is understood that the present invention shall include all such modifications and variants.

Before describing these embodiments, we must also clarify that the present description is not limited in its application to details of the construction and disposition of the components as described in the following description. The present description can provide other embodiments and can be obtained or executed in various other ways. We must also clarify that the phraseology and terminology used here is for the purposes of description only, and cannot be considered as limitative.

Terms such as“about”,“generally”,“substantially” and suchlike shall be understood with their function of modifying a term or value that is not absolute, but is not reported in the state of the art. Such terms shall be defined by the specific circumstances and by the terms that they are intended to modify according to the common acceptance of such terms in the specific field. They shall take into account at least the degree of experimental error expected, the technical error and the instrumental error for a given technique adopted to measure a value. Unless otherwise indicated, in the present description, singular forms such as“a”,“an” and“one” shall be understood to include plural forms, unless the context suggests otherwise.

All the intervals reported here shall be understood to include the extremes, including those that report an interval“between” two values, unless otherwise indicated.

The present description also includes the intervals that derive from uniting or overlapping two or more intervals described, unless otherwise indicated.

The present description also includes the intervals that can derive from the combination of two or more values taken at different points, unless otherwise indicated.

Unless otherwise defined, all the technical and scientific terms used here and hereafter have the same meaning as commonly understood by a person with ordinary experience in the field of the art to which the present invention belongs. Even if methods and materials similar or equivalent to those described here can be used in practice and in the trials of the present invention, the methods and materials are described hereafter as an example. In the event of conflict, the present application shall prevail, including its definitions. The materials, methods and examples have a purely illustrative purpose and shall not be understood restrictively.

Embodiments described here concern a method for expanding adult stem cells from blood which provides:

- growth and de-programming of adult blood stem cells of a blood sample, by means of in vitro treatment of the blood sample with a PRP-based blood derivative product which includes growth factors, including autologous MCSF, obtained from the lysis of platelets contained in the PRP obtained from a portion of the blood sample.

In particular, the blood can be whole blood, more particularly peripheral whole blood, that is, blood present in the blood vessels (arteries, veins, capillaries).

Advantageously, when the PRP-derived blood derivative product is put into contact with the remaining part of the blood sample, in order to trigger the growth and de-programming of the adult stem cells contained, the release and spread of growth factors also continues, including autologous MCSF, present in the platelets subjected to lysis, which therefore further enrich the blood sample thus treated.

Advantageously, a centrifugation operation of the first portion of the sample of whole blood can be used to obtain the lysis of the platelets, which causes the separation of the plasma and the lysis of the platelets. The centrifugation operation is also advantageously the operation with which the PRP is obtained. The lysed platelets allow to release the growth factors contained therein, advantageously including MCSF, into the blood plasma. The plasma rich in platelets and growth factors, including autologous MCSF, constitutes, in these embodiments, the PRP-based blood derivative autologous product.

In essence, the present invention provides to use mainly, instead of MCSF, or other growth factors, of extraneous origin, in particular derived from bacterial cells, MCSF contained in the growth factors released thanks to the lysis of the platelets of the PRP, that is, natively contained in the blood sample made available.

In particular, the present invention provides to use the PRP, whose platelets, subjected to lysis, release various growth factors, including MCSF and others, into the plasma obtained for example by centrifugation, as a PRP-based blood derivative autologous product. In this case, the growth factors, including MCSF, present and released in the PRP whose platelets are subjected to lysis, are advantageously obtained from autologous blood, that is, from the same patient, and are put back into contact with the sample of whole blood for a certain period of time to allow the de-programming and growth of the adult stem cells contained therein.

Advantageously, the use mainly of MCSF present in autologous growth factors released in the PRP thanks to the lysis of platelets, and not of bacterial origin, excludes, or minimizes, any minimum operation of manipulating the blood.

The present invention, which provides to use autologous growth factors contained in the PRP-based blood derivative product, including autologous MCSF, instead of using exclusively MCSF, or other growth factors, of extraneous origin to the organism, in particular obtained from bacterial cells, both avoids or considerably reduces interventions on and manipulations of the blood sample, such as the addition of an extraneous component obtained from bacterial cells, and also avoids a potential risk of possible side effects due to the use of an extraneous substance.

According to some embodiments, the lysis of the platelets present in the PRP obtained from the first portion of the blood sample can be obtained with any one of the known procedures.

According to some embodiments of the present invention, obtaining the PRP- based blood derivative product, where as a consequence of the lysis the growth factors, including MCSF, present in the platelets, have been released, can occur preferably by centrifugation of the portion of the sample of whole blood.

