ES2391111A1 - Cell regeneration compound - Google Patents

Abstract

The present invention relates to a cell regeneration compound permitting intravenous, intramuscular and local administration of stem cells for therapeutic purposes in chronic degenerative diseases, wherein are included chronic renal insufficiency and diabetes mellitus. The object of this invention is to provide a cell regeneration compound which does not exist in the market permitting the application of stem cells in the form of cell therapy, avoiding HLA gene typification studies, surgical interventions and interventional radiology employed in implantation or transplantation of stem cells. The formula of the cell regeneration compound permits the application of stem cells, without genetic modification whatsoever, cryopreserved or lyophilised in flasks or boxes of cultured cell lines, subsequent to gradual thawing from -20 DEG C to 8 DEG C, being rehydrated in the base solution of the cell regeneration compound (CRC).

Description

 Compound of cell regeneration.

BACKGROUND OF THE INVENTION

Stem cells known in English as stem cells, are also known as "stem cells," "stem cells," "precursor cells," "progenitor cells," and "stem cells." The stem cell is the precursor of approximately 250 differentiated cell lines and their stages of partial differentiation that are losing the pluripotential capacity of the original stem cell and are compromising with a certain cell lineage to form specific specialized cells, such as neurons, pancreatic beta cells, cardiomyocytes, among many others.

Stem cells are characterized by the fact that they can divide simultaneously to maintain cellular self-renewal; that is, a sustained production of stem cells similar to her. As well as the transdifferentiation and tropism that allows generating daughter cells committed to a specific cell line, in addition to having the ability to migrate to the site of injury or damage in which cell differentiation occurs, after implantation occurs, observing regeneration cell in healthy and diseased tissues (Sánchez-González DJ, Trejo-Bahena NI. Cellular and Molecular Biology. Editorial Alfil; México Distrito Federal, 2006). These characteristics make the injection of stem cells as a potential therapy, useful for carrying out cell regeneration in patients. (González-López GM, Sánchez-González DJ and Sosa-Luna CA. Cell Therapy with Stem Cells and Regenerative Medicine. Mexico 2008; Editorial Alfil).

Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92: 7844, 1995) were the first to isolate and propagate stem cells from primates. Subsequently, they derived the first human stem cell cell line from the blastocysts. Gearhart et al. Derived germ stem cells from fetal tissues of gonadal origin (Shamblott et.al. Proc. Natl. Acad. Sci. USA 95: 13726, 1998; and U.S. Pat. No. 6,090,622).

Both stem cells derived from blastocysts and those obtained from gonadal tissues have characteristics that define them as pluripotential stem cells, that is; they can be cultivated for long periods of time without them entering into differentiation, maintaining a morphology with normal karyotype and are able to differentiate a significant number of specialized cells.

The most important problem for using pluripotent stem cells in cell therapy protocols is that these stem cells are traditionally grown on a bed of cells (feeder cells) that prevent differentiation (US Pat. No. 5, 843,780; US Pat. No. 6, 090,622). According to the experiments of Thomson et al. (Science 282: 114, 1998), when the stem cells are cultured without their cell bed (feeder cells), the stem cells die and begin to differentiate in a disorderly manner. In the international patent published by Geron Corp. (WO 99/20741), entitled: "Methods and materials for the growth of stem cells derived from primary primate cells"; It refers to a cell culture medium for growing primate stem cells and remaining in a state of undifferentiation under conditions of low osmotic pressure and low level of endotoxins. This cell culture medium can combine serum to support the stem cells and the cell bed, the cell culture medium also includes nonessential amino acids, antioxidants, nucleotide growth factors and pyruvate salts. Another international patent published by Geron Corp. (WO 01/51616) entitled: "Growth and differentiation techniques of pluripotent human stem cells". And the article by Xu et al. (Nature Biotechnology 19: 971, 2001) entitled: “Growth of undifferentiated stem cells, without a bed of cells (feeder cells”). The article by Lebkowski et al. (Cancer J. 7 Suppl. 2: S83, 2001) entitled: "Human embryonic stem cells: culture, differentiation, and genetic modification for regenerative medicine applications". These publications report examples of state-of-the-art art and are always about cell culture media and methods to propagate stem cells and keep them in a state of undifferentiation. However, there is no “Cellular Regeneration Compound” that allows human cells to be injected in the form of cell therapy. Nor is there any similar pharmaceutical product that can act as a “Cellular Regeneration Compound” that has been successful in reversing the progression of chronic degenerative diseases, such as Chronic Renal Insufficiency induced by Diabetes Mellitus or other pathologies (Chirino YI, Sánchez-González DJ et. Al. Protective effects of apocynin against cisplatin-induced oxidative stress and nephrotoxicity. Toxicology 2008; 245: 18–23 and Razo-Rodríguez AC, Chirino YI, Sánchez-González DJ et. Al. Garlic powder ameliorates cisplatin-induced nephrotoxicity and oxidative stress. J Med Food 2008; 11 (3): 582-586.)

Although stem cells have been applied in some clinical research protocols in Phase I to III Medicine (Traynor A. et. Al. “Treatment of severe systemic lupus erythematosus with high-dose chemotherapy and haematopoietic stem-cell transplantation: a phase I ”published in August 2000 in The Lancet magazine Vol.

356. Andrew P. et. al., "The Adult Blood and Marrow Stem Cell Transplant Service" Hackensack. Bone Marrow Transplantation, April 2000, Sykes M. et. to the. “Treatment of severe autoimmune disease by stem-cell transplantation” Nature No. 2; Vol. 435 (7042), pp. 620-7. June 2005, Shamblott MJ et. to the. "Cell therapies for type 1 diabetes mellitus." Expert Opin Biol Ther 2004 Mar; Vol. 4 (3), pp. 269-77); In all these protocols the stem cells are cultured and sometimes expanded with the methods and culture media previously described in the

background of the present invention, and the stem cells are administered in bulk or as boluses by methods known as bone marrow transplantation, very similar to conventional blood transfusions, or injections of purified stem cells are applied, in which They include highly specialized surgical procedures, in which interventional radiology procedures can be integrated, which also require the expense of medical fees, hospitalization, anesthesia, clinical laboratory and cabinet studies. Orlic and collaborators injected stem cells in the peripheral area of acute myocardial infarction; , these cells differed in heart structures, improving the function of the infarcted heart. Cardiac repair produced by stem cells reduced mortality by 68% and infarct size by 40% (Orlic D. et. Al. Proc. Natl. Acad. Sci. USA 2001; 98 (18): 10344 –10349). Other work related to stem cells is contributing to establish that the higher the levels of circulating stem cells, the greater the body's ability to regenerate and be healthy longer (Werner et.al. N Engl J Med. 2005 ; 8; 353 (10): 999-1007). These types of inventions and investigations are changing the paradigm in the history of mankind in which it was thought that it was impossible to repair the damage caused once death or cell damage has been established in a vital organ such as infarctions. to the heart.

