Induction of neural progenitor cells, oligodendrocyte progenitor cells, and oligodendrocytes by stem cell differentiation using rat transcription factors

Abstract

To provide induction of neural progenitor cells, oligodendrocyte progenitor cells, and oligodendrocytes by stem cell differentiation using landmark transcription factors.SOLUTION: Novel methods of inducing human iPSC-derived neural progenitors, oligodendrocyte progenitors, oligodendrocytes with unprecedented efficiency and functionality. The core of the present invention is the use of experimentally discovered transcription factors in a previously unknown manner at key differentiation decision points along the pathway from pluripotency to ectoderm to neuroectoderm to NPCs to OPCs to oligodendrocytes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 532,246, filed July 13, 2017.

[0002] The present disclosure relates to inducing and / or directing the differentiation cascade of pluripotent stem cells into neural progenitor cells, oligodendrocyte progenitor cells, and oligodendrocytes through a kinetically controlled cell growth process using specific combinations and ranges of cell densities, reagent concentrations, and specific combinations of mRNA. [Background technology]

[0003] Recent efforts in the generation and subsequent differentiation of human stem cells have changed the paradigm regarding cell fate plasticity, models for human disease, and clinical treatments. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) generated from somatic cells can differentiate into an ever-growing list of specific cell types indistinguishable from their primary cell counterparts. Summary of the Invention [Means for solving the problem]

[0004] The present invention provides methods for inducing and differentiating stem cells by modulating cell growth kinetics and related parameters, thereby controlling the direction of differentiation / induction using specific combinations of cell density, reagent concentrations, and mRNA combinations. In one aspect, one benefit of the present disclosure is a newly developed protocol for providing neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs) that can become functional, mature oligodendrocytes, as well as providing oligodendrocytes directly for therapies involving various types of myelin-related diseases and injuries.

[0005] The present disclosure provides a differentiation method that utilizes the highly efficient and well-controlled expression of master control genes or key transcription factors in tissue-specific differentiation. More specifically, such factors are introduced into pluripotent stem cells in the form of appropriately modified and purified mRNA molecules, as demonstrated by the present disclosure.

[0006] In one aspect, the present disclosure provides a method for inducing cell differentiation, comprising utilizing fusions between key cell fate factors and traditional transcription factors (TFs) with transactivation domains, optimized to direct stem cells into various cell types; introducing such factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at a desired density and in a manner that results in appropriate levels of transgene expression; and maintaining the cells under optimal conditions to enhance specific differentiation efficiencies, thereby inducing the pluripotent or progenitor state of stem or progenitor cells into specific lineage or tissue cell types.

[0007] In another aspect, the present disclosure provides methods for changing the pluripotent or progenitor state of stem or progenitor cells into cell types of specific lineages or tissues, comprising at least one of the following steps: generating stem cells (collectively referred to as stem cells) that express key cell fate genes, including fusions between key cell fate factors and traditional transcription factors (TFs) with transactivation domains, optimized for directing stem cells into various cell types; introducing such factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at a desired density and in a manner that results in appropriate levels of transgene expression; and maintaining the cells under optimal conditions to enhance the efficiency of specific differentiation.

[0008] In another aspect, there is provided a method for inducing differentiation of induced pluripotent stem cells (iPSCs) and / or embryonic stem cells (ESCs) into neural progenitor cells (NPCs), comprising: a) plating iPSCs and / or ESCs onto coated, non-tissue-treated culture plates; b) transfecting the cells of step a) with mRNAs encoding one or more cell fate factors, either as individual mRNAs or combined mRNAs; c) culturing the iPSCs and / or ESCs of step b) until the cells differentiate into NPCs; d) If a large number of NPCs are required, the NPCs of c) can be expanded by transferring the cells to uncoated, ultra-low attachment conditions and growing them in suspension. Disclosed herein is a method comprising: (a) transfecting a cell fate factor in step (b) with a transfection vector; (b) transfecting a cell fate factor selected from the group consisting of one or more of Ngn2, Pax6, Sox2, Brn2, and FoxO family factors, either individually or in combination with other mRNAs; (c) transfecting a cell fate factor in step (b) with a transfection vector; (d) transfecting a cell fate factor in step (c) with a transfection vector; (e) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (f) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (g) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (h) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (i) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (ii) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (iii) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (iv) transfecting a cell fate factor in step (c) with a transactivation domain, such as, but not limited to, MyoD or VP16; (v ... Determining whether a cellular factor is expressed can be performed using, but is not limited to, immunoassays and / or qt-PCR. In yet other embodiments, one or more of SB43542, LDN193189, and PD173074 may be added to the cells in step c) as needed. In some embodiments, the culture conditions in steps a) to c) are at an oxygen concentration of about 2% to about 6%. In embodiments of the present disclosure, the medium used in steps a) to c) is selected from Minimum Essential Medium Alpha (MEMa), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F12), or DMEM with B27 supplement. The medium may also contain other components, such as knockout serum replacement (KSR), and one or more of bFGF, N2 or B27, BSA, or HSA. In another embodiment, the culture vessel is coated with Matrigel and / or PLO-laminin. In an embodiment of this method, the iPSCs and / or ESCs in step a) are cultured at about 0.75×10 in a 6-well plate. 5 Cells ~ approx. 1.5×10 6Cells are seeded at 1000 cells / well. Other sizes of culture vessels can also be used. Seeding densities for culture vessels other than 6-well plates can be determined by dividing the cell number by the growth area of ​​the culture vessel using approximately 70,000 to approximately 14,000,000 cells, as shown in Table 2, for example. In some embodiments, the cells in step c) are cultured for approximately 3 to approximately 10 days, at which time the cells have an astrocyte-like or neuron-like morphology. After approximately 3 to approximately 10 days, close to 100%, but more than approximately 75%, of the cells have an NPC cell-specific morphology. In some embodiments, approximately 40 to 60% of the cells have an NPC cell-specific morphology. If fewer than approximately 40% of the cells have an NPC cell-specific morphology, cells can be isolated using cell sorting. Further culture using a suspension culture system allows for further selection. In some embodiments, NPC confirmation can be indicated by the expression of one of the cell fate factors, such as Pax6.

[0009] Another aspect of the present disclosure is a method for generating oligodendrocyte progenitor cells (OPCs) from NPCs, the method comprising: a) Plating NPCs into coated culture vessels containing medium Steps and b) transfecting the cells of step a) with mRNAs encoding one or more cell fate factors, either as individual mRNAs or as a combination of mRNAs, to differentiate the neural progenitor cells into oligodendrocyte precursor cells; c) culturing the cells of step c) until the morphology of NPCs changes to that of OPCs; d) If a large number of OPCs are required, the OPCs of c) can be expanded by transferring them to uncoated, ultra-low attachment conditions and growing them in suspension. In an embodiment of the method, in step a), the wells of the 6-well plate contain about 0.75 x 10 5 Approximately 1.5 x 10 cells / well 6Cells are seeded at 1000 cells / well. Culture vessels of other sizes can also be used. The seeding density for culture vessels other than 6-well plates can be determined by dividing the cell number by the growth area of ​​the culture vessel using approximately 70,000 to 14,000,000 cells, as shown in Table 2, for example. In another embodiment, the mRNA used for transfection in step b) encodes a cell fate factor selected, individually or in combination, from one or more of the group consisting of Oligo2, NKX2.2, SOX10, and Oligo1 mRNA. In another embodiment, the transfection dose in step b) is approximately 20 ng to 100 ng per cell fate factor when using a 6-well culture plate, or is adjusted proportionally based on the area of ​​the vessel or the volume of culture medium when using other types of plates or culture flasks. In another embodiment, after the initial transfection, the transfection is repeated at least two more times at a dose of about 10 ng / cell fate factor to about 200 ng / cell fate factor mRNA when using a 6-well culture plate, or proportionally adjusted based on the area of ​​the vessel or the volume of culture medium when using other types of plates or culture flasks. In another embodiment, the culture vessel plate is coated with Matrigel and / or PLO-laminin. In another embodiment, the medium used in steps a) to c) is selected from DMEM / F12, DMEM, and MEMa. In some embodiments, the medium is supplemented with one or more of KSR, ascorbic acid, SAG, PDGF, HGF, IGF1, T3, insulin, RA, B27, cAMP, and / or biotin. In embodiments of this method, the culture conditions for steps a) to d) are at an oxygen concentration of about 2% to about 6%. In another embodiment, the cells in step c) are cultured for about 4 to about 20 days until cells with morphological characteristics of OPCs are observed. In another embodiment, OPC generation can be confirmed based on bipolar morphology and / or expression of one of the cell fate factors. After about 4 to about 20 days, close to 100% but more than about 75% of the cells have the characteristic OPC cell morphology.In some embodiments, approximately 40-60% of the cells have a morphology characteristic of OPC cells. If fewer than approximately 40% of the cells have a morphology characteristic of OPC cells, cell sorting can be used to isolate the cells. Further culture using a suspension culture system allows for further selection. In some embodiments, confirmation of OPCs can be indicated by the expression of one of the cell fate factors, such as Oligo2 or Sox10.

[0010] Another aspect of the present disclosure is a method for producing a method of manufacturing a computer-implemented method comprising the steps of: a) plating OPCs onto a coated culture vessel containing a culture medium; b) Cells appear with branched cell processes and / or culturing the cells of step a) until they produce myelin basic protein (MBP); In another embodiment, the OPCs in step a) are cultured at approximately 0.75 x 10 per well using a 6-well plate. 5 Cells ~ approx. 1.5×10 6 The seeding density for culture vessels other than 6-well plates can be determined by dividing the cell number by the growth area of ​​the culture vessel using approximately 70,000 to approximately 14,000,000 cells, as shown in Table 2, for example. In another embodiment, the cells in step b) are cultured for approximately 7 to approximately 30 days, at which time cells with branched cell processes are present. In another embodiment, the cell culture vessel in step a) is coated with Matrigel and / or PLO-laminin.

