Methods for generating induced oligodendrocyte lineage cells and therapies using the same

Abstract

The present invention generally relates to a method for producing induced oligodendrocyte-lineage cells (OLGs) and using the cells for treatment. The induced oligodendrocyte-lineage cells (OLGs) can be used for cell therapy, particularly for demyelination diseases.

Description

[0001] Related applications

[0002] This application claims the benefit under Section 119 of the United States Patent Act (35 USC §119) of U.S. Provisional Application No. 62 / 684,963, filed on June 14, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention generally relates to methods of generating induced oligodendrocyte lineage cells (induced OLGs) and using the cells therapeutically. Background Art

[0004] The nervous system is extremely important for transmitting signals to control voluntary and involuntary behavior. The nervous system controls muscle activity, coordinates different tissues and organs, and receives information. Neurons transmit instantaneous and rapid signals to sense and respond to changes in the environment. The nervous system of vertebrates is divided into the central nervous system (CNS) and the peripheral nervous system (PNS). The central nervous system (CNS) includes the brain and spinal cord, while the peripheral nervous system (PNS) includes muscles and glands. 1 The central nervous system (CNS) integrates and coordinates all the information received from different parts of the body. The central nervous system can be divided into gray matter and white matter. Gray matter is composed of neurons and unmyelinated nerve fibers, while white matter is composed of myelinated axons and oligodendrocytes. 2 The retina, optic nerve, olfactory nerve, and olfactory epithelium are considered part of the central nervous system (CNS), with direct synaptic connections to the brain.

[0005] The central nervous system (CNS) and the peripheral nervous system (PNS) have different types of neurons and supporting cells. The central nervous system is mainly composed of nerve cells and glial cells. Glial cells are the most abundant cell type in the central nervous system (CNS). Calculated by cell nuclei, the number of glial cells is about 10 times that of nerve cells; calculated by volume, glial cells and nerve cells both occupy about half of the brain. Nerve cells are surrounded by glial cells and interact with each other. Glial cells in the central nervous system mainly include astrocytes, oligodendrocytes (oligodendrocyte-lineage cells (OLGs)), and microglia 3,4 In the peripheral nervous system (PNS), Schwann cells and satellite cells play the same role. Glial cells in the central nervous system (CNS) have different functions. 5,6For example, astrocytes (also known as astroglial cells) are a group of star-shaped glial cells that provide biochemical support and nutrition to neural tissue and maintain extracellular ion balance. As for microglia, resident macrophages can activate immune function to clear plaques, infectious agents, and unnecessary neurons. 3 Other cell types, such as oligodendrocytes, are crucial for myelination.

[0006] Oligodendrocyte precursor / progenitor cells (OPCs) differentiate into oligodendrocytes after neurogenic / glial cell transition 3 Oligodendrocytes (also known as oligodendrocytes) are a type of glial cell that functions primarily by providing support and insulation to nerve axons. Oligodendrocytes produce myelin sheaths to wrap around axons for insulation and to increase electrical conduction. Oligodendrocytes perform the same functions as neurosheath cells in the peripheral nervous system (PNS). 3 Regarding myelination, one oligodendrocyte can wrap around about 50 axons, but it can only form one myelin sheath to each axon segment. Nerve impulses move 100 times faster in myelinated axons than in unmyelinated neurons. 7 . In addition, oligodendrocyte lineage cells help nerve regeneration by providing nutrition and an appropriate environment. The main participants in the myelination process include myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP) proteins. In addition, surface antigen O4, transcription factors Olig2 and Nkx2.2 are expressed in oligodendrocyte lineage cells (OLGs). 8 .

[0007] Central nervous system diseases are diseases that affect the spinal cord or brain. Depending on the area damaged, damage to the brain or spinal cord can lead to different disabilities. Damage to the central nervous system (CNS) caused by structural defects originates from stroke, infection, degeneration, and autoimmune diseases. One type of autoimmune disease is caused by a loss of tolerance to the body's own proteins, which causes the immune system to attack and destroy body tissues. Damage to neurons or loss of function of nerve support cells in different parts of the central nervous system (CNS) can lead to different diseases. The causes of demyelinating diseases can be divided into five categories: viral, immune, genetic, toxic, and injury. Demyelinating diseases are caused by damage to the myelin sheath in the nervous system and occur in the central nervous system (CNS) and peripheral nervous system (PNS). Myelin loss or dysfunction affects 2 to 2.5 million people worldwide. It causes a lot of morbidity and mortality 9 Several diseases belong to the group of inflammatory demyelinating diseases in the central nervous system (CNS): multiple sclerosis (MS), acute hemorrhagical leukoencephalitis (AHL), and acute-disseminated encephalomyelitis (ADEM), central pontine myelinolysis, progressive multifocal leukoencephalopathy, cerebral palsy, congenital leukodystrophies. 10,11 Among these diseases, multiple sclerosis (MS) is a common autoimmune disease in which the immune system attacks myelin or oligodendrocyte lineage cells (OLGs) leading to systemic demyelination. 12-14 MS symptoms, depending on the area of damage, can cause vision, bladder, memory / thinking, pain, spasticity, speech problems, and swallowing. In severe cases, MS can also lead to stroke, muscle weakness, or death. 15 Multiple sclerosis (MS) is a disease characterized by relapses and remissions. The latency period between relapses and remissions is unpredictable and may take many years. 16 Other neuronal diseases also involve demyelination, such as Huntington's disease and schizophrenia. Huntington's disease is caused by an increase in the CAG repeats (polyglutamine region) in the huntingtin protein. It is an autosomal dominant neurodegenerative disease. 17 Some reports indicate that in the brains of Huntington's disease patients, glial cells degenerate and pathological phenotypes are observed. 18-22 Huntington's disease is characterized by myelin breakdown and damage. 20,22,23 Furthermore, demyelination has been documented in many Huntington's disease mouse models. 24-26In addition, radiation therapy has also produced demyelination in humans and mouse models. 27-29 and can oligodendrocyte precursor cell therapy 30 .

[0008] Cell therapy is a potential and promising treatment strategy for demyelinating diseases 12-14 Currently, approximately 115 clinical trials are investigating the efficacy of glial cell-based therapies (www.clinicaltrials.gov). Some studies have shown promising results by injecting normal oligodendrocyte lineage cells (OLGs) into damaged areas to encapsulate the demyelinated areas. 31-33 However, it is difficult to obtain sufficient primary human oligodendrocyte lineage cells (OLGs) for cell therapy. 31-33 Therefore, providing sufficient numbers of oligodendrocyte lineage cells (OLGs) is a major obstacle that needs to be addressed. Currently, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide a platform for obtaining functional oligodendrocyte lineage cells (OLGs) by ectopic expression of transcription factors or stepwise differentiation of soluble factors. 34-36 Currently, human embryonic stem cells (hESCs)-derived neural stem cells (NSCs) or mesenchymal stem cells (MSCs)-derived oligodendrocyte lineage cells (OLGs) have been shown to improve the symptoms of multiple sclerosis (MS) in animal experiments. 37,38 However, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have disadvantages such as the risk of teratoma. 39 Furthermore, the differentiation process is labor-intensive, costly, and requires 45 days. On the other hand, only mouse or rat oligodendrocyte lineage cells (OLGs), not human cells, can be derived from fibroblasts transduced with transcription factor viruses, which carries the risk of insertional mutagenesis. 40,41 Furthermore, transcription factor-based reprogramming in rodent cells only achieved an efficiency of 9.2% or 15.6% of cells exhibiting O4 labeling. Summary of the Invention

[0009] The present invention discloses for the first time that induced oligodendrocyte lineage cells (OLGs) can be successfully generated by exposing skin cells, such as fibroblasts, to a chemical inducer comprising a Rho-associated protein kinase (ROCK) inhibitor and one or more auxiliary agents selected from the group consisting of a histone deacetylase (HDAC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a protein kinase C (PKC) inhibitor, and a cyclic adenosine monophosphate (cAMP) activator. According to the present invention, skin cells, such as fibroblasts, can be reprogrammed into induced oligodendrocyte lineage cells (OLGs) after such treatment, which can be used for cell therapy, particularly for demyelination diseases.