Other complementary investigations are those of Bozlar M et. The. (2005) Saudi Med J. 26 (8): 1250-4. Kong D, et. to the. (2004) Circulation. 110 (14): 2039-46. Eroglu E, et al. Agalar F, Altuntas I, Eroglu F. (2004) Tohoku J Exp Med. 204 (1): 11-6. Tomoda H, Aoki N.et.al. Clin Cardiol. 2003 Oct; 26 (10): 455-7. Krause DS et. Al. Cell 105: 369-77. Eglitis M. et. Al. Proc. Natl Acad. Sci. USA Vol. 94, pp. 4080–4085. Camargo FD, et. Al. Nature 2003 9 (12): 1520-27; and that of Ianus A, et. Al. J. Clin. Invest. (2003) 111: 843-850. Ministry of Health. Operational Procedures Manual of the Hematopoietic Transplant Unit of the National Institute of Pediatrics (2008).

DESCRIPTION OF THE INVENTION

As can be seen in the state of the art, until the moment of the present invention, the therapeutic application of stem cells in injections or transfusions does not include a "Cell Regeneration Compound", since cell therapy with stem cells has been performed directly and using fresh bone marrow cells, umbilical cord or cultured and purified pluripotential stem cell cell lines, which are implanted at the site of damage or through bone marrow transplants (similar to blood transfusion), therapeutic procedures which necessarily include the gene typification of the HLA haplotype of the donor and recipient of the transplant, procedures that raise the cost, which is also increased by highly specialized labor, such as subspecialist surgeons, such as neurosurgeons and cardiovascular surgeons, who are trained to carry stem cells in large s amounts to specific anatomical sites where they are required, through advanced surgical procedures (Sánchez-González DJ, Trejo-Bahena NI. Histology practices. Editorial Alfil; Mexico Federal District, 2007). With the purpose of suppressing these and other inconveniences, a "Cellular Regeneration Compound" (CRC) was considered in the development of the present invention, which is intended to be protected by means of the present application, since it is a compound composed of four bottles with base solutions (“A”, “B”, “C” and “D”) in which cell elements are suspended (stem cells in cryopreservation state at a temperature of minus one hundred ninety-six degrees Celsius (-196º C) and the powder derived from the same amount of dehydrated cells by lyophilization) for various therapeutic applications in Cell Therapy, Regenerative Medicine and Anti-Aging. The characteristic details of our invention entitled "Cellular Regeneration Compound" (CRC) are clearly shown in the following description and in the 3 accompanying figures. Figure 1/3 shows a scheme of the invention, Figure 2/3 refers to the results obtained after applying "Cellular Regeneration Compound" (CRC), in experimental animals Wistar rats with Diabetes Mellitus previously induced with streptozotocin, where the increase in renal function measured by creatinine clearance (ml / min) and morphological analysis of the histological sections of the glomeruli can be seen. Figure 3/3 refers to the results obtained clinically in our group of patients with chronic renal insufficiency where the statistically significant increase in renal function measured by creatinine clearance (ml / min) is clearly seen, with consistency between results in animals as in human patients, unpublished fact that has not been published until the present patent application, all of them are examples that demonstrate the effectiveness of the "Cellular Regeneration Compound" (CRC) described in the present invention patent application.

The description in Figure 1/3 refers to the way in which the CRC should be prepared; Likewise, the quantities, conditions and elements required to produce the CRC (“Cellular Regeneration Compound”) are indicated: In No. 1; refers to a cell culture box with a purified line of cryopreserved pluripotential stem cells -198 ° C, which are thawed gradually increasing the temperature at a rate of 2 degrees Celsius per minute (

LX�ºC / min) until reaching the temperature of minus twenty degrees Celsius (-20ºC).

In No. 2 of Figure 1/3; shows the second option of the source of stem cells which are in powder in a state of total dehydration obtained by a conventional lyophilization process.

In No. 3 of Figure 1/3 it is shown that both sources of stem cells must be hydrated with the solution "A" which must be found at a temperature of 8 ° C.

In No. 4 of Figure 1/3 is the basis of the “Cellular Regeneration Compound” (CRC) which is the “A” solution, it is prepared under sterile conditions and in a controlled environment at a cooling temperature less than sixteen degrees Celsius (16 ° C) and greater than four degrees Celsius (4 ° C). The base solution "A" of the "CRC" is prepared with a liter of double-distilled water in a glass or plastic bottle with a rubber stopper which is subjected to a traditional sterilization process such as the autoclave, in which previously dissolved sodium chloride (NaCl), sodium lactate (NaC3H5O3), calcium chloride (CaCl2) and potassium chloride (KCl) to reach the following ionic ratio: Na + = 130 mEq, Cl- = 109 mEq, Lactate = 28 mEq, Ca2 + = 3 mEq and K + = 4 mEq; This solution must reach an osmolarity of 273 mOsm / L, and must not exceed 300 mOsm / L which is verified using a conventional osmometer. After sterilizing this solution by any conventional sterilization method, a sterile solution with 55 mg of zinc chloride, 16.9 mg of cupric sulfate pentahydrate, 38.1 mg of magnesium sulfate, 1.3 mg of iodide is added sodium, 14 mg sodium fluoride, 163.9 mg sodium chloride, 10 mg thiamine, 250 mg pyridoxine, 1000 mg ascorbic acid, 1.0 mg activin, 0.2 mg stimulating factor pluripotential cells (C-kit), 0.6 mg of procaine derivative G1, 0.4 mg of procaine derivative G2, and 0.4 mg of procaine derivative G3.

In No. 5, the prehydrated stem cells in the base solution "A" are evaluated in the confocal microscope that uses a laser (laser) light source, which allows various transverse images of the stem cells to be made (No. 6) (Ordonez RM, Espinosa AM, Sánchez-González DJ et. Al. Enhanced oncogenicity of Asian-American human papillomavirus 16 is associated with impaired E2 repression of E6 / E7 oncogene transcription. J Gen Virol 2004; 85: 1433-1444), which are reflected and focused by the optical lenses (No. 7) emitted by the cells in suspension with the base solution "A" (No. 8) obtaining various microscopic images (No. 9) (Floriano-Sánchez E, Villanueva C, Medina-Campos ON, Rocha D, Sánchez-González DJ et al. Nordihydroguaiaretic acid is a potent in vitro scavenger of peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion, and hypochlorous acid and prevents in vivo ozone-induced tyrosine nitration in lungs Free Radic R is 2006; 40 (5): 523-533). Stem cells with healthy morphology are taken in a sterile syringe (No. 10).

In No. 11 of Figure 1/3, it refers to a thorough morphological study that rules out membrane alterations, infection, genetic alterations.

In No. 12 of Figure 1/3 refers to the quantification of the number of cells per milliliter of base solution by various methods of confocal microscopy or FACs in this approximately 1000,000,000 stem cells are suspended in 500 ml of the base solution "A" of the CRC described above.