According to preferred embodiments, the PRP-based blood derivative product can be obtained by centrifugation of the portion of blood. According to possible implementations, the method described herein may include for instance a single centrifugation. According to embodiments, the centrifugation can be carried out for a time from 3 to 9 minutes, in particular from 5 to 7 minutes, with a speed from 800 to 1200 rpm (revolutions per minute), in particular from 900 to 1100 rpm. By way of non-restrictive example, the method may include a single centrifugation of 6 minutes at a speed of 1000 rpm.

According to some embodiments, the method provides that an anticoagulant is present in the sample of whole blood made available.

According to further embodiments, the anticoagulant made available is preferably sodium citrate.

According to some embodiments, the portion used to obtain the PRP can be about half of the total blood sample made available.

According to possible embodiments, the portion of the blood sample can in any case be chosen according to the volume of the blood sample made available.

According to a variant, it is also possible to possibly introduce MCSF of an extraneous origin, in particular of bacterial origin, but advantageously in a smaller quantity than that provided in the state of the art, since autologous MCSF is already present, present natively in the autologous growth factors released in the PRP as a consequence of the lysis of the platelets. Advantageously, in this variant, it is in any case possible to reduce the quantity of MCSF extraneous to the organism, reducing possible side effects.

According to some embodiments, the method can allow to supplement the PRP-based blood derivative product with non-autologous growth factors, for example MCSF of bacterial origin. In particular, a preferred supplementation growth factor can be MCSF of bacterial origin at a concentration, for example, between 0.5 nM and 4 nM, in particular from 1 nM to 4nM.

According to some embodiments, the Platelet Rich Plasma (PRP)-based blood derivative autologous product contains the following autologous growth factors: b-NGF (Nerve Growth Factor beta), EGF (Epidermal Growth Factor), FGF-2 (basic Fibroblast Growth Factor), FGF-4 (Fibroblast Growth Factor 4), FGF-6 (Fibroblast Growth Factor 6), FGF-7 (Fibroblast Growth Factor 7), GCSF (Granulocyte Colony- Stimulating Factor), GDNF (Glial cell-derived neurotrophic factor), GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), HB-EGF (Heparin-binding EGF-like Growth Factor), HGF (Hepatocyte Growth Factor), IGF-I (Insulin-like Growth Factor), IGF-II (Insulin-like Growth Factor 2), MCSF (Macrophage Colony Stimulating Factor). Possibly, the Platelet Rich Plasma (PRP)-based blood derivative autologous product also contains the following autologous growth factors: NT-3 (Neurotrophin-3), NT-4 (Neurotrophin-4), PDGF-AA ( Platelet Derived Growth Factor- AA), PDGF-AB (Platelet Derived Growth Factor- AB), PDGF-BB (Platelet Derived Growth Factor-BB), SCF (Stem Cell Factor), TGF-a (Transforming Growth Factor-a), TGF-b (Transforming Growth Factor-b), ΊΌR-b2 (Transforming Growth Factor- b2), T01 -b3 (Transforming Growth Factor^3), VEGF (Endothelial Growth Factor); VEGF-D (Endothelial Growth Factor-D). According to some embodiments of the present invention, the PRP-derived blood derivative product which includes autologous MCSF, possibly supplemented with growth factors extraneous to the organism, for example bacterial, is combined and amalgamated to treat, in vitro, the corresponding portion of remaining whole blood. In this case, advantageously, the growth factors contained in the lysed platelets continue to be released and to spread in the blood sample.

For example, the division of the sample of whole blood can provide that the first portion is about half of the blood sample made available. The remaining part, for example about half, can in turn be divided into portions, to each of which the PRP-based blood derivative product obtained can be added, in order to trigger growth and de-programming of the adult blood stem cells present.

According to some embodiments, the treatment can provide to amalgamate the PRP-based blood derivative product with the respective remaining portion of whole blood, which can preferably occur with a circular movement in a glass container.

According to possible embodiments, which can be combined with all the embodiments described here, the method provides an in vitro treatment time comprised between 4 hours and 120 hours, in particular between 24 hours and 96 hours, more particularly between 24 hours and 72 hours, even more particularly between 36 hours and 72 hours.

According to further embodiments, the method also provides the ozonization of the blood sample.

The present invention therefore allows to obtain pluripotent adult stem cells from the blood sample made available.

Applicant has found that the stem cells obtained by applying the method described here include the stem cell markers CD90, CD90/34, CD34 and CD117, they also express some intracellular transcription factors strongly linked to pluripotency characteristics (Sox2,Oct3/4 and Nanog) and do not lose the self recognition factors following division or expansion. These stem cells do not give rise to side effects such as phenomena of rejection, infection, development of teratomas once administered in the patient, they are able to differentiate in vivo and therefore behave as pluripotent stem cells. By the expression“growth and de-programming of adult blood stem cells of a blood sample, by means of in vitro treatment of the blood sample with the PRP- based blood derivative product which includes autologous MCSF” we mean that the blood sample, which contains in itself a certain quantity of adult stem cells, is treated in vitro with the PRP-based blood derivative product which includes autologous MCSF to obtain the growth of the adult stem cells originally present in the blood sample, through de-programming of white blood cell lines, that is, white blood cells.