[0011] Another aspect of the present disclosure provides a method for differentiation of stem cells, e.g., iPSCs or embryonic stem cells, into NPCs, OPCs, or oligodendrocytes by the following steps: a) plating iPSCs and / or ESCs onto coated culture vessels at approximately 0.75×10 per well of a 6-well plate in a medium comprising Minimal Medium Alpha (MEMa) or Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12) or DMEM with B27 supplement, both with Knockout Serum Replacement (KSR); 5 Cells ~ approx. 1.5×10 6 seeding the cells, or other culture vessels, if used, proportionally; b) transfecting the cells of step a) with mRNA encoding a cell fate factor selected from the group consisting of one or more of Ngn2, Pax6, Sox2, Brn2 and FoxO family factors, either as individual mRNA or combined mRNA transfection; c) culturing the iPSCs and / or ESCs of step b) until the cells differentiate into NPCs; d) isolating the NPCs from step c); e) plating NPCs onto the coated cell culture vessel at a concentration of about 0.75×10 per well of a 6-well plate in a medium comprising MEMa or DMEM / F12 or DMEM, both having KSR and one or more of the medium supplements selected from the group consisting of ascorbic acid, SAG, PDGF, HGF, IGF1, T3, insulin, RA, B27, CAMP, and biotin. 5 Cells ~ approx. 1.5×10 6 seeding the cells, or other culture vessels, if used, proportionally; f) transfecting the cells of step e) with one or more of the group consisting of Oligo2, NKX2.2, SOX10 and Oligo1 mRNA, individually or in combination, using a transfection reagent; g) repeating the transfection of step e) at least two more times at a dose of about 10 ng to about 200 ng (per well in a 6-well plate); h) culturing the cells of step g) until the cell morphology changes from NPC cells to OPC cells; i) isolating the OPCs from step h); j) plating the OPCs isolated from step j) onto coated cell culture vessels, wherein the OPCs are plated at approximately 0.75×10 per well of a 6-well plate in a medium comprising MEMa or DMEM / F12 or DMEM, both having KSR and one or more of the medium supplements selected from the group consisting of ascorbic acid, N2, B27, biotin, Cam, T3, and insulin. 5 Cells ~ approx. 1.5×10 6 seeding the cells, or other culture vessels, if used, proportionally; K) Oligodendrocyte cells grow tree branch-looking processes. culturing the cells for at least about one week until they appear with and / or produce myelin basic protein (MBP); This is a method for sequential or direct induction using

[0012] In another aspect of the invention, the starting cells for the methods disclosed herein are obtained from the recipient, for example, from a bodily fluid or tissue.

[0013] Another aspect of the present disclosure is NPCs, OPCs, and oligodendrocytes obtained by the methods of the present disclosure. In an embodiment of this aspect, the cells may be used as a pharmaceutical composition to treat a disease, disorder, or dysplasia. Such diseases, disorders, and dysplasias may include, but are not limited to, neurodegenerative conditions, such as acute neurodegenerative conditions, such as brain injury (focal or diffuse brain injury), spinal cord injury, or peripheral nerve injury, such as those resulting from physical or chemical burns, deep cuts, or limb amputation, or cerebrovascular insufficiency. In other embodiments, this may include chronic or progressive neurodegenerative conditions, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, tumors, or chronic peripheral nerve injury, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), and the like. syndrome), multiple system degeneration (Shy-Drager syndrome), chronic epileptic conditions associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxia, corticobasal degeneration, ALS-Parkinsonism-Dementia Complex of Guam, subacute sclerosing panencephalitis, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease / spinocerebellar ataxia 3 cerebellar and olivopontocerebellar degeneration, Gilles de La Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kenneth's disease) These include, but are not limited to, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff disease, familial spastic disorders, Wohlfart-Kugelberg-Welander disease, spastic paraplegia, progressive multifocal leukoencephalopathy, familial dysautonomia (Riley-Day syndrome), and prion diseases (including, but not limited to, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, kuru, and fatal familial insomnia). In yet other embodiments, it is a disease associated with demyelination, such as multiple sclerosis, acute disseminated encephalomyelitis, neuromyelitis optica, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, central pontine myelinosis, inherited demyelinating diseases such as leukodystrophy, Charcot-Marie-Tooth disease, and Canavan disease.

[0014] Aspects of the present disclosure will now be described with reference to the drawings listed below. [Brief explanation of the drawings]

[0015] [Figure 1] Exemplary embodiments of neural progenitor cell (NPC) induction.

[0016] [Figure 2] Exemplary embodiments of oligodendrocyte precursor cell (OPC) induction.

[0017] [Figure 3] Exemplary embodiments of oligodendrocyte induction.

[0018] [Figure 4] Illustrative embodiment of further culturing and maturation of oligodendrocytes.

[0019] [Figure 5] 1 is an exemplary embodiment of human iPSC-derived oligodendrocytes exhibiting specific cell markers.

[0020] [Figure 6] Illustrative embodiments of the use of CRISPR-Cas-based gene expression regulators to achieve significant and variable TF mRNA levels in differentiating cells. DETAILED DESCRIPTION OF THE INVENTION

[0021] When describing this disclosure, all terms not defined herein have their ordinary meaning as recognized in the art. To the extent that the following description is a description of specific embodiments or specific uses of the invention, it is intended to be illustrative only and not limiting of the claimed invention. The following description is intended to encompass all alternatives, modifications, and equivalents that fall within the spirit and scope of the invention.

[0022] Due to the unlimited availability of cells, the non-invasive method of obtaining them, and the possibility of immuno-matching each treatment to an individual patient, iPSCs show particular potential in the field of personalized medicine, allowing freedom from immunosuppressive drugs.

[0023] Cell replacement therapy offers new hope for treating various injuries and preventing or preventing various human diseases that have previously lacked a true cure. For example, spinal cord injuries resulting from accidents, as well as other spinal conditions such as multiple sclerosis and various leukodystrophies, are currently treated in hospitals without any means to restore the loss of myelin and its neuronal function. Finding a reliable source of NPCs, OPCs, and oligodendrocytes remains a significant hurdle to overcome. There is a clear need for cells that can help those suffering from spinal cord injuries or diseases, yet efforts by researchers and companies have yet to provide a method that allows for the generation of fully functional oligodendrocytes with mature morphology. Whether NPCs or OPCs are used to treat spinal conditions, the ability of these precursors to give rise to mature, functional, normal oligodendrocytes remains an important measure of their potential for use in medical treatment.

[0024] function Current protocols for deriving target tissue cells from pluripotent stem cells require the use of combinations of growth factors, hormones, cytokines, signal peptides, and other intercellular signaling molecules (collectively referred to as "growth factors" for convenience in this disclosure) at each step along the differentiation cascade to generate medically applicable NPCs, OPCs, or oligodendrocytes. Unfortunately, when supplied as purified polypeptides, growth factors are generally expensive, unstable, and inconsistent from batch to batch, making their use difficult. Moreover, because growth factors function in a highly specific and combinatorial manner, each stage of differentiation is defined by a different set of growth factors, which are difficult and costly to optimize. As disclosed herein, by combining mRNAs at appropriate doses and delivery conditions at key fate transition points, NPCs, OPCs, and oligodendrocytes can be achieved with high efficiency and low cost without the use of large amounts of growth factors.

[0025] To reduce the cost burden and inconsistency, those skilled in the art often turn to the discovery of small molecules that affect signaling pathways as agonists or antagonists of growth factor receptors, thereby substituting growth factors to some extent. Small molecules are generally much cheaper than growth factors. However, one major disadvantage of small molecules is the non-specific effects they may have on unintended targets, such as cell membrane-bound receptors, intracellular organelles, or genomic components. The present disclosure also provides a novel method for achieving cell fate determination without or with reduced use of small molecules.

[0026] Presumably because a detailed roadmap, including growth factors and small molecules, that advances cell differentiation through each stage has yet to be delineated through lengthy experiments, there is currently very limited information on how to differentiate pluripotent stem cells all the way into definitive oligodendrocytes. Because mRNA is more specific in directing cellular and developmental events by encoding functional proteins, the disclosed method is far more robust than any known method in generating primary oligodendrocyte-like cells and paving the way for human therapy in treating spinal cord injuries, defects, diseases, and myelination and other related health problems.

[0027] Another important component of a typical differentiation protocol is the medium for culturing the cells, which may consist of nutrients (lipids, amino acids, carbohydrates, vitamins, etc.), appropriate concentrations of salts, pH buffers, essential elements, and general protein factors such as insulin or serum albumin. Different cell types have different requirements for nutrients and medium components, further complicated by cell-type-specific growth factors and small molecules for signaling. Therefore, specialized "differentiation media" are often painstakingly tested by removing or adding one component at a time. A major advantage of the present disclosure is the simplicity of establishing a differentiation medium through the use of appropriately supplied mRNA of differentiation-committing genes. However, the optimal combination of mRNA and the appropriate medium can further benefit the process and is an essential element of this disclosure.

[0028] For clinical applications of stem cell-derived tissue cells, most components of established differentiation media require individual certification under current good manufacturing practice (cGMP) regulations; for example, growth factors must be produced by special methods and require individual certification. Similarly, some small molecules added to specific differentiation media are produced by special chemical synthesis processes that vary in purity, stability, and toxicity. In the field of stem cell culture, some previously published protocols also rely on animal products such as serum or Matrigel. Another motivation behind the present disclosure is to create a new method based primarily on a single type of molecule that is amenable to uniform certification and quality control processes.

