Compositions of cells derived from induced pluripotent stem cells and methods of use thereof

Abstract

The present disclosure provides a multi-cell culture model for the study of neuroinflammation, including to identify novel targets, biomarkers, and therapeutic agents for the diagnosis, prognosis, and treatment of neurodegenerative diseases. Additionally, provided herein are assays for studying neuroinflammation using the cell culture models of the present invention.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 242,900, filed September 10, 2021, the entire contents of which are incorporated herein by reference.

[0002] Incorporating sequence tables This application contains a sequence listing XML that has been submitted electronically and is incorporated herein by reference in its entirety. The XML sequence listing, created on September 12, 2022, has the file name CDINP0110WO.xml and is 2,828 bytes in size. [Background technology]

[0003] 1. Field The present invention relates generally to the fields of molecular biology and medicine. More specifically, the present invention relates to compositions of cells differentiated from induced pluripotent stem cells and methods of use thereof.

[0004] 2. Description of Related Technology Immune function, especially tissue-resident macrophages, plays an essential role in disease pathogenesis. For example, the neuroimmune axis and microglia, the brain-resident macrophages, play a key role in the pathobiology of neurodegenerative diseases, including Alzheimer's disease, which is supported by both genome-wide association studies and omics studies. Furthermore, tissue-resident macrophages play a key role in the pathogenesis of NASH (Kupffer cells), AMD (subretinal microglia), asthma, COPD (alveolar macrophages), and HIV. Many studies have also identified lipid regulatory dysfunction that contributes to retinal microglial rusen formation, atherosclerotic plaque formation (peripheral macrophages), pulmonary foam cells, and brain AD neuropathology. Understanding how lipid dysfunction of tissue-resident macrophages affects homeostatic function may serve as a therapeutic avenue for numerous chronic diseases with inflammatory etiology. Summary of the Invention [Means for solving the problem]

[0005] In some embodiments, the present disclosure provides a cell culture comprising induced pluripotent stem cell (iPSC)-derived microglia, astrocytes, and / or neurons in a medium. Further provided herein are methods of producing the cell culture and methods of use thereof.

[0006] In certain embodiments, the culture comprises iPSC-derived microglia, astrocytes, and neurons. In certain embodiments, the cell culture is further defined as a tripartite culture.

[0007] In some embodiments, the neuron is an excitatory neuron or an inhibitory neuron, hi certain embodiments, the neuron is a GABAergic neuron, a dopaminergic neuron, or a glutamatergic neuron.

[0008] In certain embodiments, the microglia are derived from an isogenic iPSC line. In certain embodiments, the microglia, astrocytes and neurons are isogenic. In some embodiments, the microglia are at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) positive for TREM2, P2RY12, TMEM119, IBA-1, and / or CX3CR1. In certain embodiments, the microglia are mature microglia. In certain embodiments, the astrocytes are positive for S100beta, GFAP, and CD44. In some embodiments, the neurons are SCL1, BCL11B, Calb2, CD24, CDH1, CUX1Cux2, DCX, DLG4, Dlx, Dlx2, Emx1, Emx2, eomes, ETV1, FOXG1, FOXP2, Fut4, GABRA2, GAD1, GAD2, GAPDH, GFAP, GRIN2B, HoxB4, HTR2C, ISL1, ITGB1, LHX2, Neuro Positive for at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) markers selected from the group consisting of og1, NKX2-1, Nos1, NPY, NR4A2, PAX6, POU3F2, PVALB, RELN, SATB2, SLC17A6, SLC17A7, SLC17A8, SLC32A1, SOX1, Sox10, SST, SYN1, and Tbr1.

[0009] In certain embodiments, the microglia are derived from donors expressing disease-associated SNPs. In some embodiments, the microglia are generated from disease-associated iPSC donors with genotypes associated with TREM2, APOE, CD33, BIN, ABCA7, SNPS, or neurodegeneration. In some embodiments, the microglia comprise a disruption of TREM2, methyl-CpG binding protein 2 (MeCP2), and / or alpha-synuclein (SCNA). In certain embodiments, the microglia comprise a disruption of TREM2. For example, the disruption of TREM2 comprises a TAL nuclease-mediated disruption at amino acid 58 of exon 2 of TREM2. In some embodiments, the microglia comprise a heterozygous disruption of TREM2. In other embodiments, the microglia comprise a homozygous disruption of TREM2.