[0010] Therefore, in one embodiment, the present invention particularly provides a method for producing induced oligodendrocyte lineage cells (OLGs), comprising culturing the skin cells under conditions that allow a portion of the skin cells to be reprogrammed into induced oligodendrocyte lineage cells (OLGs), wherein the conditions include a culture medium containing a chemical inducer, the chemical inducer including a Rho-associated protein kinase (ROCK) inhibitor, and wherein the skin cells are also treated with an auxiliary agent selected from the group consisting of: a histone deacetylase (HDAC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a protein kinase C (PKC) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, and any combination thereof.

[0011] In some embodiments, the skin cells are fibroblasts.

[0012] In some embodiments, the adjuvant comprises the cyclin-dependent kinase (CDK) inhibitor.

[0013] In some embodiments, the adjuvant comprises the histone deacetylase (HDAC) inhibitor and the cAMP activator, optionally together with the cyclin-dependent kinase (CDK) inhibitor.

[0014] In some embodiments, the chemical inducer and the adjuvant are added to the culture medium simultaneously or sequentially. In some instances, the adjuvant is added to the culture medium before the chemical inducer. In some instances, the histone deacetylase (HDAC) inhibitor is added to the culture medium before the Rho-associated protein kinase (ROCK) inhibitor.

[0015] In some embodiments, the chemical inducer and / or the adjuvant is a small molecule.

[0016] In some embodiments, the chemical inducer comprises Y27632. In some embodiments, the adjuvant comprises forskolin (FSK), SU9516, VPA, and / or G06983.

[0017] In some embodiments, the induced oligodendrocyte lineage cells (OLGs) express a cell marker selected from the group consisting of platelet-derived growth factor receptor (PDGFR), myelin basic protein (MBP), oligodendrocyte transcription factor (Oligo2), SOX10, proteolipid protein (PLP) 1, A2B5, O4, and any combination thereof.

[0018] In some embodiments, the method of the present invention further comprises isolating the induced oligodendrocyte lineage cells (OLGs) from the cell culture to obtain an isolated population of induced oligodendrocyte lineage cells (OLGs).

[0019] In another embodiment, the present invention provides an isolated population of induced oligodendrocyte lineage cells (OLGs) as described herein.

[0020] In another embodiment, the present invention provides a method for treating a disease or condition, comprising administering a therapeutically effective amount of induced oligodendrocyte lineage cells (OLGs) to a subject in need of such treatment. Specifically, the induced oligodendrocyte lineage cells (OLGs) are derived from skin cells by treatment with a chemical inducer including a Rho-associated protein kinase (ROCK) inhibitor, wherein the skin cells are further treated with one or more adjuvants selected from the group consisting of: histone deacetylase (HDAC) inhibitors, cyclin-dependent kinase (CDK) inhibitors, protein kinase C (PKC) inhibitors, and cyclic adenosine monophosphate (cAMP) activators. In some specific embodiments, the adjuvant includes the cyclin-dependent kinase (CDK) inhibitor. In some instances, the (plural) adjuvant includes the histone deacetylase (HDAC) inhibitor and the cyclic adenosine monophosphate (cAMP) activator, optionally with the cyclin-dependent kinase (CDK) inhibitor. Also provided is a use of the induced oligodendrocyte lineage cells (OLGs) as described herein in the preparation of a medicament for treating a disease or disorder.

[0021] In some embodiments, the disease or disorder is associated with oligodendrocyte dysfunction.

[0022] In some embodiments, the disease or disorder is a demyelinating disease.

[0023] In some embodiments, the induced oligodendrocyte lineage cells (OLGs) are administered in an amount effective to promote neuronal myelination in the subject.

[0024] In some embodiments, the disease or disorder is selected from the group consisting of multiple sclerosis (MS), acute hemorrhagic inflammatory disease (AHL), cerebral palsy, acute disseminated encephalomyelitis (ADEM), central pontine myelinolysis, progressive multiple leukoencephalopathy, congenital leukodystrophy, Parkinson's disease, Huntington's disease, schizophrenia, and radiation-induced demyelination.

[0025] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of the induced oligodendrocyte lineage cells (OLGs) as described herein and a pharmaceutically acceptable carrier.

[0026] Also provided is a culture comprising skin cells and a culture medium comprising a chemical inducer and one or more auxiliary agents as described herein. In particular, the culture further comprises induced oligodendrocyte lineage cells (OLGs) derived from the skin cells.

[0027] The following description sets forth details of one or more specific embodiments of the present invention. Other features and advantages of the present invention will become apparent from the following description of several embodiments and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The foregoing summary of the invention and the following embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangements and means shown.

[0029] In the diagram:

[0030] Figures 1A-1B The figure shows the morphological changes of primary human skin fibroblasts induced by SU9516 and Y27632. SU9516 induces a dome-shaped morphology ( Figure 1A ), while Y27632 induced changes in dendritic morphology ( Figure 1B ).

[0031] Figure 2A and 2B Y27632 and SU9516 reprogrammed the fibroblasts into induced oligodendrocyte lineage cells (OLGs). Figure 2A Shown are Y27632 (Y) and SU9516 (S) conversion of fibroblasts into oligodendrocyte lineage cells (OLGs) with dendritic morphology. Figure 2B Shown is the expression of oligodendrocyte-specific marker (O4) in the induced cells by fluorescent staining. DAPI (4',6-diamidino-2-phenylindole) was used to stain the cell nuclei.

[0032] Figure 3 Shown are Go6983, Y27632, and SU9516 that enhance the reprogramming of inducible oligodendrocyte lineage cells (OLGs). Treatment with different combinations of Y27632 (Y), Go6983 (G), and SU9516 (S) demonstrates that each of these factors promotes oligodendrocyte lineage cell (OLG)-like morphology, dendritic outgrowth, and dome formation. Y: Y27632, S: SU9516, G: Go6983.

[0033] Figures 4A-4B It was shown that the efficiency of reprogramming was effectively enhanced by pretreatment with VPA. Figure 4A Shown is the efficiency with which pretreatment with VPA for 2 days can improve transformation efficiency. Figure 4BShown is fluorescence staining of induced cells to detect the oligodendrocyte-specific marker O4. DAPI was used to stain cell nuclei. V: VPA, Y: Y27632, S: SU9516, G: Go6983.

[0034] Figure 5 Shown are morphological changes in induced oligodendrocyte lineage cells (OLGs) induced by forskolin (FSK). Following pretreatment with VPA, 22 chemical compounds were tested to screen for drugs that promote reprogramming of YSGs. The data show that forskolin (FSK) can maintain the domed shape of cells and induce cells with more dendritic structures. V: VPA, Y: Y27632, S: SU9516, G: Go6983.

[0035] Figure 6A Shown are the expressions of various oligodendrocyte markers in induced oligodendrocyte lineage cells (OLGs) treated with five chemical compounds. Cells induced by the chemical mixture (VYSGF) expressed PDGFRα (an oligodendrocyte marker), myelin basic protein (MBP), Olig2, and O4 (oligodendrocyte-specific markers). Y: Y27632, S: SU9516, G: Go6983, F: forskolin, V: valproic acid.

[0036] Figure 6B Shown are qPCR results showing increases in oligodendrocyte-specific genes. mRNA expression of the transcription factors SOX10, myelin basic protein (MBP), and PLP1 is increased in induced cells compared to fibroblasts.

[0037] Figure 7 This study demonstrates that Go6983 is dispensable for the transformation process. Cells were treated with various chemical combinations, including VPA, Y27632, FSK, Go6983, or SU9516. The results show that VPA, Y27632, FSK, and SU9516 induce oligodendrocyte-like morphology, while Go6983 is dispensable for the transformation process.