Furthermore, it is emphasized that the expression“ozonization” here means the treatment of the blood sample with ozone, that is, the addition, delivery, administration or mixing of ozone, or of a mixture of oxygen and ozone, to/in the blood sample.

Ozone (symbol 0 ₃ ) is an allotropic form of oxygen, with a triatomic molecule and molecular weight 48. Ozone appears in normal conditions as a blue, acrid smelling gas, with strong oxidizing power. Ozone can act as a disinfectant, deodorant, bactericide, sterilizer, or as an oxidizer in numerous organic syntheses.

According to possible embodiments, the ozonization of the blood sample can be carried out before the treatment with the PRP-based blood derivative product.

According to possible embodiments, the ozonization of the blood sample can be carried out simultaneously to the treatment with the PRP-based blood derivative product.

According to further possible embodiments, the ozonization of the blood sample can be carried out after treatment with the PRP-based blood derivative product.

According to possible embodiments, it is provided to add an anticoagulant to the blood sample. Heparin, EDTA or sodium citrate are examples of possible anticoagulants.

In some embodiments, the method according to the present description can provide to use a kit for collecting blood, for the production of pluripotent stem cells according to the method described above, including at least a container, such as a test tube, suitable to contain the blood sample, containing the PRP- based blood derivative product and possibly, if provided, the anticoagulant. With a kit of this type it is possible to collect the whole blood, preferably peripheral blood, to rapidly start the growth and production of the stem cells by means of the method described above according to the present description, and therefore make their production much faster.

Embodiments described here can provide that the quantity of blood sample that is collected and subjected to the method described here, that is, growth and de-programming with the PRP-based blood derivative product, and possible ozonization of the blood sample, is of a few milliliters, for example comprised between 10 ml and 1000 ml, in particular between 10 ml and 500 ml, more particularly between 15 ml and 250 ml, even more particularly between 20 ml and 150 ml. A possible example is 100 ml.

The blood sample, once subjected to the in vitro treatment described here, can for example be injected into the circulation of the patient (intravenous or intraarterial administration), which can then possibly be subjected, subsequently, to systemic ozonization treatment.

Some embodiments, which can be combined with all the embodiments described here, can provide a method as described above which can use any type of collection container in which to introduce the whole blood and the ozone with any type of possible anticoagulant, with any concentration volume of PRP-based blood derivative product which includes autologous MCSF.

Some embodiments, which can be combined with all the embodiments described here, can provide that ozonization gives the blood sample a mixture of O2-O3.

In possible implementations, Applicant has found that the blood/0 ₂ -0 ₃ mixture volumetric ratio can preferably be 1 : 1.

In possible implementations, the quantity of 0 ₂ -0 ₃ mixture in the blood sample may be greater than, or equal to, about 1 mcg/1, in particular selected in a range from about 1 mcg/ml to about 42 mcg/ml, more particularly from about 5 mcg/ml to about 30 mcg/ml, even more particularly from about 10 mcg/ml to about 20 mcg/ml.

Embodiments described here provide that, having left the blood in these conditions possibly ozonized, preferably at room temperature, after a certain time, preferably between 4 hours and 96 hours, in particular between 4 hours and 72 hours, more particularly between 4 hours and 48 hours, the whole blood thus obtained, with the component of stem cells obtained from de-programming can be completely inoculated, both systemically (intravenously, intra-arterially), and also locally into or near a diseased tissue.

In possible implementations, the growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF can be comprised between 12 hours and 120 hours, in particular between 12 hours and 96 hours, more particularly between 12 hours and 72 hours, even more particularly between 12 hours and 36 hours.

In possible implementations, the growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF can be comprised between 24 hours and 120 hours, in particular between 24 hours and 96 hours, more particularly between 24 hours and 72 hours, even more particularly between 24 hours and 36 hours.

In possible implementations, the growth and de-programming time by means of in vitro treatment with the PRP-based blood derivative product which includes autologous MCSF can be comprised between 48 hours and 120 hours, in particular between 48 hours and 96 hours, more particularly between 48 hours and 72 hours, even more particularly between 48 hours and 60 hours.

Applicant has hypothesized that the ozonization of the blood sample subjected to growth and de-programming by means of in vitro treatment with a PRP-based blood derivative product which includes autologous MCSF stimulates the process of expansion and de-programming of adult stem cells, so that even after a few hours there is a significant number of useful adult stem cells.