[0029] This specification discloses a process that includes a method for controlling cell density and division rate to achieve desired differentiation results.Furthermore, the disclosure teaches the optimization of timing, addition order, RNA dosage and ratio between various RNAs, as well as its duration or number of repetitions during RNA transfection.The disclosure also relates to the selection of culture vessel surface and environmental conditions, such as oxygen concentration.The present invention also includes a method for selecting desired cells or enhancing their proportion in the total population, as well as a method for cryopreserving and re-cultivating differentiated cells.

[0030] The concept of a "master control" gene—i.e., one critical gene (typically a transcription factor gene, possibly a small number of genes acting together)—that can determine cell and tissue fate and ultimately the formation of entire organs during development has gained widespread acceptance based on studies of muscle (MyoD), eye (Pax6), and other areas of developmental biology. Shinya Yamanaka's discovery that viral delivery and expression of a select group of transcription factors expressed in stem cells can revert differentiated cells to a pluripotent state demonstrated the ability of a few critical transcription factors to drive cells through long-term, multistep fate changes. Other groups' work on iPSC generation has expanded the selection of reprogramming factors, demonstrating that some degree of variation can be tolerated in the selection of transcription factors for reprogramming purposes. In Yamanaka's original work, expression of reprogramming factors was achieved by applying viral vectors that integrate into the genome, because prolonged expression of such factors was required to effect cellular transformation. Concomitant genomic modifications represent a significant hurdle to the therapeutic application of iPSCs; furthermore, the possibility of reactivation of expression from integrated viral cassettes is a concern even in in vitro studies. The application of mRNA transfection to reprogramming, as recently described by the present inventors, is particularly attractive because this system allows for modulation of the expression of reprogramming cocktails and even individual components within a short timeframe by simply altering which transcripts are added to the cell culture medium. Upon termination of transfection of a specific factor, ectopic expression in target cells is quickly halted due to rapid decay of cytoplasmic mRNA. While mRNA does not persist in target cells, its ability to be directly translated in the cytoplasm, without the need for rate-limiting nuclear import as in the case of transfected DNA and integrating viral vectors, simply compensates for the short half-life of mRNA, resulting in highly efficient expression, but within a sufficiently short time window, that is critical for cell fate determination.

[0031] Persistent DNA vectors, such as episomal plasmids, when used to alter cell fate, need to be weaned to reduce any risk of random genome integration. RNA viruses or virus derivatives, such as Sendai virus, Sendai virus or Venezuelan equine encephalitis (VEE) virus can transmit viral elements even after modification into non-infectious RNA replicons. However, the viral vectors still retain their markers and tend to recombine with hidden viral elements in the host genome. It is always difficult to be completely certain that cells will eliminate the viral vector without the hassle of searching for evidence in the form of negative data. This disclosure discloses multiple inventive steps aimed at applying the benefits of mRNA-based cell fate determination to direct differentiation. In summary, this disclosure teaches single or multiple rounds of ectopic transcription factor expression in a streamlined manner to direct cell differentiation.

[0032] Nevertheless, technical barriers to mRNA-based stem cell differentiation exist. Not all stem cell types and culture media are equally conducive to efficient mRNA delivery, which currently constitutes an obstacle to mRNA-based differentiation. Furthermore, stem cells, particularly most human stem cell lines, are somewhat difficult to culture without forming transfection-resistant patches. As disclosed herein, pluripotent stem cells can be grown under conditions that allow mRNA transfection into most cells. In another embodiment, the dose of RNA and transfection reagent (both of which are toxic) is provided to cells at a level that exerts master control gene effects and maintains target cell viability in the face of pro-apoptotic and cytostatic forces generated by the cell fate change process.

[0033] Therefore, in consideration of the problems associated with previously known stem cell differentiation methods, novel methods, materials, and protocols described herein generate a variety of cell types from iPSCs or embryonic stem cells (ESCs) with improved process efficiency and cell quality. This disclosure demonstrates significant improvements by enhancing TF mRNA delivery to target stem cells. This disclosure also provides a novel protocol useful for generating footprint-free tissue cells from human stem cells without the use of feeder cells or any other potentially xenogeneic reagents. The new protocol extends the benefits of modified mRNA and helps overcome remaining barriers to the therapeutic application of stem cell induction technology.

[0034] Given that differentiation from a pluripotent state to a terminally differentiated state often involves multiple steps and requires a time frame of weeks to months, stepwise growth factor-based strategies are inherently inefficient and tedious. Thus, embodiments of the present disclosure essentially eliminate the need for growth factors to direct the generation of neural progenitor cells to oligodendrocyte precursor cells to oligodendrocyte cells.

[0035] More specifically, the present disclosure relates to converting the pluripotent or progenitor state of stem or progenitor cells (collectively referred to as stem cells) into cell types of specific lineages or tissues by expressing key cell fate genes optimized for directing stem cells into various cell types, including fusions between traditional transcription factors (TFs) with transactivation domains, such as MyoD or VP16, and many other transactivation domains commonly known in the art, which result in strong transcriptional activation activity; introducing such factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at a desired density and by a method that results in appropriate levels of transgene expression; and maintaining the cells under optimal conditions to produce specific differentiation efficiencies not previously achievable. Factors expressed by the introduction of mRNA can also include growth factors, cytokines, hormones, signal peptides, and other secreted factors or modifying enzymes that affect cell fate. Because microRNAs (miRNAs) or other non-protein-coding RNAs can regulate or influence TFs and produce essentially the same effects as direct transfection of TF mRNA, similar methods can be used to direct differentiation by introducing such non-coding RNAs into cells under cell state transitions. Furthermore, modified CRISPR-Cas9 systems in which engineered Cas9 (e.g., non-cleaving Cas9) or other Cas family member proteins are fused to a transcriptional activation domain (and in some cases, a transcriptional repression domain for repressing the TF through the opposite action of the desired TF), a chromatin-modifying enzyme or its enzymatic domain, or other epigenetic modifying enzyme or its enzymatic domain, along with guide RNA(s) that direct such an action domain to the vicinity of the target TF gene on the chromosome, can be used to activate TFs important for differentiation, as described in the Examples. Disclosed herein is a CRISPR-Cas system for cell differentiation that preferably uses only RNA as the molecule delivered into target cells, for the same advantages as described above, except that it requires a DNA molecule as a template for modifying the genome sequence.Compared to other methods known in the art, the methods disclosed herein dramatically reduce the time, cost, and effort required to differentiate stem cells into NPCs, OPCs, and oligodendrocytes.

[0036] The disclosure herein particularly describes in detail methods for changing the pluripotent or progenitor state of stem or progenitor cells into cell types of specific lineages or tissues, comprising at least one of the following steps: expressing key cell fate genes, including key cell fate factors, optimized to direct stem cells into various cell types; introducing such factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at a desired density and in a manner that results in appropriate levels of transgene expression; and maintaining the cells under optimal conditions to enhance specific differentiation efficiency.

[0037] In certain embodiments, fully stable and expanded human ESCs or iPSCs are provided.

[0038] In certain embodiments, iPSCs can be passaged 10 or more times after isolation without requiring removal of episomes or RNA viruses (e.g., Sendai).

[0039] In certain embodiments, the process is feeder-free.

[0040] In certain embodiments, the process is xeno-free, involving any synthetic or human reagents, and is free of non-human animal-derived components.

[0041] In certain embodiments, the process is footprint-free and does not involve random integration of DNA into the genome (which often occurs with episomal DNA constructs).

[0042] In certain embodiments, mRNA function can be achieved by using non-coding RNAs (including miRNAs) or guide RNA / Cas fusion systems, along with understanding which TF activities are important for specific differentiation steps as disclosed in the Examples below.

[0043] In certain embodiments, the process results in a fully customized genetic background via patient-specific starting tissue and / or genome editing. definition

[0044] As used herein, the term "about" means within 20%, more preferably within 10%, and most preferably within 5%. When used in the context of days, the term "about" means around 1 or 2 days. Thus, if a cell culture is cultured for about 3 to about 10 days, this can mean 3 to 10 days, 1 to 12 days, or 2 to 11 days.

[0045] The term "oligodendrocyte-like cells" is intended to mean cells that share characteristics with oligodendrocytes. Oligodendrocyte-like cells are further defined by morphological characteristics as well as by specific marker characteristics. Because induced pluripotent stem cell-derived oligodendrocyte-like cells share similar characteristics (including marker and hormonal characteristics) with primary oligodendrocytes, they are often referred to as induced pluripotent derived oligodendrocytes. Oligodendrocyte-like cells may be used interchangeably with induced pluripotent stem cell-derived oligodendrocytes, or simply oligodendrocytes.

[0046] "Embryoid body" refers to an aggregate of cells derived from pluripotent cells, where cell aggregation can be induced by any method that prevents cells from adhering to a surface and forming typical colony growth.As used herein, "embryoid body" refers to a three-dimensional spherical aggregate of pluripotent stem cells, including but not limited to embryonic stem cells derived from blastocyst-stage embryos from mammalian sources.Embryoid bodies can be formed from embryonic stem cells obtained by any technique commonly known in the art, including but not limited to somatic cell nuclear transfer or somatic cell reprogramming to obtain induced pluripotent stem cells.

[0047] As used herein, the term "induced pluripotent stem cells" refers to pluripotent stem cells derived from somatic cells (e.g., adult somatic cells). Induced pluripotent stem cells are similar to embryonic stem cells in their ability to form any adult cell type, but are not derived from embryos, i.e., cells that can differentiate into multiple cell types, but are artificially derived from non-pluripotent cells (non-naturally derived).

[0048] As used herein, "cell," "cell line," and "cell culture" include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property as screened for in the originally transformed cell are included.