[0038] Figure 8 This figure shows that a low dose of the 4C chemical cocktail (10 μM of Y27632, FSK, and SU9516, 3 mM of VPA) is sufficient for the transformation process. Cells were treated with various concentrations of Y27632, FSK, and SU9516 in the 4C chemical cocktail. The results show that 10 μM of these chemicals can induce the oligodendrocyte lineage cells (OLGs).

[0039] Figure 9AShown are markers for oligodendrocyte lineage cells (OLGs) induced by four chemical treatments. Cells induced by a small molecule (VYSF) express the oligodendrocyte lineage cell (OLG) markers Olig2, O4, and PDGFRa. Y: Y27632, S: SU9516, F: Forskolin, V: Valproic acid.

[0040] Figure 9B Compared to fibroblasts, iOLGs express oligodendrocyte markers. The cells were induced using a chemical cocktail (4C, VYSF). The data show that iOLGs express oligodendrocyte-specific markers GalC, GPR17, myelin basic protein (MBP), and O1.

[0041] Figure 9C Shown are qPCR results showing increases in different oligodendrocyte-specific genes: Sox10, myelin basic protein (MBP), and PLP1. mRNA expression shows that the transcription factor SOX10, myelin forming proteins myelin basic protein (MBP), and PLP1 are increased in iOLGs (4C, VYSF) compared to fibroblasts.

[0042] Figure 10 This image shows that induced oligodendrocyte lineage cells (OLGs) myelinate neurons. Co-culture of induced oligodendrocyte lineage cells (OLGs) with mouse DRG cells revealed colocalization of the oligodendrocyte-specific marker myelin basic protein (MBP) and the neural marker neurofilament (NF), demonstrating that induced oligodendrocyte lineage cells (OLGs) have the ability to myelinate.

[0043] Figure 11 This image shows that induced oligodendrocyte lineage cells (OLGs) have the ability to proliferate. Immunofluorescence staining shows that O4-positive cells also express the proliferation marker Ki67.

[0044] Figure 12A 、 12B 12C and 12C show that induced oligodendrocyte lineage cells (OLGs) express oligodendrocyte precursor cell markers A2B5 and O4. Figure 12A Shown is increased A2B5 expression in induced oligodendrocyte lineage cells (OLGs) compared to fibroblasts. Figure 12B Shown is increased O4 expression in induced oligodendrocyte lineage cells (OLGs) compared to fibroblasts. Figure 12C Shown are the quantitative results of A2B5 and O4 expression analyzed by flow cytometry.

[0045] Figures 13A-13BShown that VPA can be replaced by other histone deacetylase (HDAC) inhibitors to generate inducible oligodendrocyte lineage cells (OLGs). Figure 13A Shown are that motinositol and pasinositol can replace VPA and induce fibroblasts to reprogram into the more mature morphology of induced oligodendrocyte lineage cells (OLGs). Figure 13B Shown are the results of immunofluorescence staining, which show that cells induced with motinomastat and pasinomastat have stronger expression of oligodendrocyte markers O4 and myelin basic protein (MBP).

[0046] Figure 14A and 14B Shown are 3C (three compounds)-induced iOLGs expressing the oligodendrocyte precursor cell marker A2B5. Figure 14A Shown is the quantification of oligodendrocyte marker A2B5 by flow cytometry. Cells treated with 3C (VPA, forskolin, Y27632) showed increased A2B5 expression compared to fibroblasts. Cells treated with 4C (VPA, forskolin, Y27632, SU9516) served as a positive control. Figure 14B Shown is immunofluorescence staining showing increased expression of the oligodendrocyte lineage cell (OLG) transcription factor Olig2 in cells treated with 3C. Cells treated with 4C served as a positive control.

[0047] Figure 15 Shown is a summary of the chemical conversion process of induced oligodendrocyte lineage cells (OLGs) from human dermal fibroblasts. DETAILED DESCRIPTION

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] 1. Definition

[0050] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" includes a plurality of such components and equivalents thereof known to those skilled in the art.

[0051] The terms "comprise" or "comprising" are generally used in the sense of including / comprising, which indicates that one or more features, ingredients or components are allowed to be present. The terms "comprise" or "comprising" include the terms "consists of" or "consisting of."

[0052] As used herein, the term "oligodendrocyte lineage cells (OLGs)" may include oligodendrocytes, oligodendrocytes, and oligodendrocyte precursors / pioneer cells (OPCs). Oligodendrocytes in the central nervous system (CNS) produce myelin, which wraps around nerve axons to insulate and increase electrical conduction, and is also important for axonal integrity and survival.

[0053] As used herein, the term "induced oligodendrocyte lineage cells (induced OLGs)" refers to oligodendrocyte lineage cell (OLGs)-like cells (i.e., cells with oligodendrocyte lineage cell (OLGs)-like characteristics) that are generated (or reprogrammed) from other cell types, such as skin cells.

[0054] As used herein, "skin cell" refers to a cell found in the skin, such as an epithelial cell or a fibroblast.

[0055] As used herein, the term "reprogramming" refers to the process of converting a cell into a different cell type with some different properties or biological function.

[0056] As used herein, the term "culture" refers to a population of cells cultured in a medium, preferably to maintain the cells alive or to allow the cells to grow.

[0057] As used herein, the term "small molecule" refers to an organic or inorganic molecule synthesized or found in nature, typically having a molecular weight of less than 10,000 g / mole, particularly less than 5,000 g / mole, particularly less than 2,000 g / mole, and particularly less than 1,000 g / mole. In some embodiments, a small molecule refers to a non-polymeric chemical molecule, such as a non-protein or nucleic acid.

[0058] As used herein, the term "about" refers to plus or minus 10% of the numerical value of the quantity to which it is applied. Thus, about 1% represents a range of 0.9% to 1.1%.

[0059] As used herein, a Rho-associated protein kinase (ROCK) inhibitor may refer to an agent that downregulates, reduces, or inhibits the amount and / or activity of a Rho-associated protein kinase. Examples of Rho-associated protein kinase (ROCK) inhibitors described herein include, but are not limited to, Y-27632, AS 1892802, GSK 269962, GSK 429286, H 1152 dihydrochloride, HA 1100 hydrochloride, OXA 06 dihydrochloride, RKI 1447 dihydrochloride, SB 772077B dihydrochloride, and the like.

[0060] As described herein, a cyclin-dependent kinase (CDK) inhibitor may refer to an agent that downregulates, reduces, or inhibits the amount and / or activity of a cyclin-dependent kinase. Examples of cyclin-dependent kinase (CDK) inhibitors as described herein include, but are not limited to, SU9516, PD-0332991, Roscovitine, SNS-032, Dinaciclib, Flavopiridol, AT7519, Flavopiridol, JNJ-7706621, AZD5438, MK-8776, PHA-793887, BS-181, Palbociclib, Palbociclib (PD0332991), sodium isethionate, A-674563, abemaciclib, BMS-265246, PHA-767491, Milciclib, R547, NU6027, P276-00, MSC2530818, Senexin A, LY2857785, LDC4297, ON123300, Kenpaullone, K03861, THZ1 2HCl, AT7519 HCl, Purvalanol A, Ro-3306, XL413, LDC000067, ML167, TG003, Ribociclib, Wogonin, BIO, AZD1080, 1-Azakenpullone, and others.

[0061] As used herein, a cyclic adenosine monophosphate (cAMP) activator may refer to an agent that increases the intracellular level of cyclic adenosine monophosphate (cAMP) compared to the background physiological intracellular level in the absence of the agent. Examples of cyclic adenosine monophosphate (cAMP) activators include, but are not limited to, forskolin, rolipram, NKH477, PACAP1-27, PACAP1-38, and others.

