METHOD FOR OBTAINING A CO-CULTURE OF OLIGODENDROCYTES AND NEURONS WITH ENHANCED DIFFERENTIATION CAPACITY, CULTURE MEDIUM AND ITS USE AS A NEUROREGENERATIVE RE-MYELINIZING GRAFT OF PERIPHERAL NERVES

Abstract

The present invention describes a method for obtaining a co-culture of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) with enhanced differentiation capacity, a culture medium, and their use as a neuroregenerative re-myelinating cell graft for peripheral nerves. The method comprises the use of a specific ratio of OLCs and NLCs, growth factors added to culture media to promote enhanced differentiation of up to 81.54 ± 8.26% for myelin basic protein (MBP), up to 89.28% for proteolipid protein (PLP), and up to 22.26 ± 5.12% for neurofilament (NF).The present invention further demonstrates the use of co-culture as a cell graft for re-myelinating neuroregeneration of peripheral nerves. They managed to myelinate it in a manner similar to the control nerve (without decellularization) at 3, 7 and 21 days, thus allowing a possibility for the treatment of demyelinating diseases or the repair of peripheral nervous system (PNS) injuries or spinal cord injuries.

Description

The present invention relates to a method for obtaining a co-culture of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) with enhanced differentiation capacity, a culture medium, and their use as a re-myelinating neuroregenerative graft for peripheral nerves. The present invention belongs to the technical field of medicinal preparations containing nerve cells. BACKGROUND The central nervous system (CNS) is composed of two types of cells: neurons, whose functions are the reception and transmission of information through electrical impulses, and glial cells, which are responsible for the metabolic and mechanical support and protection of neurons. Neurons constitute the most important component of the CNS, with oligodendrocytes playing a key role (He Z, Jin Y. Intrinsic Control of Axon Regeneration. Neuron [Internet]. May 4, 2016; 90(3):437-51. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 27151637). Oligodendrocytes located alongside axons are responsible for myelin synthesis and the protection of neuronal axons by wrapping them with multiple layers of myelin. The main proteins present in myelin are myelin basic protein (MBP) and proteolipid protein (PLP), which together represent 80% of the total myelin proteins. Myelination allows axons to conduct action potentials faster and over longer distances (Mekhail M, noRAnn / ίζηζ / E / γίΛΐ Almazan G, Tabrizian M. Oligodendrocyte-protection and remyelination post-spinal cord injuries: A review. Prog Neurobiol [Internet]. March 2012;96(3):322-39. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 22307058). Numerous growth factors have been studied that promote the proliferation, maturation, and survival of oligodendrocytes, such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), triiodothyronine, progesterone, insulin, and transferrin, among others. Mature myelinating oligodendrocytes express markers sequentially: galactocerebroside (GalC), PLP, PBM, myelin-associated glycoprotein (MAG), and finally oligodendrocyte myelin glycoprotein (MOG), which allow for their identification and / or characterization. (Elbaz B, Popko B. Molecular Control of Oligodendrocyte Development. Trends Neurosci [Internet]. April 1, 2019; 42(4):263-77. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 30770136; Girolamo F, Strippoli M, Errede M, Benagiano V, Roncali L, Ambrosi G, et al.Characterization of oligodendrocyte lineage precursor cells in the mouse cerebral cortex: a confocal microscopy approach to demyelinating diseases. Ital J Anat Embryol [Internet]. 2010;115(1 — 2):95-102. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 21072997; Santos AK, Vieira MS, Vasconcellos R, Goulart VAM, Kihara AH, Resende RR. Decoding cell signaling and regulation of oligodendrocyte differentiation. Semin Cell Dev Biol [Internet]. May 23, 2018, Available online: https: / / www.sciencedirect.com / science / article / abs / pii / S1084952118300569?via%3 Dihub. However, although there are numerous protocols that allow the predifferentiation of oligodendrocytes, these have the limitation that the percentage of differentiation achieved is less than 40%, and the oligodendrocytes are not functional and therefore high values ​​in myelin production are not reached (Czepiel M, Balasubramaniyan V, Schaafsma W, Stancic M, Mikkers H, Huisman C, et al.).Differentiation of induced pluripotent stem cells into functional oligodendrocytes. Glia [Internet]. Jun 2011; 59(6):882-92. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 21438010; Tracy ET, Zhang CY, Gentry T, ηοβΑηη / ίζηζ / Ε / γίΛΐ. Shoulars KW, Kurtzberg J. Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood. Cytotherapy [Internet]. Jul 2011; 13(6):722-9. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 21341973; Zhang Y, Lu XY, Casella G, Tian J, Ye ZQ, Yang T, et al. Generation of Oligodendrocyte Progenitor Cells From Mouse Bone Marrow Cells. Front Cell Neurosci [Internet]. 2019; 13:247. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 31231194. Therefore, the search for culture methods that allow the maturation of oligodendrocytes and achieve high levels of myelin expression remains a challenge. It is very important to have alternatives that allow the use of oligodendrocyte therapies, since there are a large number of diseases that damage this oligodendrocyte-myelin-axon interaction. This is the case with demyelinating diseases and other more serious conditions such as spinal cord injury. Multiple sclerosis (MS) is a chronic, often progressive, inflammatory disease of the central nervous system (CNS) characterized pathologically by primary demyelination, generally without initial axonal injury. The etiology and pathogenesis of MS are unknown. Several immunological features of MS, and its moderate association with certain alleles of major histocompatibility complexes, have led to speculation that MS is an autoimmune disease and may originate through an allergic trigger; where the injection of certain myelin components into genetically susceptible animals leads to T-cell-mediated CNS demyelination. However, specific autoantigens and pathogenic myelin-reactive T cells have not been definitively identified in the CNS of MS patients, nor is it known whether MS is associated with other autoimmune diseases (International Multiple Sclerosis Genetics Consortium).Multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility. Science (80) [Internet]. 27 September 2019; 365(6460): eaav7188. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 3160424). ηοβρηη / ίζηζ / Ε / γίΛΐ Spinal cord injury (SCI) refers to damage to the spinal cord caused by an external mechanism. It is a chronic, and so far irreversible, disease characterized by the degeneration of nerve tissue. Symptoms range from the loss of motor and / or sensory function in the limbs and trunk to impaired breathing, urination, and digestion, which can be life-threatening for patients. SCI generates high costs for healthcare systems and is a devastating disease for patients and their families, with collateral damage to society (Alizadeh A, Dyck SM, Karimi-Abdolrezaee S. Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. Front Neurol [Internet]. March 22, 2019; 10:282. Available online: https: / / www.frontiersin.Org / article / 10.3389 / fneur.2019.00282 / ful I; Badhiwala JH, Wilson JR, Fehlings MG. Global burden of traumatic brain and spinal cord injury. Lancet Neurol [Internet]. January 1, 2019; 18(1):24-5. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 30497967). The limited efficacy of current therapies for MS, demyelinating diseases, and spinal cord injury (SCI) has spurred interest in new therapies to improve these conditions. However, as mentioned in US7807166B2, due to the seemingly complex etiopathogenesis of these diseases, which potentially involves both environmental and autoimmune factors, there remains a need for effective treatment of these demyelinating disorders. Cell-based regenerative therapies are a field currently being explored, as transplanted cells are capable of fulfilling many roles, including providing support, modulating the inflammatory response, regenerating lost neuronal circuits, and remyelinizing axons. For example, mesenchymal stem cells stand out for being pluripotent, meaning they are capable of differentiating into various cell types, including cells of the central nervous system (CNS) or peripheral nervous system (PNS) (Lotfy A, Ali NS, Abdelgawad M, Salama M. Mesenchymal stem cells as a treatment for multiple sclerosis: a focus on experimental animal studies. Rev ηοβΑηη / ίζηζ / E / γίΛΐ Neurosci [Internet]. 12 de octubre de 2019; 0(0). Disponible en línea: http: / / www.ncbi.nlm.nih.gov / pubmed / 31605598; Gene B, Bozan HR, Gene S, Gene K. Stem Cell Therapy for Múltiple Sclerosis. In: Advances in experimental medicine and biology [Internet]. 2018. p. 145-74. Disponible en línea: http: / / www.ncbi.nlm.nih.gov / pubmed / 30039439; Mi R, Tammia M, Shinn D, Li Y, Martin R, Mao H, et al. Oligodendrocyte precursors gain Schwann cell-like phenotype and remyelinate axons upon engraftment into peripheral nerves. J Tissue Eng Regen Med [Internet]. 30 de julio de 2019; term.2935. Disponible en línea: https: / / onlinelibrary.wiley.eom / doi / abs / 10.1002 / term.2935). There are numerous patent applications based on cell therapy strategies with the aim of remyelinizing neuronal axons, such as the Chinese patent CN103396993B, which seeks to repair spinal cord injuries through primate embryonic stem cells using the supplement B27, insulin, progesterone, putrescine dihydrochloride, sodium selenite, transferrin, FGFb, EGF and T3. Laree Hiser's patent number US20100069254A1 proposes to demyelinate and remyelinate structures using mouse embryonic cells by inducing them with leukemia inhibitory factor and 2-mercaptoethanol, retinoic acid, neurobasal medium supplemented with B27, and glutamine. US patent 7098027B2 describes methods for obtaining stem cells from various sources (bone marrow cells, umbilical cord blood cells, or embryonic liver tissue) that can differentiate into neural cells and cell fractions containing such cells. The team hopes that these cells and cell fractions can be used to treat neurological diseases, particularly in autologous transplant therapy. US patent application 20200197487A1 relates to a system that uses genetically induced adult neural stem cells (NSCs) to simultaneously produce a cocktail of signaling factors containing IL-10, neurotrophin 3 (NT-3), and soluble protein noRAnn / Lznz / E / YiAi LINGO-1 (LINGO-I-Fc), under the control of the Tet-on system for the purpose of treating multiple sclerosis and other demyelinating diseases. The FDA refers to regenerative medicine advanced therapy (RMAT) as therapy that: • Meets the definition of regenerative medicine therapy which includes cell therapies, tissue-engineered therapeutic products, human cell and tissue products, and combination products that use such therapies or products. • It is intended to treat, modify, reverse, or cure a serious condition; and preliminary clinical evidence indicates that regenerative medicine therapy has the potential to address unmet medical needs for such a condition. (Guidance for Industry, Expedited Programs for Regenerative Medicine Therapies for Serious Conditions 2019 FDA) (Expedited Programs for Regenerative Medicine Therapies for Serious Conditions | FDA [Internet], [accessed October 13, 2019]. Available online: https: / / www.fda.gov / regulatory-information / search-fda-guidancedocuments / expedited-programs-regenerative-medicine-therapies-seriousconditions). Unlike what is disclosed in the prior art, the present invention allows the differentiation of human adipose tissue mesenchymal stem cells (hAT-MSCs) into oligodendrocyte-like cells (OLCs, abbreviation used throughout the detailed description of the invention) by culturing them with neuron-like cells (NLCs, abbreviation used throughout the detailed description of the invention), which are capable of remyelinating peripheral nerves, where the peripheral nerve scaffolds provide a support structure for cell growth that guides axonal regeneration, employing the principles of the characteristics of an ideal scaffold: having a structure similar to the spinal cord, facilitating cell adhesion, and having compatibility with the cell component noRAnn / ί�ηζ / E / γίΛΐ (Girolamo F, Strippoli M, Errede M, Benagiano V, Roncali L, Ambrosi G, et al.Characterization of oligodendrocyte lineage precursor cells in the mouse cerebral cortex: a confocal microscopy approach to demyelinating diseases. Ital J Anat Embryol [Internet]. 2010; 115(1-2):95-102. Available online:. (http: / / www.ncbi.nlm.nih.gov / pubmed / 21072997). Thanks to numerous studies, the use of cell-free scaffolds, derived from nerve tissue and prepared through chemical methods, has become increasingly popular. In these scaffolds, the cellular component is removed, while the extracellular matrix (ECM) is preserved. Among the advantages offered by this technology are: unlimited availability, low immunogenicity, and an intact three-dimensional structure similar to that of nerve tissue. The compositions and methods of the invention can also be applied to promote remyelination in demyelinating conditions of the peripheral nervous system or to reconnect and repair lesions thereof, such as multiple sclerosis, spinal cord injury, brachial plexus injuries, and injuries from trauma or compression of peripheral nerves. Because nerve transfers (autografts) have suboptimal results that are evident only in the long term (after several years and with rehabilitation), and because commercially available scaffolds do not surpass autografts (Fathi SS, Zaminy A. Stem cell therapy for nerve injury. World J Stem Cells [Internet]. September 26, 2017; 9(9):144. Available from: http: / / www.ncbi.nlm.nih.gov / pubmed / 29026460). BRIEF DESCRIPTION OF THE FIGURES Figure 1. Diagram showing the steps of the method for obtaining a co-culture of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) with enhanced differentiation capacity and their use in a decellularized peripheral nerve. Figure 2. Morphological characteristics and surface markers in OLCs and NLCs differentiated from MCC-TAh. A) Unstained third-pass MCC-TAh. B) OLCs cultured in Matrigel. C) Myelin basic protein (MBP) immunohistochemistry in MCC-TAh cultures differentiated to OLCs (positive cells are observed as brown and indicated by a white arrow). D) Neurofilament (NF) immunohistochemistry in MCC-TAh cultures differentiated to NLCs (positive cells are brown and indicated by a yellow arrow). E) MCP immunohistochemistry in co-cultures of OLCs (positive cells are brown and indicated by a white arrow) and NLCs (yellow arrow). Figure 3. Comparative graph of the expression of the characteristic markers of OLCs (PBM and PLP) and NLCs (NF) in individual and co-culture (present invention). Figure 4. Morphological characteristics and surface markers NF and MBP in OLCs and NLCs differentiated from CMM-TAh. The first row shows the expression of the surface marker NF in NLC (20X), NLC (40X), co-culture (20X) and CMM-TAh (positive cells are observed in brown), while the second row shows the expression of the surface marker MBP in OLC (20X), OLC (40X), co-culture (20X) and CMM-TAh (positive cells are observed in brown). Figure 5. Recellularization of sciatic nerves with oligodendrocytes and neurons (10:1) co-cultured for 3, 7, and 21 days. A) Decellularized nerve and B) recellularized nerve with DAPI-labeled OLCs and NLCs (4x). C) Comparative graph of PBM marker levels between demyelinated and remyelinated nerves. The DF series shows the PBM titration of control (non-decellularized) nerves. The Gl series represents the result of the present invention: recellularized (with OLCs / NLCs co-culture) and remyelinated nerves, where the PBM titration is evident over time. The JL series shows demyelinated nerves labeled with PBM. Images at 15X magnification. The positive signal is observed in different intensities of brown. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining a co-culture of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) with enhanced differentiation capacity, culture medium and its use as a re-myelinating neuroregenerative graft of peripheral nerves, wherein certain compositions and combinations of matter have been identified that can act as re-myelinating agents. The method for obtaining a co-culture of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) with enhanced differentiation capacity comprises the following steps: a) Take individual cultures of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) and perform a co-culture in a 10:1 ratio of OLCs and NLCs in a co-culture medium consisting of DMEM-F12 supplemented with 1% fetal bovine serum (FBS), 35 ng / mL triiodothyronine (T3), 100 ng / mL basic fibroblast growth factor (bFGF), 5 ng / mL platelet-derived growth factor (PDGF-AA), 200 ng / mL heregulin (HRG) and 10 μM forskolin at 37°C in an atmosphere with 5% CO2 for 7 days to increase the degree of differentiation of both cell populations. The present method includes a co-culture medium called DMEM / F12co comprising: DMEM-F12 supplemented with 1% Fetal Bovine Serum (FBS), 35 ng / mL of triiodothyronine (T3), 100 ng / mL of basic fibroblast growth factor (bFGF), 5 ng / mL of platelet-derived growth factor (PDGFAA), 200 ng / mL of heregulin (HRG) and 10 μM of Forskolin and allows increasing the degree of differentiation of both cell populations, compared to the expression of myelin proteins of the individual cultures. Finally, the present invention describes the use of OLC and NLC co-culture as a re-myelinating neuroregenerative graft of peripheral nerves for the treatment of demyelinating diseases or repair of peripheral nervous system (PNS) lesions or spinal cord injuries. noRAnn / Lznz / E / YiAi EXAMPLE 1. OBTAINING A CO-CULTURE OF OLIGODENDROCYTE-LIKE CELLS (OLCs) AND NEURON-LIKE CELLS (NLCs) WITH ENHANCED DIFFERENTIATION CAPACITY. Figure 1 shows a general diagram of the stages of the entire process of obtaining a co-culture of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) with enhanced differentiation capacity and its use, which is described below: Obtaining mesenchymal stem cells derived from human adipose tissue (CMM-TAh) The present invention requires the prior isolation of mesenchymal stem cells obtained from human adipose tissue (MSC-HT) by mechanical and enzymatic disaggregation, and their expansion to the third passage. The MSC-HT are obtained from abdominal fat aspirate via liposuction, disaggregated, and characterized using protocols already described in the literature, such as surface markers positive for CD105 and CD90 and negative for CD45; and multilineage differentiation into osteocytes, adipocytes, and chondrocytes. The isolation and characterization are described below: Isolation of MSC-TAh. Lipoaspirate (35 mL) was taken from a healthy adult who underwent plastic surgery and voluntarily donated the sample. It was incubated with 5 mL of a collagenase I solution (200 IU / mL) (Sigma-Aldrich, Mexico) at 37°C in a 5% CO2 atmosphere for 1 h. The sample was homogenized with a Pasteur pipette, and the cells were separated from tissue debris and adipose cells by centrifugation at 1000 × g for 15 min at 21°C. The supernatant was discarded. The cells were washed twice, resuspended in 2 mL of phosphate-isotonic saline solution (PBS), pH 7.4, and centrifuged at 1000 × g for 15 min. The cells were resuspended in 2mL of DMEM / F12 culture medium (Sigma-Aldrich of Mexico) supplemented with 10% fetal bovine serum ([FBS] v / v), 2.5 pg / ml of amphotericin B and 100 pg / ml of gentamicin (w / v) (this medium will henceforth be referred to as complete medium).The cells were seeded in 25 cm2 polystyrene bottles with a gas-permeable stopper (CORNING® noRAnn / ίζηζ / E / γίΛΐ. Corning Incorporated, Corning, NY, USA) containing 5 mL of DMEM / F12. The cultures were incubated at 37°C in a 5% CO2 atmosphere for 48 h. Non-attached cells and used medium were transferred to a new bottle. To the original bottle containing the attached cells, 5 mL of fresh complete medium was added. Both bottles were incubated. This procedure was repeated once more, and the substrate-attached cells from all three plates (CMM-TAh) were incubated in a 5% CO2 atmosphere at 37°C until the CMM-TAh reached 80% confluence. Culture and expansion of CMM-TAh. Upon reaching 80% confluence, 1 mL of 0.25% trypsin in PBS was added to 25 cm² primary culture plates, and the plates were incubated at 37°C in a 5% CO₂ atmosphere for 5 min. The cells were placed in a 15 mL tube, 1 mL of complete medium was added, and the cells were pelleted at 4,000 x g. The cell pack was resuspended in 1 mL of fresh complete medium, and the cell suspension was divided into two aliquots. Each aliquot was placed in two new 25 cm² bottles; the volume of the bottles was brought up to 5 mL with fresh complete medium, and the bottles were incubated at 37°C in a 5% CO₂ atmosphere. Every third day, the used culture medium was replaced with an equal volume of fresh complete medium and when the CMM-TAh monolayers reached a confluence of 80%, the cells were reseeded for use and / or maintenance. Characterization. The human mesenchymal stem cells (HMSCs) were characterized by protocols already described in the literature with surface markers positive for CD105, CD90 and negative for CD45; multilineage differentiation to osteocytes, adipocytes and chondrocytes. (Jacobo Arreóla, Selene. (2015) Differentiation to oligodendrocytes of human mesenchymal stem cells originating in adipose tissue. Master's thesis. Autonomous University of Nuevo León. (available online: http: / / eprints.uanl.mx / 4204 / ). Figure 2A shows a microscopic photograph of HMSCs from the third unstained passage. noRAnn / Lznz / E / YiAi Obtaining individual cultures of oligodendrocyte-like cells and neuron-like cells Subsequently, the differentiation process of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) was carried out as previously reported by Abbaszadeh and Jang, respectively. Individual cultures of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) were obtained, and differentiation was induced using previously reported growth factors to achieve a differentiation percentage of at least 10.5% for myelin basic protein (MBP) expression and at least 15.46% for proteolipid protein (PLP) expression in OLCs; and at least 22.26% for neurofilament (NF) expression in NLCs (the limit of differentiation percentages reported in the prior art to date). Individual culture of oligodendrocyte-like cells (OLCs) The Abbaszadeh protocol (Abbaszadeh HA, Tiraihi T, Delshad AR, Saghedi Zadeh M, Taheri T. Bone marrow stromal cell transdifferentiation into oligodendrocyte-like cells using triiodothyronine as an inducer with expression of platelet-derived growth factor as a maturity marker. Iran Biomed J [Internet]. 2013;17(2):62-70. Available online: http: / / www.ncbi.nlm.nih.gov / pubmed / 23567847) was modified to obtain the OLCs; for this, the following methodology was followed: Pre-induction: The CMM-TAh obtained in the previous stage in the 3rd pass were seeded in a microchamber culture (Nunc™ Lab-Tek™ II Chamber Slide™ System, Thermo Scientific, NY, USA), placing 10,000 cells / cm2 in each of the four wells of the microchamber or in 25 cm2 flasks and 50,000 cells were also seeded in 35 mm x 10 mm petri dishes (Corning, NY, USA) to carry out the differentiation process. A medium was prepared containing 2% dimethyl sulfoxide (DMSO) (SIGMA, St. Louis, MO, USA) dissolved in DMEM / F12 medium (GIBCO®-BRL, Grand Island, NY, USA) without fetal bovine serum (FBS) (GIBCO®-BRL, Grand Island, NY, USA), with 2.5 pg / ml of amphotericin B. B and 100 pg / ml of gentamicin (w / v). 500 pL was added to each well of the microchamber and 1.5 mL to the petri dishes. The preparations were incubated at 37 °C in a 5% CO2 atmosphere for 24 h. The medium with DMSO was changed the following day to DMEM / F12 (GIBCO ®-BRL) with 15% FBS and 1 μM of total trans-retinoic acid (TTRA) (Calbiochem®, © Merck KGaA Darmstadt, Germany) and the microchambers were reincubated for 72 h under the same conditions as before. Induction. The pre-induction medium was removed from the microchambers and plates, which were then washed with PBS. The cells were incubated for 48 h in DMEM / F12 medium supplemented with 5 ng / mL of PDGF-AA, 10 ng / mL of bFGF and 200 ng / mL of HRG (PREPOTECH, INC., Rocky Hill, NJ, USA) for two days, and induction was performed with 35 ng / mL of T3 (Sigma-Aldrich) in addition to the previously added factors (induction medium). The cells were incubated in this medium for two to three days until oligodendrocyte-like cells (OLCs) were obtained. The culture media used in all cases were kept at 37°C for 2-24 h prior to their use on the cells to prevent them from detaching. Characterization. It was confirmed by RT-PCR that the OLCs derived from human mesenchymal stem cells (HMSCs) expressed oligodendrocyte-characteristic genes (Jacobo Arreóla, Selene. (2015) Differentiation to oligodendrocytes of human mesenchymal stem cells originating from adipose tissue. Master's thesis. Autonomous University of Nuevo León, (available online: http: / / eprints.uanl.mx / 4204 / ). A densitometric analysis of the nestin, CNPase, and PDGFR-α genes showed a significantly greater difference in oligodendrocytes than in uninduced HMSCs (p < 0.05). The expression of the Olig2, MOG, NG2, and O4 genes was not detected in either HMSCs or induced differentiation cells (IDCs). Meanwhile, the expression of the PBM and PLP genes was higher in cells similar to oligodendrocytes (OLCs) that in non-induced CMM-TAh (p<0.05), which allowed confirmation of obtaining oligodendrocyte-like cells (OLCs).A 3D culture of the cells differentiated into OLCs was performed, showing multiple fine extensions and a small, round body. For their preparation, DMEM / F12s and Matrigel (1:4) were used, incubating at 37°C in a 5% CO2 atmosphere for 3 days. The cells were then released from the Matrigel, and the cultures were incubated with 200 IU / mL of collagenase I (Sigma-Aldrich) for 4 hours, gently agitating with a pipette. Figure 2B shows a photograph of the OLCs cultured in Matrigel. An immunocytochemical test with anti-myelin basic protein (MBP) antibodies was performed to observe the differentiation of the cultures of CMM-TAh differentiated into OLCs. Positive cells appear brown and are indicated by a white arrow in Figure 2C. Individual culture of neuron-like cells (NLCs) Differentiation. For differentiation into neuron-like cells (NLCs), the CMM-TAh culture was exposed to a cocktail of neurotrophic factors according to the protocol described by Jang et al., (2010) (Jang, S., Cho, H. H., Cho, YB, Park, JS, & Jeong, HS (2010). Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin. BMC Cell Biology, 11. https: / / doi.org / 10.1186 / 1471-2121-11-25). The THA-MMCs were incubated in culture bottles containing DMEM / F12 medium with 1% fetal bovine serum (FBS), 100 ng / mL basic fibroblast growth factor (bFGF), 50 pg / mL gentamicin, and 50 pg / mL antifungal at 37°C in a 5% CO2 atmosphere for 7 days. To further neuronal differentiation, the cells were exposed to induction medium containing 1% BFS, 100 ng / mL bFGF, 10 pM forskolin, and 50 pg / mL DMEM / F12 and incubated at 37°C in a 5% CO2 atmosphere for 7 days. Characterization.Cells with neuron-like cell (NLC) morphology were subjected to immunohistochemical staining for identification and quantification of the typical marker Neurofilament (NF), a characteristic marker of neuronal lineages, where a differentiation percentage of at least 22.26% was obtained and Figure 2D shows CMM-TAh cultures differentiated to NLCs, NF-positive cells are brown and are indicated with a yellow arrow. ηοβΑπη / ίζηζ / Ε / γίΛΐ Co-culture of oligodendrocyte-like cells and neuron-like cells Once the individual cultures of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) were obtained, a co-culture was carried out in a 10:1 ratio of OLCs and NLCs in a co-culture medium described in the present invention to increase the degree of differentiation of both cell populations. The oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) (1x106 / 100,000) previously differentiated individually were cultured in a 10:1 ratio in each of the four compartments of a Chamber Slide System microchamber.Each compartment contained the cells in 1 ml of co-culture medium consisting of DMEM-F12 supplemented with (following the combination of factors at maximum concentration) 1% fetal bovine serum (FBS), 35 ng / mL triiodothyronine (T3), 100 ng / mL basic fibroblast growth factor (bFGF), 5 ng / mL platelet-derived growth factor (PDGF-AA), 200 ng / mL heregulin (HRG), 10 μM forskolin (referred to throughout this text as DMEM-F12 co-culture medium) at 37°C in an atmosphere with 5% CO2 for 7 days. The used medium was replaced with fresh medium every three days. For both individual cultures and for the co-culture of OLCs and NLCs, a morphometric analysis was performed by quantifying the cells that were positive or negative for staining with the anti-PBM, anti-PLP, and anti-NF antibodies. For this purpose, three slides were used, and eight fields were randomly quantified. Using these quantifications, the percentage of cells positive for each of the analyzed markers was calculated with respect to the total number of cells quantified, obtaining a degree of differentiation of at least 10.5% for myelin basic protein (MBP) expression and at least 15.46% for proteolipid protein (PLP) expression in OLCs; and at least 22.26% for neurofilament (NF) expression in NLCs.The factors added to the co-culture medium were greater than previously reported and these improved the degree of differentiation in both cell populations subjected to the method and culture medium described in the present invention to up to 81.54 ± 8.26% positivity for PBM, 89.28 ± 8.23% positivity for PLP in OLCs and 22.26 ± 5.12% for NF in NLCs. The results are shown in Figure 3, which allows us to conclude that a percentage of enhanced differentiation has been achieved for both cell populations. Figure 4 shows more detailed images of the morphological characteristics and surface markers NF and MBP in OLCs and NLCs differentiated from CMM-TAh in single and co-cultures. The first row shows the expression of the surface marker NF in NLCs (20X) and NLCs (40X), in co-culture (20X), and in CMM-TAh (positive cells appear brown), while the second row shows the expression of the surface marker MBP in OLCs (20X), OLCs (40X), in co-culture (20X), and in CMM-TAh (positive cells appear brown). Recellularization of peripheral nerves Once the co-culture of OLCs and NLCs was obtained, peripheral nerve recellularization was performed to confirm the co-culture's capacity to serve as a remyelinating neuroregenerative graft using rat and human peripheral nerves as a 3D scaffold. Scaffold preparation first required decellularization with detergents. To confirm the absence of nuclei or cellular debris that could indicate graft rejection, histological sections of some nerves were examined before and after the decellularization protocol using H&E and DAPL staining. Finally, the myelinating capacity of the OLC and NLC co-culture, when implanted into a decellularized rat and human peripheral nerve, was evaluated using various factors on days 3, 7, and 21. The obtained nerves were decellularized and demyelinated following the protocol of Sondell et al. (1998) using the chemical detergents 4% deoxycholate and 3% Triton. The nerves were cleaned, removing as much connective tissue as possible. This tissue was handled as infectious biological hazardous waste (RPBI), as indicated by NOM-087-ECOL-SSA1-2002, under sterile conditions. The nerves were cut into fragments of approximately 20 mm and decellularized according to the method described (Sondell M, Lundborg G, Kanje M. Regeneration of the rat sciatic nerve into allografts made acellular through Chemical extraction. Brain Research. 1998; 1;795.p 44-54. https: / / doi.org / 10.1016 / s0006-8993(98)00251-0).Briefly: The nerve segments were washed with sterile distilled water for 10 hours, replacing the used water with fresh water every 2 hours. At room temperature, they were then immersed in 3% Triton™ X-100 overnight and in 4% sodium deoxycholate for 24 hours. This process was repeated for 15 days with constant agitation at 500 rpm. The material was washed with distilled water and stored in PBS at 4°C until use. Decellularized and control (unprocessed) nerves were fixed with 2.5% glutaraldehyde and stained with hematoxylin and eosin (H&E) to visualize cell nuclei. Type I collagen fibers (blue) were visualized using Masson's trichrome stain, contrasting with the red cytoplasm and the lilac / brown nucleus. Immunohistochemistry was performed to assess the degree of nerve demyelination using myelin basic protein (MBP) antibody (1:250, Abcam), and nuclei were stained with DAPI using fluorescence microscopy. Once it was confirmed that more than 90% of the fields lacked nuclei, these were used as scaffolds to evaluate the co-culture medium after the nerves were recellularized with oligodendrocyte-like cells and neurons. 50 pL of a cell suspension (as a cell graft) containing a co-culture of viable oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) in a 10:1 ratio (1 x 10⁶ / 100,000) in DMEM / F12co medium was gently injected into previously decellularized human and rat nerves using an insulin syringe. Immediately afterward, the recellularized nerves were incubated separately in co-culture medium (DMEM / F12co) at 37°C in a 5% CO₂ atmosphere for 3, 7, and 21 days in 24-well plates. The used co-culture medium was replaced with fresh medium every three days. The nerves were fixed with Carnoy's solution (ethanol, acetic acid, chloroform, 6:3:1, v / v) for 24 h. Transverse and longitudinal sections were obtained from these preparations, which were stained with H&E, and labeled by immunohistochemistry, using monoclonal antibodies specific for oligodendrocytes and neurons.All experiments were performed in triplicate at each of the time points used. Density analysis. Image J and Graphpad Prism software were used to measure the density units in control nerves and experiments for subsequent comparative analysis. After culturing the nerves for 3, 7, and 21 days, remyelination of the structures was achieved, as shown in Figure 5. Figure 5A shows a typical histological section of a scaffold—decellularized sciatic nerve—stained with DAPI, where the complete absence of genetic material is evident. On the other hand, Figure 5B shows that 3 days after recellularizing a scaffold—obtained exactly like the empty scaffold in Figure 5A—with oligodendrocyte-like and neuron-like cells, the sections are filled with well-preserved cell nuclei (blue stippling), distributed homogeneously along the scaffold, indicating successful implantation within the nerve. Furthermore, Figures 4D-F,They show that when evaluating the presence of PBM in a nerve used as a positive control, it was detected that it has 74.3 ± 6.3 DUs (Optical Density Units) and that it is arranged in tracts in the longitudinal section, while when quantifying PBM in the demyelinated / decelled nerve, 0.47 ± 0.14 DUs were presented. On the other hand, after the implantation of the OLCs and NLCs co-culture of the present invention (cell graft), the PBM showed a value of 8.9 ± 0.88 DUs during the first 3 days, at 7 days it was 7.34 ± 0.11 and managed to detect PBM for up to 21 days, showing 5.10 ± 0.84 DUs (Figure 5C); This was a statistically significant increase compared to the demyelinated nerve (P < 0.001) on each of the days when a Student's t-test was performed. In the first 3 days, the myelin shows a disorganized noRAnn / izηζ / E / γίΛΐ distribution; as the days pass, it acquires a tract-like arrangement similar to that of the control.and remains so until day 21 (Figure 5C). Figure 5 also shows the DF series, the PBM titration of control (non-decellularized) nerves. The Gl series represents the result of the present invention, with respect to re-cellularized and remyelinated nerves with the OLCs / NLCs co-culture, where the PBM titration is evident over the days, and the JL series shows demyelinated nerves labeled with PBM. Images at 15X magnification. The positive signal is observed in different intensities of brown. No spontaneous remyelination was observed in the cell-free nerves used as a control with co-culture medium after immunohistochemical analysis at 21 days of culture. These results confirm that the OLCs and NLCs co-culture of the present invention has remyelinating capacity when tested in a three-dimensional culture.promoting its use as an agent that promotes remyelination of nerves for the treatment of demyelinating diseases such as neurodegenerative diseases, immune-mediated demyelinating diseases, hereditary and acquired metabolic disorders, post-infectious encephalitis, and demyelinating lesions, for example: multiple sclerosis, acute disseminated encephalomyelitis, transverse myelitis, Schilder's disease, Balo's disease, Alexander's disease, Canavan's disease, Cockayne syndrome, Pelizaeus-Merzbacher disease, optic neuritis, neuromyelitis optica, HTLV-I-associated myelopathy, hereditary leukoencephalopathy, Guillain-Barret syndrome, central pontine myelosis, deep white matter ischemia, progressive multifocal leukoencephalopathy, HIV demyelinating encephalitis, demyelinating radiation injury, acquired toxic-metabolic disorders, syndrome of posterior reversible encephalopathy,metachromatic leukodystrophy.

Claims

1. A method for obtaining a co-culture of oligodendrocyte-like cells and neuron-like cells with enhanced differentiation capacity, characterized in that it comprises the step of: a) Taking individual cultures of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) and performing a co-culture in a 10:1 ratio of OLCs and NLCs in a co-culture medium consisting of DMEM-F12 supplemented with 1% fetal bovine serum (FBS), 35 ng / mL of triiodothyronine (T3), 100 ng / mL of basic fibroblast growth factor (bFGF), 5 ng / mL of platelet-derived growth factor (PDGF-AA), 200 ng / mL of heregulin (HRG), and 10 μM of forskolin at 37°C in an atmosphere with 5% CO2 for 7 days to increase the degree of differentiation of both cell populations.

2. The method according to claim 1, characterized in that in step a) the individual culture of oligodendrocyte-like cells (OLCs) has a differentiation percentage of at least 10.5% for the expression of myelin basic protein (MBP) and at least 15.46% for the expression of proteolipid protein (PLP).

3. The method according to claim 1, characterized in that in step a) the individual culture of neuron-like cells (NLCs) has a differentiation percentage of at least 22.26% for neurofilament (NF) expression.

4. The method according to claim 1, characterized in that the individual oligodendrocyte-like cell (OLC) culture is derived from noRAnn / Lznz / E / YiAi mesenchymal stem cells obtained from human adipose tissue (CMMTAh).

5. The method according to claim 1, characterized in that the individual culture of neuron-like cells (NLCs) is derived from mesenchymal stem cells obtained from human adipose tissue (CMMTAh).

6. A culture medium for enhancing the differentiation percentage of a co-culture of oligodendrocyte-like cells (OLCs) and neuron-like cells (NLCs) described in claim 1, characterized in that it comprises: DMEM-F12 culture medium supplemented with 1% fetal bovine serum (FBS), 35 ng / mL triiodothyronine (T3), 100 ng / mL basic fibroblast growth factor (bFGF), 5 ng / mL platelet-derived growth factor (PDGF-AA), 200 ng / mL heregulin (HRG), and 10 μM forskolin.

7. A co-culture of oligodendrocyte-like cells and neuron-like cells with enhanced differentiation capacity obtained by the method described in claims 1 to 5 characterized in that it has a degree of positivity of up to 81.54 ± 8.26% for myelin basic protein (MBP).

8. The co-culture according to claim 7 characterized in that it has a degree of positivity of up to 89.28% ± 8.23% for the proteolipid protein (PLP).

9. The co-culture according to claim 7 characterized in that it has a degree of positivity of up to 22.26 ± 5.12% for neurofilament (NF).

10. The co-culture of oligodendrocyte-like cells and neuron-like cells with enhanced differentiation capacity described in claims 7 to 9 for use as a remyelinating neuroregenerative graft for the treatment of demyelinating diseases or repair of peripheral nervous system (PNS) lesions or spinal cord lesions.

11. The use of co-culture according to claim 10 wherein the demyelinating diseases are selected from the list comprising: neurodegenerative diseases, immune-mediated demyelinating diseases, hereditary and acquired metabolic disorders, post-infectious encephalitis, and demyelinating lesions.

12. The use of co-culture according to claim 10 wherein said demyelinating disease is selected from the list comprising: multiple sclerosis, acute disseminated encephalomyelitis, transverse myelitis, Schilder's disease, Balo's disease, Alexander's disease, Canavan's disease, Cockayne syndrome, Pelizaeus-Merzbacher disease, optic neuritis, neuromyelitis optica, HTLV-I-associated myelopathy, hereditary leukoencephalopathy, Guillain-Barret syndrome, central pontine myelosis, deep white matter ischemia, progressive multifocal leukoencephalopathy, HIV demyelinating encephalitis, demyelinating radiation injury, demyelinating acquired toxic-metabolic disorders, posterior reversible encephalopathy syndrome, metachromatic leukodystrophy.