In No. 13 of Figure 1/3 it refers to the second base solution "B" of the CRC; useful for inducing cell regeneration by chondrogenesis, hematopoiesis and osteogenesis. Which is prepared as follows: 500 ml of CRC base solution “A” with a concentration of 2000,000 cell elements (stem cells) quantified and verified previously by morphological analysis in a confocal microscope, at this volume (500 ml) also adds: L-cysteine 1.0 mg, 0.5 μg of interleukin 7 (IL-7), 0.5XIgX of Xinterleukin X3X (IL-3), 0.1 mg of Mesodermal Growth Factor A1 (FGM), 0.1 mg monocyte colony stimulating factor (M-CSF), and 0.1 mg granulocyte colony stimulating factor (GM-CSF); all added and mixed in a controlled manner under sterile conditions stored at eight degrees Celsius (8º C) for no more than one week, or at least four degrees Celsius (-4º C) for no more than five weeks or at least twenty degrees Celsius (-20 ºC) for no more than twenty four weeks.

In No. 14 of Figure 1/3 it refers to the third base solution "C" of the CRC; useful for inducing cell regeneration by angiogenesis and vasculogenesis. Which is prepared as follows, 500 ml of base solution "B" of the CRC previously prepared to which the following is added: to which is added 0.3 mg of beta growth transformation factor (TGF- B) and 0.2 mg of K2 endodermal growth factor (FGE); all added and mixed in a controlled manner under sterile conditions stored at eight degrees Celsius (8º C) for no more than one week, or at least four degrees Celsius (-4º C) for no more than five weeks

or at least twenty degrees Celsius (-20 ° C) for no more than twenty four weeks. In No. 15 of Figure 1/3 it refers to the fourth base solution "D" of the CRC; useful for inducing cell regeneration by neurogenesis, angiogenesis and vasculogenesis. Which is prepared as follows: 500 ml of CRC base solution “C” described above to which the following is added: 100 mg retinol, 0.5 mg fibroblast growth factor (FGF) and 0, 2 mg of neurological growth factor (FGFN).

Finally, the “Cellular Regeneration Compound” CRC for application in Cellular Therapy, Regenerative Medicine and Anti-Aging is integrated by the four base solutions (“A”, “B”, “C” and “D”), forming a liquid composition composed of a concentration of 2 x 109 hydrated stem cells, previously analyzed and quantified by confocal microscopy, which is prepared by adding various substances in the concentrations and conditions already described (oligometals, activin, fibroblast growth factors, etc.).

The formula and the procedure to prepare the “CRC” allow to apply pluripotential stem cells without any genetic modification, which are cryopreserved or lyophilized in bottles or boxes of cell lines in culture, after gradually defrosting them from -20ºC to 8ºC, they are prehydrated in the base solution of the cell regeneration compound (base solution "A") which is the base solution for preparing the cell regeneration compound (bottles "B", "C" and "D").

The "CRC" (in its base solutions "B", "C" and "D") serve as stem cell inducing compounds in cell therapy protocols, favoring their regenerative and anti-aging capacity. The application of the procedure to prepare the cell regeneration compound ("A", "B", "C" and "D" bottles) includes sterility conditions, morphological analysis and quantification of stem cells.

The application of this “CRC” in cell therapy No. 16 and No. 17, figure 1/3 (bottles “A”, “B”, “C” and “D”) allows cell therapy with stem cells to be very safe and does not require gene typing studies for HLA haplotypes because both the base and the cell regeneration compound (bottles “A”, “B”, “C” and “D”) serve as a vehicle to transport the stem cells in the protocols of cell therapy, regenerative medicine and anti-aging useful for various chronic degenerative diseases that require cell regeneration such as: Chronic Renal Failure; Diabetes Mellitus, osteoarthritis, Parkinson's Disease, Vitiligo, etc. For which it is required to apply a minimum of 5 intravenous application every 7 days of 6 ml of the cell regeneration compound using “2 ml of bottle“ B ”, 2 ml of bottle“ C ”and 2 ml of bottle“ D ”, in addition to apply a volume of 1.0 ml intramuscularly in the deltoid region of compound "B" and if necessary 3 ml more of the cell regeneration compound, using a volume of 1 ml of bottle "B", 1 ml of bottle "C" and 1 ml of the bottle "D" this volume is applied locally at the site to be regenerated.

Finally, the “Cellular Regeneration Compound” CRC is prepared as a compound in a transparent color liquid state with various reddish tones, for application in the form of injections, which is useful in Cellular Therapy, Regenerative Medicine and Anti-Aging, and which is integrated by the four base solutions ("A", "B", "C" and "D"), described extensively in the description of the invention. The "CRC" forms a liquid composition composed of a concentration of 2 x 109 pluripotential stem cells, previously analyzed and quantified by confocal microscopy, which is prepared by adding various substances in the concentrations and conditions already described (oligometals, activin, factors of Fibroblast growth (FGF), Beta Transformation Factor (TGF-�), Ascorbic X-acid, thiamine, riboflavin, Neurological Growth Factor, retinol, procaine derivative G1, procaine derivative G2, procaine derivative G3, L-Cysteine, Growth Factor Mesodermal A1 (FGM), Endodermal Growth Factor K2 (FGE), Pluripotential Cell Stimulating Factor (cKit), Interleukin 7 (IL7), Interleukin 3 (IL-3), Monocyte Colony Stimulating Factor (M-CSF) and Granulocyte Colony Stimulating Factor (GM-CSF). These compositions are extracted from the container bottle of the "CRC" always using a syringe with sterile needles, applying conventional procedures of asepsis and antisepsis (cleaning of the rubber stopper with swab with alcohol of 96 °), and immediately a volume of 5 to 6 ml “CRC” is applied intravenously, (see Figure 1/3 No. 16) and intramuscularly in the same patient deltoid region (see Figure 1/3 No. 17). The “CRC” must be stored at a refrigeration temperature at 4ºC to 8ºC, it has an expiration period of 10 days and should not be used after one week. In Figure 1/3 in No. 16 and No. 17, they refer to the protocol that must be followed to apply the injections with the “CRC”, once the four base solutions “A” have been prepared, ”B "," C "and" D "; described above.

The "CRC" can be applied by various routes of paraenteral administration, at a dose of 0.1 milliliters per kilogram of weight; generally, five applications are used intravenously with a volume of 5 milliliters of the cell regeneration compound and five injections intramuscularly preferably in the deltoid region (No. 17), injecting 0.5 milliliters of the same compound every 7 days, obtaining effects of cell regeneration to induce: neurogenesis, hematopoiesis, chondrogenesis, osteogenesis, vasculogenesis and angiogenesis that can be assessed in the skin, renal function tests, as well as the disappearance of neurological symptoms, inflammation and joint pain that occur due to biological aging and for the development of chronic degenerative diseases. This cycle of ten injections of the compound can be used twice to three times to complete thirty injections over a period of four months. The CRC application is the safest, since most of our patients (plus 700 patients with 10 CRC applications each) have not had to take surgical risks and have not required the support of interventional radiology, and only in some cases procedures requiring an operating room, and subspecialized surgeons and anesthesiologists have been required. Intensive therapy is generally not required, and after three years of application no complications have been observed, however, all patients improve markedly enough by appreciating clinically, with signs and symptoms of cell regeneration. The "CRC" is currently the only real cell regeneration option for incurable diseases that do not have current treatment. The "CRC" is a source of replacement of dead, devitalized cells and cell regeneration in diseased tissues, prolonging and improving the quality of life in healthy or sick people. Circulating stem cells released by the “CRC” can be incorporated into undamaged organs, and act as anti-aging agents, by increasing the average life of the cells of the organ and the person in question (Méndez-Bolaina E, Sanchez-Gonzalez DJ et al. Effect of caveolin-1 scaffolding peptide and 17f-estradiol on intracellular Ca2 + kinetics evoked by angiotensin II in human vascular smooth muscle cells. Am J Physiol Cell Physiol 2007; 293: C1953 - C1961).

The object of this invention is to provide a cell regeneration compound that does not exist in the market, which allows the application of stem cells in the form of cellular therapy, avoiding studies of HLA typing gene, and surgical and interventional radiology interventions used in the implant. or cell transplant. The "CRC" acts using the principle of cell regeneration, which naturally occurs in body tissues. If we consider that throughout life organisms suffer from continuous wear, if there is no cell regeneration, the life expectancy of living beings would be significantly reduced. On the other hand, a large part of the wide range of diseases that affect the human being have their origin in the degeneration and death of the different types of differentiated cells that make up the tissues and organs of our body, which are adversely affected by acute way as usually occurs in an acute myocardial infarction or chronically as in degenerative diseases and biological aging. It is widely described that the invention referred to in the present invention patent application is a "Cellular Regeneration Compound" (CRC) that allows intravenous, intramuscular and local administration of stem cells for therapeutic purposes in chronic degenerative conditions in which Chronic renal failure and Diabetes Mellitus are included. After describing its composition and way of preparing the “Cellular Regeneration Compound”, inventors Gerardo Martín González-López, Dolores Javier Sánchez- González and Carlos Armando Sosa-Luna, claim the “Compound of Cellular Regeneration” (CRC) as its invention, stating that to its real knowledge and understanding they have described in the simplest way, the way to carry out their invention, in addition to including the results of their investigations and even reaching the development of the "CRC", which confirms the operation of the present invention which they have named I swim "Compound of Cellular Regeneration" "CRC" stating that it is the safest way to take pluripotential stem cells to the organism of human beings, avoiding hospitalization and laboratory studies in most cases (p. and. genotyping such as HLA haplotypes).

Chronic, degenerative diseases, aging and the aggressions of the environment contaminated by human industrialization considerably accelerate the reserve of stem cells of living beings by limiting the intrinsic cell regeneration capacity of organisms (Trejo-Bahena NI, Pérez-Astudillo LH , Orjuela-Henry DJ, Balderas-Cornelio A, Salinas-Cano F, Martínez-Martínez CM, Sánchez-González DJ Comparative study to determine hippuric acid (HA) in urine using Ogata and Astudillo colorimetric methods Rev Sanid Milit Mex 2008; 62 (1): 35-41).

In several recent scientific publications noted above in the background of the present patent application, it has been concluded that the number of stem cells in the blood is one of the best indicators for human health. Therefore, a compound that allows the introduction of healthy stem cells into the blood is required, this is achieved safely by applying the CRC "Cell Regeneration Compound", widely described in the present invention application.

The functioning of the “Cellular Regeneration Compound” (CRC) is explained because it increases the reserves of stem cells by increasing the intrinsic capacity of cellular regeneration in patients and healthy people, obtaining a better state of health and quality of life in many ways, There is no doubt about this as the number of investigations that prove that circulating stem cells are the most important factor in people's health is increasing every day, which allows cell regeneration; The greater the amount of stem cells in circulation, the greater the body's ability to regenerate. The operation of our invention called "Cellular Regeneration Compound" (CRC) works by inducing the regenerative capacity of stem cells in a sterile liquid suspension that allows a sufficient number of stem cells to be carried in a suitable volume (6 to 0.5 ml). (10,000,000 of cellular elements) that allow in a safe and practical way to increase the number of stem cells in the systemic circulation or locally using various routes of pharmacological administration, such as: intravenous, intramuscular, local.

By applying intravenously and intramuscularly the “Compound of Cellular Regeneration” (CRC), allows the intelligent release of pluripotential stem cells and bioactive molecules, which promote the activation of the elements necessary for cell regeneration to take place, which facilitates its entry into the circulation to damaged tissues and accelerates transdifferentiation, that is, the stem cells acquire the characteristics of the tissue cells where they are implanted more quickly. At the site of injury, changes in the microenvironment are formed that attract the stem cells released and activated by the "CRC" that are circulating. The circulating stem cells of the "CRC" can be incorporated into undamaged organs, acting as anti-aging agents by providing bioactive molecules of cellular regeneration, which increase the average life of the cells of the organ and the person in question.

The best example that the “Cellular Regeneration Compound” (CRC) is an inducer of hematopoiesis, vasculogenesis, angiogenesis, myogenesis and neurogenesis, is the regeneration effect in chronic renal failure, which is broadly exemplified in the present application of patent. However, the "Cellular Regeneration Compound" (CRC) is also useful for treating various diseases and diseases of a chronic, degenerative nature or biological cell aging, obtaining good results in patients in whom the disease or condition is the result of the destruction and / or dysfunction of specific differentiated cells, such as: Parkinson's disease that is the result of the destruction or degeneration of dopaminergic neurons in the brain (González López GM et. al. Neurol Neurocir Psiquiat 2007; 40 ( 3): 80-91). We have observed how patients who are treated with 10 intravenous injections of “CRC” decrease levels of oxidative stress and increase the levels of neurotransmitters mainly dopamine, quantified by modern techniques of computed transcutaneous impedance measurement with the Interstitial Scanner (EIS) system; Diabetes Mellitus, which is the result of the destruction or degeneration of pancreatic B cells and their vascular complications, which can be treated with CRC since cell regeneration is induced by inducing vasculogenesis, angiogenesis, neurogenesis and myogenesis that allows to reverse the natural history or clinical evolution of patients with chronic renal failure, with loss or dysfunction of endothelial cells, manifesting with arterial hypertension, and diabetic nephropathy; In addition to peripheral vasculopathy that presents with venous, arterial insufficiency, ulcers and infections of difficult scarring in the lower extremities, a condition known as diabetic foot. (Sosa Luna CA et. Al. Rev Sanid Milit Mex 2005 59 (1): 32-50 and Rev Sanid Milit Mex 2006 60 (5): 324-333), sickle cell anemia, thalassemia, muscular dystrophy, Alzheimer's dementia, Epilepsy , Schizophrenia, Cerebrovascular Disease, (Sosa Luna CA et. Al. Rev Sanid Milit Mex 1999; 53 (2): 123-128 and Sosa Luna CA et. Al. Rev. Neurology, Neurosurgery and Psychiatry 2002; 35 (4 ): 183-202) traumatic injury of the spine, degeneration of Purkinje cells, cardiac ischemia, liver failure, Duchenne muscular dystrophy, osteoarthritis, osteogenesis imperfecta, Down syndrome, and as support regeneration in cancer patients who He is receiving radiotherapy and chemotherapy schemes, etc. (Vásquez-Moctezuma I, Meraz-Ríos MA, Magaña M, Villanueva-López GC, Sánchez-González DJ. Chemo resistance in melanoma. Preliminary report. Proceedings Dermatol Dermatopatol 2006; 6: 8-10 and Torres-Salazar JJ, Sánchez-González DJ et. Al. Distribution and status d and maturation of dendritic cells and activation of CD4 + lymphocytes in prostate adenocarcinoma. Rev Mex Urol 2007; 67: 140-146)