[0062] As used herein, a protein kinase C (PKC) inhibitor can refer to an agent that downregulates, reduces, or inhibits the amount and / or activity of a PKC kinase. Examples of PKC inhibitors as used herein include, but are not limited to, Go6976, Go66850, Go6983, schizotoxin, bisindolylmaleimide II, C-1, calreticulin C, bee venom, GF 109203X, dihydrosphinganine, chelerythrine, chloride, CGP 53353, CID 2858522, dihydrosphinganine, GF 109203X, Go 6976, Go 6983, [Ala107]-MBP (104-118), Ala 113 ]-MBP(104-118), (±)-palmitoylcarnitine chloride, PKC(19-36) (pseudosubstrate peptide; PKC inhibitor), PKC 412, PKC pseudosubstrate, Ro 32-0432 hydrochloride, sphingomyelin, D-erythro-sphingosine (synthetic), ZIP, Ro 31-8220 methanesulfonic acid, and others.

[0063] As used herein, a histone deacetylase (HDAC) inhibitor may refer to an agent that downregulates, reduces, or inhibits the amount and / or activity of histone deacetylase to remove acetyl groups from lysine residues on histones. Examples of histone deacetylase (HDAC) inhibitors include, but are not limited to, valproic acid (VPA, 2-propylpentanoic acid), Apicidin, CI 994, FK 228, LMK 235, M 344, MC 1568, MC 1742, MI 192, NCH 51, NSC 3852, PCI 34051, sodium 4-phenylbutyrate, Pyroxamide, SAHA, SBHA, Scriptaid, sodium butyrate, TC-H106, TCS HDAC6 20b, Trichostatin A, Tubacin, UF 010, Motinostat, Pasinostat, and others.

[0064] As used herein, the term "isolated or purified cell population" or "isolated or purified cells" refers to a preparation of cells that has been separated from other cellular components or other cells associated with the cells. For example, the isolated cells may have been removed from their natural environment or cell population, or may have been produced by the propagation of cells that have been removed from a cell population. When cells are described as "isolated" or "purified," it should be understood that they are not absolutely isolated or purified, but relatively isolated or purified. For example, a preparation containing isolated cells may contain cells at an amount of 0.5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the total number of cells in the preparation. In some specific embodiments, a preparation containing isolated cells may contain cells at an amount of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the total number of cells in the preparation.

[0065] As used herein, the term "subject" includes humans and non-human animals, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), or experimental animals (e.g., rats, mice, guinea pigs, etc.).

[0066] As used herein, the term "treating," when referring to therapeutic treatment, refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a disease, a sign or symptom of the disease, or an exacerbation of the disease, for the purpose of curing, healing, alleviating, slowing, altering, remedying, improving, enhancing, or affecting the disease, the sign or symptom of the disease, the disability caused by the disease, or the exacerbation of the disease. On the other hand, the term "treating" can refer to an application or process unrelated to the therapeutic treatment of a disease, such as the administration of one or more ingredients or agents to contact a cell to change its fate, for example, to revert to a different cell type.

[0067] As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient that confers a therapeutic effect in a treated subject. The therapeutically effective amount may vary depending on various factors, such as the route and frequency of administration, the body weight and type of individual receiving the drug, and the purpose of administration. As used herein, when referring to applications or procedures unrelated to the therapeutic treatment of a disease, the term "effective amount" may refer to the amount of an ingredient or agent used to achieve the intended purpose, for example, the amount of an ingredient or agent administered to contacted cells, such as fibroblasts, for the purpose of reprogramming.

[0068] 2. Use of Chemical Reagents to Generate Inducible Oligodendrocyte Lineage Cells (OLGs)

[0069] The present invention is based on the unexpected discovery that skin cells, such as fibroblasts, can be reprogrammed into induced oligodendrocyte lineage cells (OLGs) by culturing with chemical inducers and one or more auxiliary agents, without the need for genetic modification by transduction of transcription factors.

[0070] According to the present invention, skin cells can be cultured in a culture medium containing a chemical inducer, wherein the chemical inducer includes a Rho-associated protein kinase (ROCK) inhibitor and an auxiliary agent selected from the group consisting of: a histone deacetylase (HDAC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a protein kinase C (PKC) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, and any combination thereof, in an amount effective to induce reprogramming, so that the skin cells are converted into oligodendrocyte lineage cells (OLGs). In some embodiments, the auxiliary agent includes the cyclin-dependent kinase (CDK) inhibitor. In some embodiments, the auxiliary agent includes the histone deacetylase (HDAC) inhibitor and the cyclic adenosine monophosphate (cAMP) activator, and may optionally contain the cyclin-dependent kinase (CDK) inhibitor. The chemical inducer and the auxiliary agent(s) can be added to the culture medium simultaneously or sequentially. For example, the auxiliary agent is first added to the culture medium for pretreatment (pre-culture), and then the chemical inducer is added for subsequent culture. In some embodiments, the histone deacetylase (HDAC) inhibitor (e.g., VPA) is added to the culture medium before the Rho-associated protein kinase (ROCK) inhibitor (e.g., Y27632). The pretreatment (pre-incubation) time is about one-tenth (1 / 10), one-fifth (1 / 5), one-quarter (1 / 4), one-third (1 / 3), or longer of the total culture period. In one example, the pretreatment (pre-incubation) time is about two-thirds (2 / 3) of the total culture period.

[0071] Culture media suitable for culturing the skin cells according to the present invention are available in the art, such as DMEM, MEM, DMEM / F12, DMEM-depleted, or IMEM. Culture can be performed under normal conditions, such as 37°C and 1-10% CO2. Specifically, the culture medium can be serum-free.

[0072] In some embodiments, the culture medium used for transformation (transformation medium) contains cut-out DMEM, AlbuMAXI, N2 supplement, and nonessential amino acids (NEAA).

[0073] In some embodiments, the culturing is performed for at least 1 day or longer (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or longer), whereby a portion of the skin cells are converted into induced oligodendrocyte lineage cells (OLGs). In a specific example, the culturing is performed for 3 days or longer, including pretreatment with an adjuvant for about 2 days (two-thirds of the total culturing period) and culturing with a chemical inducer and optional adjuvant(s) for an additional day (one-third of the total culturing period).

[0074] In some embodiments, the culture medium may include one or more growth factors and / or culture supplements that facilitate oligodendrocyte differentiation. Examples of growth factors include, but are not limited to, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and neurotrophin-3 (NT3). Examples of culture supplements include, but are not limited to, N2 and B27.

[0075] Table A illustrates some examples of chemical inducers used herein.

[0076]

[0077]

[0078]

[0079] According to the present invention, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more of the skin cells in culture are reprogrammed into induced oligodendrocyte lineage cells (OLGs). In some embodiments, about 20% or more, such as 30% to 90% (e.g., 40% to 80%, 50% to 75%) of the skin cells in culture are reprogrammed into induced oligodendrocyte lineage cells (OLGs).

[0080] In a specific embodiment, the skin cells are first treated with a histone deacetylase (HDAC) inhibitor (e.g., VPA), then treated with a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y27632) and a cyclic adenosine monophosphate (cAMP) activator (FSK), and optionally further treated with a cyclin-dependent kinase (CDK) inhibitor (SU9516).

[0081] In certain embodiments, the skin cells are treated with a Rho-associated protein kinase (ROCK) inhibitor (eg, Y27632) and a cyclin-dependent kinase (CDK) inhibitor (SU9516).

[0082] 3. Skin cells for reprogramming

[0083] Skin cells such as fibroblasts can be used herein to generate the induced oligodendrocyte lineage cells (OLGs) of the present invention. Fibroblasts used for reprogramming herein can be obtained from neonatal or adult donors.

[0084] Skin biopsies can be obtained from suitable autologous or allogeneic donors by skin puncture or circumcision, and skin fibroblasts can be grown from the skin biopsies. Typically, a skin biopsy of approximately 4 mm can produce 15-20 million fibroblasts. In some embodiments, fibroblasts can be purchased commercially. Preferably, the fibroblasts used for conversion into induced oligodendrocyte lineage cells (OLGs) as used herein are of mammalian origin, most preferably of human origin.

[0085] 4. Inducible Oligodendrocyte Lineage Cells (OLGs)

[0086] According to the present invention, the generated induced oligodendrocyte lineage cells (OLGs) have oligodendrocyte lineage cell (OLGs)-like features. Specifically, the induced oligodendrocyte lineage cells (OLGs) have an oligodendrocyte lineage cell (OLGs)-like morphology (cell body is dome-shaped and solid, bipolar or multipolar branches). More specifically, the generated induced oligodendrocyte lineage cells (OLGs) can show typical oligodendrocyte lineage cell (OLGs) markers.

[0087] In some embodiments, the oligodendrocyte lineage cell (OLGs) marker is selected from the group consisting of platelet-derived growth factor receptor (PDGFR), myelin basic protein (MBP), oligodendrocyte transcription factor (OLIG2), SOX10, proteolipid protein (PLP) 1, glial fibrillary acidic protein (GFAP), Ki67, A2B5, O4, and any combination thereof.

[0088] After culturing in some specific embodiments, in order to further enrich the induced oligodendrocyte lineage cells (OLGs), the cells can be sorted using one or more oligodendrocyte lineage cells (OLGs) markers. Cell sorting can be achieved by various techniques known in the art. Examples of cell sorting techniques include fluorescence-activated cell sorting (FACS), immunoaffinity column separation, or immunomagnetic separation (MACS) or any technique that can obtain enrichment of a specific cell type based on physical characteristics (density) or structural characteristics (particularly specific antigens).

[0089] 5. Applications using induced oligodendrocyte lineage cells (OLGs)

[0090] Based on our findings, autologous or allogeneic induced oligodendrocyte lineage cells (OLGs) can be generated from accessible skin biopsies, which can be easily obtained in the clinic. This process does not require surgery or any other painful procedures.

[0091] Induced oligodendrocyte lineage cells (OLGs) can effectively promote neuronal myelination and thus can be used for treatment, particularly for the treatment of demyelinating diseases or diseases or conditions associated with oligodendrocyte dysfunction.

[0092] Examples of such diseases or disorders include, but are not limited to, multiple sclerosis (MS), acute hemorrhagic inflammatory disease (AHL), cerebral palsy, acute disseminated encephalomyelitis (ADEM), central pontine myelinolysis, progressive multiple leukoencephalopathy, congenital leukodystrophy, Parkinson's disease, Huntington's disease, schizophrenia, and radiation-induced demyelination.

[0093] The therapeutic use of induced oligodendrocyte lineage cells (OLGs) includes transplanting the induced oligodendrocyte lineage cells (OLGs) into a subject in need thereof. The cells can be injected or transplanted into the central nervous system (CNS), such as the corpus callosum or cerebellum, in an amount effective to promote neuronal myelination.

[0094] In summary, the present invention provides a novel technique for generating inducible oligodendrocyte lineage cells (OLGs), which includes the following features and advantages:

[0095] (i) The first method to generate induced oligodendrocyte lineage cells (OLGs) from human differentiated somatic cells.

[0096] (ii) The first chemical cocktail capable of reprogramming / transdifferentiating somatic cells into oligodendrocyte lineage cells (OLGs). No previous technology can generate oligodendrocyte lineage cells (OLGs) from any other somatic cell type using chemicals.

[0097] (iii) Inducible oligodendrocyte lineage cells (OLGs) were generated within 3 days using a small amount of chemicals (with or without growth factors).

[0098] (iv) The conversion efficiency of induced oligodendrocyte lineage cells (OLGs) from skin fibroblasts was high, with an average efficiency exceeding 30%.

[0099] (v) Inducible oligodendrocyte lineage cells (OLGs) are expandable and resemble natural cells.

[0100] (vi) The components of the mixture used to generate induced oligodendrocyte lineage cells (OLGs) are well-defined and animal serum-free, suitable for clinical application and highly reproducible.

[0101] (vii) Inducible oligodendrocyte lineage cells (OLGs) express specific markers of glial cells and oligodendrocytes.

[0102] (viii) This is a chemical reprogramming process without retrovirus / lentivirus / plasmid infection process, avoiding insertional mutagenesis or other biosafety issues.

[0103] The present invention can provide the following various applications:

[0104] (i) Commercial kit for generating induced oligodendrocyte lineage cells. Since this is the first and only method for generating oligodendrocyte lineage cells (OLGs) from somatic cells, it can be used as a kit for generating oligodendrocyte lineage cells for basic research as well as cell therapy.

[0105] (ii) Production of induced oligodendrocyte lineage cells for disease treatment. Based on our research results, it is possible to generate autologous or allogeneic oligodendrocyte lineage cells (OLGs) from accessible somatic cells, such as skin biopsies, which can be easily obtained clinically. This process does not require surgery or any other painful procedures. Patients can then obtain their own induced oligodendrocyte lineage cells without taking immunosuppressive drugs. Alternatively, it may be easier to find healthy donors willing to donate skin cells for the production of induced oligodendrocyte lineage cells (OLGs) than to obtain oligodendrocyte lineage cells (OLGs) that form the central nervous system (CNS) through surgery. Given the myelin regeneration and neural support capabilities of glial cells, induced oligodendrocyte lineage cells (OLGs) can be used to treat diseases including demyelinating diseases and neurodegenerative diseases (multiple sclerosis (MS) and acute hemorrhagic encephalitis (AHL), cerebral palsy, Parkinson's disease, schizophrenia, and radiation-induced demyelinating diseases). We can also use the pharmaceutical formulation of the present invention to directly convert somatic cells into functional oligodendrocyte lineage cells (OLGs) in vivo to promote the repair of neural tissue.

[0106] (iii) Oligodendrocyte lineage cells in tissue engineering and regenerative medicine. Induced oligodendrocyte lineage cells (OLGs) can support the growth and differentiation of neurons and may help repair nerve damage in the treatment of neurological diseases. The conversion method can be combined with appropriate biomedical materials to support nerve growth, such as in the construction of neural tubes in tissue engineering and regenerative medicine.

[0107] (iv) Personalized treatment platform. We can use induced oligodendrocyte lineage cells (OLGs) for drug screening to identify appropriate drug targets for different patients. Patients can use their own somatic cells or fibroblasts to generate induced oligodendrocyte lineage cells (OLGs) to establish optimal personalized treatments for diseases related to glial cell lines.

[0108] Specifically, the present invention provides a culture comprising skin cells and a culture medium comprising a chemical inducer and one or more auxiliary agents as described herein. In particular, the culture further comprises induced oligodendrocyte lineage cells (OLGs) derived from the skin cells.

[0109] According to the present invention, the induced oligodendrocyte lineage cells (OLGs) or cultures thereof as described herein can be used as active ingredients for treating diseases of subjects in need. In some specific embodiments, a therapeutically effective amount of the active ingredient can be formulated into a pharmaceutical composition in an appropriate form with a pharmaceutically acceptable carrier for the purpose of delivery and absorption. Depending on the mode of administration, the pharmaceutical composition of the present invention preferably comprises an active ingredient in an amount of about 0.1% by weight to about 100% by weight, wherein the weight percentage is calculated based on the weight of the entire composition. The composition can be directly used as an implant or further modified to a form suitable for transplantation.

[0110] As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, and preferably can stabilize the active ingredient and is safe for the subject being treated. Examples of pharmaceutically acceptable carriers include conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), physiological saline, sterile water, antioxidants (such as ascorbic acid), isotonic agents, and preservatives.

[0111] In some embodiments, the composition according to the present invention is formulated into a dosage form suitable for injection, wherein the cells are suspended in a pharmaceutically acceptable carrier, for example, sterile water or normal saline or stored frozen before use. In some embodiments, the composition may further comprise a biodegradable polymer, which can be used to stabilize, support and fix the cell cluster after local injection into the defect site. The composition according to the present invention can be formulated into a unit dosage form or incorporated into a multidose container. The dosage form can be a suspension, solution or emulsion in an oily or aqueous medium, or a powder, granules, tablet or capsule. The composition of the present invention can be delivered by a physiologically acceptable route, typically by injection.

[0112] The present invention is further illustrated by the following examples, which are provided for the purpose of illustration and not limitation. Based on the disclosure of the present invention, it will be understood by those skilled in the art that many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining the same or similar results.

[0113] Examples

[0114] Many demyelinating diseases, such as multiple sclerosis (MS) and acute hemorrhagic inflammatory disease (AHL), are associated with oligodendrocyte dysfunction. Some demyelinating diseases are induced by radiation therapy. Furthermore, several neurodegenerative diseases, such as cerebral palsy, Parkinson's disease, Huntington's disease, and schizophrenia, are also oligodendrocyte diseases. Therefore, glial cells have the potential to treat neurodegenerative diseases and demyelinating diseases. Currently, approximately 115 clinical trials are investigating the efficacy of glial cell-based therapies (www.clinicaltrials.gov). To date, it is known that oligodendrocyte lineage cells (OLGs) can be derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, the differentiation process requires more than 45 days, which is labor-intensive and costly. Furthermore, deriving ESCs or induced pluripotent stem cells (iPSCs) carries the risk of teratomas. Only mouse or rat oligodendrocyte lineage cells (OLGs), not human cells, can be derived from fibroblasts induced with transcription factors. Until now, no program has used chemical cocktails to export any oligodendrocyte lineage cells (OLGs). Our program provides the first method to generate human induced oligodendrocyte lineage cells (OLGs) from dermal fibroblasts using chemicals. This method is highly efficient and avoids the insertion of viral genes. Most importantly, it is the fastest reprogramming method to date, taking only three days. This discovery may benefit stem cell biology, cell therapy, and regenerative medicine.

[0115] 1. Materials and Methods

[0116] 1.1 Generation of Inducible Oligodendrocyte Lineage Cells (OLGs)

[0117] The cells (1×10 4) were seeded in each well of a 24-well plate. Cells were cultured for 2 days in HG-DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Hyclone). The cells were then replaced with either a knockout medium (treatment medium; all reagents were from Life Technologies) supplemented with 1% N2 supplement, 1% Albumax, and 1% non-essential amino acids (NEAA) or DMEM / F12 medium supplemented with 1% N2, 1% B27, 20 ng / mL PDGF-AA, EGF, bFGF, and 10 ng / mL NT3, and various treatment chemical mixtures. The final conditions were 3 mM valproic acid for 2 days, 10 μM Y27632, 10 μM SU9516, 10 μM forskolin (FSK) (Tocris) for 1 day; or 10 μM modinomastat or pasinomastat for 2 days, 10 μM Y27632, 10 μM SU9516, 10 μM forskolin (FSK) (Tocris) for 1 day.

[0118] 1.2 Quantitative real-time polymerase chain reaction (qRT-PCR).

[0119] RNeasy Micro (Qiagen, Hilden, Germany) or Total RNA was isolated using LS reagent (Thermo Fisher Scientific). RNA was treated with DNase I to remove contaminated DNA (Promega, Madison, Wisconsin, USA) and reverse transcribed using Superscript III. The resulting cDNA (80 ng / sample) was used as a template for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) using SYBR GREEN2X master mix (KAPA Biosystems, Wilmington, MA, USA). Relative cDNA amounts were measured and quantified using an ABI 7900 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The relative amounts of the target genes were standardized relative to the RNA content of glyceraldehyde 3-phosphate dehydrogenase (GADPH).

[0120] 1.3 Immunofluorescence analysis

[0121] Induced oligodendrocyte lineage cells (OLGs) were fixed with 4% formaldehyde for 15 minutes at room temperature. After washing with 1× PBS, the cells were permeabilized with 0.03% Triton X-100 for 5 minutes. The cells were washed twice with 1× PBS and then incubated with primary antibodies (anti-Olig2 antibody (1:100; Merck Millipore), Nkx2.2 (1:100; Standard Cruz), O4 antibody (1:100; Merck Millipore), myelin basic protein (MBP) antibody (1:100; Proteintech), and PDGFRα antibody (1:100; R&D Systems)) and 4',6-diamidino-2-phenylindole (DAPI) (1:1500; Life Technologies) in blocking buffer (PBS containing 0.02% BSA) overnight at 4°C. The cells were washed twice in 1×PBS and then incubated with CF555 goat anti-rabbit secondary antibody (1:200; Life technologies), CF488 goat anti-rabbit secondary antibody (1:200; Biotium), CF555 goat anti-mouse secondary antibody (1:200; Life technologies), CF488 goat anti-mouse secondary antibody (1:200; Biotium), and CF555 goat anti-rat secondary antibody (1:200; Life technologies) in blocking buffer at room temperature for 1 hour in the dark. The cells were washed twice with 1×PBS and kept in 1×PBS. Fluorescence was analyzed by using a fluorescence microscope. The fluorescence intensity of each image was analyzed by image analysis software (Image-Pro plus 4.5 software, Maryland, USA). More than 300 cells / field were quantified for each immunofluorescence image.

[0122] 1.4 Flow cytometric analysis

[0123] Cells were sequentially treated with primary and secondary antibodies and analyzed using FACSCanto (Becton Dickinson, Franklin Lakes, NJ, USA). Data were further quantified using FACSDiva software (BD Biosciences).

[0124] 1.5 Myelin co-culture assay

[0125] Dorsal root ganglion neuron (DRGN) culture was isolated and purified from mice. The cells were maintained in maturation medium (DMEM-F12, supplemented with N1, 0.01% fetal bovine serum albumin, and B27 supplement (Invitrogen), 10ng / mL basic fibroblast factor (bFGF), and 2nmol / L triiodothyronine (T3), 10ng / mL platelet-derived growth factor (PDGF)-AA, 12.5ng / mL nerve growth factor (NGF)) for 3 weeks. The cells formed a neuronal network. Oligodendrocyte lineage cells (0.7×10 5 cells / cm 2 ) were suspended in dorsal root ganglion neuron (DRGN) cultures in a defined medium. The defined medium consisted of DMEM-F12 supplemented with N1 (Sigma-Aldrich), 0.01% fetal bovine serum albumin, 1% penicillin-streptomycin, B27 supplement (Invitrogen), 10 ng / mL platelet-derived growth factor (PDGF)-AA, 10 ng / mL basic fibroblast growth factor (bFGF), and 2 nmol / L triiodothyronine (T3) (Sigma-Aldrich) (referred to as DM+GF). Cell differentiation and myelination were analyzed after two weeks of co-culture.

[0126] 2. Results

[0127] 2.1 Optimization of the composition of the mixture of five chemical agents

[0128] To optimize the induction method of oligodendrocyte lineage cells (OLGs) in skin fibroblasts, we first measured Y27632 and SU9516. The results showed that SU9516 was associated with dome formation ( Figure 1A ), Y27632 caused changes in dendritic morphology ( Figure 1B The combination of Y27632 and SU9516 effectively transformed most fibroblasts into oligodendrocyte lineage cells (OLGs)-like cells ( Figure 2A The results showed that the morphology of the induced cells changed rapidly within two days, with O4 showing a significant change ( Figure 2B In addition, we included the PKC inhibitor Go6983, which has been reported to improve oligodendrocyte formation. 42 Go6983 and Y27632 promote the formation of dendrites, while SU9516 has the function of maintaining the dome shape ( Figure 3Following previous studies, we inhibited histone deacetylase (HDAC) by pre-treating with valproic acid (VPA) for 2 days and successfully improved the efficiency of reprogramming ( Figure 4A 、 4B ). Next, we screened for supporting factors for mixture-based drug screening. Among 22 drugs, we found that forskolin (FSK) significantly promoted transformation ( Figure 5 Overall, the optimized protocol treated cells with VPA, Y27632, Go6983, FSK, and SU9516 to guide reprogramming ( Figure 6A 、 6B ). The optimized mixture can transform skin fibroblasts into oligodendrocyte lineage cells (OLGs)-like cells with corresponding cell morphology and marker expression. In our case, the five chemicals can effectively transform cells within three days ( Figure 6A 、 6B ).