[0049] As used herein, a "composition" refers to a combination of an active agent and at least one other compound or molecule, inert (e.g., a detectable agent or label) or active, e.g., an adjuvant. In the context of the present disclosure, a composition also includes differentiated cells produced by the methods described herein, and may include NPCs, OPCs, and oligodendrocyte cells.

[0050] As used herein, "culturing" refers to maintaining cells as a population of cells under conditions that allow them to proliferate and avoid senescence. "Culturing" can also include conditions under which the cells may alternatively differentiate.

[0051] As used herein, "differentially expressed" refers to a difference between the level of RNA produced by the same gene or regulator region in normal or control cells. It refers to the differential production of RNA, including but not limited to mRNA, tRNA, miRNA, siRNA, snRNA, and piRNA transcribed from a regulatory region of a gene or genome, or a protein product encoded by a gene, when compared. In other contexts, "differentially expressed" also refers to a nucleotide sequence or protein in a cell or tissue that has a different temporal and / or spatial expression profile when compared to normal or control cells.

[0052] As used herein, "highly expressed" or "high expression" refers to an increase in the expression level of an RNA or protein product encoded by a gene compared to the expression level of the RNA or protein product in a normal or control cell.

[0053] As used herein, "underexpressed" or "underexpression" refers to a decrease in the expression level of an RNA or protein product encoded by a gene compared to the expression level of the RNA or protein product in a normal or control cell.

[0054] As used herein, "differentiate" or "differentiation" refers to the process by which a precursor or progenitor cell (i.e., a neural progenitor cell) differentiates into a specific cell type, for example, an oligodendrocyte.

[0055] As used herein, an "effective amount" is an amount sufficient to produce a beneficial or desired biological, emotional, medical, or clinical response in a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term also includes within its scope an amount effective to enhance normal physiological function.

[0056] As used herein, "expansion" or "expanded" "Expansion," in the context of cells, refers to an increase in the number of one or more characteristic cell types, which may or may not be identical, from an initial population of cells. The initial cells used for expansion need not be identical to the cells produced by expansion. For example, expanded cells may be produced by ex vivo or in vitro growth and differentiation of the initial population of cells.

[0057] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into an RNA transcript. In the context of mRNA and other translated RNA species, "expression" also refers to the process or processes by which the transcribed RNA is subsequently translated into a peptide, polypeptide, or protein.

[0058] As used herein, "non-integrated iPS cells" refer to iPS cells that do not contain an exogenous transgene integrated into the genome of the non-pluripotent cell.

[0059] As used herein, "isolated" means that a polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof is separated from components, cells, and the like, with which it is normally associated in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to distinguish it from its naturally occurring counterpart. With respect to NPCs, OPCs, and oligodendrocytes described herein, isolated means separation from undifferentiated cells and isolation of a pure population of the cells of interest.

[0060] As used herein, "enriched" refers to molecules, including but not limited to, cells, specific cell types, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof, that are distinguishable from their naturally occurring counterparts in that the concentration or number of molecules per volume is greater than the concentration or number of molecules per volume of their naturally occurring counterparts.

[0061] As used herein, "diluted" refers to molecules, including but not limited to cells, specific cell types, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof, that are distinguishable from their naturally occurring counterparts in that the concentration or number of molecules per volume is less than the concentration or number of molecules per volume of their naturally occurring counterparts.

[0062] As used herein, "isolated" refers to a state in which a compound, cell, agent, particle, or molecule is physically separated from an original source or population such that the separated compound, cell, agent, particle, or molecule can no longer be considered to be part of the original source or population.

[0063] As used herein, "mammal" for purposes of treatment refers to any animal classified as a mammal, including, but not limited to, humans, domestic and farm animals, non-human primates, and zoo animals, sport animals, and pets, such as dogs, horses, cats, and cows.

[0064] As used herein, "stem cell" refers to any self-renewing totipotent, pluripotent, or multipotent cell or progenitor or precursor cell, which is capable of differentiating into multiple cell types.

[0065] As used herein, "totipotent" refers to a cell that is capable of differentiating into and giving rise to all cell types of an organism, as well as extraembryonic or placental cells.

[0066] As used herein, "pluripotent" refers to a cell that can differentiate into and give rise to all of the cell types that make up an organism, including any fetal or adult cell type except extraembryonic or placental cells.

[0067] As used herein, "multipotent" refers to cells that can develop into more than one cell type, but are more restricted in the cell types they can develop into than pluripotent cells.

[0068] As used interchangeably herein, a "subject," "individual," or "patient" refers to a vertebrate organism.

[0069] As used herein, a "substantially pure cell population" refers to a population of cells that have a particular cell marker characteristic and differentiation potential, with about 50%, preferably about 75-80%, more preferably about 85-90%, and most preferably at least about 95% of the cells constituting the total cell population. Thus, a "substantially pure cell population" refers to a population of cells that contains less than about 50%, preferably less than about 20-25%, more preferably less than about 10-15%, and most preferably less than about 5% of cells that do not exhibit a particular marker characteristic and differentiation potential under specified assay conditions.

[0070] As used herein, "pre-differentiation" refers to the process by which precursor or progenitor cells (e.g., pluripotent stem cells) differentiate into intermediate cell types, e.g., neural progenitor cells or oligodendrocyte precursor cells, that have the potential to further differentiate into ultimate effector cells (e.g., oligodendrocytes).

[0071] As used herein, "therapeutic" refers to treating, curing, and / or ameliorating a disease, disorder, condition, or side effect, or reducing the rate of progression of a disease, disorder, condition, or side effect. The term also includes within its scope the enhancement of normal physiological function, palliative treatment, and partial treatment of a disease, disorder, condition, or side effect.

[0072] The terms "treating" and "treatment," as used herein, generally refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of preventing or partially preventing the disease, symptom, or condition, and / or therapeutic, in terms of partially or completely curing the disease, condition, symptom, or adverse effects caused by the disease. The term "treatment," as used herein, encompasses any treatment in mammals, particularly humans, and includes (a) preventing the onset of a disease in a subject who may be predisposed to, but has not yet been diagnosed with, the disease; (b) inhibiting the disease, i.e., arresting its progression; or (c) palliating the disease, i.e., reducing or reversing the disease and / or its symptoms or condition. The term "treatment," as used herein, refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include those already with the disorder, as well as those in whom the disorder is to be prevented.

[0073] As used herein, "prophylactic" refers to preventing or halting a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in an asymptomatic stage.

[0074] As used herein, "active agent" refers to a substance, compound, or molecule that is biologically active or otherwise induces a biological or physiological effect in a subject to which it is administered. In the context of the present disclosure, NPCs, OPCs, and oligodendrocytes produced by the methods described herein are "active agents."

[0075] As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle administered in conjunction with an active agent, a chondrocyte of the present disclosure, or a composition comprising an oligodendrocyte of the present disclosure, that is approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals and / or humans.

[0076] As used herein, "FoxO" refers to a subclass of forkhead transcription factor family members. The experiments disclosed herein may use mRNA from one or more of FoxO family members, such as FoxO1, FoxO2, FoxO3a, and FoxO4.

[0077] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0078] Cell types: Examples of cell types may include, for example, ectodermal cells, neural progenitor cells, oligodendrocyte precursor cells, oligodendrocytes.

[0079] Examples of suitable surfaces for iPSC culture vessels include, but are not limited to, vitronectin, E-cadherin, Corning® Synthemax® II, or Matrigel. Suitable surfaces for neuroectoderm and neural progenitors include, but are not limited to, Matrigel, PLO-laminin. Suitable surfaces for neural progenitors and oligodendrocyte precursors and oligodendrocytes include, but are not limited to, Matrigel, PLO-laminin, or collagen.

[0080] In one embodiment, the exemplary method for dedifferentiating or reprogramming somatic cells can include using any one or more of the synthetic mRNA reprogramming factors and transactivation domains selected from Oct4, Sox2, Klf4, cMyc, Nanog and Lin28, thereby reprogramming or dedifferentiating somatic cells.The method and composition for modulating iPSCs are described in US Patent Application Nos. 13 / 893,166 and 14 / 292,317, and such iPSCs can be used in the methods described herein.The contents of which are incorporated herein by reference.

[0081] Suspension systems, when available, are more practical for large-scale production for clinical applications. Therefore, in this protocol, each step was tested to see if the cells could be sufficiently expanded in suspension. In certain embodiments, there is a protocol for suspension cell culture and the use of low cell attachment culture plates and vessels that can be used for such suspension culture.

[0082] In certain embodiments, environmental conditions, for example, oxygen concentration, can be modulated to optimal induction conditions.

[0083] In certain embodiments, processes and methods are provided for the selection of desired cells or for enhancing their confluency or cell density in a whole cell culture population.

[0084] In certain embodiments, a method for cryopreserving NPC, OPC or oligodendrocyte-like cells is provided.Some differentiated cells may need to be cryopreserved to ensure optimal cell viability during storage.For example, 2.5% HSA plus 10% DMSO may be used in culture medium.This application can be used to optimize cell number to further improve viability during storage.

[0085] Also provided is a method for reculturing differentiated cells.Cells can be recultivated in most culture vessels, such as, but not limited to, T75 flasks, T25 flasks, 4-well plates, 6-well plates, 8-well plates, 24-well plates, 48-well plates, and 96-well plates.Cells can be recultivated at various cell densities for various applications.

[0086] In certain embodiments, the present disclosure also provides methods for managing physical stress on cells, thereby improving their survival rate during differentiation. Certain cell types, such as iPSCs, are very small during differentiation. These small cells are highly sensitive to centrifugal forces. To maintain these cells, they can be cultured as colonies and then dissociated as clusters rather than as single cells. If single cells are required for differentiation, the cells can be detached after dissociation is complete, the dissociation solution can be removed, and the remaining dissociation solution can further dissociate the cells. This protocol is commonly used in cell culture. iPSCs are highly sensitive to excessive centrifugal forces. Some cell types, like iPSCs, are highly adhesive during differentiation. These cells are highly sensitive to sheer forces. When processing these cells, we avoided using any small tip and used a 10 mL pipette to avoid repeatedly pipetting the cells up and down. Oligodendrocytes have a unique cell morphology, including many branches and processes capable of attaching to axons. Transferring oligodendrocytes from one container to another requires careful manipulation and unconventional techniques that the inventors of the present disclosure have mastered through trial and error.

[0087] NPCs, OPCs, and oligodendrocytes generated by the methods of the present disclosure can be used to treat spinal cord injuries and other injuries. For example, highly enriched or purified populations of NPCs, OPCs, and / or oligodendrocytes generated using the methods of the present disclosure can be used for transplantation into various conditions of brain and spinal cord disease and injury. For background information, see (Liu K, et al., "Neuronal Intrinsic Mechanisms of Axon Regeneration." Annu Rev Neurosci. 2011; 34:131-152); Tuszynski MH, Steward O., Concepts and Methods for the Study of Axonal Regeneration in the CNS. Neuron. 2012; 74:777-791); (Gage, "Mammalian Neural Stem Cells," Science 287:1433-1438 (2000), all of which are incorporated herein by reference in their entireties. (Goldman, "Adult Neurogenesis: From Canaries to the Clinic," J. Neurobiology 36:267-286 (1998); Pincus et al., "Neural Stem and Progenitor Cells: A Strategy for Gene Therapy and Brain Repair," Neurosurgery 42:858-868 (1998); Svendsen et al., "Neural Stem Cells in the Developing Central Nervous System: Implications for Cell Therapy Through Transplantation," Prog. Brain Res. 127:13-34 (2000); and Svendsen et al., "New Prospects for Human Stem-Cell Therapy in the Nervous System," Trends Neurosci. 22:357-364 (1999), all of which are incorporated by reference in their entirety. (Snyder et al., "Multipotent Neural Precursors Can Differentiate Toward Replacement of Neurons Undergoing Targeted Replacement," incorporated herein by reference in its entirety.) "Apoptotic Degeneration in Adult Mouse Neocortex," Proc. Natl. Acad. Sci. USA 94:11663-11668 (1997), which is incorporated herein by reference in its entirety), and for remyelinated regions of brains demyelinated by any chemical, see (Windrem et al., "Progenitor Cells Derived from the Adult Human Subcortical White Matter Disperse and Differentiate as Oligodendrocytes Within Demyelinated Regions of the Rat Brain," J. Neurosci. Res. 69:966-975 (2002), which is incorporated herein by reference in its entirety), or for autoimmune demyelination, see (Pluchino et al., "Injection of Adult Neurospheres Induces Recovery in a Chronic Model of Multiple Sclerosis," Nature 422:688-694 (2003), which is incorporated herein by reference in its entirety), (Yandava See, e.g., S. et al., "Global Cell Replacement is Feasible Via Neural Stem Cell Transplantation: Evidence from the Dysmyelinated Shiverer Mouse Brain," Proc. Natl. Acad. Sci. USA 96:7029-7034 (1999), which is incorporated herein by reference in its entirety. I want to be illuminated.

[0088] In some embodiments, cells generated and administered according to the present disclosure differentiate into neurons, oligodendrocytes, and astrocytes, thereby reconstituting lost cellular elements in the injured brain and spinal cord. In some embodiments, transplantation of the NPCs, OPCs, and oligodendrocytes of the present disclosure into the affected area mediates structural regeneration and repair. This may include, but is not limited to, the reconstitution of brain tissue lost due to stroke and injury. In addition, neural stem cells are highly migratory in the perinatal brain, and as a result, they may also be used to replenish enzymes that are deficient or mutated due to genetic and metabolic diseases.

[0089] To date, most experimental therapeutic studies of neural stem and progenitor cell engraftment have been performed using fetal or adult tissue as a source of neural stem and progenitor cells. Such approaches have required the continuous generation of new fetal and adult tissue from both animal and human donors. The latter is particularly problematic as a source, as human tissue-derived cells, whether of fetal or adult origin, are difficult to obtain and are more difficult to standardize and scale up (Keyoung et al., "Specific Identification, Selection and Extraction of Neural Stem Cells from the Fetal Human Brain," Nature Biotech. 19:843-850 (2001);Pincus et al., "Fibroblast Growth Factor-2 / Brain-Derived Neurotrophic Factor-Associated Maturation of New Neurons Generated from Adult Human Subependymal Cells," Ann. Neurol. 43:576-585 (1998);Roy et al., "Promoter-Targeted Selection and Isolation of Neural Progenitor Cells from the Adult Human Ventricular Zone," J. Neurosci. Res. 59:321-331 (2000); and Roy et al., "In Vitro Neurogenesis by Progenitor Cells Isolated from the Adult Human Hippocampus," Nature Med. 6:271-277 (2000), which are incorporated herein by reference in their entireties. (The disclosure of which is incorporated herein by reference.) In contrast, the NPCs, OPCs, and oligodendrocytes generated using the methods of the present disclosure allow for rapid acquisition, scalable expansion, and directed differentiation of neural stem and progenitor cells.

[0090] Furthermore, the present disclosure allows for the use of autologous NPCs, OPCs, and oligodendrocytes in some embodiments. Alternatively, heterologous NPCs, OPCs, and oligodendrocytes can be used in any of the treatment methods of the present disclosure. ES cell-derived neural stem cells and progenitor cells, both mouse and human, have already been found to be capable of both remyelination in the case of ES cell-derived glial cells and oligodendrocytes, and dopamine system replenishment in experimental Parkinson's disease in the case of ES cell-derived midbrain dopaminergic neurons. Now that the purification of neural stem cells from ES cell cultures has been achieved, the present invention substantially increases both the reliability and safety of their use, thereby eliminating the need to obtain human tissue.

[0091] Another aspect of the disclosure features a method of treating a subject having a neurodegenerative condition, the method comprising administering to the patient the postnatal-derived cells described above in an amount effective to treat the neurodegenerative condition. In certain embodiments, the neurodegenerative condition is an acute neurodegenerative condition, e.g., resulting from brain injury (focal or diffuse brain injury), spinal cord injury or peripheral nerve injury, e.g., physical or chemical burns, deep cuts or limb amputations, or cerebrovascular insufficiency. In other embodiments, it is a treatment for a chronic or progressive neurodegenerative condition, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, tumors, or chronic peripheral nerve injury, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steele-Richardson syndrome), multiple system degeneration (Shy-Drager syndrome), chronic epileptic conditions associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxias, corticobasal degeneration, ALS-Parkinson-Dementia Complex of Guam, subacute sclerosing panencephalitis, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease / spinocerebellar ataxia. These include tonicity syndrome type 3 and olivopontocerebellar degeneration, Gilles de la Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff disease, familial spastic disorders, Wohlfart-Kugelberg-Welander disease, spastic paraplegia, progressive multifocal leukoencephalopathy, familial dysautonomia (Riley-Day syndrome), and prion diseases (including, but not limited to, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, kuru, and fatal familial insomnia).

[0092] In yet other embodiments, it is a disease associated with demyelination, such as multiple sclerosis, acute disseminated encephalomyelitis, neuromyelitis optica, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, central pontine myelinolysis, inherited demyelinating diseases such as leukodystrophy, Charcot-Marie-Tooth disease, and Canavan disease.

[0093] In certain embodiments, the iPSCs, ESCs, NPCs, OPCs, and oligodendrocytes of the present disclosure can be administered separately, together, or in combination of two or more. In yet other embodiments, the cells are administered with at least one other agent, such as a neurotherapeutic drug or another beneficial adjunct, such as an anti-inflammatory agent, an anti-apoptotic agent, an antioxidant, or a growth factor.

[0094] In certain embodiments, the cells are administered to a predetermined site in the patient's central or peripheral nervous system, which can be by injection or infusion, or by encapsulation in an implantable device, or by implanting a matrix or scaffold containing the cells.

[0095] Another aspect of the invention features a pharmaceutical composition for treating a patient having a neurodegenerative condition, the composition comprising one or more of the cell types of the present disclosure and a pharmaceutically acceptable carrier. The neurodegenerative condition to be treated can be an acute neurodegenerative condition, or it can be a chronic or progressive condition.

[0096] In certain embodiments, the pharmaceutical composition comprises at least one other agent, such as a neurotherapeutic drug, or another beneficial adjunct, such as an anti-inflammatory agent, an anti-apoptotic agent, an antioxidant, or a growth factor.

[0097] In certain embodiments, the pharmaceutical composition is formulated for administration by injection or infusion. Alternatively, it may comprise an implantable device that encapsulates the cells, or a matrix or scaffold that contains the cells. The pharmaceutical composition may comprise other substances, such as natural biological materials, such as gelatin, collagen, or carboxymethylcellulose. For delivery by injection, a saline vehicle may be used. [Example]

[0098] The present invention will now be described with reference to the following examples. Such examples are provided for illustrative purposes only, and the present invention is not limited to such examples, but rather encompasses all variations that become apparent as a result of the teachings presented herein. The following tables include experimental conditions for use in some embodiments, including conditions that have been verified by experimentation. Any one of the parameters, parameter ranges, or exemplary parameters presented in the following tables or herein can be used in conjunction with any other of the parameters, parameter ranges, or exemplary parameters presented in the following tables or herein.