[0010] In some embodiments, the cell culture is a two-dimensional (2D) culture. In certain embodiments, the medium further comprises IL-34 and M-CSF, or an analog or mimetic thereof. For example, the medium further comprises IL-34 at a concentration of 50-200 ng / mL (e.g., 50, 75, 100, 150, 175, or 200 ng / mL, particularly 100 ng / mL), and M-CSF at a concentration of 10-50 ng / mL (e.g., 10, 25, 30, 40, or 50 ng / mL, particularly 25 ng / mL). In further embodiments, the medium further comprises TGFβ or an analog or mimetic thereof, for example at a concentration of 10-100 ng / mL (e.g., 10, 25, 30, 40, 50, 75, 80, 90, or 100 ng / mL, particularly 50 ng / mL).

[0011] In some embodiments, the cells are cultured or placed on a cell surface. In certain embodiments, the cells are cultured on a surface coated with polyethyleneimine (PEI). In some embodiments, the cells are cultured on a surface coated with an extracellular matrix protein. In certain embodiments, the extracellular matrix is ​​basement membrane extract (BME) purified from mouse Engelbreth-Holm-Swarm tumors. In some embodiments, the extracellular matrix protein is MATRIGEL®, GELTREX™, collagen, or laminin. For example, the extracellular matrix protein is GELTREX™ or laminin.

[0012] In some embodiments, the cell culture is a three-dimensional (3D) culture.In certain embodiments, the 3D culture is a brain organoid culture.In some embodiments, the 3D culture comprises functional neuronal network.In certain embodiments, the functional neuronal network comprises calcium oscillation.

[0013] In some embodiments, the culture comprises microglia and astrocytes present in a ratio of about 3:1 to 1:3, particularly about 2:1 to 1:2, such as about 1:1. In particular embodiments, the microglia and astrocytes are seeded in the culture in a ratio of 3:1 to 1:3, particularly about 2:1 to 1:2, such as about 1:1. In particular embodiments, the culture comprises microglia, astrocytes, and neurons in a ratio of about 1:1:5 to 1:1:10, such as 1:1:6, 1:1:7, 1:1:8, 1:1:9, or 1:1:10. In particular embodiments, the culture comprises microglia, astrocytes, and neurons seeded in a ratio of about 1:1:5 to 1:1:10, such as 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, or 1:1:10, particularly about 1:1:5. In some embodiments, microglia, astrocytes, and neurons are in a ratio of about 3:1:5 to 1:3:10, e.g., about 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:2:5, 3:2:6, 3:2:7, 3:2:8, 3:2:9, 3:2:10; 1:3:5, 1:3:6, 1:3:7, 1:3:8, 1:3 2:1:5, 1:2:6, 1:2:7, 1:2:8, 1:2:9, 1:3:10, 2:3:5, 2:3:6, 2:3:7, 2:3:8, 2:3:9, 2:3:10, 2:1:5:2:1:6, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 1:2:5, 1:2:6, 1:2:7, 1:2:8, 1:2:9, or 1:2:10 or any range derivable therein. In some embodiments, microglia, astrocytes, and neurons are in a ratio of about 3:1:5 to 1:3:10, e.g., about 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:2:5, 3:2:6, 3:2:7, 3:2:8, 3:2:9, 3:2:10; 1:3:5, 1:3:6, 1:3:7, 1:3:8, 1:3: 1:2:5, 1:2:6, 1:2:7, 1:2:8, 1:2:9, or 1:2:10, or any range derivable therein.In some embodiments, the cultures include microglia seeded at a cell density of 5,000 cells / well to 7,500 cells / well. In certain embodiments, the cultures include neurons seeded at a cell density of 40,000 cells / well to 50,000 cells / well. In some embodiments, the cultures include astrocytes seeded at a cell density of 8,000 cells / well to 10,000 cells / well. The cultures include 15,000 cells / cm. 2 ~25,000 cells / cm 2 , e.g. 15,625 cells / cm 2 ~23,438 cells / cm 2 In some embodiments, the cultures contain microglia at a cell density of 125,000 cells / cm. 2 ~160,000 cells / cm 2 , e.g. 125,000 cells / cm 2 ~156,250 cells / cm 2 In certain embodiments, the cultures contain neurons at a cell density of 25,000 cells / cm. 2 ~35,000 cells / cm 2 , e.g. 25,000 cells / cm 2 ~31,250 cells / cm 2 containing astrocytes at a cell density of .

[0014] In certain embodiments, the microglia, astrocytes, and neurons are in culture for at least 10 days, e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days, or more. In certain embodiments, the microglia, astrocytes, and neurons are in culture for at least 14 days. In some embodiments, the microglia, astrocytes, and neurons are in culture for at least 14 days and are in a ratio of 2:6:1. In some embodiments, the microglia, astrocytes, and neurons are in culture for at least 14 days and are in a ratio of about 15:1:1 to 1:15:30, e.g., about 10:1:1 to 1:10:20, about 8:1:1 to 1:8:20, about 5:1:1 to 1:5:20, or about 3:1:1 to 1:3:20.

[0015] In some embodiments, the iPSCs are human. In certain embodiments, the culture is xeno-free, feeder-free, and / or conditioned medium-free. In certain embodiments, the medium is a defined medium.

[0016] Another embodiment provides a method of culturing microglia and neurons. In some embodiments, the microglia and neurons are isogenic. In some embodiments, the neurons are GABAergic neurons or glutamatergic neurons. In certain embodiments, co-culturing GABAergic or glutamatergic neurons and astrocytes results in enhanced phagocytic activity by microglia compared to monoculture of microglia. In some embodiments, the method further comprises detecting soluble TREM2. In some embodiments, the astrocytes are isogenic.

[0017] Further embodiments provide a cell culture comprising brain microvascular endothelial cells (BMECs), pericytes, and astrocytes in a sandwich format, hi certain aspects, the culture is further defined as a blood-brain barrier model.

[0018] In some embodiments, the sandwich format comprises an extracellular matrix layer between two cell layers. In certain embodiments, the extracellular matrix layer comprises at least two extracellular matrix proteins. For example, the at least two extracellular matrix proteins are collagen IV and fibronectin. In certain embodiments, the sandwich format comprises BMECs on the apical side, an extracellular matrix layer in the middle, and astrocytes and pericytes on the basolateral side. In some embodiments, the extracellular matrix layer further comprises gelatin.

[0019] In certain embodiments, the sandwich format further comprises a permeable membrane insert. In some embodiments, the permeable membrane insert is a polytetrafluoroethylene (PFTE), polycarbonate, or polyethylene terephthalate (PTE) insert. In some embodiments, the permeable membrane insert is a PFTE insert. For example, the PFTE insert is a TRANSWELL™ insert. In some embodiments, the permeable membrane insert is coated on the apical side with human collagen IV and human fibronectin. In some embodiments, the permeable membrane insert is coated with human collagen IV at a concentration of 200-400 μg / mL, e.g., 200, 250, 300, 350, or 400 μg / mL, and human fibronectin at a concentration of 50-100 μg / mL, e.g., 50, 75, or 100 μg / mL. In certain embodiments, the basolateral side of the permeable membrane insert is coated with gelatin, e.g., 0.01-0.2% gelatin, in particular about 0.1% gelatin.

[0020] In some embodiments, the astrocytes and pericytes are present in a ratio of about 2:1 to 1:2, such as a ratio of about 2:1. In some embodiments, the astrocytes and pericytes are seeded into the culture in a ratio of about 2:1 to 1:2, such as a ratio of about 2:1. In some embodiments, the astrocytes and pericytes are on the basolateral side of the permeable membrane insert. In certain embodiments, the BMECs are on the apical side of the permeable membrane insert.

[0021] In certain embodiments, the BMECs are in medium that includes EFRA2. In some embodiments, the BMECs, astrocytes and pericytes are in medium that includes a ROCK inhibitor, such as Y-27632.

[0022] In some embodiments, the BMECs, astrocytes, and pericytes are present in a ratio of about 5:1:1 to 1:3:3, e.g., 4:1:2, 4:1:1, 4:2:1, or 2:1:1. In some embodiments, the BMECs, astrocytes, and pericytes are seeded in a ratio of about 5:1:1 to 1:3:3, e.g., 4:1:2, 4:1:1, 4:2:1, or 2:1:1. In some embodiments, the BMECs are seeded at a ratio of about 1×10 6 cells / cm 2 ~1.5×10 6 cells / cm 2 , for example, about 1.3 × 10 6 cells / cm 2 In certain embodiments, the astrocytes are seeded at a cell density of 300,000 cells / cm. 2 ~700,000 cells / cm 2 , for example, about 333,000 cells / cm 2 In certain embodiments, the pericytes are seeded at a cell density of 300,000 cells / cm. 2 ~700,000 cells / cm 2 , for example, about 666,000 cells / cm 2 The cells are seeded at a cell density of 1000×.

[0023] Another embodiment provides a method for screening for therapeutic compounds for treating a neurodegenerative disease comprising contacting a test compound with a culture of this embodiment and aspects thereof (e.g., a cell culture comprising induced pluripotent stem cell (iPSC)-derived microglia, microglia, and / or neurons in medium, or a cell culture comprising brain microvascular endothelial cells (BMECs), pericytes, and astrocytes in a sandwich format); and measuring functional activity of the cell.

[0024] In some embodiments, an increase in functional activity indicates that the test compound can treat a neurodegenerative disease. In certain embodiments, measuring the functional activity includes measuring dendritic area (MAP2), synapse number, cell number, or axon area. In certain embodiments, measuring the functional activity includes detecting the release of complement C3 from microglia. In certain embodiments, a decrease in complement C3 released from microglia indicates that the therapeutic compound can treat a neurodegenerative disease.

[0025] In certain embodiments, the method further comprises contacting the culture with LPS. In some embodiments, measuring the functional activity comprises measuring an analyte released into the medium with or without stimulation with LPS. For example, the analyte is an M1 factor, such as TNF alpha, IL-6, CCL2, CCL3, CCL4, IL-1 beta, IL-12, IL-13, IL-8, Interferon gamma, IL1-Alpham FAS Ligand, IL-2, GMCSF, Granzyme B, ICAM-1, and / or CXCL11. In certain embodiments, the analyte is an M2 factor, such as IL-4, IL-10, IL-21, VEGF, CCL5, IL-17, IL1-RII, GCSG, CXCL5.

[0026] In certain embodiments, the methods include cultures with TREM2 wild-type microglia, cultures with TREM2 heterozygous knockout microglia, and / or cultures with TREM2 homozygous knockout microglia.

[0027] In some embodiments, measuring the functional activity comprises measuring neuronal function by calcium signaling or microelectrode array (MEA). In certain embodiments, measuring the functional activity comprises measuring amyloid-β phagocytosis function.

[0028] In certain embodiments, the neurodegenerative disease is Alzheimer's disease or multiple sclerosis.

[0029] Further embodiments provide for the use of cultures of this embodiment and aspects thereof (e.g., cell cultures comprising induced pluripotent stem cell (iPSC)-derived microglia, microglia, and / or neurons in media, or cell cultures comprising brain microvascular endothelial cells (BMECs), pericytes, and astrocytes in a sandwich format) as models for neurodegenerative disease.

[0030] In some embodiments, the models include cultures with TREM2 wild-type microglia, cultures with TREM2 heterozygous knockout microglia, and / or cultures with TREM2 homozygous knockout microglia. In certain embodiments, the models include engineered or patient-specific iPSC-derived astrocytes, neurons and / or microglia carrying disease-associated genotype SNPs or mutations in APOE4 / 4, CD33, ABCA, BIN1, or R47H.

[0031] Another embodiment provides a method of screening for a neurodegenerative disease comprising detecting levels of soluble TREM2 in a culture of this embodiment and aspects thereof (e.g., a cell culture comprising induced pluripotent stem cell (iPSC)-derived microglia, microglia, and / or neurons in medium, or a cell culture comprising brain microvascular endothelial cells (BMECs), pericytes, and astrocytes in a sandwich format).

[0032] In some embodiments, the cells in the culture are derived from an isogenic iPSC line or a donor expressing a disease-associated SNP or a mutation associated with neurodegeneration. In certain embodiments, the level of soluble TREM2 is detected in the conditioned medium. In some embodiments, the detection comprises performing an ELISA. In some embodiments, detecting an increase in soluble TREM2 levels compared to a control indicates the presence of a neurodegenerative disease. In some embodiments, the method further comprises detecting the levels of COMT, NRXN2 and / or SST in microglia. In certain embodiments, the neurodegenerative disease is Alzheimer's disease or multiple sclerosis.

[0033] Further embodiments of the present disclosure provide methods and compositions for an in vitro method for differentiating induced pluripotent stem cells (iPSCs), the in vitro method comprising: (a) culturing iPSCs on a charged surface in the absence of extracellular matrix proteins; and (b) differentiating the iPSCs into endothelial cells, mesenchymal stem cells (MSCs) or hematopoietic progenitor cells (HPCs).

[0034] In some embodiments, the charged surface is positively charged. In certain embodiments, the positively charged surface is an amine surface or a poly-L-lysine surface. In certain embodiments, the positively charged surface comprises a nitrogen-containing functional group. In other embodiments, the charged surface is negatively charged. In certain embodiments, the negatively charged surface is a carboxyl surface. In certain embodiments, the negatively charged surface comprises an oxygen-containing functional group. In some embodiments, the charged surface is a polymeric surface. For example, the polymeric surface is a polystyrene surface. In certain embodiments, the charged surface comprises a positively charged group and a negatively charged group. In some embodiments, the positively charged group is a nitrogen-containing group and the negatively charged group is an oxygen-containing group.

[0035] In certain embodiments, the iPSCs are cultured in a serum-free defined medium. In some embodiments, the differentiating comprises culturing in the presence of a ROCK inhibitor, such as blebbistatin or H1152. In certain embodiments, the method is free or essentially free of extracellular matrix proteins, such as laminin, fibronectin, vitronectin, MATRIGEL™, tenascin, entactin, thrombospondin, elastin, gelatin, or collagen.

[0036] In further embodiments, the method further comprises engineering the iPSCs to have disrupted expression of TREM2, MeCP2 and / or SCNA prior to step (a). In certain embodiments, the engineering comprises introducing an indel into exon 2 of TREM2 using a TAL nuclease. In some embodiments, the disrupted expression of MeCP2 is further defined as a truncated variant of the MeCP2 protein. In certain embodiments, the disrupted expression is due to a missense point mutation, such as A53T.

[0037] In some embodiments, the method comprises differentiating the progenitor cells into endothelial cells. In certain embodiments, step (a) comprises culturing on an amine surface to generate progenitor cells, and step (b) comprises culturing on a carboxyl surface in the presence of an endothelial differentiation medium to produce endothelial cells. In certain embodiments, the endothelial cells are positive for CD31.

[0038] In a further embodiment, the method further comprises differentiating the endothelial cells into brain microvascular endothelial cells (BMECs).

[0039] In some embodiments, the method further comprises differentiating the endothelial cells into lymphatic endothelial cells.

[0040] In certain embodiments, the method comprises differentiating the progenitor cells into MSCs. In certain embodiments, the differentiating comprises culturing the progenitor cells on an amine surface in the presence of MSC medium. In some embodiments, the MSCs are positive for CD73, CD44 and CD105. In certain embodiments, at least 90% of the differentiated cells are positive for CD73.

[0041] In further embodiments, the method further comprises differentiating the MSCs into pericytes. In certain embodiments, the MSCs are cultured in the presence of pericyte medium in the absence of extracellular proteins. In some embodiments, the pericytes are positive for NG2, PDGFRβ and CD146.

[0042] In some embodiments, the method comprises differentiating the progenitor cells into HPCs. In certain embodiments, the method further comprises differentiating the HPCs into microglia. In certain embodiments, the differentiating comprises culturing the HPCs on a neutrally charged surface or an ultra-low attachment surface in the presence of microglia differentiation medium. In certain embodiments, the microglia differentiation medium comprises IL34, TGF and MCSF, or analogs or mimetics thereof. In some embodiments, the differentiating comprises culturing in normoxia. In some embodiments, the differentiating is for 20-25 days. In certain embodiments, the microglia are positive for CD45, CD11b and CD33. In certain embodiments, at least 50% (e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 50-60%, 60-70%, or 80-90%) of the differentiated cells are positive for CD11b. In some embodiments, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the differentiated cells are positive for CD33.

[0043] In certain embodiments, the method does not include purification of the cells. In some embodiments, purification is further defined as performing MACS.

[0044] In certain embodiments, the method is Good Manufacturing Practice (GMP) compliant. In some embodiments, the method is performed under hypoxic conditions. In certain embodiments, the iPSCs are human.

[0045] In another embodiment, a composition is provided that includes a microglial cell population that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 90-93%, 93-96% or 96-100%) positive for P2RY12, CX3CR1, TMEM119, IBA-1 and TREM2. In some aspects, the microglial cell population is produced by the method of the embodiments. In some aspects, the microglial cell population is generated from a disease-related iPSC donor that has a genotype associated with TREM2, APOE, CD33, BIN, ABCA7, SNPS or neurodegeneration. In certain aspects, the microglial cell population has disrupted expression of TREM2, MeCP2 and / or SCNA. In some aspects, the disrupted expression of TREM2 is further defined as a homozygous knockout of TREM2 expression. In certain embodiments, the disrupted expression of MeCP2 is further defined as a truncated mutant of the MeCP2 protein. In some embodiments, the disrupted expression of SCNA is due to a missense point mutation, such as A53T.

[0046] Further embodiments provide a method for screening a test compound, comprising introducing the test compound into the microglial cell population of the present embodiment. In some embodiments, the microglial cell population is further introduced into amyloid beta. In certain embodiments, the microglial cell population is further introduced into LPS.

[0047] Another embodiment provides a composition comprising a pericyte population produced by the method of this embodiment.

[0048] In yet another embodiment, provided herein is a blood-brain barrier model comprising microglia, pericytes and BMECs produced according to the present embodiments.

[0049] Further embodiments provide a method for generating microglia, comprising: (a) differentiating iPSCs into HPCs; and (b) sorting the HPCs for CD34 positive cells; and (c) culturing the HPCs in microglia differentiation medium, thereby generating a population of microglia. In some embodiments, HPCs are differentiated according to this embodiment. In certain embodiments, sorting comprises using CD34 magnetic beads. In certain embodiments, the method does not comprise sorting the HPCs for CD43 positive cells. In certain embodiments, the method does not comprise ECM proteins.

[0050] In some embodiments, the microglial differentiation medium comprises IL-34, TGFβ1 or M-CSF or an analog or mimetic of each. In certain embodiments, the microglial differentiation medium comprises 200 ng / mL IL-34, 100 ng / mL TGFβ1 and 50 ng / mL M-CSF. In some embodiments, the cells are fed with microglial differentiation medium every 48 hours.

[0051] In certain embodiments, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 90-93%, 93-96%, or 96-100%) of the cells in the microglial population are TREM positive. In some embodiments, at least 10% (e.g., 15%, 20%, 25%, 30%, 10-15%, 15-20%, or 20-30%) of the HPCs are differentiated into microglia.

[0052] In certain embodiments, the culturing of step (b) is performed in a 96-well format. In certain embodiments, the culturing of step (b) is performed on a charged surface. In some embodiments, the charged surface is positively charged. For example, the positively charged surface is an amine surface. In other embodiments, the charged surface is negatively charged. For example, the negatively charged surface is a carboxyl surface.

[0053] In further embodiments, the method further comprises maturing the population of microglia in a medium comprising CD200 and / or fractalkine or an analog or mimetic thereof, hi some embodiments, the method further comprises cryopreserving the microglia.

[0054] In some embodiments, HPCs are differentiated from iPSCs that have been engineered to have disrupted expression of TREM2. In certain embodiments, the engineering comprises using a TAL nuclease.

[0055] In certain embodiments, cryopreserved microglia retain phagocytic function against pHrodo bacterial particles, hi certain embodiments, cryopreserved microglia mature after thawing and are able to respond to stimuli and secrete interleukins, chemokines and immunomodulatory ligands into the supernatant medium.

[0056] A further embodiment provides an in vitro method for producing neural progenitor cells (NPCs) from iPSCs, comprising: (a) preconditioning iPSCs in a medium comprising a glycogen synthase kinase 3 (GSK3) inhibitor; and (b) differentiating the iPSCs into NPCs, the in vitro method not including inhibition of SMAD signaling.

[0057] In some embodiments, the iPSCs are maintained under hypoxic conditions prior to step (a). In certain embodiments, the iPSCs are seeded in the presence of a ROCK inhibitor and then cultured in the absence of a ROCK inhibitor prior to step (a).

[0058] In certain embodiments, the GSK3 inhibitor is CHIR99021, BIO or SB-216763. In certain embodiments, the GSK3 inhibitor is CHIR99021, such as at a concentration of 1 μM, 2 μM, 3 μM, 4 μM or 5 μM, particularly 3 μM. In some embodiments, the preconditioning is for 2-4 days, such as 1, 2 or 3 days.