[0129] 2.2 Expression of multiple oligodendrocyte-specific markers on induced oligodendrocyte lineage cells (OLGs) generated by five chemicals

[0130] Based on preliminary data, we designed a cocktail of five chemicals to induce fibroblasts to generate oligodendrocyte lineage cells (OLGs). Here, we examined the expression of various oligodendrocyte-specific markers ( Figure 6A Marker staining results showed that the expression levels of PDGFR (oligodendrocyte precursor / pioneer cell (OPCs) marker), myelin basic protein (MBP) (a marker of mature myelinating oligodendrocytes), and Olig2 (a transcription factor constitutively expressed in oligodendrocyte precursor / pioneer cell (OPCs)) were increased in induced oligodendrocyte lineage cells (OLGs) ( Figure 6A In addition, we examined the expression levels of SOX10 (a transcription factor associated with oligodendrocyte precursor / pioneer cell (OPCs) differentiation) and PLP1 (a myelin protein expressed on mature oligodendrocytes) by RT-qPCR ( Figure 6B The results showed that SOX10 and PLP1 were highly expressed. Overall, these data demonstrate the consistency between molecular characteristics and morphological changes.

[0131] 2.3 Low doses of the four chemicals are sufficient to induce the transformation of oligodendrocyte lineage cells (OLGs)

[0132] Furthermore, we found that Go6983 was unnecessary for the conversion process ( Figure 7). In addition, a low dose of the mixture of Y27632, SU9516, and FSK (10 μM) was sufficient to obtain similar results as the high dose (50 μM) ( Figure 8 The results showed that 10 μM was sufficient to trigger reprogramming and had better conversion efficiency. Oligodendrocyte-specific markers oligo2, O4, PDGFRα, GlaC, GRP17, myelin basic protein (MBP), O1 ( Figure 9A and 9B ) were expressed in large quantities by immunofluorescence assay. qRT-PCR analysis showed that Sox10, myelin basic protein (MBP), and PLP1 were significantly increased by more than 3 times ( Figure 9C ).

[0133] 2.4 Co-culture experiments confirmed that induced oligodendrocyte lineage cells (OLGs) can myelinate neurons.

[0134] To further characterize myelination in vitro, we performed a co-culture assay of induced oligodendrocyte lineage cells (OLGs) (generated from skin cells by treatment with four compounds 4C (VYSF)) with mouse neurons (dorsal root ganglion cells, mDRGs). For analysis, we differentiated induced cells from mouse neurons (mDRGs) by staining with human nuclei (HuNu). The results demonstrated co-localization of neurofilaments (NF) and myelin basic protein (MBP) on mouse neurons (mDRGs) and induced oligodendrocyte lineage cells (OLGs). Figure 10 This indicates that neurons can be myelinated by inducible oligodendrocyte lineage cells (OLGs).

[0135] We also investigated the proliferative capacity of induced oligodendrocyte lineage cells. To determine whether the induced cells still had sufficient proliferative capacity after we treated them with chemicals, we stained for mitotic markers and detected Ki67 expression on O4-positive induced oligodendrocyte lineage cells (OLGs) generated from skin cells by treating them with four compounds, 4C (VYSF). Our findings showed that these induced cells expressed Ki67 and had a certain proliferative capacity ( Figure 11 ). In addition, we planned to use A2B5 (oligodendrocyte precursor cell marker) to isolate oligodendrocytes at early developmental stages through flow cytometric analysis. These A2B5 positive cells reached 78% viability and were able to expand and show O4 labeling up to 56.4% ( Figure 12A 、 12B , 12C). The results showed that A2B5-positive cells exhibited oligodendrocyte lineage characteristics and had high conversion efficiency.

[0136] 2.5 Using modinstat and pasinstat instead of VPA to generate induced oligodendrocyte lineage cells (OLGs).

[0137] In particular, like VPA, motin and pacin are histone deacetylase (HDAC) inhibitors. We tried replacing VPA with motin and pacin in the first step of induction. The experimental results showed that compared with the original mixture, these two histone deacetylase (HDAC) inhibitors can promote the differentiation of fibroblasts into more mature oligodendrocytes with more branches and complex structures ( Figure 13A Immunofluorescence staining results showed that cells treated with these two histone deacetylase (HDAC) inhibitors expressed oligodendrocyte-specific markers O4 and myelin basic protein (MBP) ( Figure 13B ).

[0138] 2.6 Three chemicals (3C) are sufficient to convert fibroblasts into oligodendrocyte lineage cells (OLGs).

[0139] To understand which chemicals are key factors in the conversion of oligodendrocyte lineage cells (OLGs), we tried to test and reduce one or two factors. The experimental results showed that three chemicals containing VPA, Y27632 and FSK can lead to the conversion of oligodendrocyte lineage cells (OLGs) specific marker A2B5 ( Figure 14A )、O4( Figure 14B ), and myelin basic protein (MBP) ( Figure 14B This indicates that 3C can form induced oligodendrocyte lineage cells (OLGs) with lower efficacy.

[0140] 3. Summary

[0141] In summary, we demonstrate that the chemical combination of Y27632 (a Rho-associated protein kinase (ROCK) inhibitor) with VPA (a histone deacetylase (HDAC) inhibitor) and forskolin (a cyclic adenosine monophosphate (cAMP) activator), optionally combined with SU9516 (a cyclin-dependent kinase (CDK) inhibitor) or Y27632 (a Rho-associated protein kinase (ROCK) inhibitor), can reprogram fibroblasts into inducible oligodendrocyte lineage cells (OLGs) expressing multiple oligodendrocyte markers. The conversion efficiency was high, with 56.4% of OLGs positive for the marker O4, compared with 73.9% for A2B5. These induced oligodendrocyte lineage cells (OLGs) expressing multiple oligodendrocyte lineage markers are functional and efficient in myelinating neurons and thus can be used for cell therapy, particularly for demyelinating diseases.

[0142] References

[0143] 1.O'Rahilly, R. & F. Basic human anatomy: a regional study of human structure. (Saunders, Philadelphia; 1983).

[0144] 2. Purves, D. & Williams, SM Neuroscience, Edn. 2nd. (Sinauer Associates, Sunderland, Mass.; 2001).

[0145] 3. Allen, NJ & Barres, BANeuroscience: Glia-more than just brainglue. Nature 457, 675-677 (2009).

[0146] 4.Burns,TC,Verfaillie,CM&Low,WCStem cells for ischemic braininjury:a critical review.J Comp Neurol 515,125-144(2009).

[0147] 5.Thoma,E.C.et al.Chemical conversion of human fibroblasts intofunctional Schwann cells.Stem Cell Reports 3,539-547(2014).

[0148] 6.Ohara,P.T.et al.Gliopathic pain:when satellite glial cells gobad.Neuroscientist15,450-463(2009).

[0149] 7.Kier,L.B.&Tombes,R.M.Proton hopping:a proposed mechanism formyelinated axon nerve impulses.Chem Biodivers 10,596-599(2013).

[0150] 8.Bradl,M.&Lassmann,H.Oligodendrocytes:biology and pathology.ActaNeuropathol 119,37-53(2010).

[0151] 9.Inglese,M.Multiple sclerosis:new insights and trends.AJNR Am JNeuroradiol27,954-957(2006).

[0152] 10.Love,S.Demyelinating diseases.J Clin Pathol 59,1151-1159(2006).