[0099] [Table 1-1] [Table 1-2]

[0100] In some embodiments, the number of cells used for seeding is, for example, about (7.1250 x 10 4 , 1.187500×10 6 , 2.375000×10 6 , 4.750000×10 6 , 7.125000×10 6 , 9.500000×10 6 , 1.1875000×10 7 or 1.4250000×10 7 ) or any number of cells between any two of the listed cell numbers. 2 Such seeding density as can be determined for any culture vessel by dividing the number of cells by the growth area of ​​the culture vessel (see Table 2), e.g., for a 9.5 cm 2 Using 6-well plates with a growth area of ​​1000, the seeding density was approximately (0.75 x 10) per 6-well plate. 5 , 1×10 5 , 1.25×10 5 , 2.50 x 10 5 , 5.00×10 5 , 7.50 x 105 , 1×10 6 , 1.25×10 6 Or 1.5 x 10 6 cells / cm 2 ), or any density between any two of the listed densities. Thus, the cell density required for any culture vessel can be determined in a similar manner. [Table 2-1] [Table 2-2]

[0101] In some embodiments, the oxygen concentration can be, for example, about (2, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, or 6.0%) oxygen, or any range between any two of the recited percentages. Example 1 Generation of neural progenitor cells from iPSCs

[0102] iPSCs were cultured in standard-sized 6-well cell culture plates (growth area approximately 9.5 cm). 2 / well) or a standard-sized 12-well cell culture plate (growth area approximately 3.8 cm 2 The cells were seeded onto a 6-well or 12-well plate (1 / well) and differentiation was initiated. Other sizes of culture vessels are also applicable as needed, and larger than 6-well or 12-well plates may be more preferable due to more efficient use of reagents and time. Applicant has confirmed that the disclosed method can also be used with culture vessels of other sizes.

[0103] Approximately 1 x 10 cells per well in a 6-well plate 5 ~Approx. 1×10 6Cells with population sizes of 100-1500 μm have been successfully used. When sufficient typical iPSC colonies, with clearly defined and compact cells, were present and the colonies were not overgrown, the iPSCs were considered ready for differentiation. While Applicant was able to successfully derive iPSCs generated by other researchers using non-mRNA methods, e.g., the NCL2 iPSC line generated by NIH / Lonza (data not shown), the quality of the iPSCs of the present invention generated using such criteria proved important for differentiation when comparing iPSC lines of the present invention with iPSC lines generated by others using other methods. Meanwhile, the disclosed techniques can also be applied at any stage during the process, for example, starting from NPCs and inducing them to OPCs, astrocytes, or other glial cells, or starting from OPCs and inducing them to astrocytes or other glial cells. In one exemplary experiment, Applicant used primary NPCs isolated from donated tissue and further differentiated them into OPCs and oligodendrocytes using the same protocol, alongside NPCs differentiated directly from iPSCs. Results using the two different sources of NPCs were generally similar, with iPSC-derived NPCs being slightly more efficient and more likely to form spheres when Applicant attempted to grow the cells in suspension at that stage (data not shown).

[0104] iPSCs at this stage were induced to differentiate into ectodermal lineage cells. Although most current protocols for differentiation prefer the use of adherent monolayer cells, we have found that a suspension culture system is very useful for scale-up at this stage. We have found that iPSCs grown in suspension for induction are more resistant to chemical toxicity and easier to re-plated at later stages. When the suspension culture route is chosen for a particular differentiation "run," we have found that Ultra- Low Attachment plates (Sigma-Aldrich) or other low attachment plates were used to promote iPSC suspension cell growth.

[0105] When iPSCs need to be passaged, it is important to dissociate iPSC colonies using a protocol that is less cytotoxic and produces smaller iPSC clusters. This allows for the rapid formation of spheres when suspension culture is desired. iPSCs were dissociated using TripLE™ (ThermoFisher), Accutase (Life Technology), or EDTA in DPBS (Fisher Scientific) at about 37°C for 5 minutes with about 0.1 mM, optionally about 0.5 mM, or about 1 mM EDTA. Various dissociation times have been successfully used for this step, including about 1 to about 2 minutes, and optionally about 10 to about 20 minutes. In some embodiments, dissociation times can range from about (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) or any time between any two of the listed times.

[0106] Regarding the culture medium, we tested MEMa, DMEM / F12, or DMEM+B27 containing 5% KSR and were able to achieve the desired results in this differentiation stage. Furthermore, the medium supplements N2, BSA, or HAS and bFGF were used in the culture medium depending on the basal medium used in this stage. The iPSCs were then induced to leave the pluripotent stage and differentiate into ectoderm as needed, for example, in the presence of SB431542, LDN193189, or PD173074 at approximately (5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM) or any range between any two of the listed concentrations. LDN-193189 is a highly potent small molecule inhibitor of the bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3. LDN193189 is a selective, transcriptionally active bone morphogenetic protein (BMP) type I receptor inhibitor. It inhibits activin receptor-like kinase 2 (ALK2) and ALK3. SB431542 is a selective and potent inhibitor of the TGF-β / activin / NODAL pathway and a potent and selective inhibitor of ALK5. PD173074 is a potent FGFR1 inhibitor and also inhibits VEGFR2. In other embodiments, other inhibitors of such pathways may also be used.

[0107] In one experiment, cells were then transfected with mRNA for Ngn2, Pax6, Sox2, Brn2, or FoxO (one or more members of the subclasses), individually or in combination, at a dose of 20 ng per well in a 6-well plate using Stemgent Transfection Reagent (Stemgent) or other transfection reagents. The mRNA dose can be adjusted proportionally based on the area of ​​the culture vessel or the volume of culture medium when using other types of culture plates. This transfection was repeated 3, 4, 5, or 6 times, sometimes using approximately 10-fold higher mRNA doses, using Stemgent Transfection Reagent or other commercially available transfection reagents. In the examples disclosed herein, repeat transfections can be performed approximately 24 hours plus or minus 2-3 hours after the initial transfection, or overnight after the initial transfection, or at approximately (6-24 hours, 7-24 hours, 8-24 hours, 9-24 hours, 10-24 hours, 11-24 hours, 12-24 hours, 13-24 hours, 14-24 hours, 15-24 hours, 16-24 hours, 17-24 hours, 18-24 hours, 19-24 hours, 20-24 hours, 21-24 hours, 22-24 hours) or any time range between the two listed time points after the initial transfection. While cells at this stage exhibited morphology more similar to neural progenitor cells than mesenchymal cells, these NPCs generated by TF expression appeared to yield higher cell yields in the oligodendrocyte pathway compared to NPCs generated by other methods, primarily chemical-based methods. NPCs at this stage were Pax6 positive by antibody staining, demonstrating their identity in the neural progenitor lineage (Figure 1). The mRNA, DNA, and protein sequences of Ngn2, Pax6, Sox2, Brn2, and FoxO are known in the art, e.g., in GenBank. Methods for preparing mRNA for transfection for use in the methods described herein are known in the art, e.g., U.S. Patent Application Nos. 13 / 893,166 and 14 / 292,317 (the contents of which are incorporated herein by reference). Example 2 Generation of oligodendrocyte precursor cells from neural progenitor cells

[0108] Neural progenitor cells, or NPCs, were plated in commercially available cell culture vessels. Six-well plates were used for the experiments shown in Figure 2, although other well sizes are also applicable. Plates were pre-coated with Matrigel (BD Biosciences) and PLO-laminin, and then approximately 1 × 10 cells were plated. 5 ~1×10 6 Cells were plated in DMEM / F12 or DMEM or MEM3 supplemented with KSR and ascorbic acid, SAG, PDGF, HGF, IGF1, T3, insulin, RA, B27, CAMP, and biotin at commonly used concentrations. NPCs generated using chemical-based protocols commonly known in the art were also used to generate OPCs in the following steps, and it was observed that NPCs generated by the introduction of TF activity generated more numerous and much better OPCs.

[0109] In one experiment, cells were then transfected in culture medium with one or various combinations of Oligo2, NKX2.2, SOX10, and Oligo1 mRNA, either individually or in combination, at a dose of approximately 20 ng per well / cell fate factor using Stemgent Transfection Reagent (Stemgent), and repeated two, three, four, or more times over approximately 24 hours using Stemgent Transfection Reagent or other commercially available transfection reagents at doses as low as approximately 10 ng per well and as high as approximately 200 ng per well. Cells at this stage appeared to have transformed from the typical morphology of NPCs to that of OPCs and stained positive for A2B5 and O4 (Figure 2). Example 3 Generation of oligodendrocytes from OPCs

[0110] Oligodendrocyte precursor cells were cultured in DMEM / F12, MEMa, or DMEM B27 in 6-well plates or other plates pre-coated with Matrigel (BD Biosciences) and / or PLO-laminin. Similar adherent cell culture media are also suitable for use. The medium was typically supplemented with ascorbic acid, N2, B27, biotin, cAMP, T3, insulin, and KSR at commonly used concentrations. Cells were cultured for approximately 1 week, sometimes for about 2, 3, or even 4 weeks, during which time mature-appearing oligodendrocytes emerged, migrated from the OPC clusters, and then diffused into the space, where they formed increasing numbers of dendritic arborization-like processes (Figure 3). Example 4 Upon further culture, oligodendrocytes form new clusters.