Embodiments of the method described here can also provide, in addition to the container where the PRP-based blood derivative product which includes autologous MCSF is present and where the expansion of adult stem cells occurs, the use of a second container to contain the stem cells obtained as described above, for example in the case of intravenous or intra-arterial use, and possibly a third container, of a different size, for local use. The stem cells produced and preserved in the containers can be used immediately, or they can be preserved, for example in liquid nitrogen, to be used subsequently, when there is a need.

The ozonization according to variants of the embodiments described here can be carried out on blood samples containing the stem cells, before, during or after these have been expanded and de-programmed with the PRP-based blood derivative product which includes autologous MCSF, contained in any one of the containers mentioned.

According to possible embodiments, the blood just taken from the patient can be immediately put into the test tube with the anticoagulant. The anticoagulant can block the beginning of the coagulation. A first portion is removed from this sample, which is processed to obtain the PRP-based blood derivative product which includes autologous MCSF. The latter is put into the remaining blood sample to allow the expansion process to rapidly begin, and to guarantee that the time to start treating the patient is minimized. Furthermore, the sample can possibly be subjected to ozonization according to the present description.

According to further possible embodiments an anticoagulant can be added to the blood taken from the patient to block the coagulation of the blood; the blood is subjected to a preservation procedure which does not alter its ability to produce stem cells. When the need arises, the blood is taken from where it is stored and is subjected to the stem cell expansion procedure described above, that is, taking a first portion thereof which is processed to obtain the PRP-based blood derivative product which includes autologous MCSF, which is then added to the remaining part of the blood sample, rapidly obtaining the necessary quantity of stem cells. Furthermore, also in this case the sample is subjected to ozonization according to the present description.

The method according to the present description allows to overcome the disadvantages of the state of the art and entails many advantages.

For example, the present invention allows to prepare whole blood and greatly simplify the therapy, avoiding any type of cellular manipulation made in the laboratory, as well as avoiding, according to advantageous variants, the addition of components that are extraneous to the organism, such as growth factors derived from bacterial cells. In particular, the present invention eliminates the need or the possibility to carry out treatments for example to eliminate red blood cells, to purify the stem cells with respect to all the other blood components, to obtain a greater quantity of pluripotent stem cells with respect to the other two stem cell components, hematopoietic and mesenchymal, to cultivate them, to differentiate them into other cell types. These additional treatments can normally stress the stem cells obtained, leaving them alive, but with less information and energy potential. Instead, the present invention advantageously avoids all further processing and manipulations of the blood sample, so that the adult stem cells present in the whole blood maintain their characteristics better because they are not stressed, and because they can benefit from the presence in the blood of other elements assisting in the regenerative process.

Applicant has found that incurable pathologies such as myocardial degeneration already treated with good results with stem cells obtained from blood by de-programming, as described in WO-A-2008/034370, have had a decidedly more positive evolution with stem cells de-programmed in whole blood, avoiding the additional operations and manipulations mentioned above, thus adopting a method that can even consist exclusively of growth and de programming with the PRP-based blood derivative product which includes autologous MCSF, without additional processing steps, including purification, subsequent expansions, or which can include growth and de-programming with the PRP-based blood derivative product which includes autologous MCSF and ozonization.

The preparation of stem cells according to the method described here avoids complex laboratory preparations, allowing any hospital, clinic or doctor to prepare stem cells by possessing a simple test tube with the preferably minimum quantity of the PRP-based blood derivative product which includes autologous MCSF obtained as described above. In other words, with a single test tube in which to place an adequate quantity of blood sample with the PRP-based blood derivative product which includes autologous MCSF, it is possible to treat and improve even serious pathologies, such as for example the after-effects of a heart attack or Parkinson's disease. Therefore, these results support the fact that, according to possible embodiments, a method for expanding adult stem cells from blood can even consist exclusively of growth and de-programming of the adult blood stem cells of a blood sample, by means of in vitro treatment of the blood sample with the PRP-based blood derivative product which includes autologous MCSF.

Furthermore, in some embodiments, the addition or supply of ozone to the blood sample, deriving from the ozonization treatment of the blood sample, can have a catalyzing effect on the de -programming process of the adult stem cells and on the quality of the stem cells obtained and on the their information and energy content, such that it positively influences the cellular regeneration of damaged tissues, as well as giving the product additional safety with regard to sterility. In fact, as we said, ozone can act as a disinfectant or bactericide.

It is clear that modifications and/or additions of parts may be made to the method for expanding adult stem cells from whole blood as described heretofore, without departing from the field and scope of the present invention.

It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art shall certainly be able to achieve many other equivalent forms of method for expanding adult stem cells from whole blood, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby.