iPSC-derived immune cells in the prevention and treatment of age-related and neurodegenerative diseases

Abstract

Induced pluripotent stem cell-derived mononuclear phagocytes, designated as iMPs, are provided, further comprising monocytes generated from induced pluripotent stem cells, and optionally macrophages generated from induced pluripotent stem cells, for use in improving cognitive function, improving neurological health, and / or alleviating or treating neurodegenerative disorders in mammals. In various embodiments, the iMPs generate macrophages and / or express macrophage markers after transplantation or after in vitro stimulation. The inventors have shown that the iMPs, upon administration, improve cognitive and neurological health in rodent models of aging, Alzheimer's disease, and amyotrophic lateral sclerosis. Treatment methods are also provided using mononuclear phagocytes generated from autologous stem cells or from induced pluripotent stem cells obtained from autologous cells in patients in need of treatment or prevention of neurodegenerative disorders. TIFF2024529729000003.tif63170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 234,984, filed August 19, 2021, which is incorporated by reference herein in its entirety.

[0002] Technical Field The present invention relates to pluripotent stem cell derived therapies for neurodegenerative diseases and aging, and to improved protocols for generating monocytes and / or macrophages from pluripotent stem cells. [Background technology]

[0003] background All publications in this specification are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.The following description includes information that may be useful for understanding the present invention.This is not an admission that any of the information provided herein is prior art or related to the invention claimed herein, or that any publication specifically or implicitly referenced is prior art.

[0004] Neurodegenerative diseases affect nerve cells in the brain or peripheral nervous system, which are believed to lose function over time. As one example, Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects cognition and function, with patients experiencing symptoms that affect multiple aspects of life, such as cognitive function, behavior, mood, and psychological state. However, there are no known disease-modifying therapies, making drug discovery an unaddressed area of ​​medicine. As another example, amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a common, devastating, and invariably fatal adult neurodegenerative disease. In addition to the loss of upper and lower motor neurons, ALS is now considered a disorder with immune dysregulation, characterized by alterations / activation of inflammatory cells that enhance the disease burden and rate of disease progression. Unfortunately, there are no treatments currently available to block or substantially delay these relentless inflammatory responses in patients with ALS.

[0005] Previous studies using young plasma and bone marrow have shown some improvement in cognitive ability and neurological health in aging adults or those with neurodegeneration. However, the risks associated with the administration of young plasma and bone marrow make it an inappropriate treatment. For example, plasma infusions carry risks such as allergies and transfusion-associated circulatory overload, which can result in pulmonary edema (swelling) and respiratory distress.

[0006] It is therefore an object of the present invention to provide new therapies for the treatment or alleviation of neurodegenerative disorders. Summary of the Invention

[0007] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods that are intended to be exemplary and illustrative, without limiting scope.

[0008] In various aspects, methods are provided for treating or providing prophylaxis to a subject having a neurodegenerative disorder, experiencing cognitive impairment, or in need of improved cognitive function, comprising administering to the subject a therapeutically effective amount of mononuclear phagocytes generated from pluripotent stem cells. The mononuclear phagocytes can include monocytes, macrophages, or a mixture of monocytes and macrophages. A therapeutically effective amount of mononuclear phagocytes for a human subject is about 1×10 given in one or more doses. 6 , 1×10 7 , or 1 × 10 8 Thus, mononuclear phagocytes generated from the pluripotent stem cells of the invention herein, in particular from induced pluripotent stem cells (iPSCs), provide a superior supply of large numbers than their naturally occurring counterparts, since the latter are difficult, if not impossible, to grow in vitro.

[0009] In various embodiments, the mononuclear phagocytes for use in the treatment disclosed herein are generated from pluripotent stem cells, preferably from iPSCs, in a process that includes culturing pluripotent stem cells in cell culture medium under conditions that induce myeloid differentiation, resulting in the generation of mononuclear phagocytes.In various embodiments, the process of inducing myeloid differentiation to generate mononuclear phagocytes does not include causing cells to become microglia or dendritic cells.In some additional embodiments, the process of inducing myeloid differentiation to generate mononuclear phagocytes does not include causing cells to become macrophages in vitro; while in other additional embodiments, the process of inducing myeloid differentiation to generate mononuclear phagocytes includes causing cells to become macrophages in vitro.

[0010] In some embodiments, the process of inducing myeloid differentiation to generate mononuclear phagocytes comprises the first one, two, three or all four of the following steps: contacting iPSCs in a medium with a first composition comprising bone morphogenetic protein 4 (BMP-4); after culturing the iPSCs in the presence of the first composition, contacting the cell culture medium with a second composition comprising one or more of bFGF, VEGF, and SCF; after culturing the iPSCs in the presence of the second composition, contacting the cell culture medium with a third composition comprising one or more of SCF, IL-3, thrombopoietin (TPO), macrophage colony stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand); and after culturing the iPSCs in the presence of the third composition, contacting the cell culture medium with a fourth composition comprising one or more of M-CSF, GM-CSF, and FLT3 ligand.

[0011] In a preferred embodiment, the first composition comprising BMP-4 is in a medium comprising bFGF and TGFβ, and optionally further comprising aminobutyric acid (GABA), pipecolic acid, and lithium chloride.Preferably, the first composition is in mTeSR1 medium.In an additional embodiment, the second composition, the third composition, and / or the fourth composition are in a hematopoietic cell medium, for example, StemPro-34 medium.Preferably, the medium is a serum-free medium, for example, serum-free mTeSR1 medium or StemPro-34 serum-free medium.

[0012] Additional embodiments provide that the mononuclear phagocytes for use in the treatments disclosed herein are generated from iPSCs reprogrammed from blood cells, such as peripheral blood mononuclear cells, or from fibroblasts or another somatic cell source.In some embodiments, the mononuclear phagocytes for use in the treatments disclosed herein are autologous, i.e., generated from iPSCs reprogrammed from autologous somatic cells.In some embodiments, the mononuclear phagocytes for use in the treatments disclosed herein are generated from iPSCs reprogrammed from autologous somatic cells obtained from a subject.

[0013] In various embodiments, the generated mononuclear phagocytes are for use in aging mammalian subjects, or in subjects with neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, multiple sclerosis (MS), schizophrenia, and autism spectrum disorder, or in subjects in need of reducing inflammation associated with neurodegenerative disorders. In various embodiments, the generated mononuclear phagocytes as disclosed herein provide an improvement in the cognitive function of the subject in one or more behavioral assessments, in the level of synaptic transporters, VGLUT1, microglial branch length, or another molecular analysis. In some aspects, the improvement is compared to the subject's baseline state prior to administration of the mononuclear phagocytes. In some aspects, the improvement results in a level comparable or similar to that of a young or healthy subject without a neurodegenerative disorder.

[0014] Also provided are mononuclear phagocytes generated from pluripotent stem cells, which may be present in a composition further comprising one or more phagocytes that are phagocytes that are phagocytes that are generated from iPSCs that are reprogrammed from blood cells or fibroblasts.

[0015] Additional embodiments provide methods for drug screening using the mononuclear phagocytes generated herein, including but not limited to high-throughput screening methods. In some embodiments, methods are provided for identifying compounds useful for treating or preventing diseases or disorders associated with deficiencies or deficiencies of monocytes and / or macrophages, or neurodegenerative diseases or disorders, comprising contacting the mononuclear phagocytes generated by the methods disclosed herein with a candidate compound, and determining whether the candidate compound ameliorates the deficiencies or deficiencies of monocytes or macrophages, or neurodegenerative diseases or disorders, respectively.

[0016] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of aspects of the invention. [Brief description of the drawings]

[0017] Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein should be considered illustrative and not limiting. [Figure 1] Figure 1 is a schematic diagram of the mouse model in which iPSC-derived mononuclear phagocytes are administered every 3 days and behaviorally tested for several cognitive tasks. "83iGFP" represents the iPSC line used to generate iMPs. "SAB" represents spontaneous alternation behavior test. "FC" represents fear conditioning. "NOP" represents novel object placement test. "NOR" represents novel object recognition test. "EPM" represents elevated plus maze. Young mice are 3-4 months old, while aged mice are 11-13 months old. [Diagram 2] FIG. 2 is a schematic depicting the spontaneous alternation behavior (SAB) task testing spatial working memory, in which mice with good spatial working memory would rotate from arm A → B → C, whereas mice with poor memory would move more frequently between the arms they had just emerged from (e.g., moving between arms A and B and back to A). [Diagram 3] Figure 3A shows that aged animals treated with vehicle without iMP (designated as "Aged Veh"; green dots) performed worse in the spontaneous alternation task, whereas aged mice treated with iMP (designated as "Aged iMP"; red dots) performed no worse compared to young animals (designated as "Young"; blue dots). Figure 3B shows that both aged groups made significantly fewer arm entries than young animals, indicating that the effect of iMP in Figure 3A is cognitive and not due to changes in locomotion. [Figure 4A]Figure 4A depicts a "novel object recognition" (NOR) study in which mice are exposed to two novel objects that they have not encountered before, and after a 30 minute retention delay, the mice are then exposed to one novel object, and if the mice remember having seen the previous object before, they will spend more time with the novel object. There is no effect of age or treatment in the novel object recognition assay, and the results are not statistically different between young mice and aged mice treated with vehicle and iMP. [Figure 4B] Figure 4B depicts a "novel object location" (NOP) study in which one of two objects is moved and animals are expected to spend more time with the object located in the novel position if they remember where the previous object was located. The results show that aged mice are significantly impaired in recognizing the object whose location has been moved, and that iMP treatment (the "aged iMP" group) significantly improved the performance of aged mice in this task. [Diagram 5] FIG. 5A depicts that the number of Neun+ cells does not change with age or treatment in cornu ammonis areas 1 and 3 (CA1, CA3). Neun is a marker for neuronal nuclei and therefore indicates the number of neurons. FIG. 5B depicts that VGLUT1 is decreased in aged animals, but not in those treated with iMP, compared to young animals. VGLUT1 is a glutamate transporter located at synapses that is essential for normal synaptic function and has been shown to be decreased in Alzheimer's disease. [Figure 6]FIG. 6A depicts that in CA3, the branch length of microglia is reduced in aging animals, but not in iMP-treated aging animals. The iMP-treated aging animals are designated as the "Aged iMP" group. FIG. 6B depicts that in CA1, the branch length is again reduced in aging animals, but significantly increased in iMP-treated aging animals. Given that microglia are the primary immune cells of the brain and their function is to survey the surroundings for damage or injury, they normally have long branched processes. However, upon activation, microglia retract these processes, resulting in shorter branch lengths per cell. This is known to occur with both aging and Alzheimer's disease. Furthermore, in both cases, there is an increase in the overall number of microglia. [Figure 7] Figure 7 depicts that LAMP1 is increased in CA1 in both aging groups. LAMP1 is a lysosomal marker that has been shown to increase with aging and Alzheimer's disease. [Figure 8] Figure 8 depicts that astrocyte numbers increase in aging animals but not in iMP-treated aging animals. GFAP is a marker for astrocytes, whose numbers and soma size normally increase with aging and in disease. [Figure 9A] Figure 9A depicts the timeline of the study of iMPs administered to a mouse model of Alzheimer's disease, starting with 3-month-old 5xFAD mice, when they first develop pathology (AD mouse model with amyloid & microglial activation begins at about 2 months). Cyclosporine A is administered by intraperitoneal injection 3 days prior to the first cells, then via drinking water. 83iGFP mononuclear cells are injected at 500,000 cells / injection over 8 injections as shown in the figure. We will repeat this with 7-month-old animals, which already have extensive pathology. [Figure 9B]FIG. 9B depicts that at 3 months, 5×FAD mice treated with iMP (“iMP”) show no changes in spatial working memory and short-term memory tasks compared to vehicle-treated 5×FAD mice. [Figure 9C] FIG. 9C depicts that iMP-treated animals show improvement in the "novel object recognition" study. [Figure 10A] 10A is a hierarchical clustering of bulk RNA sequencing data comparing iPSCs (designated as number 1, with three lineages designated as 1A, 1B, and 1C), iMPs grown in well plates (as described in Example 2; designated as number 3, with three lineages designated as 3A, 3B, 3C), cryopreserved iMPs collected from culture in well plates (designated as number 2, with three lineages designated as 2A, 2B, 2C), and iMPs produced in bioreactors at early (day 15; designated as number 4, with three lineages designated as 4A, 4B, 4C) and late (day 55; designated as number 5, with three lineages designated as 5A, 5B, 5C) times. All of the differentiated iMPs (groups 2-5) are very similar to each other. [Figure 10B] Figure 10B is a principal component analysis plot depicting the 2000 most variable genes across the assays depicted in Figure 10A. Similar to the clustering in Figure 10A, proximity indicates similarity, and thus iMPs in groups 2-5 were shown to be similar to each other. [Figure 11] Figure 11 shows the results of RNA-seq for the expression (transcripts per kilobase million, TPM) of several key monocyte / macrophage markers (CD14, CD16, CD64, CD11b, CD11c, and CD71) in groups 2-5 of cells depicted in Figure 10A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Description of the invention All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0019] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0020] "Subject" means a human or an animal. Usually, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, and canine species, such as dogs, foxes, and wolves. The terms "patient," "individual," and "subject" are used interchangeably herein. In one aspect, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. In one aspect, the subject is a human. In a further embodiment, the subject is a human who exhibits symptoms of a neurodegenerative condition or disease, e.g., symptoms of loss of memory (such as short-term memory, working memory, etc.), confusion with time or place, tremors.