[0111] If growth of stage 2 oligodendrocyte-like cells is allowed to continue for about 1 week to about 4 more weeks, or even longer, they form new clusters with overlapping processes, somewhat mimicking sheath formation in vivo (Figure 4). Example 5 Exemplary embodiments of human iPSC-derived oligodendrocytes exhibiting specific cell markers

[0112] To demonstrate that human pluripotent stem cell-derived oligodendrocytes are mature oligodendrocytes, we removed them from differentiation culture plates by chemical or physical means, formed them into spheres, fixed them, and stained them with anti-MBP antibodies. All cells generated in this manner appeared to stain positive for MBP, a commonly known mature oligodendrocyte marker (Fig. 5). Example 6 Function of iPSC-derived oligodendrocytes in animal models

[0113] To further test the functionality of mature oligodendrocytes generated according to the present invention, the spinal cord repair function of iPSC-derived oligodendrocytes could be tested in a rat model of spinal cord injury, such as that described by Mark Tuszynski's group. Oligodendrocytes could be delivered as cells, cell sheets, cell clusters, as isolated cell types, or in combination with other cells, such as OPCs or NPCs. OPCs derived from iPSCs by the methods of the present disclosure are also tested as therapeutic candidates for treating spinal cord injury or other central nervous system diseases and conditions. In another embodiment, oligodendrocytes, OPCs, NPCs, or combinations thereof could be presented on a hydrogel, and in some cases, in the presence of cytotrophic factors, such as BDNF, NGF, etc.

[0114] Non-human primate models, such as monkeys and chimpanzees, could also be used to test human cells, such as oligodendrocytes of the central nervous system, for safety and efficacy with immunosuppressive factors or with corresponding non-human primate iPSC-derived NPCs, OPCs, or oligodendrocytes using essentially the same methods as those disclosed herein. Example 7 iPSC-derived oligodendrocytes in the treatment of human patients with spinal cord injury or other central nervous system diseases and conditions

[0115] Clinical trials using human iPSC-derived oligodendrocytes, OPCs, NPCs, neurons, or combinations of the above, using the disclosed protocols as suitably adapted, under cGMP procedures, are conducted in accordance with animal studies for other cell therapies. The produced cells can be delivered to other parts of the human central nervous system, such as the spinal cord, or possibly the brain. Example 8 Use of the CRISPR-Cas9 system to drive the expression of critical genes and thereby determine cell fate using mRNA generated by intracellular transcription.

[0116] As an example demonstrating that functional proteins can be directed by gRNA to target regions of interest within the genome via Cas9 fusion, we conjugated dCas9, a nuclease-deactivated version of Cas9, to the fluorescent protein mNeonGreen (Allele Biotechnology), as shown in Figure 6. The same strategy can be applied to functional domains, proteins, peptides, etc., to drive gene expression (i.e., mRNA production in cells that will become committed cells). An example is the MyoD transcriptional activation domain shown in Figure 6, and other examples include VP16 and many other such domains. When such transcriptional activation fusion Cas9 fusion proteins are placed by gRNA into regions that turn on key TFs, such as Ngn2, Pax6, Sox2, Brn2, FoxO, Oligo2, NKX2.2, or SOX10, transfection of in vitro transcribed mRNA can be achieved. The same strategy can be applied to enzymatically active domains or proteins that can affect gene expression by epigenetically modifying specific regions on chromosomes, for example, using DNA (cytosine-5)-methyltransferase 1 or DMNT1. Nucleotide converting enzymes fused to Cas9 could be used to switch specific nucleotides for other nucleotides, thereby changing the encoded protein or causing premature termination of protein production. If the modified gene is a critical TF, such as those listed above or other genes, the gene expression profile of the cell can be altered in this way, and cell fate can be determined, according to the general strategy disclosed in this application.

[0117] Nuclease-inactivated Cas9 and methods for fusing transcription activation domains with targets using guide RNAs are known in the art (Didovyk et al., Current Opinion in Biotechnology (2016), 40:177-184), the contents of which are incorporated herein by reference. The guide RNA sequence targeting the cell fate factor promoter can be prepared as described in Balboa et al., Stem Cell Reports (2015) 5:448-459, the contents of which are incorporated herein by reference. The present invention provides, for example, the following items. (Item 1) 1. A method for inducing differentiation of induced pluripotent stem cells (iPSCs) and / or embryonic stem cells (ESCs) into neural progenitor cells (NPCs), comprising: a) plating iPSCs and / or ESCs onto a coated, non-tissue-treated culture vessel containing culture medium; b) transfecting the cells of step a) with mRNAs encoding one or more cell fate factors, either as individual mRNAs or as a combination of mRNAs; and c) culturing the iPSCs and / or ESCs of step b) until the cells differentiate into NPCs. A method comprising: (Item 2) Item 1. The method according to item 1, wherein the cell fate factors in step b) are selected from the group consisting of one or more of Ngn2, Pax6, Sox2, Brn2 and FoxO family factors, as individual mRNA or combined mRNA transfection. (Item 3) 3. The method according to item 2, wherein the cell fate factor in step b) is Ngn2. (Item 4) 3. The method according to item 2, wherein the cell fate factor in step b) is Pax6. (Item 5) 3. The method according to item 2, wherein the cell fate factor in step b) is Sox2. (Item 6) 3. The method of claim 2, wherein the cell fate factor in step b) is Brn2. (Item 7) 3. The method according to item 2, wherein the cell fate factor in step b) is a FoxO family factor. (Item 8) 3. The method of claim 2, wherein one or more of the cell fate factors of step b) are fused to a transactivation domain. (Item 9) 2. The method of claim 1, wherein the cells of step c) are cultured until the cells express one or more of the cell fate factors of step b). (Item 10) Item 1, further comprising adding one or more of SB43542, LDN193189 and PD173074 to the cells of step c). (Item 11) Item 1, wherein the culture conditions in steps a) to c) are at an oxygen concentration of about 2% to about 6%. (Item 12) 2. The method according to item 1, wherein the culture medium used in steps a) to c) is selected from the group consisting of Minimum Essential Medium Alpha (MEMa), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F12), or DMEM with B27 supplement. (Item 13) 13. The method according to item 12, wherein the medium used also contains knockout serum replacement (KSR). (Item 14) 14. The method of claim 13, wherein the culture medium further comprises one or more of bFGF, N2 or B27, BSA or HSA (human serum albumin). (Item 15) 2. The method according to item 1, wherein the culture vessel is coated with Matrigel and / or PLO-laminin. (Item 16) 2. The method of claim 1, wherein the iPSCs and / or ESCs in step a) are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or per culture well plate. (Item 17) 2. The method according to item 1, wherein the cells in step c) are cultured for about 3 to about 10 days. (Item 18) 2. The method according to item 1, wherein the transfection in step b) is repeated at least once. (Item 19) 1. A method for generating oligodendrocyte precursor cells (OPCs) from neural precursor cells, comprising: a) plating NPCs onto a coated cell culture vessel containing a medium; b) transfecting the cells of step a) with mRNAs encoding one or more cell fate factors, either as individual mRNAs or as a combination of mRNAs, to differentiate the neural progenitor cells into oligodendrocyte precursor cells; c) culturing the cells of step b) until the morphology of the NPCs changes to that of OPCs; A method comprising: (Item 20) 20. The method of item 19, wherein the NPCs of step a) are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or per culture well plate. (Item 21) 20. The method according to item 19, wherein the mRNA used for transfection in step b) encodes a cell fate factor selected individually or in combination from one or more of the group consisting of Oligo2, NKX2.2, SOX10 and Oligo1 mRNA. (Item 22) 22. The method of claim 21, wherein the mRNA encodes Oligo2. (Item 23) 22. The method of claim 21, wherein the mRNA encodes NKX2.2. (Item 24) 22. The method of claim 21, wherein the mRNA encodes Sox10. (Item 25) 22. The method of claim 21, wherein the mRNA encodes Oligo1. (Item 26) 20. The method according to item 19, wherein a 6-well culture plate is used and the dose for transfection in step b) is about 20 ng per cell fate factor. (Item 27) 20. The method of item 19, wherein after the initial transfection, the transfection is repeated at least two more times. (Item 28) Item 20. The method according to Item 19, wherein the culture conditions in steps a) to c) are at an oxygen concentration of about 2% to about 6%. (Item 29) 20. The method of claim 19, wherein the culture vessel is coated with Matrigel and PLO-laminin. (Item 30) 20. The method according to item 19, wherein the medium used in steps a) to c) is selected from the group consisting of DMEM / F12, DMEM, and MEMa. (Item 31) 31. The method of claim 30, wherein the medium is supplemented with one or more of KSR, ascorbic acid, SAG, PDGF, HGF, IGF1, T3, insulin, RA, B27, CAMP, and biotin. (Item 32) a) plating OPCs onto a coated cell culture vessel containing a culture medium; b) culturing the cells of step a) until the cells appear with dendritic branch-like processes or produce myelin basic protein (MBP); 1. A method for generating oligodendrocytes from OPCs, comprising: (Item 33) Item 33. The method according to Item 32, wherein the medium used in steps a) to b) is selected from the group consisting of DMEM / F12, DMEM, and MEMa. (Item 34) 34. The method of claim 33, wherein the medium is supplemented with one or more medium supplements selected from the group consisting of KSR, ascorbic acid, N2, B27, biotin, CAM, T3 and insulin. (Item 35) 33. The method of claim 32, wherein the OPCs of step a) are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or per culture well plate. (Item 36) 33. The method according to item 32, wherein the cells of step b) are cultured for about 7 to about 30 days. (Item 37) 33. The method according to item 32, wherein the cell culture vessel in step a) is coated with Matrigel and / or PLO-laminin. (Item 38) A cell obtained by the method according to item 1, 19 or 32. (Item 39) 39. A composition for treating a disease, disorder or dysplasia, comprising the cells of item 38. (Item 40) 39. A method for treating a disease, disorder, and / or dysplasia, comprising administering the cell and / or composition of item 38 to a subject in need of such treatment. (Item 41) 1. A method for sequentially or directly inducing differentiation of stem cells into NPCs, OPCs, and oligodendrocytes, comprising performing the steps of Item 1, followed by the steps of Item 19, followed by the steps of Item 32. (Item 42) 1. A method for sequentially or directly inducing differentiation of stem cells into NPCs, OPCs, and oligodendrocytes, comprising: a) plating iPSCs and / or ESCs onto a coated culture vessel, using about 70,000 to about 14,000,000 cells per culture flask or per culture well plate in a medium comprising Minimal Medium Alpha (MEMa) or Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12) or DMEM with B27 supplement, both with Knockout Serum Replacement (KSR); b) transfecting the cells of step a) with mRNA encoding a cell fate factor selected from the group consisting of one or more of Ngn2, Pax6, Sox2, Brn2 and FoxO family factors, either as individual mRNA or combined mRNA transfection; c) culturing the iPSCs and / or ESCs of step b) until the cells differentiate into NPCs; d) isolating the NPCs from step c); e) plating NPCs onto the coated cell culture vessel, seeding the NPCs at about 70,000 to about 14,000,000 cells per culture flask or per culture well plate in a medium comprising MEMa or DMEM / F12 or DMEM, both having KSR and one or more of the following media supplements selected from the group consisting of ascorbic acid, SAG, PDGF, HGF, IGF1, T3, insulin, RA, B27, CAMP, and biotin; f) transfecting the cells of step e) with one or more of the group consisting of Oligo2, NKX2.2, SOX10 and Oligo1 mRNA, individually or in combination, using a transfection reagent; g) repeating the transfection of step f) at least two more times; h) culturing the cells of step g) until the morphology of the cells changes from NPC cells to OPC cells; i) isolating the OPCs from step h); j) plating the OPCs isolated from step h) onto coated cell culture vessels, seeding the OPCs at about 70,000 to about 14,000,000 cells per culture flask or per culture well plate in a medium comprising MEMa or DMEM / F12 or DMEM, both having KSR and one or more of the following medium supplements selected from the group consisting of ascorbic acid, N2, B27, biotin, Cam, T3, and insulin; k) culturing the oligodendrocyte cells for at least about one week until the cells appear with dendritic branch-like processes and / or produce myelin basic protein (MBP); A method comprising: (Item 43) 43. The method of items 1, 19, 41 or 42, wherein the NPCs are derived from a recipient subject. (Item 44) 43. The method of items 1, 19, 41 or 42, wherein the starting cells are obtained from a body fluid or body tissue. (Item 45) 43. The method of items 1, 19, 41 or 42, wherein the starting cells are harvested from the recipient. (Item 46) Next step d): d) expanding the NPCs or OPCs of step c) by transferring them to uncoated ultra-low attachment plates. 20. The method of claim 1 or 19, further comprising: (Item 47) 28. The method according to Item 27, wherein a 6-well culture plate is used and the transfection dose is about 10 ng / cell fate factor to about 200 ng / cell fate factor mRNA dose. (Item 48) 20. The method according to item 19, wherein the cells of step c) are cultured for about 4 to about 20 days. (Item 49) A method for regenerating cells or tissues in vivo, comprising administering an effective amount of the NPC described in item 1 to a subject in need of regenerating cells or tissues in vivo, wherein the NPCs transform after transplantation or administration in an in vivo environment, thereby regenerating the cells or tissues. (Item 50) A pharmaceutical composition comprising the NPC according to item 1. (Item 51) 51. A kit comprising the pharmaceutical composition of item 50 and instructions for use.