The "CRC" has a biological anti-aging effect, this is explained because as time passes the stem cells become scarcer, perhaps this decrease is what determines the greatest vulnerability of the elderly. If we inject the “CRC” into the circulation of these patients, they will have more capacity for cell regeneration, since in the circulation the number of stem cells activated and released by the “CRC” is increased they have the ability to migrate from the circulation to the organs, devices and systems that are aging. The stem cells of the “CRC” have the ability to transdifferentiate into different types of differential cells such as: the endothelial and others that make up the organs and tissues of the body, observing a cellular regeneration of the skin, greater hydration, luminosity, turgencies of the skin, as well as the elderly perceive a state of revitalization (energy recovery) (Tapia E, Sánchez-González DJ, et. al. Treatment with pyrrolidine dithiocarbamate improves proteinuria, oxidative stress and glomerular hypertension in overload proteinuria. Am J Physiol Renal Physiol 2008 Aug 27).

In the chromosomes of people with biological aging, DNA cross-links are observed and breaks of a single chain frequently occur; DNA methylations decrease and telomeric sequences are lost in said nucleic acid. Biological aging also includes environmental aggression (which includes various infections, accidents, trauma and other diseases whose origin and pathophysiology is not yet known), as well as endogenous aggression which is divided into DNA alterations inherited by our parents, which is associated with the appearance of chronic degenerative diseases such as diabetes mellitus, hypertension, cancer etc. (Pou-López VC, Gallardo-Ollervides FJ, García-Mendoza JA, Sánchez-González DJ. Transtympanic dexamethasone in sudden late sensorineural hearing loss. Rev Sanid Milit Mex 2008; 62 (2): 57-65). These aggressions continue with the series of harmful impacts, although the primary structure of the proteins is not altered with the aging of the DNA, the environmental and endogenous aggressions increase the post-translation changes (Sánchez-González DJ et. Al. Ozone exposure induces iNOS expression and tyrosine nitration in rat aorta Environ Toxicol Pharmacol 2004; 17: 1-7 and Sánchez-González DJ et. al. Production of iNOS and protein nitration in the aorta of rats exposed to ozone Rev Sanid Milit Mex 2005 ; 59 (4): 223-240).

Protein damage can be best explained by poor diet (rich in carbohydrates, poor in amino acids and proteins) and damage to mitochondria, which also deteriorate over time, although they do not always do so constantly. When the damage exists, it is possible to structurally demonstrate it with high-resolution confocal microscopy (Sánchez-González DJ et. Al. Confocal microscope in the dermatological diagnosis of autoimmune bullous diseases. Actas Dermatol Dermatopatol 2007; 7: 9-15 and Microscopy images confocal in the diagnosis of autoimmune bullous diseases Rev Sanid Milit Mex 2006; 60 (2): 82-90).

In aging and in organisms that receive this type of nutrition rich in carbohydrates and poor in proteins, we have observed how mitochondria are more susceptible to suffer from alterations in the DNA of the same condition conditions of oxidative stress cause alterations in the biochemical processes of deamination, oxidation, cross-linking and non-enzymatic glycosylation, in the enzymes prostacyclin synthase and thromboxane synthase, causing an imbalance in the bioavailability of thromboxane released in the circulation and a reduction in prostacyclin, causing a greater predisposition to thrombosis and vasoconstriction in the blood vessels of Patients with Diabetes Mellitus and Hypertension (Sosa Luna CA et. al. Rev Sanid Milit Mex 2005 59 (1): 32-50 and Rev Sanid Milit Mex 2006 60 (5): 324-333).

The effect of cell regeneration is most evident when, due to some disease, a differentiated cell line is under great challenge or stress; such is the case of the insulin-producing cells known as B cells of the islets of Langerhans of the pancreas, which are diminished

or destroyed in patients with Diabetes Mellitus, causing a feeling of chronic fatigue, and when insulin insufficiency is caused, the increase in blood glucose is observed, appearing increase in urine, thirst and increased appetite, characteristic symptomatology of this disease (Shamblott MJ; Clark GO, Cell therapies for type 1 diabetes mellitus. Expert Opin Biol Ther 2004 Mar; Vol. 4 (3), pp. 269-77).

In Figure 2/3 some results of our experiments are presented applying “Cellular Regeneration Compound” CRC (Cell Therapy) in animals with renal insufficiency in an experimental model of Diabetes Mellitus of our laboratory with insulin insufficiency, in rats of the strain wistar hyperglycemic (blood glucose> 300 mg / dl), by induction with streptozotocin. The application of Cellular Regeneration Compound ”CRC (Cellular Therapy) allowed the regeneration of damaged kidneys, evidenced by the increase in creatinine clearance and morphological analysis. We can see that the first bar starting on the left (healthy or control laboratory animals) has an average creatinine clearance of 2.84 ml / min equivalent to a renal function of 94.66%; while the laboratory animals represented by the second bar, patients with Diabetes Mellitus, without any treatment during the evolution of hyperglycemia), presented an average creatinine clearance of 1.84 ml / min, which is equivalent to a renal function of 61 , 3%, value that allows the diagnosis of chronic renal failure (<70%). Sick animals represented by the third bar with Diabetes Mellitus who received a standard nephroporotective treatment with an angiotensin-converting enzyme inhibitor Ramipril at a dose of 1 mg / kg daily orally had a creatinine clearance of 2.6 ml / min equivalent to a renal function of 86.6% and the sick animals with Diabetes Mellitus represented by the fourth bar that received the “Cellular Regeneration Compound” (Cellular Therapy) presented a creatinine clearance of 3.7 ml / min equivalent to a renal function of 123.3%, observing cellular regeneration. Animals with Diabetes Mellitus referenced in the fifth bar were treated with a selective cyclooxygenase inhibitor drug, Celecoxib at a dose of 4 mg / kg of weight daily orally. These animals had a nephrotoxic effect, observing the presence of frank proteinuria in Bowman's capsule, and a creatinine clearance of 1.4 ml / min equivalent to a renal function of 46.66% in frank renal failure.