[0021] "Neurological disorder" refers to disorders affecting the brain and nerves found throughout the body and spinal cord, including, but not limited to, epilepsy, learning disabilities, neuromuscular disorders, autism, attention deficit disorder, brain tumors, and cerebral palsy.

[0022] "Neurodegenerative disease or disorder" generally describes a condition in which nerve cells in the brain or peripheral nervous system lose function over time and eventually die. The risk of being affected by neurodegenerative diseases increases dramatically with age. Alzheimer's disease and Parkinson's disease are common neurodegenerative diseases. Examples of neurodegenerative diseases include Alzheimer's disease and other dementias, Parkinson's disease and related disorders, Huntington's disease, prion diseases, motor neuron diseases, spinocerebellar ataxias, spinal muscular atrophy, and amyotrophic lateral sclerosis.

[0023] "Spatial working memory" involves the ability to actively hold spatial information in working memory for short periods of time.

[0024] "Short-term memory," also known as primary or active memory, is the ability to store small amounts of information in the mind and keep it readily available for a short period of time. Usually, short-term memory is very short-lived. If short-term memory is not rehearsed or actively held, it may last only a few seconds.

[0025] The term "treat" or "treatment" or "treating" refers to a therapeutic measure that cures, slows down, relieves symptoms, and / or stops the progression of a diagnosed pathological disease or disorder.Thus, those who need treatment include those who already have the disorder.In certain embodiments, a subject is "treated" successfully for a disease or disorder when the subject shows, for example, a total, partial, permanent, or temporary alleviation or elimination of any symptoms associated with the disease or disorder.

[0026] The term "about" or "approximately", when used in connection with a referenced numerical indication (percentage), means up to the referenced numerical indication (percentage) plus or minus 5% of the referenced numerical indication (percentage), unless otherwise specified herein. For example, the term "about 50%" covers a range of 45% to 55%. In various embodiments, the term "about", when used in connection with a referenced numerical indication, can mean up to the referenced numerical indication plus or minus 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referenced numerical indication, if specifically specified in the claims. In other embodiments, "about" or "approximately", when used in connection with a referenced numerical indication of a period of at least days (e.g., days, weeks, or months), means up to the referenced numerical indication plus or minus at least one day, or up to at least one day and up to 10% of the indicated period, if 10% of the indicated period exceeds one day. For example, the term "approximately" 4 days covers a range of 3 to 5 days; the term "approximately" 60 days or "approximately" 2 months covers a range of 54 to 66 days.

[0027] The term "pluripotent stem cell" or "PSC" refers to a self-renewing cell that has the ability to develop into any of endodermal, ectodermal, and mesodermal cells, and the ability to proliferate. Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), germline stem cells ("GS cells"), embryonic germ cells ("EG cells"), and induced pluripotent stem cells ("iPS cells" or "iPSCs"). In some embodiments, the PSC is a human PSC. Preferred examples of PSC include ES cells and iPS cells.

[0028] ES cells are stem cells established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans or mice, and have the ability to proliferate by pluripotency and self-renewal. ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal, and then culturing the inner cell mass on fibroblasts as feeders. The cells can be maintained by subculturing using a medium supplemented with substances such as leukemia inhibitory factor (LIF) and / or basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described, for example, in US 5,843,780 B; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. USA. 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1 145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; and Klimanskaya I, et al. (2006), Nature. 444:481-485.

[0029] Induced pluripotent stem (iPS) cells can be prepared by introducing certain reprogramming factors into somatic cells, and the reprogramming factors are in the form of DNA or proteins. iPS cells are somatic cell-derived artificial stem cells that have almost the same characteristics as those of ES cells, such as pluripotency and the ability to proliferate by self-renewal. The reprogramming factors can be composed of genes or their gene products, or non-coding RNAs that are specifically expressed in ES cells; or genes or their gene products, non-coding RNAs, or low molecular weight compounds that play an important role in maintaining the undifferentiated state of ES cells. Examples of reprogramming factor genes include Oct3 / 4, Sox2, Soxl, Sox3, Soxl5, Soxl7, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbxl5, ERas, ECAT15-2, Tell, beta-catenin, Lin28b, Salll, Sall4, Esrrb, Nr5a2, and Tbx3, and these reprogramming factors can be used either alone or in combination.

[0030] The term "serum" refers to human serum, monkey serum, fetal bovine serum, bovine serum, porcine serum, horse serum, donkey serum, chicken serum, quail serum, sheep serum, goat serum, dog serum, cat serum, rabbit serum, rat serum, guinea pig serum, mouse serum, etc. Examples of serum-free media include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Iscove's modification of Dulbecco's medium (IMDM), StemPro-34SFM (Invitrogen), Stemline II (Sigma-Aldrich), etc., supplemented with ITS; medium for culturing primate ES cells to which serum replacement has been added beforehand (medium for primate ES / iPS cells, ReproCELL); and serum-free medium (mTeSR, Stemcell Technology). The serum-free medium or "serum-free" is more preferably mTeSR1 medium or StemPro-34 serum-free medium.

[0031] "Hematopoietic factors" refer to factors that promote the differentiation and proliferation of blood cells. Examples of these include stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin (EPO), thrombopoietin (TPO), interleukins, and Flt3 ligand. Interleukins are proteins secreted by white blood cells and can be divided into various types, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL-9.

[0032] The phrase "substantially pure" refers to a population of cells in which at least 95% of the cells have the described phenotype or expression marker profile. In all embodiments that refer to a "substantially pure" cell population, alternative embodiments are also contemplated in which the cell population has a lower or higher level of purity. For example, in some embodiments, instead of a given cell population being "substantially pure", the cell population may be one in which at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells, or 100% of the cells have the described phenotype or gene expression profile.

[0033] Various embodiments provide a method for improving cognitive function of a subject, or treating a subject with a neurodegenerative disorder, or alleviating a neurodegenerative disorder, treating a neurodegenerative disorder, or delaying the onset of a neurodegenerative disorder, or reducing inflammation in a subject with a neurodegenerative disorder, comprising administering to the subject a therapeutically effective amount of a composition comprising a population of mononuclear phagocytes generated from pluripotent stem cells. In some aspects, the population of mononuclear phagocytes is differentiated from induced pluripotent stem cells (iPSCs). In other aspects, the population of mononuclear phagocytes is differentiated from autologous iPSCs. In yet another aspect, the population of mononuclear phagocytes is differentiated from embryonic stem cells. In various aspects, the mononuclear phagocytes generated from pluripotent stem cells include monocytes generated from pluripotent stem cells. In various aspects, the mononuclear phagocytes generated from pluripotent stem cells are monocytes generated from pluripotent stem cells. In some embodiments, the mononuclear phagocytes generated from pluripotent stem cells further comprise macrophages; the macrophages are generated after transplantation of monocytes generated from pluripotent stem cells or by in vitro or ex vivo stimulation of monocytes generated from pluripotent stem cells.In further embodiments, the population of mononuclear phagocytes is myeloid lineage cells generated from iPSCs by one or more differentiation methods disclosed herein.In additional embodiments, the mononuclear phagocytes of the present invention herein comprise monocytes and can further comprise macrophages, but exclude neutrophils.

[0034] In some embodiments, a method for improving cognitive function of a subject, or treating a subject with a neurodegenerative disorder, or alleviating a neurodegenerative disorder, treating a neurodegenerative disorder, or delaying the onset of a neurodegenerative disorder, or reducing inflammation in a subject with a neurodegenerative disorder is provided, comprising administering to the subject a therapeutically effective amount of a composition comprising monocytes generated from iPSCs by one or more differentiation methods disclosed herein. In some embodiments, a method for improving cognitive function of a subject, or treating a subject with a neurodegenerative disorder, or alleviating a neurodegenerative disorder, treating a neurodegenerative disorder, or delaying the onset of a neurodegenerative disorder, or reducing inflammation in a subject with a neurodegenerative disorder is provided, comprising administering to the subject a therapeutically effective amount of a composition comprising cells consisting of monocytes generated from iPSCs by a differentiation method disclosed herein, wherein the differentiation method does not include differentiating the generated monocytes into macrophages in vitro, for example, the differentiation method does not include culturing the generated monocytes in the presence of M-CSF and one or both of IFN-gamma or IL-4. In other aspects, a method for improving cognitive function in a subject, or treating a subject with a neurodegenerative disorder, or alleviating, treating or delaying the onset of a neurodegenerative disorder, or reducing inflammation in a subject with a neurodegenerative disorder comprises administering to the subject a therapeutically effective amount of a composition comprising mononuclear phagocytes generated from iPSCs, where the mononuclear phagocytes can be (1) a mixture of monocytes generated from iPSCs and macrophages generated from iPSCs, if the differentiation method further leads to macrophage differentiation in vitro, or (2) substantially pure macrophages generated from iPSCs, if the differentiation method further leads to macrophage differentiation in vitro and further sorting / purification is performed to obtain only iPSC-generated macrophages, or (3) substantially pure monocytes generated from iPSCs, if the differentiation method does not lead to differentiation of monocytes generated from iPSCs.