Claims

1. A method for inducing differentiation of induced pluripotent stem cells (iPSCs) and / or embryonic stem cells (ESCs) into neural progenitor cells (NPCs), comprising: a) plating iPSCs and / or ESCs onto a coated, non-tissue-treated culture vessel containing culture medium; b) transfecting the cells of step a) with mRNA encoding Ngn2, Pax6, Sox2, Brn2, and FoxO family factors; c) culturing the iPSCs and / or ESCs of step b) until the cells differentiate into NPCs; Including, The method, wherein the induced pluripotent stem cells (iPSCs) and / or embryonic stem cells (ESCs) are seeded at a density of 1x105 to 1x106 cells per well of a 6-well plate, and the mRNA dose per transfection is 20ng to 200ng / well.

2. The method described in claim 1, wherein one or more of the Ngn2, Pax6, Sox2, Brn2, and FoxO family factors in step b) are fused to a transactivation domain.

3. The method described in claim 1, wherein the cells of step c) are cultured until the cells express one or more of the Ngn2, Pax6, Sox2, Brn2, and FoxO family factors of step b).

4. The method of claim 1, further comprising adding one or more of SB43542, LDN193189 and PD173074 to the cells of step c).

5. The method described in claim 1, wherein the culture conditions for steps a) to c) are at an oxygen concentration of about 2% to about 6%.

6. The method of claim 1, wherein the culture medium used in steps a) to c) is selected from Minimum Essential Medium Alpha (MEMa) or Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F12) or DMEM with B27 supplement.

7. The method described in claim 6, wherein the culture medium used also contains knockout serum substitute (KSR).

8. The method described in claim 7, wherein the culture medium further contains one or more of bFGF, N2 or B27, BSA or HSA (human serum albumin).

9. The method of claim 1, wherein the culture vessel is coated with Matrigel and / or PLO-laminin.

10. The method of claim 1, wherein the iPSCs and / or ESCs in step a) are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or per culture well plate.

11. The method of claim 1, wherein the cells in step c) are cultured for about 3 to about 10 days.

12. The method described in claim 1, wherein the transfection of step b) is repeated at least once.

13. A method for generating oligodendrocyte progenitor cells (OPCs) from neural progenitor cells (NPCs), comprising: a) plating NPCs onto a coated cell culture vessel containing a culture medium; b) transfecting the cells of step a) with mRNA of Oligo2, NKX2.2, SOX10 and Oligo1; c) culturing the cells of step b) until the morphology of the NPCs changes to that of OPCs; Including, The method, wherein said NPCs are seeded at a density of 1×10 5 to 1×10 6 cells per well of a 6-well plate, and the mRNA dose is 10 ng / well to 200 ng / well.

14. The method of claim 13, wherein the NPCs in step a) are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or per culture well plate.

15. The method of claim 13, wherein after the initial transfection, the transfection is repeated at least two more times.

16. The method described in claim 13, wherein the culture conditions for steps a) to c) are at an oxygen concentration of about 2% to about 6%.

17. The method of claim 13, wherein the cell culture vessel is coated with Matrigel and PLO-laminin.

18. The method of claim 13, wherein the culture medium used in steps a) to c) is selected from DMEM / F12, DMEM, and MEMa.

19. The method of claim 18, wherein the culture medium is supplemented with one or more of knockout serum substitute (KSR), ascorbic acid, sonic hedgehog agonist (SAG), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), triiodothyronine (T3), insulin, retinoic acid (RA), B27, cyclic adenosine monophosphate (CAMP), and biotin.

20. A method for inducing differentiation of NPCs into oligodendrocytes, comprising carrying out the steps of claim 13, followed by: seeding OPCs onto the coated cell culture vessel containing the medium, the OPCs being plated at a seeding density of 1×10 5 to 1×10 6 cells per well of a 6-well plate; culturing the OPCs until the cells emerge with dendrite-like processes or produce myelin basic protein (MBP); A method comprising:

21. A method for inducing differentiation of stem cells into oligodendrocytes, comprising carrying out the steps of claim 1, followed by the steps of claim 13, followed by: seeding OPCs onto the coated cell culture vessel containing the medium, the OPCs being plated at a seeding density of 1×10 5 to 1×10 6 cells per well of a 6-well plate; culturing the OPCs until the cells emerge with dendrite-like processes or produce myelin basic protein (MBP); A method comprising:

22. A method for inducing differentiation of stem cells into oligodendrocytes, comprising: a) plating iPSCs and / or ESCs onto a coated culture vessel, seeding the iPSCs and / or ESCs in a medium comprising Minimal Medium Alpha (MEMa) or Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12), both with Knockout Serum Replacement (KSR), or DMEM with B27 supplement, using about 70,000 cells to about 14,000,000 cells per culture flask or per culture well plate; b) transfecting the cells of step a) with mRNA encoding Ngn2, Pax6, Sox2, Brn2 and FoxO family factors, wherein the mRNA is 20 ng to 200 ng per 1×10 5 to 1×10 6 cells; c) culturing the iPSCs and / or ESCs of step b) until the cells differentiate into NPCs; d) isolating the NPCs from step c); e) plating NPCs onto the coated cell culture vessel, seeding the NPCs at about 70,000 to about 14,000,000 cells per culture flask or per culture well plate in a medium comprising MEMa or DMEM / F12 or DMEM supplemented with KSR and one or more of the following media supplements selected from ascorbic acid, SAG, PDGF, HGF, IGF1, T3, insulin, RA, B27, CAMP, and biotin; f) transfecting the cells of step e) with mRNA of Oligo2, NKX2.2, SOX10 and Oligo1 using a transfection reagent, wherein the mRNA dose is 20 ng to 200 ng per 1×10 5 to 1×10 6 cells; g) repeating the transfection of step f) at least two more times; h) culturing the cells of step g) until the morphology of the cells changes from that of NPC cells to that of OPC cells; i) isolating the OPC from step h); j) plating the OPCs isolated from step h) onto coated cell culture vessels, seeding the OPCs at about 70,000 to about 14,000,000 cells per culture flask or culture well plate in a medium comprising MEMa or DMEM / F12 or DMEM, both having KSR and one or more of the following medium supplements selected from ascorbic acid, N2, B27, biotin, Cam, T3, and insulin; k) culturing the oligodendrocyte cells for at least about one week until the cells appear with dendritic branch-like processes and / or produce myelin basic protein (MBP); A method comprising:

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