[0153] 11.Lepeta,K.et al.Synaptopathies:synaptic dysfunction in neurologicaldisorders.J Neurochem(2016).

[0154] 12.Najm,F.J.et al.Drug-based modulation of endogenous stem cellspromotes functional remyelination in vivo.Nature 522,216-220(2015).

[0155] 13.Imani,A.&Golestani,M.Cost-utility analysis of disease-modifyingdrugs in relapsing-remitting multiple sclerosis in Iran.Iran J Neurol 11,87-90(2012).

[0156] 14.Gallo,P.,Van Wijmeersch,B.&Paradig,M.S.G.Overview of themanagement of relapsing-remitting multiple sclerosis and practicalrecommendations.Eur J Neurol 22Suppl 2,14-21(2015).

[0157] 15.Compston,A.&Coles,A.Multiple sclerosis.Lancet 372,1502-1517(2008).

[0158] 16.Grigoriadis,N.,van Pesch,V.&Paradig,M.S.G.A basic overview ofmultiple sclerosis immunopathology.Eur J Neurol 22Suppl 2,3-13(2015).

[0159] 17.Zoghbi,H.Y.&Orr,H.T.Glutamine repeats and neurodegeneration.AnnuRev Neurosci 23,217-247(2000).

[0160] 18.Rosas,H.D.et al.Evidence for more widespread cerebral pathology inearly HD-An MRI-based morphometric analysis.Neurology 60,1615-1620(2003).

[0161] 19.Fennema-Notestine,C.et al.In vivo evidence of cerebellar atrophyand cerebral white matter loss in Huntington disease.Neurology 63,989-995(2004).

[0162] 20.Bartzokis,G.et al.Myelin breakdown and iron changes in Huntington's disease:Pathogenesis and treatment implications.Neurochem Res 32,1655-1664(2007).21.Di Paola,M.et al.Multimodal MRI Analysis of the Corpus CallosumReveals White Matter Differences in Presymptomatic and Early Huntington'sDisease.Cereb Cortex 22,2858-2866(2012).

[0163] 22.Di Paola,M.et al.MRI measures of corpus callosum iron and myelinin early Huntington's disease.Hum Brain Mapp 35,3143-3151(2014).

[0164] 23.Phillips,O.et al.Deep White Matter in Huntington's Disease.PlosOne 9(2014).

[0165] 24. Wade, A., Jacobs, P. & Morton, A. J. Atrophy and degeneration in sciatic nerve of presymptomatic mice carrying the Huntington's disease mutation. Brain Res 1188, 61 - 68 (2008).

[0166] 25. Xiang, Z. M. et al. Peroxisome - Proliferator - Activated Receptor Gamma Co - activator 1alpha Contributes to Dysmyelination in Experimental Models of Huntington's Disease. J Neurosci 31, 9544 - 9553 (2011).

[0167] 26. Huang, B. et al. Mutant huntingtin downregulates myelin regulatory factor - mediated myelin gene expression and affects mature oligodendrocytes. Neuron 85, 1212 - 1226 (2015).

[0168] 27. Kurita, H. et al. Radiation - induced apoptosis of oligodendrocytes in the adult rat brain. Neurol Res 23, 869 - 874 (2001).

[0169] 28. Oi, S. et al. Brain tumors diagnosed in the first year of life in five Far - Eastern countries. Statistical analysis of 307 cases. Childs Nerv Syst 6, 79 - 85 (1990).

[0170] 29.Panagiotakos,G.et al.Long-term impact of radiation on the stemcell and oligodendrocyte precursors in the brain.PLoS One 2,e588(2007).

[0171] 30.Piao,J.et al.Human embryonic stem cell-derived oligodendrocyteprogenitors remyelinate the brain and rescue behavioral deficits followingradiation.Cell Stem Cell 16,198-210(2015).

[0172] 31.Kawabata,S.et al.Grafted Human iPS Cell-Derived OligodendrocytePrecursor Cells Contribute to Robust Remyelination of Demyelinated Axonsafter Spinal Cord Injury.Stem Cell Reports 6,1-8(2016).

[0173] 32.Tontsch,U.,Archer,D.R.,Dubois-Dalcq,M.&Duncan,I.D.Transplantationof an oligodendrocyte cell line leading to extensive myelination.Proc NatlAcad Sci U S A 91,11616-11620(1994).

[0174] 33.Groves,A.K.et al.Repair of demyelinated lesions by transplantationof purified O-2A progenitor cells.Nature 362,453-455(1993).

[0175] 34.Guarino,A.T.&McKinnon,R.D.Reprogramming cells for brainrepair.Brain Sci 3,1215-1228(2013).

[0176] 35.Ogawa,S.,Tokumoto,Y.,Miyake,J.&Nagamune,T.Immunopanning selectionof A2B5-positive cells increased the differentiation efficiency of inducedpluripotent stem cells into oligodendrocytes.Neurosci Lett 489,79-83(2011).

[0177] 36.Biswas,D.&Jiang,P.Chemically Induced Reprogramming of SomaticCells to Pluripotent Stem Cells and Neural Cells.Int J Mol Sci 17,226(2016).

[0178] 37.Aharonowiz,M.et al.Neuroprotective effect of transplanted humanembryonic stem cell-derived neural precursors in an animal model of multiplesclerosis.PLoS One 3,e3145(2008).

[0179] 38.Mikaeili Agah,E.,Parivar,K.&Joghataei,M.T.Therapeutic effect oftransplanted human Wharton's jelly stem cell-derived oligodendrocyteprogenitor cells(hWJ-MSC-derived OPCs)in an animal model of multiplesclerosis.Mol Neurobiol 49,625-632(2014).

[0180] 39.Bulic-Jakus,F.,Katusic Bojanac,A.,Juric-Lekic,G.,Vlahovic,M.&Sincic,N.Teratoma:from spontaneous tumors to the pluripotency / malignancyassay.Wiley Interdiscip Rev Dev Biol 5,186-209(2016).

[0181] 40.Najm,F.J.et al.Transcription factor-mediated reprogramming offibroblasts to expandable,myelinogenic oligodendrocyte progenitor cells.NatBiotechnol 31,426-433(2013).

[0182] 41.Yang,N.et al.Generation of oligodendroglial cells by directlineage conversion.Nat Biotechnol 31,434-439(2013).

[0183] 42.Baer,A.S.et al.Myelin-mediated inhibition of oligodendrocyteprecursor differentiation can be overcome by pharmacological modulation ofFyn-RhoA and protein kinase C signalling.Brain 132,465-481(2009).

Claims

1. A method for generating induced oligodendrocyte-lineage cells (OLGs), comprising culturing skin cells in a culture medium containing valproic acid, motinositol, or pasinositol, and then culturing them in a culture medium containing Y27632, SU9516, and forskolin for 3 days, thereby reprogramming the skin cells into induced oligodendrocyte-lineage cells; wherein the final conditions are culturing with 3 mM valproic acid for 2 days and culturing with 10 μM Y27632, 10 μM SU9516, and 10 μM forskolin for 1 day; or culturing with 10 μM motinositol or pasinositol for 2 days and culturing with 10 μM Y27632, 10 μM SU9516, and 10 μM forskolin for 1 day. The method of claim 1 , wherein the skin cells are fibroblasts.

3. The method of claim 1, wherein 40% to 80% of the skin cells are reprogrammed into induced oligodendrocyte lineage cells.

4. The method of any one of claims 1 to 3, wherein the skin cells are human cells.

5. The method of claim 1, wherein the induced oligodendrocyte lineage cells (OLGs) express a cell marker selected from the group consisting of platelet-derived growth factor receptor (PDGFR), myelin basic protein (MBP), oligodendrocyte transcription factor (OLIG2), SOX10, proteolipid protein (PLP) 1, Ki67, A2B5, O4, and any combination thereof.

6. The method of claim 1, further comprising isolating the inducible OLGs from the cell culture to obtain an isolated population of inducible OLGs.

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