[0035] In various embodiments, in the process disclosed herein, particularly by culturing in a bioreactor, a clinically significant amount of mononuclear phagocytes (or bone marrow monocytic cells) is generated from pluripotent stem cells (e.g., induced pluripotent stem cells). In contrast to having to isolate and use mononuclear phagocytes (or monocytes) from patients, the clinically significant amount of mononuclear phagocytes generated from pluripotent stem cells, particularly from iPSCs, can be stored (e.g., frozen) or maintained in culture for use in administering to patients in significant amounts. Starting from pluripotent stem cells, particularly induced pluripotent stem cells, the generated mononuclear phagocytes described herein may be less prone to genetic mutations and may have fewer disease mutations compared to autologous monocytes or macrophages obtained from patients or subjects in need of treatment.

[0036] In some embodiments, a method for improving cognitive function in a subject, particularly an aging subject (e.g., a human aged 40-50 years, 50-60 years, 60-70 years, 70-80 years, 80-90 years, 90-100 years, or over 100 years) comprises administering to the subject a therapeutically effective amount of a composition comprising mononuclear phagocytes generated from the subject's autologous iPSCs. In some embodiments, a method for treating a subject having a neurodegenerative disorder comprises administering to the subject a therapeutically effective amount of a composition comprising mononuclear phagocytes differentiated from the subject's autologous iPSCs. In some embodiments, a method for alleviating, treating, or delaying the onset of a neurodegenerative disorder in a subject comprises administering to the subject a therapeutically effective amount of a composition comprising mononuclear phagocytes differentiated from the subject's autologous iPSCs. In further embodiments, a method for treatment or prevention comprises administering to a subject having, suspected of having, or at risk of developing a disease or disorder associated with a deficiency or deficiency of mononuclear phagocytes generated from autologous iPSCs. In some embodiments, the method for treatment or prevention comprises administering mononuclear phagocytes generated from autologous iPSCs to a subject having, suspected of having, or at risk of developing a disease or disorder associated with a deficiency or deficiency of macrophages, where after administration to the subject, the administered mononuclear phagocytes generate macrophages. In yet additional embodiments, the method for reducing inflammation comprises administering mononuclear phagocytes generated from autologous iPSCs to a subject having, suspected of having, or at risk of developing a disease or disorder associated with inflammation; optionally, after administration to the subject, the administered mononuclear phagocytes generate macrophages. The disease or disorder associated with inflammation can be a neurodegenerative disease or disorder.

[0037] In other embodiments, methods for improving cognitive function in a subject, particularly an aging subject (e.g., a human aged 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or over 100 years of age), include administering to the subject a therapeutically effective amount of a composition comprising myelomonocytic cells or monocytes generated from the subject's autologous iPSCs, where the myelomonocytic cells or monocytes are not stimulated to differentiate into microglia, dendritic cells, or macrophages prior to administration to the subject. In some embodiments, methods for treating a subject having a neurodegenerative disorder include administering to the subject a therapeutically effective amount of a composition comprising myelomonocytic cells or monocytes generated from the subject's autologous iPSCs, where the myelomonocytic cells or monocytes are not stimulated to differentiate into microglia, dendritic cells, or macrophages prior to administration to the subject. In some embodiments, a method for alleviating, treating, or delaying the onset of a neurodegenerative disorder in a subject comprises administering to the subject a therapeutically effective amount of a composition comprising myelomonocytic cells or monocytes generated from the subject's autologous iPSCs, where the myelomonocytic cells or monocytes are not stimulated to differentiate into microglia, dendritic cells, or macrophages prior to administration to the subject. In further embodiments, a method for treatment or prevention comprises administering to a subject having, suspected of having, or at risk of developing a disease or disorder associated with a deficiency or deficiency of monocytes or mononuclear phagocytes, myelomonocytic cells or monocytes generated from the subject's autologous iPSCs, where the myelomonocytic cells or monocytes are not stimulated to differentiate into microglia, dendritic cells, or macrophages prior to administration to the subject. In some embodiments, methods for treatment or prevention include administering to a subject having, suspected of having, or at risk of developing a disease or disorder associated with a deficiency or deficiency of macrophages, bone marrow monocytic cells or monocytes generated from the subject's autologous iPSCs, where after administration to the subject, the administered bone marrow monocytic cells or monocytes generate macrophages.In yet additional embodiments, the method for reducing inflammation comprises administering to a subject having, suspected of having, or at risk of developing a disease or disorder associated with inflammation, wherein the myelomonocytic cells or monocytes are not stimulated to differentiate into microglia, dendritic cells, or macrophages prior to administration to the subject. The disease or disorder associated with inflammation can be a neurodegenerative disease or disorder. In alternative embodiments, the method for treating or preventing or reducing inflammation comprises administering to the subject myelomonocytic cells derived from the subject's autologous iPSCs, wherein the administered myelomonocytic cells are further differentiated into macrophages prior to administration to the subject.

[0038] In various implementations, the method comprising administering pluripotent stem cells, preferably mononuclear phagocytes differentiated from iPSCs, does not comprise administering plasma or bone marrow to the subject; or the subject in these methods does not receive plasma or bone marrow transplantation. In alternative implementations, the method comprising administering pluripotent stem cells, preferably mononuclear phagocytes differentiated from iPSCs, is added to plasma or bone marrow transplantation therapy for the subject. In another implementation, the method comprising administering mononuclear phagocytes differentiated from pluripotent stem cells is for subjects who respond ineffectively to plasma infusion or bone marrow transplantation therapy or who are complicated by a pathological condition.

[0039] In various aspects of the method, the mononuclear phagocyte is generated from the pluripotent stem cell in a process comprising culturing the pluripotent stem cell in a cell culture medium under conditions inducing myeloid differentiation to generate myeloid lineage cells (preferably monocytes), and the process does not include contacting the generated cells with or culturing them in a microglia differentiation medium or a dendritic cell differentiation medium.For example, the process of generating mononuclear phagocytes (or myeloid lineage cells) from the pluripotent stem cell does not include culturing or contacting the generated cells in the presence of (a) IL-34, (b) IL-34 and GM-CSF, (c) IL-4, or (d) IL-4 and GM-CSF.Therefore, the mononuclear phagocyte (preferably monocyte) generated from the pluripotent stem cell for use in one or more methods disclosed herein is not a microglia cell or a dendritic cell. The methods disclosed herein for improving cognitive function of a subject, or treating a subject with a neurodegenerative disorder, or alleviating a neurodegenerative disorder in a subject, treating a neurodegenerative disorder, or delaying the onset of a neurodegenerative disorder, or treating or preventing a subject with, suspected of, or at risk of developing a disease or disorder associated with macrophage or monocyte deficiency or deficiency, do not include administering microglia or dendritic cells generated from pluripotent stem cells. In additional embodiments, the process further excludes contacting or culturing the generated cells in macrophage differentiation medium or in the presence of a macrophage differentiation stimulant. For example, in these additional examples, the process for generating mononuclear phagocytes (or myeloid lineage cells) from pluripotent stem cells does not include culturing or contacting the generated cells in the presence of either or both of (a) IL-34, (b) IL-34 and GM-CSF, (c) IL-4, (d) IL-4 and GM-CSF, or (e) M-CSF and IFN-gamma and IL-4.That is, the process of generating mononuclear phagocytes does not include culturing the generated cells with any of: (a) IL-34, (b) a combination of IL-34 and GM-CSF, (c) IL-4, (d) a combination of IL-4 and GM-CSF, (e) a combination of M-CSF and IFN-gamma, and (f) a combination of M-CSF and IL-4. The multiple reagents in the "combination" can be added to the medium simultaneously or sequentially. Alternatively, the process can further include contacting or culturing the generated cells in a macrophage differentiation medium or in the presence of a macrophage differentiation stimulant. For example, the process of generating mononuclear phagocytes (or myeloid lineage cells) from pluripotent stem cells does not include culturing in the presence of or contacting the generated cells with (a) IL-34, (b) IL-34 and GM-CSF, (c) IL-4, or (d) IL-4 and GM-CSF, but does include culturing or contacting the generated cells with M-CSF and either or both of IFN-gamma and IL-4.

[0040] In particular, in some examples, a process for generating mononuclear phagocytes from pluripotent stem cells includes culturing pluripotent stem cells in a cell culture medium under conditions inducing myeloid differentiation, the culturing including contacting the pluripotent stem cells with a first composition comprising BMP-4 in a serum-free medium comprising bFGF, comprising bFGF and TGFβ, or comprising bFGF, TGFβ, aminobutyric acid, pipecolic acid, and lithium chloride. In various aspects, the culturing includes contacting the pluripotent stem cells with a first composition comprising BMP-4 in mTeSR1 medium comprising all of bFGF, TGFβ, aminobutyric acid, pipecolic acid, and lithium chloride, or comprising one, two, three, or four of them.

[0041] The step of culturing pluripotent stem cells in cell culture medium under conditions inducing myeloid differentiation can further include contacting the cells obtained from the step with the first composition with a second composition comprising one or more or all factors including bFGF, VEGF, SCF, or a combination thereof. In various aspects, contacting the cells with the second composition refers to changing the cell culture medium to one comprising the second composition or contacting the cell culture medium with the second composition. In various aspects, the second composition comprises hematopoietic factors consisting of bFGF, VEGF, or SCF, or a combination of bFGF, VEGF, and SCF; preferably in serum-free medium.

[0042] The step of culturing pluripotent stem cells in cell culture medium under conditions inducing myeloid differentiation can further include contacting the cells obtained from the step with the second composition with a third composition comprising one or more or all of the factors including SCF, IL-3, thrombopoietin, M-CSF, FLT3 ligand, or a combination thereof. In various aspects, contacting the cells with the third composition refers to changing the cell culture medium to one comprising the third composition, or contacting the cell culture medium with the third composition. In various aspects, the third composition comprises hematopoietic factors consisting of SCF, IL-3, thrombopoietin, M-CSF, or FLT3 ligand, or a combination of SCF, IL-3, thrombopoietin, M-CSF, and FLT3 ligand; preferably in serum-free medium.

[0043] The step of culturing pluripotent stem cells in cell culture medium under conditions inducing myeloid differentiation can further include contacting the cells obtained from the step with the third composition with a fourth composition comprising one or more or all of the factors, including M-CSF, GM-CSF, FLT3 ligand, or a combination thereof. In various aspects, contacting the cells with the fourth composition refers to changing the cell culture medium to one comprising the fourth composition, or contacting the cell culture medium with the fourth composition. In various aspects, the fourth composition comprises hematopoietic factors consisting of M-CSF, GM-CSF, or FLT3 ligand, or a combination of M-CSF, GM-CSF, and FLT3 ligand.

[0044] In one embodiment, the process for generating mononuclear phagocytes from pluripotent stem cells includes the steps of: (a) culturing pluripotent stem cells in adherent culture with a first composition comprising BMP-4 but not serum; (b) culturing the cells obtained by step (a) in adherent culture with a second composition comprising bFGF, VEGF, and SCF but not serum; (c) culturing the cells obtained by step (b) in adherent culture with a third composition comprising SCF, IL-3, thrombopoietin, M-CSF, and FLT3 ligand but not serum; and (d) culturing the cells obtained by step (c) in adherent culture with a fourth composition comprising M-CSF, GM-CSF, and FLT3 ligand, whereby macrophages and / or monocytes are produced / collected. Alternatively, the process for generating mononuclear phagocytes from pluripotent stem cells can be performed in suspension culture. For example, step (d) can be performed in suspension culture, such as in a bioreactor containing a fourth composition comprising M-CSF, GM-CSF, and FLT3 ligand. Table 1 shows that the cells cultured in suspension culture in a bioreactor are viable. The step of culturing in suspension culture includes a step of collecting cells present in the culture supernatant when the cell culture medium is changed, and a step of returning the collected cells to the cell culture.

[0045] In some embodiments, a process for generating mononuclear phagocytes from pluripotent stem cells comprises performing one or more of the following four steps: first, contacting the cell culture with a first composition comprising BMP4 in a culture medium, where the cell culture comprises pluripotent stem cells when the cell culture is initially contacted with the first composition; second, contacting the cell culture with a second composition comprising one or more of bFGF, SCF, and VEGF-A (e.g., each of bFGF, SCF, and VEGF-A) in a hematopoietic cell medium; Third, contacting the cell culture with a third composition comprising one or more of SCF, IL-3, TPO, M-CSF, and FLT3 ligand (e.g., each of SCF, IL-3, TPO, M-CSF, and FLT3 ligand) in hematopoietic cell medium; and fourth, contacting the cell culture with a fourth composition comprising one or more of M-CSF, FLT3 ligand, and GM-CSF (e.g., each of M-CSF, FLT3 ligand, and GM-CSF) in hematopoietic cell medium, thereby generating mononuclear phagocytes. In some embodiments, all of the above four steps are performed in order. In various embodiments, the generated mononuclear phagocytes are not further differentiated or stimulated into microglia or dendritic cells. In additional embodiments, the generated mononuclear phagocytes are not further differentiated or stimulated into macrophages; whereas instead, the generated mononuclear phagocytes can be differentiated to obtain at least some macrophages. In some such embodiments, the medium used for any of these four steps is a serum-free medium. In some such embodiments, the medium used for any of these four steps is a chemically-defined medium. In some such embodiments, all or any of the above four steps are performed in a dish or well plate coated with extracellular matrix. In some aspects, the extracellular matrix is ​​a reconstituted basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma cells.

[0046] In one aspect, a process for generating mononuclear phagocytes from pluripotent stem cells includes the steps of incubating pluripotent stem cells in a first medium supplemented with bone morphogenetic protein 4 (BMP-4), thereby forming first medium-treated cells; incubating the first medium-treated cells in a second medium supplemented with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF), thereby forming second medium-treated cells; and incubating the first medium-treated cells in a second medium supplemented with SCF, interleukin 3 (IL-3), thrombopoietin (TP), and erythrocyte proliferation and differentiation (ICD)-induced cell death. O), incubating the second medium-treated cells in a third medium supplemented with macrophage colony-stimulating factor (M-CSF), and FLT3 ligand, thereby forming third medium-treated cells; and incubating the third medium-treated cells in a fourth medium supplemented with M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and FLT3 ligand, thereby forming fourth medium-treated cells, which are mononuclear phagocytes differentiated from pluripotent stem cells.

[0047] In one embodiment, a process for generating monocytes from pluripotent stem cells includes incubating iPSCs in a first medium supplemented with BMP-4, thereby forming first medium-treated cells; incubating the first medium-treated cells in a second medium supplemented with bFGF, VEGF, and SCF, thereby forming second medium-treated cells; incubating the second medium-treated cells in a third medium supplemented with SCF, IL-3, TPO, M-CSF, and FLT3 ligand, thereby forming third medium-treated cells; and incubating the third medium-treated cells in a fourth medium supplemented with M-CSF, GM-CSF, and FLT3 ligand, thereby forming fourth medium-treated cells, which are monocytes differentiated from pluripotent stem cells. The resulting monocytes generated from iPSCs are suitable for use in transplantation, transfer, or otherwise administered to a patient in need thereof.

[0048] Some aspects provide that the first medium is a feeder-free culture medium, mTeSR, and is supplemented with BMP-4. BMP-4 can be added to the first medium to a final concentration of 10-200 ng / mL, or 40-160 ng / mL, or 60-120 ng / mL, or about 80 ng / mL. In some aspects, the concentration of BMP-4 is 5 ng / mL-150 ng / mL. In some aspects, the concentration of BMP-4 is 10 ng / mL-100 ng / mL. In some aspects, the concentration of BMP-4 is 20 ng / mL-80 ng / mL. In further aspects, the first medium is standard mTeSR medium rather than mTeSR custom medium, and thus the first medium is mTeSR with bFGF, TGFβ, GABA, pipecolic acid, and lithium chloride. This first medium containing supplements can be used in one or more fresh portions to culture the stem cells for about 3 days, or from days 1 to 4. In some aspects, a tissue culture medium suitable for maintaining stem cells is used as the first medium. In other aspects, a tissue culture medium suitable for differentiating stem cells is used as the first medium.

[0049] "TeSR" is a serum-free, xeno-free medium shown to support the derivation and long-term feeder-independent culture of hPSCs, developed by Tenneille Ludwig and coworkers (Ludwig TE et al., Nat Biotechnol. 24: 185-7, 2006). The "TeSR" formulation contains high levels of bFGF along with TGF, GABA, pipecolic acid, and lithium chloride. This original publication by Ludwig et al. describes the use of a cell support matrix consisting of four human components (collagen IV, fibronectin, laminin, and vitronectin). Ludwig and coworkers further developed a modified version of the medium ("mTeSR1") that, despite containing some proteins of animal origin, retains the advantages of being fully-defined and serum-free, and supports the self-renewal of hPSCs without the need for feeder cells (Ludwig TE, et al., Nat Methods 3: 637-46, 2006). mTeSR1 medium according to Ludwig TE, et al., Nat Methods 3: 637-46, 2006, contains DMEM / F12, Stock B (containing dissolved bovine serum albumin, thiamine, reduced glutathione, L-ascorbic acid 2-phosphate magnesium salt, selenium, trace element B, trace element C, insulin, holo-transferrin), zebrafish bFGF, TGFβ1, pipecolic acid, GABA, lithium chloride, lipids, L-glutamine-β mercaptoethanol, MEM NEAA, and NaHCO3, which upon mixing is adjusted to a pH of 7.4 using NaOH and an osmolality of 340-350 mOsMol using crystalline NaCl, and is preferably filter sterilized before use.

[0050] Some aspects provide that the second medium is a serum-free medium, e.g., StemPro-34, and is supplemented with (1) bFGF at a final concentration of 5-100 ng / mL, or 10-50 ng / mL, or 20 ng / mL-35 ng / mL, or about 25 ng / mL; (2) VEGF at a final concentration of about 10-200 ng / mL, about 40-120 ng / mL, about 60-100 ng / mL, or about 80 ng / mL; and (3) SCF at a final concentration of about 10-500 ng / mL, or 30-300 ng / mL, or 50-150 ng / mL, or 80-120 ng / mL, or about 100 ng / mL. This second medium containing the supplements can be used in one or more fresh doses to culture the cells for about 2 days, up to days 4-6.

[0051] In some aspects, the third medium is a serum-free medium, e.g., StemPro-34, and contains: (1) SCF at a final concentration of 5 to 100 ng / mL, or 25 to 75 ng / mL, or 40 to 60 ng / mL, or about 50 ng / mL; (2) IL-3 at a final concentration of 5 to 100 ng / mL, or 25 to 75 ng / mL, or 40 to 60 ng / mL, or about 50 ng / mL; (3) IL-3 at a final concentration of 0.5 to 20 ng / mL, 1 to 10 ng / mL, (4) TPO at a final concentration of 5-100 ng / mL, or 25-75 ng / mL, or 40-60 ng / mL, or about 50 ng / mL; (5) FLT3 (or FLT3 ligand) at a final concentration of 5-100 ng / mL, or 25-75 ng / mL, or 40-60 ng / mL, or about 50 ng / mL. This third medium containing the supplements can be used in one or more fresh doses to culture the cells for about 6 or 7 days, e.g., from day 6 to day 12 or day 13.

[0052] Some aspects provide that the fourth medium is a serum-free medium, e.g., StemPro-34, and is supplemented with: (1) M-CSF at a final concentration of 5-100 ng / mL, or 25-75 ng / mL, or 40-60 ng / mL, or about 50 ng / mL; (2) GM-CSF at a final concentration of about 5-50 ng / mL, or 10-40 ng / mL, or 20-30 ng / mL, or about 25 ng / mL; and (3) FLT3 (or FLT3 ligand) at a final concentration of 5-100 ng / mL, or 25-75 ng / mL, or 40-60 ng / mL, or about 50 ng / mL.

[0053] A further aspect provides that any suitable hematopoietic cell medium can be used as the second, third, and fourth medium in stages 2-4. In one embodiment, the hematopoietic cell medium is "StemPro-34". The composition of StemPro-34 medium is known in the art and is described, for example, in EP0891419 (or US20040072349, US20100297090) and WO1997033978 (or US20040072349, US20100297090), entitled "Hematopoietic Cell Culture Nutrient Supplement", the contents of which are incorporated herein by reference. However, the skilled artisan will recognize that there are several other types of media that are equivalent to StemPro-34 medium in terms of suitability for use in culturing hematopoietic cells, any of which could be used.

[0054] In various examples, the concentration of cytokines, including hematopoietic factors, used in each step is not limited as long as the cells of interest can be obtained at that concentration. In some aspects, the concentration of bFGF in the cell culture medium in each step is 10 ng / mL to 100 ng / mL. In some aspects, the concentration of bFGF in the cell culture medium in each step is 20 ng / mL to 50 ng / mL. In some aspects, the concentration of bFGF in the cell culture medium in each step is about 25 ng / mL. In some aspects, the concentration of VEGF in the cell culture medium in each step is 20 ng / mL to 100 ng / mL. In some aspects, the concentration of VEGF in the cell culture medium in each step is 30 ng / mL to 70 ng / mL. In some aspects, the concentration of VEGF in the cell culture medium in each step is about 50 ng / mL. In some aspects, the concentration of SCF in the cell culture medium in each step is 20 ng / mL to 100 ng / mL. In some aspects, the concentration of SCF in the cell culture medium in each step is 30ng / mL to 70ng / mL. In some aspects, the concentration of SCF in the cell culture medium in each step is about 50ng / mL. In the case of IL-3, the concentration is, in some examples, 5ng / mL to 100ng / mL. In some aspects, the concentration of IL-3 can be 30ng / mL to 70ng / mL. In other aspects, the concentration of IL-3 can be about 50ng / mL. In the case of TPO, the concentration is 1ng / mL to 25ng / mL. In some aspects, the concentration of TPO is preferably 1ng / mL to 10ng / mL. In some aspects, the concentration of TPO is about 5ng / mL. In the case of Flt3-ligand (FLT3L), the concentration is, in various aspects, 10ng / mL to 100ng / ml. In some aspects, the concentration of FLT3L is 30ng / ml to 70ng / ml. In some aspects, the concentration of FLT3L is about 50ng / mL. In the case of GM-CSF, the concentration is, in various aspects, 5ng / mL to 100ng / mL. In some aspects, the concentration of GM-CSF is preferably 10ng / mL to 50ng / mL. In some aspects, the concentration of GM-CSF is about 25ng / mL.In the case of M-CSF, the concentration is in various aspects between 5 ng / ml and 100 ng / ml, hi some aspects, the concentration of M-CSF is preferably between 30 ng / ml and 70 ng / ml, or more preferably 50 ng / ml.