Naturally, the stem cells are released from the bone marrow and other reservoirs, crossing the bloodstream to those tissues that are most in need. When an organ is subject to disease requirements, this organ fires compounds that emit a signal so that the stem cells are released into the circulation. The organ starts the cascade of inflammation by releasing compounds that attract stem cells to this particular organ. Then the stem cells that were released by our cell regeneration compound, follow the path of concentration of those compounds and leave the bloodstream to migrate to the organ where they are proliferating and begin to differentiate into cells of that particular organ. The ability of the "CRC" to release the activated stem cells in the circulation through our invention and the described protocols of cell therapy for regenerative and anti-aging medicine are of great relevance since the circulating stem cells decrease with increasing age and age. chronic and degenerative diseases (Pacheco-Ramírez MA, Rodríguez-Perales MA, López-Chavira A, Canul-Andrade LP, Martínez-Martínez CM, Sánchez-González DJ. Expression of nitric oxide synthases in head and neck glomic tumors Rev Sanid Milit Mex 2006; 60 (6): 369-378). In this way, it could be concluded that children and babies have a very effective “stem cell system” and do not need the support of the “CRC”. However, we have applied “CRC” in children under 10 years of age in support of chemotherapy and radiotherapy treatment in acute leukemia, as well as in pediatric patients with Genetic Syndromes such as Down, Turner, etc. Also in children with neurodevelopmental problems, autistic, mental weakness, childhood cerebral palsy, attention deficit and young people with schizophrenia. We are receiving testimonies with information about very strong experiences indicating that cell therapy with stem cells could provide significant benefits for young children (Aguilera-Hernández P, Chánez-Cárdenas ME, Sánchez-González DJ et. Al. Regulation of the synthases of nitric oxide in the rat striatum in the quinoline-induced model of Huntington's disease Arch Neurocien Mex 2006;

11:31).

Cell therapy using "CRC" is the safest way to treat the damage suffered by the genetic material found in the cell nucleus and mitochondria. These damages are one of the most relevant factors of the biological aging process and cellular senescence. In the elderly there are a greater number of structural alterations of chromosomes (genetic material) which are composed of deoxyribonucleic acid (DNA) that manifest with the symptoms of aging such as deterioration of cardiovascular, musculoskeletal, presbiacusia and presbyopia, etc. (Béjar-Cornejo F, Olivares D, Cantero MA, Sánchez-González DJ. Corneal thickness determined by corneal topography ORBSCAN in patients diagnosed with glaucoma in the Mexican population. Rev Sanid Milit Mex 2007; 61 (5): 310-319). Stem cells contain intact DNA as well as healthy mitochondria and many proteins that have the ability to reverse many of the effects of aging. Stem cells are rich in the telomerase enzyme which can decrease the loss of telomeric DNA sequences in aged or senescent cells. From the point of view of "gene therapy", we can consider that stem cells are the best vector, and can be considered as "intelligent micro-spheres" (since they are microscopic living organisms, with the full capacity of self-replication as well as of tropism, that is, they recognize and travel to the site of damage or injury, as well as transdifferentiation of differences in the missing or injured cell type) (Sánchez-González DJ et. al. Biological effects of industrial frequency electromagnetic fields. Model in rats. Rev Sanid Milit Mex 2007; 61 (6): 371-380). Since they contain within them the complete human genome, which, unlike the gene therapy protocols, is intact, undifferentiated and can give rise to all the proteins that give rise to the various cell types that constitute the human body, It should be noted that in cell therapy with stem cells, the stem cells have not been modified, altered or even touched in their genetic machinery (DNA), they have only been protected from all the harmful agents mentioned above such as irradiation, infectious agents , etc. So the cells that are transplanted have a biological age of zero (new) years.

Below we present several examples of the utility in the use of “Cellular Regeneration Compound (CRC):

Example 1: Chronic and neurodegenerative diseases are a group of clinical entities that are increasingly diagnosed in our country and in the world. These diseases include: Alzheimer's dementia, Parkinson's disease, Huntington's disease and Amyotrophic Lateral Sclerosis (Aguilera P, Sánchez-González DJ et. Al. Time-related changes in constitutive and inducible nitric oxide synthases in the rat striatum in a model of Huntington's disease Neurotoxicology 2007; 28 (6): 1200-1207). The particularity of these clinical entities is that they do not have a form of treatment that limits or reverses the severe deterioration and disability they produce. They have an invariably progressive, disabling and short-term mortality course in some, and significant disability in all of them. Given the little curative effect of current treatments, the use of cell therapy as a therapeutic alternative has been explored. Brain repair through the application of cell transplants is the focus of attention in recent research. Additionally, the nervous system has immunological privileges that reduce the need for regular immunosuppressants in transplants, even in heterologists. Additionally, autologous stem cell transplantation favors that these cells can be established in the brain and transdifferentiated to neurons.

Example 2: Patients with refractory angina and a history of myocardial ischemia or infarction, when they can no longer be treated with conventional therapies such as coronary angioplasties and Sten placements, with ejection fraction of 37.5% after receiving cell therapy With “CRC” they can improve their ejection fraction in months, leaving 47% and reducing medication requirements and symptoms. Patients with heart failure with functional class III and 30% basal ejection fraction improve after five to fifteen sessions of cellular therapy with “CRC”. All patients will improve to a functional class II. Some of the SPECT Sestamibi Tc99m studies after the “CRC” treatment during clinical follow-up show improvement due to cellular regeneration resulting in an increase in myocardial vascularity and viability. One of our patients with three coronary vessels disease out of treatment for coronary angioplasty after being evaluated by the specialist Dr. Hugo Gutierrez Leonard at the Central Military Hospital, improved myocardial perfusion SPECT Sestamibi Tc99m after receiving five intravenous injections of the “ CRC ”finding a notable improvement evidenced by the same family members, since now he could walk more blocks without presenting chest pain, tiredness and shortness of breath; The study showed ischemia in a single coronary branch, that is, of the three vessels with ischemia, two were regenerated, with the application of stem cells.

Example 3: Chronic Renal Failure and Complications of Diabetes Mellitus, Figure 3/3 shows a graph with the average creatinine clearance result before and after treatment with “CRC” in our patients with chronic renal failure. obtained from creatinine clearance studies before and after receiving ten applications of "CRC" intravenously and intramuscularly. They presented reversal in the progression of renal function deterioration in their clinical laboratory studies and symptomatology, unprecedented (Cruz C, Correa-Rotter R, Sánchez-González DJ et. Al. Renoprotective and antihypertensive effects of Sallylcysteine in 5/6 nephrectomized rats Am J Physiol Renal Physiol 2007; 293: F1691 – F1698 and Chirino YI, Trujillo J, Sánchez-González DJ et. al. Selective iNOS inhibition reduces renal damage induced by cisplatin. Toxicol Lett 2008; 176 (1): 48- 57). Figure 2/3 showed the graph, elaborated with the results of our experiments performed in laboratory animals, wistar strain rats with diabetic nephropathy due to Diabetes Mellitus; induced with an intraperitoneal injection of streptozotocin.

Changes can be observed at 5 to 10 weeks after treatment with “CRC”, which corroborate the results of creatinine clearance consistent with those obtained in humans in clinical records (Pedraza-Chaverri J, Yam-Canul P, Chirino YI, Sánchez-González DJ et. Al. Protective effects of garlic powder against potassium dichromate-induced oxidative stress and nephrotoxicity. Food Chem Toxicol 2008; 46 (2): 619-627 and Segoviano-Murillo S, Sanchez-Gonzalez DJ et. al. S-allylcysteine ameliorates ischemia and reperfusion induced renal damage. Phytother Res 2008; 22 (6): 836-840). The animal experiments figure 2/3, not only allowed us to evaluate the function, but we were also able to observe the changes in the microscope of the glomeruli in the renal histology sections. The cuts of the control animals were similar to the cuts of diabetic rats treated with our Regenerative Medicine treatment offered to patients at SITECEM (Orozco-Ibarra M, Medina-Campos ON, Sanchez-Gonzalez DJ et. Al. Pedraza -Chaverri J. Evaluation of oxidative stress in dserine induced nephrotoxicity.Toxicology 2007; 229: 123-135; Yam-Canul P, Chirino YI, Sánchez-González DJ et. Al. PJ34, a poly adenosine diphosphate-ribose polymerase inhibitor, attenuates chromate-induced nephrotoxicity Basic Clin Pharmacol Toxicol 2008; 102 (5): 483-488 and Yam-Canul P, Chirino YI, Sánchez-González DJ et. al. Nordihydroguaiaretic acid attenuates potassium dichromate-induced oxidative stress and nephrotoxicity. Food Chem Toxicol 2008; 46 (3): 1089-1096).