[0055] In various aspects, the process of generating mononuclear phagocytes from pluripotent stem cells (preferably from iPSCs) comprises using each of the factors in the following combinations: In the first cell culture medium, BMP-4 is 60ng / mL-100ng / mL; In the second cell culture medium, bFGF is 15ng / mL-30ng / mL, VEGF is 60ng / mL-100ng / mL, and SCF is 80ng / mL-120ng / mL; In the third cell culture medium, SCF is 40ng / mL-60ng / mL, IL-3 is 40ng / mL-60ng / mL, TPO is 4ng / mL-6ng / mL, M-CSF is 40ng / mL-60ng / mL, and FLT3L is 40ng / mL-60ng / mL; and In the fourth cell culture medium, M-CSF is 40ng / mL to 60ng / mL, GM-CSF is 15ng / mL to 30ng / mL, and FLT3L is 40ng / mL to 60ng / mL.

[0056] Various embodiments also provide that, in terms of the duration of each step, step (a) above (or "stage 1") is carried out over 2 days or more, preferably over 2 to 6 days, more preferably over 4 days. Step (b) above (or "stage 2") is carried out over 1 day or more, preferably over 1 to 5 days, more preferably over 2 days. Step (c) above (or "stage 3") is carried out over 5 days or more, preferably over 6 to 14 days, more preferably over 9 days. Step (d) above (or "stage 4") is carried out over 3 days or more, preferably over 3 to 90 days. In some embodiments, step (d) (or "stage 4") is carried out over at least 55 or 60 days, up to about 90 days.

[0057] In some embodiments, the generated mononuclear phagocytes are at least 1×10 6 The cells are further cultured in a bioreactor to expand to clinically relevant numbers of around 1000 cells. In some embodiments, the process of generating mononuclear phagocytes from pluripotent stem cells is carried out in a bioreactor starting from any one of steps (a) ("Stage 1"), (b) ("Stage 2"), (c) ("Stage 3"), or (d) ("Stage 4"). As shown in Example 3, mononuclear phagocytes generated from pluripotent stem cells and expanded in a bioreactor (for various days, 1-10 days, 11-20 days, 21-30 days, 31-40 days, 41-50 days, 50-60 days or more) show similar gene expression profiles and expression levels of monocyte / macrophage markers as those generated in well plates.

[0058] Bioreactors known in the art are generally suitable for growing iMPs to obtain clinically relevant numbers for administration. Exemplary bioreactors include stirred flasks, also called stirrer tank bioreactors, where mixing by an impeller keeps the cells in suspension and fluid movement aids in the bulk transport of nutrients and waste products. In addition to stirred flasks, the inventors also envision using rocker bag systems and / or G-REX® systems for scale-up production of iMPs. For example, a rocker bag system includes a rocker (which includes a base, provides a platform such as the shape of a tray, and optionally further includes a heater and / or thermocouple) and one or more cell culture rocker bags (for enclosing the cell culture and suitable for placement on the platform). The rocker provides a smooth rocking undulating motion that results in gentle and efficient mixing and gas transfer. Cell culture rocker bags usually include ports for the ingress and egress of fluids and / or gases into and out of the bag. G-REX® stands for gas permeable rapid expansion. The G-REX bioreactor gives cells unlimited and unhindered access to nutrients and oxygen to produce large quantities of cells, eliminating the need for media exchange and complex hardware required in integrated systems.

[0059] In various embodiments, the myelomonocytic cells or mononuclear phagocytes generated from pluripotent stem cells (e.g., iPSCs) in the processes disclosed herein are called iMPs (mononuclear phagocytes generated from iPSCs) because they are positive for monocyte / macrophage markers such as CD14, CD16, CD64, CD11b, CD11c, CD71, or in various examples, include iPSC-generated monocytes and / or iPSC-generated macrophages (called iMACs in priority application US63 / 234,984), and no longer or have little expression of the hematopoietic stem cell marker CD34. In various embodiments, the mononuclear phagocytes generated from stem cells (e.g., from iPSCs) are not microglia, because the method of differentiation does not include culturing any of the generated cells in microglia medium. The differentiated mononuclear phagocytes can be cultured in a new volume of the four media (including supplements) until harvest. In various embodiments, the differentiation method further comprises or involves amplifying the cells by supplementing with a new amount of medium at each stage and optionally passage the cells. In some implementations, the harvested cells are directly administered to a subject, optionally at some dilution or concentration. In various embodiments, at least 50%, 60%, 70%, 80%, or 90% of the cells harvested from the cells treated with the fourth medium are monocytes. Preferably, at least 50% of the cells harvested after the fourth medium are monocytes. More preferably, at least 70% or about 70% of the cells harvested after the fourth medium are monocytes. To determine the percentage of cells expressing monocyte / macrophage markers, the cells can be analyzed using antibodies targeting antigens such as CD34, CD11b, CD11c, CD14, and CD16.

[0060] In other implementations, the harvested mononuclear phagocytes derived from stem cells are cryopreserved to maintain the stability of the product during storage and delivery steps. In some aspects, the harvested mononuclear phagocytes generated from the process are purified, for example, by marker CD14. Purification of CD14 positive cells can be performed by methods well known to those skilled in the art, and the method is not limited thereto. For example, CD14 microbeads or flow cytometer can be used to purify cells. In some aspects, the mononuclear phagocytes are harvested without further selection by one or more markers, in particular without selection by marker C3CR1.

[0061] In various embodiments, a quantity of mononuclear phagocytes generated from pluripotent stem cells is cultured in a bioreactor, e.g., at least 1×10 6 , 1×10 7 , or 1 × 10 8 Clinically meaningful quantities of around 10 ...

[0062] In particular, in various embodiments, the generated iMPs have differential gene expression compared to naturally occurring monocytes or naturally occurring macrophages or naturally occurring mononuclear phagocytes. The generated iMPs are positive for similar markers as their naturally occurring counterparts, and in particular behave similarly to said counterparts in in vivo functional tests, and may thereby have therapeutic effects as shown in the Examples. Although neither iPSCs nor naturally occurring macrophages / monocytes are proliferative, as detailed in Examples 2 and 3, the present invention provides cysts differentiated from iPSCs, such that iMPs blastema and detach from the cysts, thereby allowing production to be scaled up to therapeutic (or clinically meaningful) amounts.

[0063] In various embodiments of the method, the composition comprising the population of mononuclear phagocytes is administered in two or more exposures to the subject. In one embodiment, the composition comprising the population of mononuclear phagocytes is at least administered to the subject over at least three doses. In one embodiment, the composition comprising the population of mononuclear phagocytes generated from iPSCs is administered to the subject over 4-10 doses. In one embodiment, the composition comprising the population of mononuclear phagocytes generated from iPSCs is administered to the subject over 6-12 doses. In some implementations, the composition is administered weekly, biweekly, bimonthly, or monthly, or as needed by the subject. In some implementations, the composition of each exposure to the subject is administered over at least 10 doses. 6 cells, 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, or 10 11 The cells can include 100% soluble cellulose. In various implementations, the therapeutically effective amount of cells depends on the patient's requirements, age, physiological state, and health status, as well as the tissue size and therapeutic goal to be reached, the implantation site, the degree of pathology (neuronal level deterioration), the selected transfer mode and therapeutic strategy. In some implementations, low doses of cells are repeatedly implanted. These cells can be used to treat acute or chronic nerve damage, and / or to delay the onset of, alleviate, or treat neurodegenerative and neuronal diseases.

[0064] Various embodiments of the methods of treatment are for aging mammals, for example humans at least 50 years of age, at least 60 years of age, at least 70 years of age, at least 80 years of age, or at least 90 years of age.

[0065] In another embodiment, the method disclosed herein is for a subject who develops or is diagnosed with a neurodegenerative disease such as Alzheimer's disease.In a further embodiment, the method disclosed herein is for a subject who first shows the pathology of Alzheimer's disease, such as when amyloid and microglial activation are detected.In yet another embodiment, the method disclosed herein is for a subject who is significantly affected by Alzheimer's disease or has been diagnosed with Alzheimer's disease for at least 6 months, 1 year, 2 years, or more, and the method alleviates or reverses the pathology. In certain embodiments, the neurodegenerative or neuronal disease of the method is selected from the group consisting of Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Rett syndrome, diffuse leukoencephalopathy with spheroid formation, hereditary diffuse leukoencephalopathy with axonal spheroid formation, frontotemporal lobar degeneration (FTLD), familial FTLD, schizophrenia, autism spectrum disorder, Huntington's disease, dementia with Lewy bodies, cerebellar ataxia, Stein-Leventhal syndrome, spinal cord injury, epilepsy, and stroke accompanied by focal ischemia.

[0066] In additional aspects, the methods disclosed herein are for a subject who has Alzheimer's disease (e.g., exhibits signs or symptoms of or has been diagnosed with Alzheimer's disease), but the subject does not have amyotrophic lateral sclerosis (ALS).

[0067] In additional aspects, the methods disclosed herein are for a subject having a deficiency in macrophages or a disease or disorder associated with a lack or deficiency of macrophages, and thus administration of mononuclear phagocytes generated from iPSCs may result in the production of macrophages in the subject following administration.

[0068] Various embodiments provide that the method disclosed herein further comprises selecting a subject having, showing symptoms of, or at risk of developing a neurodegenerative disease to receive mononuclear phagocytes generated from pluripotent stem cells. Various embodiments provide that the method disclosed herein further comprises obtaining autologous somatic cells (e.g., fibroblasts, blood cells) from a subject having, showing symptoms of, or at risk of developing a neurodegenerative disease, and then generating iPS cells from the autologous somatic cells by a reprogramming process known in the art to obtain mononuclear phagocytes generated from iPS cells for administration to the subject. Additional embodiments provide that the method disclosed herein further comprises growing the generated mononuclear phagocytes in a bioreactor to produce at least 1×10 6 or 1×10 7 The method further comprises the step of obtaining the cells.

[0069] An additional embodiment provides a method for generating mononuclear phagocytes (or myelomonocytic or myeloid lineage cells) from pluripotent stem cells, the method comprising the steps of: incubating the stem cells in a first medium supplemented with bone morphogenetic protein 4 (BMP-4), thereby forming first medium treated cells; incubating the first medium-treated cells in a second medium supplemented with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF), thereby forming second medium-treated cells; incubating the second medium-treated cells in a third medium supplemented with SCF, interleukin 3 (IL-3), thrombopoietin, macrophage colony-stimulating factor (M-CSF), and FLT3 ligand, thereby forming third medium-treated cells; and incubating the third medium-treated cells in a fourth medium supplemented with M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and FLT3 ligand, thereby forming fourth medium-treated cells, which are macrophages or monocytes differentiated from stem cells. Including, The method does not include the step of culturing in the presence of IL-34, or a combination of IL-34 and GM-CSF, or IL-4, or a combination of IL-4 and GM-CSF, or a combination of M-CSF and IFN-gamma, or a combination of M-CSF and IL-4.

[0070] In some aspects of the method for differentiation, the method does not include incubating the cells treated with the fourth medium in microglia differentiation medium or dendritic cell differentiation medium. In some aspects, at least 50% of the cells treated with the fourth medium are monocytes; or the cells obtained from step (d) (or "stage 4") are substantially pure mononuclear phagocytes (including monocytes and macrophages) and are characterized for expression of markers CD14, CD16, CD64, CD11b, CD11c, and CD71. In some aspects of the method for differentiation, the generated mononuclear phagocytes are not microglia, are not dendritic cells, and the generated mononuclear phagocytes are positive for one or more markers of CD11b, CD11c, CD14, and CD16.

[0071] In some aspects, the mononuclear phagocytes are differentiated from iPSCs prepared by reprogramming blood cells, preferably peripheral blood mononuclear cells (PBMCs), from a subject, e.g., a healthy human subject, a young human (e.g., a human aged within the age groups 5-11, 12-16, 17-18, 19-21, 22-34, or 35-49 years), or a young healthy human subject. In further embodiments, the mononuclear phagocytes are differentiated from iPSCs prepared by reprogramming fibroblasts obtained from the subject.

[0072] In some embodiments, methods for reprogramming blood cells into iPSCs are disclosed in WO2017219000, U.S. Patent No. 10,221,395, and U.S. Patent No. 10,745,671, which are hereby incorporated by reference. For example, a method of generating blood cell-derived iPSCs includes delivering an amount of EBNA1 and reprogramming factors including Oct-4, Sox-2, Klf-4, 1-Myc, Lin-28, SV40 large T antigen ("SV40LT"), and a short hairpin RNA targeting p53 ("shRNA-p53") into a quantity of blood cells; and culturing the blood cells in reprogramming medium for at least four days, where the delivery of EBNA1 and the reprogramming factors and culturing in the reprogramming medium generates blood cell-derived induced pluripotent stem cells, where the reprogramming factors are encoded by four oriP / EBNA1-derived vectors including a first vector encoding Oct4, Sox2, SV40LT, and Klf4, a second vector encoding Oct4 and shRNA-p53, a third vector encoding Sox2 and Klf4, and a fourth vector encoding 1-Myc and Lin-28; and a fifth oriP / EBNA1-derived vector encoding EBNA1.

[0073] In some aspects, mononuclear phagocytes are differentiated from stem cells or induced pluripotent stem cells from a species and used to treat subjects of the same species.In some aspects, mononuclear phagocytes are differentiated from stem cells or induced pluripotent stem cells from a species at a young age, for example, the stem cells are obtained or reprogrammed from somatic cells obtained from a species at a younger age than the first half of the average lifespan of the species.In some aspects, mononuclear phagocytes are differentiated from mouse stem cells obtained from mice that are less than 4 months old, for example, 3-4 months old, 2-3 months old, 1-2 months old.In some aspects, mononuclear phagocytes are generated from iPSCs reprogrammed from somatic cells of humans in their early teens, 20s, or 30s, or 40s, and used when humans show aging or neurodegenerative diseases or disorders. In another aspect, the mononuclear phagocytes are differentiated from a human subject having cognitive impairment or a neurodegenerative disease / disorder, or from an aged human subject (e.g., at least 40 years old, at least 50 years old, at least 60 years old, at least 70 years old, or at least 80 years old). In another aspect, the mononuclear phagocytes are differentiated from an aged mouse, e.g., about 11-13 months old.

[0074] In various aspects, the present invention provides pharmaceutical compositions. The pharmaceutical compositions comprise a population of mononuclear phagocytes derived from stem cells, for example, differentiated from induced pluripotent stem cells. In some aspects, the patient's own (autologous) cells are used to derive the mononuclear phagocytes. In other aspects, donated (allogeneic) cells are used to derive the mononuclear phagocytes. The mononuclear phagocytes differentiated from stem cells can be maintained in a liquid suspension or formulation until administration.

[0075] The disclosed method can improve cognitive function and / or neurological health. For example, the treated subject can have improved spatial working memory and / or improved short-term memory compared to the state of the subject before treatment. The treated subject can also have increased levels of synaptic transporters, increased levels of microglia, and / or increased levels of astrocytes compared to the control. In some aspects, the control can be a subject with a neurodegenerative disorder that is not treated with the cell therapy disclosed herein. In other aspects, the control can be the baseline level of the subject before treatment. Alternatively, the treated subject can show cognitive function or neurological health comparable to that of a young and / or healthy subject.

[0076] Also provided is the mononuclear phagocyte differentiated from stem cell by the differentiation method disclosed herein.In various aspects, provided is the monocyte generated from iPSC by the process disclosed herein.In various implementations, the mononuclear phagocyte differentiated from induced pluripotent stem cell is provided in a composition or pharmaceutical composition together with one or more excipients.

[0077] Preferably, the mononuclear phagocytes are differentiated from the autologous stem cells of the subject to which the generated mononuclear phagocytes are administered, often after expansion. For example, blood cells or fibroblasts or other somatic cells from a mammal are reprogrammed into induced pluripotent stem cells by the differentiation method disclosed herein, which are then differentiated into mononuclear phagocytes; the resulting mononuclear phagocytes are infused / transplanted or otherwise injected into the mammal in need of cognitive function improvement or suffering from a neurodegenerative disorder. In another example, somatic cells of a healthy or young mammal are reprogrammed into induced pluripotent stem cells; the resulting mononuclear phagocytes are infused, transplanted or otherwise injected into the mammal in need of cognitive function improvement or suffering from a neurodegenerative disorder. In one embodiment, the multipotent stem cells are mouse, pig, monkey, sheep or human embryonic stem cells. In another embodiment, the experimenter is a patient, more preferably a human patient, and the multipotent stem cells are reprogrammed from the human patient's own tissue cells. Additional procedures for reprogramming somatic cells into induced pluripotent stem cells (or multipotent stem cells) are known, for example, as described in Zhao et al., iScience 23, 101192, 2020, and U.S. Patent Nos. 9,534,205, 9,394,524, 9,540615, and 9,771,563, which are incorporated by reference herein.

[0078] Some embodiments provide a method for reducing inflammation in a subject or treating a subject having an inflammation-associated disease, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising mononuclear phagocytes generated from iPSCs, wherein the mononuclear phagocytes are generated from iPSCs by a process comprising or consisting essentially of the steps of: incubating the iPSCs in a first culture medium supplemented with bone morphogenetic protein 4 (BMP-4), thereby forming first culture medium treated cells; incubating the first medium-treated cells in a second medium supplemented with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF), thereby forming second medium-treated cells; incubating the second medium-treated cells in a third medium supplemented with SCF, interleukin 3 (IL-3), thrombopoietin, macrophage colony-stimulating factor (M-CSF), and FLT3 ligand, thereby forming third medium-treated cells; and Incubating the third medium-treated cells in a fourth medium supplemented with M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and FLT3 ligand, thereby forming fourth medium-treated cells, which are mononuclear phagocytes differentiated from iPSCs.

[0079] Some embodiments provide a method for improving cognitive function in a subject, or treating a subject having, or alleviating, treating, or delaying the onset of a neurodegenerative disorder in a subject, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising mononuclear phagocytes generated from iPSCs, wherein the mononuclear phagocytes are differentiated from iPSCs by a process comprising or consisting essentially of the steps of: incubating the iPSCs in a first culture medium supplemented with bone morphogenetic protein 4 (BMP-4), thereby forming first culture medium treated cells; incubating the first medium-treated cells in a second medium supplemented with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF), thereby forming second medium-treated cells; incubating the second medium-treated cells in a third medium supplemented with SCF, interleukin 3 (IL-3), thrombopoietin, macrophage colony-stimulating factor (M-CSF), and FLT3 ligand, thereby forming third medium-treated cells; and Incubating the third medium-treated cells in a fourth medium supplemented with M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and FLT3 ligand, thereby forming fourth medium-treated cells, which are mononuclear phagocytes differentiated from iPSCs.

[0080] In some embodiments, the subject is an aged human. In some embodiments, the subject has Alzheimer's disease and / or amyotrophic lateral sclerosis. In some embodiments, the pharmaceutical composition comprising iMPs is administered intravenously. In some embodiments, the pharmaceutical composition comprising iMPs is administered by intraperitoneal injection. In some embodiments, the method further comprises one or more of behavioral assays (learning and memory studies), neurological health tests, and measuring inflammation levels. In some embodiments, after administration of the pharmaceutical composition comprising mononuclear phagocytes differentiated from stem cells, the subject shows improved neurological health, cognitive function, and / or reduced inflammation levels compared to the subject's baseline prior to treatment, as assayed by one or more behavioral studies.

[0081] In various aspects, one or more methods disclosed herein result in improved cognitive function (e.g., as characterized by one or more behavioral tests) or improved levels of synaptic trafficking (e.g., VGLUT1) compared to a control subject having a neurodegenerative disorder but not receiving macrophage or monocyte treatment.

[0082] In other aspects, one or more methods disclosed herein result in improved cognitive function (e.g., as characterized by one or more behavioral tests) or improved levels of synaptic trafficking (e.g., VGLUT1) compared to a control level, which is the subject's baseline level prior to treatment with macrophages and / or monocytes generated from pluripotent stem cells.

[0083] Pharmaceutical compositions can include pharma- ceutically acceptable excipients or carriers, such as buffers, salts, polymers, proteins, and preservatives, that are added to stabilize cells or provide physiological osmolality. "Pharmaceutically acceptable excipients" generally refer to excipients that are safe, non-toxic, and useful in the preparation of desirable pharmaceutical compositions, including excipients that are acceptable for veterinary use as well as human pharmaceutical use. Such excipients can be solid, liquid, or semi-solid. Residual amounts of cell culture supplements may also be retained in the final harvest of cells prior to formulation and patient use. Thus, compositions disclosed herein can include excipients, which refer to components used in formulation and auxiliary materials (e.g., cell culture supplements) that may remain in the final product. Examples of excipients include, but are not limited to, human serum albumin, dimethyl sulfoxide (DMSO), calcium chloride, potassium chloride, sodium chloride, sodium lactate, water, dextran, and combinations thereof. "Pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid filler, diluent, excipient, solvent, or encapsulant, or a combination thereof. The carrier is suitable for use in contact with any tissue or organ with which it may come into contact, i.e., the carrier should not involve toxicity, irritation, allergic reaction, immunogenicity, or any other risk of complications that unduly outweigh its therapeutic benefits.

[0084] In various embodiments, the pharmaceutical composition according to the present invention can be formulated for delivery via any route of administration. "Parenteral" refers to a route of administration generally associated with injection, including intraorbital, infusion, intraarterial, intravesical, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the composition may be in the form of a solution or suspension for infusion or injection. Typically, the composition is administered by injection. Methods for these administrations are known to those skilled in the art.

[0085] In a further embodiment, the disclosed treatment and / or prevention method further comprises administering to a subject with a neurodegenerative disease, such as Alzheimer's disease, one or more drugs or standard therapies to the subject. In some implementations, the pharmaceutical composition of the present invention is administered simultaneously with one or more drugs or standard therapies. In some implementations, the pharmaceutical composition of the present invention is administered separately from one or more drugs or standard (currently approved) therapies. Suitable drugs or currently approved therapies include galantamine, rivastigmine, donepezil, memantine, aducanumab (a human antibody that targets aggregated forms of amyloid-β).

[0086] Additional embodiments provide methods of drug screening using mononuclear phagocytes as generated as described herein or produced using the methods described herein, including, but not limited to, high-throughput screening methods. For example, in one embodiment, the present invention provides a method for identifying a compound useful for treating or preventing a disease or disorder associated with a deficiency or deficiency of monocytes and / or macrophages, comprising the steps of: contacting a mononuclear phagocyte generated by the methods disclosed herein with a candidate compound, and determining whether the candidate compound improves the deficiency or deficiency of monocytes or macrophages. In some embodiments, the method for identifying a compound is a high-throughput method. In some embodiments, the identified compound is useful for treating or preventing a disease or disorder in a human or mammal. In some embodiments, the mononuclear phagocytes are autologous or are generated from autologous cells, including iPSCs reprogrammed from autologous somatic cells. In some embodiments, the mononuclear phagocytes are allogeneic or are generated from allogeneic cells, including iPSCs reprogrammed from allogeneic cells. In some embodiments, the disease or disorder associated with a deficiency or deficiency of macrophages and / or monocytes is Alzheimer's disease. In some embodiments, the disease or disorder associated with a deficiency or deficiency of macrophages and / or monocytes is Parkinson's disease. EXAMPLES

[0087] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, this is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0088] Example 1 The use of blood, plasma, or bone marrow from young subjects to restore cognitive function in aging subjects has significant practical drawbacks that limit its potential therapeutic value. Induced pluripotent stem cells (iPSCs) offer the ability to provide autologous therapy.

[0089] Herein, we sought to identify the cell type responsible for the beneficial effects observed in studies using young plasma and bone marrow, generating mononuclear phagocytes (iMPs) from iPSCs, which express low levels of the hematopoietic stem cell marker CD34 and high levels of the monocyte / macrophage markers CD11b, CD14, and CD16; which we administered via tail vein injection to genetically immunocompromised aged NOD-scid-gamma (NSG) mice (young mice are 3-4 months old; aged mice are 11-13 months old). Mice were treated every 3 days for 22 days (Figure 1) and tested for several behavioral assays. We found significant improvements in spatial working memory in the spontaneous alternation test (Figures 3A, 3B) and hippocampal-dependent short-term memory in the novel object placement assay (Figure 4B). Furthermore, treatment with iMPs had significant effects on several important neuronal health indicators, including the synaptic transporter, VGLUT1 (Figure 5B), which is reduced in aged mice but is restored by treatment; and on the number and morphology of microglia and astrocytes (Figures 6A, 6B, and 8). The increased number of astrocytes and microglia and the decreased length of microglial branches seen in aged animals are all reversed by treatment with iMPs. These findings indicate that immune cells, particularly monocytes and macrophages, may be involved in the regenerative effects previously observed after young plasma transfer or bone marrow transplantation. Importantly, we find that iMPs have significant regenerative potential in aging.

[0090] Furthermore, we have begun to test this potential of iMP in 5xFAD mice, a mouse model of Alzheimer's disease (Figure 9A) and found improvement in the novel object recognition test even at 3 months of age, when these mice were first developing pathology (Figure 9C). We plan to also test 8-month-old 5xFAD mice, which have significant pathology, and predict that iMP treatment will more significantly affect behavioral and neurohealth / inflammatory outcomes in these older 5xFAD mice.

[0091] These studies demonstrate the potential benefits of iPSCs for improving cognitive and neurological health in aging and neurodegeneration. iPSC-derived mononuclear phagocytes can mimic the effects of young plasma and bone marrow transplants and can be used in subjects to restore cognitive function as a therapeutic agent for aging and Alzheimer's disease.

[0092] Example 2 The following differentiation protocol is used to differentiate iMPs from iPSCs. Passage iPSCs at low density using the EZ passaging tool; aim for 120 colonies in each well. Use the EZ passaging tool to chop only the center of the iPSC well. Aspirate the medium. Use 3ml of fresh medium to blow the colonies off the plate. Do not scrape! Remove the colony suspension and transfer to a new 15 ml conical tube. Dilute the colony suspension to approximately 120 colonies / ml (eyeball). NOTE: It may be necessary to dilute the colony suspension significantly (add up to an additional 10 ml of medium to the suspension). NOTE: If necessary, adjust the concentration of the colony suspension for each cell line (i.e., increase the concentration for poorly adhering strains and decrease it for strongly adhering strains). Matrigel is aspirated from the plates and replaced with 1 ml of medium / well. 1 ml of colony suspension is added to each well, transferred to an incubator and shaken horizontally and vertically (z&X axis) 4 times in each direction. Colonies are allowed to attach overnight.

[0093] Daily maintenance of the colony Remove all any differentiating cells and colonies that meet any of the following criteria: Much smaller or larger than the majority of colonies -There are any differentiated cells next to it Too close to other colonies (i.e. they will grow into each other over time) Wells with more than 15 colonies (ideal wells have 10-12 colonies) Any oddly shaped colonies (i.e. not quite close to crescent / circular)

[0094] It is important to have an even distribution of colonies that are uniform in diameter.

[0095] Brightfield culture images (5x) representative of each differentiation stage should show similar colony morphology if differentiation is done correctly. Note also that as differentiation progresses, the colonies become very "messy" as they should be.

[0096] Start of differentiation When all colonies are a minimum of 0.7 mm in diameter and the majority are 1.0 mm in diameter (7 cm in diameter in evos 4x), culture on stage 1 medium (mTeSR * + 80 ng / mL BMP4), which is D0 (day 0). *Unlike Douvaras et al., who used mTeSR custom medium as described in Stem Cell Reports, vol.8, 1516-1524, 2017, we use standard mTeSR (e.g., STEMCELL Technology, Catalog No. 85850, containing at least bFGF and TGFβ). Douvaras et al. described in Stem Cell Reports, vol.8, 1516-1524, 2017 (Supplemental) that his mTeSR custom medium is mTeSR1 medium (Stem Cell Technologies) that does not contain lithium chloride, GABA, pipecolic acid, basic fibroblast growth factor (bFGF), and transforming growth factor β (TGFβ1).

[0097] Stage 1: Stage 1 begins at D0. Feed with 1 ml / well of Stage 1 medium daily until day 4. That is, aspirate the supernatant daily and add 1 mL of fresh medium daily until day 4. At the end of stage 1, just before stage 2, the medium is changed completely to stage 2 medium.

[0098] Stage 2: On day 4, cells are switched to Stage 2 medium (StemPro-34 SFM + 25 ng / mL basic fibroblast growth factor (bFGF), 80 ng / mL vascular endothelial growth factor (VEGF), 100 ng / mL stem cell factor (SCF)), 2 ml / well. At the end of stage 2, just before stage 3, perform a complete medium change.

[0099] Stage 3: On day 6, cells are switched to stage 3 (StemPro-34 SFM + 50 ng / mL SCF, 50 ng / mL IL-3, 5 ng / mL thrombopoietin (TPO), 50 ng / mL macrophage CSF (M-CSF), 50 ng / mL FLT3-ligand (FLT3L)), 2 ml / well. Change the medium completely on day 10. On D10, the medium was changed and the cells were refed with 2 ml / well of Stage 3 medium (supernatant was aspirated and freshly fed). Just before stage 4, at the end of stage 3, perform a complete medium change. Note: Cyst formation begins to occur (a good sign)

[0100] Stage 4 (Collection Phase): On D12 or D13 or D14, switch cells to Stage 4 medium (StemPro-34 SFM + 50 ng / mL M-CSF, 25 ng / mL GM-CSF, 50 ng / mL FLT3L) 2 ml / well (aspirate supernatant completely, then add Stage 4 medium). Since the cysts were loosely attached to the well plates, no further aspiration was performed thereafter, but the cells floating in suspension were collected and the cells were fed with 2 ml / well of stage 4 twice a week (i.e., every Monday or Tuesday and Friday) without aspiration. NOTE: Cells were fed twice a week, one feeding after collection. Cysts were loosely attached to the plate, and monocytes or mononuclear phagocytes had blastulated and separated from the cysts, floating in suspension. Therefore, once a week, the medium containing the floating cells was collected by a serological pipette and spun down, leaving a cell pellet, which could then be used for dosing. Feeding had to be spaced 3 or 4 days apart; the floating cells were collected, spun down, and then the old medium was aspirated, and the cell pellet was resuspended in fresh medium and returned to the plate. At this point in the protocol, Douvaras et al. (Stem Cell Reports, vol. 8, pp:1516-1524, June 6, 2017) performed weekly sorting to collect only CD14+ / CX3CR1+ cells, and then continued to further differentiate these cells into microglia by exposing them to GM-CSF and IL34 for 2 weeks. Conversely, we did not sort these cells, but instead collected this more immature cell type for use in treating our animal models of aging and neurodegeneration. We did not culture the cells in microglial medium (RPMI-1640 with 2mM GlutaMAX-I, 10ng / mL GM-CSF, and 100ng / mL IL-34).

[0101] Example 3. Scale-up process and characterization in bioreactors to produce clinically relevant numbers of iMPs Once stage 4 was reached (approximately D14 in Example 2), the cells had formed cysts that were loosely attached to the plate. iMPs detached from these cysts by blastema and then floated in suspension. iMP cysts were lifted, for example using a cell scraper, and then stirred on a slow speed magnetic stir plate (e.g., DURA-MAG (商標) The cultured cysts were then transferred to a stirred flask bioreactor (e.g., CORNING®) on a 400°C (100°F) rack. The lifted cysts were allowed to adapt to suspension culture for 24 hours, after which the stir plate was turned on and set at 30 revolutions per minute. In the bioreactor, the cysts floated and the iMPs continued to separate from the cysts as blastemas, and the resulting iMPs were collected for further analysis or procedures.

[0102] We set the starting density, i.e., monolayer seeding density, at approximately 100,000 cells / cm 2 A polydimethylsiloxane (PDMS) stamp was used to plate small "islands" of proteins for the cells to attach to (e.g., extracellular matrix proteins or Matrigel), i.e., "seeded" into separate / isolated islands so that the cysts would be spaced apart during the differentiation process, rather than all over the surface of the culture device.

[0103] We also used RNA sequencing techniques to compare gene expression in iMPs cultured in bioreactors for 15 days, iMPs cultured in bioreactors for 55 days, iMPs cultured in well plates as shown in Example 2, and iMPs recovered from frozen vials of cultured cells from well plates as shown in Example 2, as well as iPSCs as a control. Figures 10A and 10B show that these four conditions of iMPs were similar to each other in terms of hierarchical clustering analysis and principal component analysis. Figure 11 shows the relative expression ratio of each gene in each of the four groups.

[0104] The total number of cells (including live and dead cells) collected from the bioreactor culture on different days was counted, showing a consistently high cell viability of over 65% over at least 48 days (Table 1).

[0105] Table 1. Number of live cells vs. number of dead cells and calculated percentage of viability (viability = number of live cells ÷ (number of live cells + number of dead cells)) for each given harvest date from the bioreactor. TIFF2024529729000002.tif64131

[0106] We have so far used either 125 mL or 500 mL flasks, but the process can be scaled beyond these volumes if necessary.

[0107] In addition to RNA-Seq analysis, flow cytometry, Western blotting, and phagocytosis assays (bead uptake) were also performed.

[0108] Various aspects of the present invention are described above in the detailed description. Although these descriptions directly describe the above aspects, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be included therein as well. Unless otherwise stated, it is the intention of the inventors that the words and phrases in this specification and claims are given the ordinary and customary meaning to those skilled in the applicable technical field.

[0109] The foregoing description of various aspects of the present invention known to the applicant at the time of filing this application is presented and is intended for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teachings. The described aspects serve to explain the principles of the present invention and its practical application, and to enable others skilled in the art to utilize the present invention in various aspects and with various modifications suited to the particular use contemplated. Therefore, it is not intended that the present invention be limited to the specific aspects disclosed for carrying out the invention.