Example 4: In neurodegenerative diseases, from 2001 and 2003, the results of controlled studies of embryonic dopamine neuron transplantation in patients with mild-moderate Parkinson's disease were reported. In these studies it was concluded that the application of multiple implants, including the nigra substance and striatum in animal models produce better results. Likewise, these findings were confirmed in patients, who showed clinical improvement as well as with the use of positron emission tomography with fluorodopa (PET), and without developing motor complications. The objective of implanting stem cells in Parkinson's disease is to reconstruct the neural neural pathway with precursors of neural stem cells or grafts of dopamine neurons. The transplantation of dopaminergic neurons derived from stem cells has been attempted in order to stimulate local synapse or cytokine release by grafting. Patients with neurodegenerative diseases such as Parkinson's improve clinically with the treatment of human stem cells, reducing generalized clumsiness and slowness in performing movements; Improving the lack of spontaneous motility, reduces resting tremor and stiffness (Sánchez-González DJ et. al. Nitric oxide in the central nervous system. Nitrurgical neurons. Neurol Neurocir Psiquiat 2004; 37 (2): 7378). Patients recover the response to pharmacological administration of the precursor (levodopa). Today the application of the "CRC" is an alternative for Parkinson's patients. MRI studies can be performed at the beginning and 6 months after cell therapy, with N-acetylaspartate (NAA) / creatine (Cr) spectroscopy and SPECT perfusion studies with Tc 99m sestamibi. The clinical results have been very satisfactory. No complications have occurred and all patients improve their clinical parameters.

Example 5: in dementias that are clinical syndrome characterized by severe loss of acquired cognitive and emotional skills that interfere with daily performance and quality of life. Dementia can be caused by more than 55 diseases, some not progressive and occurs mainly in late stages of life. The prevalence is one% in people 60 years of age and doubles every five years, to increase to 30 or 50% in people 85 years and older.

There are multiple forms of dementia; However, the most common is Alzheimer's dementia, which accounts for between 80 and 85% of the causes of dementia. Other forms are vascular dementia (due to multiple cerebral infarctions); dementia in Huntington's disease, fronto-temporal dementia, dementia with Lewy bodies and AIDS dementia, among others. Of all cases with dementia, less than 15% are susceptible to treatment with the “CRC”

Although there are several medications useful in the treatment of dementia, none of them has been effective in stopping the inevitable progression of this neurodegenerative disease. The frequency and prevalence of this disorder is gradually increasing in the world. This fact is mainly due to the increase in the life expectancy of the population. An estimate made by our group of researchers in 1999 reported that in Mexico there was a prevalence of 814 thousand people with cognitive disorders associated with age, and more than half a million Mexicans with Alzheimer's dementia. Alzheimer's patients improve their intellectual capacity and memory. Autistic and children with Down syndrome improve their ability to speak, articulate words, improving communications with their parents and teachers.

Example 6: We have high expectations due to the improvement observed in patients with neurological conditions, facial paralysis, which is why we have used the “CRC” in patients with Down syndrome, autism, deafness, muteness, sequelae due to cerebral vascular events and all they have presented improvement in the symptoms and signs they present, all of these have been treated with external protocols and we are in the process of building an internment area to apply cell therapy with “CRC” in patients with severe mental and psychiatric conditions, such as Chronic Schizophrenia, and we have a cooperation and research agreement with the Mexican Association of Friends of Schizophrenic Patients AC (AMAPE).

Example 7: In traumatic injuries, osteoarthritis, sprains, for faster recovery from surgery or during traumatic spinal cord injury is not a neurodegenerative disease. However, in our laboratory we have been working on the search for the most appropriate methodology to look for damaged spinal cord repair. Unfortunately, young and young adults have sequelae of different magnitude ranging from herniated discs with an excellent response to treatment with “CRC” to the consequence of paralysis due to spinal injury caused by accidents or degenerative and congenital diseases. Therefore, we are clinically investigating the effect of “CRC” to try to repair the damaged spinal cord. In our country there have been no studies on the frequency and prevalence of traumatic spinal cord injury; nevertheless, it has been estimated that the annual frequency of people with spinal cord damage is 40 cases per one million inhabitants (United States), which represents an incidence of 11 thousand new cases each year. This estimate does not include cases of people who die in the accident. The most frequent causes of spinal cord injury are consistently vehicle accidents (41%), violence injuries (22%) and falls (21%). Because the age of presentation of the spinal injury is in young people and young adults, with potential for prolonged survival, the prevalence of this clinical entity is quite high and increases every year. It has been estimated that more than a quarter of a million people in the United States currently live with disabilities, due to sequelae due to spinal cord injury. Most cases of spinal cord trauma occur in young adults. The average age of these cases is 28.7 years; most of the spinal traumas occurred between 16 and 30 years of age. 77.8% of cases of spinal injury are male, usually young or young adults who are in stage

productive or at the beginning of it. It has been observed that more than half of the patients (64.2%) were employed

before suffering the spinal cord injury. After the traumatic event, the tenth year of evolution only a third

part of the patients with Paraplegia returned to gain paid employment. Therefore, it is considered that this

5 clinical entity has an important economic impact on the patient and his family, due to the direct costs and

indirect that it produces. The average annual expenditure for the patient's health care and direct costs vary from

according to the severity of the injury. However, indirect costs that include loss of salary, loss of

Supplemental benefits and productivity, are estimated to be on average around 60 thousand dollars per year;

However, this figure may vary depending on the educational level of the patient, the type of employment and work history before the accident, in addition to the severity of the spinal cord injury. Therefore, we consider the

application of "CRC" as an alternative therapeutic option for this type of problem.

Claims (2)

1) Procedure for the preparation of a Compound for Cellular Regeneration comprising the following steps: a) provide a purified line of pluripotential stem cells from either cryopreserved stem cells at -198 ° C that are defrosted gradually increasing the temperature at a rate of 2 ° C / min until reaching the temperature of -20 ° C, or stem cells powder in a state of total dehydration obtained by a conventional lyophilization process, where the stem cells have not been obtained by methods that destroy or use human embryos as raw material; b) prepare a base solution “A” according to the following procedure: i) dissolve sodium chloride (NaCl), sodium lactate (NaC3H5O3), calcium chloride (CaCl2) and potassium chloride (KCl) in double distilled water until reaching the following ionic ratio: Na + = 130 mEq, Cl- = 109 mEq , lactate = 28 mEq, Ca2 + = 3 mEq and K + = 4 mEq, reaching an osmolarity of at least 273 mOsm / L and not exceeding 300 mOsm / L in sterile conditions and in a controlled environment at a cooling temperature less than 16 ° C and greater than 4 ° C; ii) sterilize the sub-stage solution i) through a traditional sterilization process such as autoclave; iii) add to the solution of sub-stage ii) a sterile solution with 55 mg of zinc chloride, 16.9 mg of cupric sulfate pentahydrate, 38.1 mg of magnesium sulfate, 1.3 mg of sodium iodide , 14 mg of sodium fluoride, 163.9 mg of sodium chloride, 10 mg of thiamine, 250 mg of pyridoxine, 1000 mg of ascorbic acid, 1.0 mg of activin, 0.2 mg of stimulatory factor of pluripotential cells (C-kit), 0.6 mg of procaine derivative G1, 0.4 mg of procaine derivative G2, and 0.4 mg of procaine derivative G3; c) hydrate the stem cell sources of stage a) in the base solution "A" obtained in stage b) at a temperature of 8 ° C; d) select prehydrated stem cells with healthy morphology and collect them in a sterile syringe; e) suspend approximately 1,000,000,000 stem cells in 500 ml of the base solution "A", thus providing a base solution "A" enriched with stem cells; f) prepare a base solution "B" by mixing in a controlled manner and under sterile conditions 500 ml of solution "A" with a concentration of 2,000,000 stem cells, with 1.0 mg of L-cysteine, 0.5 μg of interleukin 7 (IL7), X0.5XIgXdeXinterleucine X3X (IL-3), 0.1 mg of Mesodermal Growth Factor A1 (FGM), 0.1 mg of Monocyte Colony Stimulating Factor (M-CSF), and 0.1 mg granulocyte colony stimulating factor (GM-CSF), stored at 8º C for no more than one week, or -4º C for no more than five weeks, or at -20 ° C for no more than twenty four weeks; g) prepare a base solution "C" by mixing in a controlled manner and under sterile conditions 500 ml of solution "B" with 0.3 mg of beta growth transformation factor (TGF-B) and 0.2 mg of endodermal growth factor K2 (FGE), stored 8º C for no more than one week, or -4º C for no more than five weeks, or -20ºC for no more than twenty four weeks; h) prepare a base solution "D" by mixing 500 ml of solution "C" with 100 mg retinol, 0.5 mg fibroblast growth factor (FGF) and 0.2 mg neurological growth factor (FGFN). i) provide the Compound for Cellular Regeneration composed of the four base solutions "A", "B", "C" and "D". 2) Compound for Cell Regeneration obtained by the method of claim 1. 3) Use of the Compound for Cellular Regeneration obtained according to the method of claim 1 in the manufacture of a medicament for treating chronic degenerative conditions that require cell regeneration. 4) Use according to claim 3, wherein the chronic degenerative conditions are selected from the group consisting of chronic renal failure, heart failure, ischemic heart disease, diabetes mellitus, osteoarthritis, fibromyalgia, dementia, Alzheimer's disease, rheumatoid arthritis, lupus erythematosus systemic, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, vitiligo, herniated discs, Down syndrome, autism, schizophrenia, hearing loss and other senses, paralysis and arterial or venous vascular insufficiency. SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201190033 SPAIN Date of submission of the application: 11.11.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

US 20050181502 A1 (FURCHT ET AL.) 08/18/2005, see summary; paragraphs [0019], [0027], [00350037], [0089-0097], [0110-0115], [0128-0141], [0231-0238], [0246-0264]; and figures 5-14. 1-4

TO

WO 2004085630 A1 (MEDVET SCIENCE PTY LTD & ANGIOBLAST SYSTEMS INC) 07/10/2004, see summary; page 22, lines 19-28; claims 2, 3, 11, 17, 66 and 67; figures. 1-4

TO

TARE, R.S., et al. Skeletal stem cells: phenotype, biology and environmental niches informing tissue regeneration. Molecular and cellular endocrinology. . Vol. 288, No. 1-2, pages 11-21. ISSN 0303-7207. See summary, introduction, paragraph 4.1, figure 1 and conclusions. 1-4

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 05.11.2012

Examiner B. Pérez Esteban Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201190033 CLASSIFICATION OBJECT OF THE APPLICATION C12N5 / 071 (2010.01) A61K35 / 12 (2006.01) A61P43 / 00 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61K, A61P Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, TXTE, MEDLINE, BIOSIS, NPL, EMBASE, XPESP. State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201190033 Date of Written Opinion: 05.11.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-4 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-4 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201190033  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

US 20050181502 A1 (FURCHT et al.) 18.08.2005

D02

WO 2004085630 A1 (MEDVET SCIENCE PTY LTD & ANGIOBLAST SYSTEMS INC) 07.10.2004

D03

TARE, R.S., et al. Skeletal stem cells: phenotype, biology and environmental niches informing tissue regeneration. Molecular and cellular endocrinology. . Vol. 288, No. 12, pages 11-21. ISSN 0303-7207. See summary, introduction, paragraph 4.1, figure 1 and conclusions.

 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present patent application describes and claims a method of preparing a cell regeneration compound that consists in preparing a purified line of pluripotential stem cells, to which are added to different solutions containing inorganic compounds and growth factors, which, used in certain combinations, they allow the differentiation of the mentioned stem cells towards processes of chondrogenesis, hematopoiesis and osteogenesis in one case, of angiogenesis and vasculogenesis in another, and of neurogenesis, angiogenesis and vasculogenesis in another. The application also claims the compound prepared by this procedure, and its use to prepare a medicament for the treatment of various degenerative diseases. Despite the existence of numerous documents in the state of the art in which the differentiation of stem cells to the different cell lineages, and the culture media and factors necessary to obtain each type of tissue are disclosed, no document has found composition of the invention as described and claimed, whereby claims 1 to 4 of the present application are considered new and inventive according to articles 6 and 8 of Patent Law 11/1986. In document D01, for example, the therapeutic use of stem cells capable of forming cells of multiple types of tissue is described. This document details the culture media used, and the factors necessary for cell differentiation to occur in one way or another, and differentiation is achieved to mesodermal tissue (osteoblasts and chondroblasts) and endothelial, hematopoietic and neuronal cells. However, although some of the factors and compounds used in D01 coincide with those of the international application, the compositions used are totally different, so D01 does not affect the novelty or the inventive activity of the application. In document D02, the inventors isolate mesenchymal stem cells from perivascular niches, identify the surface markers that characterize them, and differentiate them in vitro to osteocytes, adiposites and chondrocytes (see page 22, lines 19 to 28). Again, the compositions described for each cell differentiation differ from those of the application, which, therefore, would have novelty and inventive activity in light of what was disclosed in D02. In document D03, human bone marrow stem cells are obtained and the growth factors and matrix components necessary for their differentiation are studied, with special emphasis on chondrogenic differentiation and its use for musculoskeletal regeneration. Nor in this case is the composition claimed in the application used to favor the differentiation of the stem cells, so D03 does not affect the novelty or the inventive activity of the application. State of the Art Report Page 4/4