[0110] While particular embodiments of the present invention have been shown and described, it will be apparent to one skilled in the art that, based on the teachings herein, changes and modifications can be made without departing from the present invention and its broader aspects, and therefore the appended claims are intended to encompass within their scope all such changes and modifications as are within the true spirit and scope of the present invention. In general, it will be understood by those skilled in the art that the terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "includes but is not limited to", etc.).

[0111] Although the open-ended term "comprising" as a synonym for terms such as including, containing, or having is used herein to describe and claim the invention, the invention or embodiments thereof may instead be described using alternative terms, such as "consisting of" or "consisting essentially of."

Claims

1. A pharmaceutical composition for improving cognitive function in a subject or treating a subject with a neurodegenerative disorder, comprising mononuclear phagocytes generated from induced pluripotent stem cells (iPSCs), administering the pharmaceutical composition to the subject improves cognitive function in the subject or treats the subject for a neurodegenerative disorder; the mononuclear phagocytes are generated from iPSCs in a process comprising culturing iPSCs in cell culture medium under conditions that induce myeloid differentiation, resulting in the generation of mononuclear phagocytes; and the culturing does not include contacting the generated mononuclear phagocytes in a microglial differentiation medium containing interleukin-34 (IL-34) or containing IL-34 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the culturing does not include contacting the generated mononuclear phagocytes in a dendritic cell differentiation medium containing interleukin-4 (IL-4) or containing IL-4 and GM-CSF. The pharmaceutical composition.

2. the mononuclear phagocytes comprise monocytes; Optionally, after said administration, said mononuclear phagocytes generate macrophages in said subject, or optionally, said mononuclear phagocytes are further cultured in cell culture medium under conditions that induce macrophage differentiation.

10. The pharmaceutical composition of claim 1.

3. 2. The pharmaceutical composition of claim 1, wherein the culturing comprises contacting the iPSCs with a first composition comprising bone morphogenetic protein 4 (BMP-4).

4. The culturing After culturing the iPSCs in the presence of the first composition, contacting the cell culture medium with a second composition comprising one or more factors selected from the group consisting of bFGF, vascular endothelial growth factor (VEGF), stem cell factor (SCF), and combinations thereof.

4. The pharmaceutical composition of claim 3, further comprising:

5. The culturing contacting the cell culture medium with a third composition comprising one or more factors selected from the group consisting of SCF, interleukin 3 (IL-3), thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), Fms-like tyrosine kinase 3 ligand (FLT3 ligand), and combinations thereof; 5. The pharmaceutical composition of claim 4, further comprising:

6. The culturing contacting the cell culture medium with a fourth composition comprising one or more factors selected from the group consisting of M-CSF, GM-CSF, and FLT3 ligand, and combinations thereof.

6. The pharmaceutical composition of claim 5, further comprising:

7. The culture contacting the cell culture medium with the first composition for approximately 4 days, contacting the cell culture medium with the second composition for approximately 2 days, contacting the cell culture medium with the third composition for approximately 6-8 days, and / or contacting the cell culture medium with the fourth composition for approximately 3-90 days.

7. The pharmaceutical composition of claim 6, comprising:

8. A pharmaceutical composition described in any one of claims 3 to 7, wherein the first composition containing BMP-4 is in mTeSR1 medium containing one or more of bFGF, TGFβ, GABA, pipecolic acid, and lithium chloride; the first composition is serum-free, and the second composition, the third composition, and / or the fourth composition are independently in serum-free hematopoietic cell medium, optionally being StemPro-34 serum-free medium.

9. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the mononuclear phagocytes express markers including CD11b, CD14, CD16, CD64, CD11c, CD71, or a combination thereof.

10. The process comprises: 6 , 5×10 6 , or 1 × 10 7 8. The pharmaceutical composition of any one of claims 1 to 7, further comprising culturing in a bioreactor to obtain said mononuclear phagocytes.

11. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the iPSCs are derived from peripheral blood mononuclear cells (PBMCs) or fibroblasts; and optionally, the PBMCs and fibroblasts are obtained from the subject.

12. The pharmaceutical composition of any one of claims 1 to 7, wherein the subject is a human aged 50 or older.

13. The pharmaceutical composition of any one of claims 1 to 7, wherein the subject has a neurodegenerative disorder selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, multiple sclerosis (MS), Rett syndrome, diffuse leukoencephalopathy with spheroid formation, hereditary diffuse leukoencephalopathy with axonal spheroid formation, frontotemporal lobar degeneration (FTLD), familial FTLD, schizophrenia, and autism spectrum disorder.

14. 8. The pharmaceutical composition of any one of claims 1 to 7, further comprising measuring one or more of the following in the subject after said administration compared to a control subject, or compared to the respective levels in the subject before said administration: improved spatial working memory, improved short-term memory, increased synaptic transporter levels, increased microglial branch length in the subject.

15. 1. A method for generating mononuclear phagocytes, comprising: Culturing induced pluripotent stem cells in a cell culture medium under conditions that induce myeloid differentiation, resulting in the production of mononuclear phagocytes, wherein the culturing comprises contacting the induced pluripotent stem cells with a first composition comprising bone morphogenetic protein 4 (BMP-4); After culturing the induced pluripotent stem cells in the presence of the first composition, contacting the cell culture medium with a second composition comprising one or more factors selected from the group consisting of bFGF, vascular endothelial growth factor (VEGF), and stem cell factor (SCF), and combinations thereof; After culturing in the presence of the second composition, contacting the cell culture medium with a third composition comprising one or more factors selected from the group consisting of SCF, interleukin 3 (IL-3), thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand), and combinations thereof; and after culturing in the presence of the third composition, contacting the cell culture medium with a fourth composition comprising one or more factors selected from the group consisting of M-CSF, GM-CSF, and FLT3 ligand, and combinations thereof, thereby generating the mononuclear phagocytes. and the culturing does not include contacting the generated mononuclear phagocytes in a microglial differentiation medium containing interleukin-34 (IL-34) or containing IL-34 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the culturing does not include contacting the generated mononuclear phagocytes in a dendritic cell differentiation medium containing interleukin-4 (IL-4) or containing IL-4 and GM-CSF. The method.

16. 16. The method of claim 15, wherein the first, second, third, and fourth compositions are in serum-free media, and optionally, the first composition is in mTeSR1 media and the second, third, and fourth compositions are in StemPro-34 media.

17. 16. The method of claim 15, comprising contacting the cell culture medium with the first composition for approximately 4 days, contacting the cell culture medium with the second composition for approximately 2 days, contacting the cell culture medium with the third composition for approximately 6-8 days, and / or contacting the cell culture medium with the fourth composition for approximately 3-90 days.

18. The culture contains at least 1 x 10 6 , 5×10 6 , or 1 × 10 7 18. The method of any one of claims 15 to 17, comprising culturing in a bioreactor to obtain a population of said mononuclear phagocytes, optionally wherein said bioreactor is a stirred tank bioreactor.

19. (1) culturing pluripotent stem cells in adherent culture in a first cell culture medium containing bone morphogenetic protein 4 (BMP-4); (2) culturing the cells obtained by step (1) in adherent culture in a second cell culture medium containing basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF); (3) culturing the cells obtained by step (2) in adherent culture in a third cell culture medium containing SCF, interleukin 3 (IL-3), thrombopoietin, macrophage colony-stimulating factor (M-CSF), and FLT3 ligand; and (4) culturing the cells obtained by step (3) in suspension culture in a fourth cell culture medium containing M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and FLT3 ligand, thereby generating macrophages and / or monocytes.

1. A mononuclear phagocyte produced using a method comprising: the method does not include contacting the generated mononuclear phagocytes in a microglial differentiation medium containing interleukin-34 (IL-34) or containing IL-34 and GM-CSF, and the culturing does not include contacting the generated mononuclear phagocytes in a dendritic cell differentiation medium containing interleukin-4 (IL-4) or containing IL-4 and GM-CSF. The mononuclear phagocyte.

20. 20. The mononuclear phagocyte of claim 19 produced by said method, wherein said pluripotent stem cells are human induced pluripotent stem cells.

21. 20. The mononuclear phagocyte of claim 19, produced by the method wherein each of the first, second, third, and fourth cell culture media is serum-free; optionally, the first composition is in mTeSR1 medium containing one or more or all of basic fibroblast growth factor (bFGF), transforming growth factor beta (TGFβ), aminobutyric acid (GABA), pipecolic acid, and lithium chloride; and optionally, the second, third, and fourth compositions are in StemPro-34 medium.

22. 20. The mononuclear phagocyte of claim 19, which is positive for CD11b, CD14, CD16, CD64, CD11c, CD71, or a combination thereof.

23. The mononuclear phagocyte of claim 19, produced by the method, wherein the culturing in any one or more of steps (4), (3), (2), and (1) comprises culturing in a bioreactor.

24. A pharmaceutical composition for treating a subject having a disease or disorder associated with a deficiency or deficiency of macrophages, comprising the mononuclear phagocytes described in any one of claims 19 to 23, wherein after administration of the pharmaceutical composition to the subject, the mononuclear phagocytes produce macrophages.

25. 1. A pharmaceutical composition for treating a subject with a neurodegenerative disorder or a subject in need of improved cognitive function, comprising mononuclear phagocytes, the mononuclear phagocytes are generated by culturing induced pluripotent stem cells in a cell culture medium under conditions that induce myeloid differentiation; The culture contacting the induced pluripotent stem cells with a first composition comprising bone morphogenetic protein 4 (BMP-4); after culturing the induced pluripotent stem cells in the presence of the first composition, contacting the cell culture medium with a second composition comprising one or more factors selected from the group consisting of bFGF, vascular endothelial growth factor (VEGF), and stem cell factor (SCF), and combinations thereof; After culturing in the presence of the second composition, contacting the cell culture medium with a third composition comprising one or more factors selected from the group consisting of SCF, interleukin 3 (IL-3), thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand), and combinations thereof; and after culturing in the presence of the third composition, contacting the cell culture medium with a fourth composition comprising one or more factors selected from the group consisting of M-CSF, GM-CSF, and FLT3 ligand, and combinations thereof, thereby generating the mononuclear phagocytes. Including, the culturing does not include contacting the generated mononuclear phagocytes in a microglial differentiation medium containing interleukin-34 (IL-34) or containing IL-34 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the culturing does not include contacting the generated mononuclear phagocytes in a dendritic cell differentiation medium containing interleukin-4 (IL-4) or containing IL-4 and GM-CSF; and administering to the subject a pharmaceutical composition comprising the generated mononuclear phagocytes to treat the subject with the neurodegenerative disorder or improve the cognitive function of the subject. The pharmaceutical composition.

26. 26. The pharmaceutical composition of claim 25, wherein the culturing does not include contacting the generated mononuclear phagocytes in a macrophage differentiation medium containing M-CSF and one or both of interferon gamma (IFN-γ) and IL-4.

27. 26. The pharmaceutical composition of claim 25, wherein said culturing further comprises contacting said generated mononuclear phagocytes in a macrophage differentiation medium.

28. 28. The pharmaceutical composition of claim 27, wherein the macrophage differentiation medium comprises M-CSF and IL-4.

29. 28. The pharmaceutical composition of claim 27, wherein the macrophage differentiation medium comprises M-CSF and interferon gamma (IFN-γ).

30. For administration of the generated population of mononuclear phagocytes to the subject, the culture is cultured to a concentration of at least 1 x 10 6 , 5×10 6 , or 1 × 10 7 30. The pharmaceutical composition of any one of claims 25 to 29, comprising culturing in a bioreactor to generate said population of mononuclear phagocytes.

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