[0059] In some embodiments, the iPSCs of step (a) and / or step (b) are cultured on a surface coated with an extracellular matrix (ECM) protein. In certain embodiments, the ECM protein is MATRIGEL™, laminin, or vitronectin. In certain embodiments, the ECM protein is laminin or vitronectin.

[0060] In certain embodiments, steps (a) and (b) are performed under normoxic conditions. In some embodiments, the differentiating comprises culturing the iPSCs on a surface coated with an ECM protein. In certain embodiments, the ECM protein is laminin or vitronectin. In some embodiments, the differentiating comprises culturing the iPSCs in an ultra-low attachment plate or spinner flask in the presence of a ROCK inhibitor. In certain embodiments, step (b) is performed for 5-10 days, such as 6 days, 7 days, 8 days, 9 days or 10 days.

[0061] In further embodiments, the method further comprises detecting expression of Tra-162, CD56, CD15, Sox1, Nestin, β3 microglobulin, and / or Pax-6 in the NPCs. In some embodiments, at least 70% (e.g., 80%, 85%, 90%, 95%, 70-80%, 80-90%, or 90-100%) of the NPCs are positive for CD56.

[0062] In a further embodiment, the method comprises further differentiating the NPCs into astrocytes or neurons.

[0063] Another embodiment provides a method for screening for a neurodegenerative disease, comprising detecting levels of soluble TREM2 in microglia conditioned medium. In some embodiments, the detecting comprises performing an ELISA. In certain embodiments, the microglia are derived from isogenically engineered iPSCs or donors expressing a disease-associated SNP or mutation. In some embodiments, the microglia are produced by the method of this embodiment or an aspect thereof. In certain embodiments, an increased level of soluble TREM2 compared to a control detects a neurodegenerative disease, such as Alzheimer's disease or multiple sclerosis.

[0064] A further embodiment provides a method for performing high throughput screening to identify therapeutic agents, comprising contacting microglia produced by the method of this embodiment or aspect thereof with a plurality of candidate agents and measuring cytokine and / or chemokine levels and / or amyloid beta phagocytic function.

[0065] In some embodiments, the microglia are cryopreserved, isogenically engineered iPSC lines derived microglia, donors expressing disease-associated SNPs, or donors expressing mutations associated with neurodegeneration. In some embodiments, the cytokines and / or chemokines are selected from the group consisting of IL6, IL10, IL3, TNFα, IL13, CCL2 / MCP-1, CCL20 / MIP-3α, CCL4 / MIP-1β, CCL5 / RANTES, CX3CL1 / fractalkine, CXCL1 / GROα, CXCL10 / IP-10, CXCL2 / GROβ, and IL-8 / CXCL8.

[0066] Also provided herein is a co-culture comprising the microglia and aspects thereof of the present embodiment with endothelial cells, pericytes, astrocytes and / or neural progenitor cells. Another embodiment provides the use of the co-culture to mimic human brain development.

[0067] Other objects, features and advantages of the present invention will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0068] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0069] [Figure 1] Schematic of the 2D and 3D HPC differentiation process. [Diagram 2] Schematic representation of endothelial cell derivation from day 6 HPCs derived from 2D or 3D HPC differentiation process. [Diagram 3] Characterization of CD31+ endothelial cells generated using serial passage purification without MACS. [Figure 4A-4B] (FIG. 4A) Cell morphology at the end of replating 3. Endothelial cells can be cryopreserved at the end of replating passages 2 or 3. (FIG. 4B) Media formulation used for endothelial cell induction. [Figure 5A-5D] (FIG. 5A) Overview of MSC differentiation process. (FIG. 5B) Media formulation for generating MSCs. (FIG. 5C) Schematic showing tri-lineage differentiation of MSCs into adipocytes, osteocytes and chondrocytes. (FIG. 5D) Phenotypic characterization of MSC precursors at day 6. [Figure 6] Emergence of pure populations of MSCs on amine surfaces. Cryopreserved day 6 HPCs or live cultures at the end of differentiation on day 6 are placed in the presence of MSC medium in the presence of 1 uM H1152 (or blebbistatin) on amine charged plate surfaces. Cultures were transferred to normoxic and normal tissue culture plates at P4. MSC purity specifications were achieved at P5. [Figure 7]Cells stained for surface MSC markers CD73, CD44, CD105, CD49d and absence of endothelial markers CD31 and CD144. Cryopreserved day 6 HPCs or live cultures at the end of differentiation on day 6 are placed in the presence of MSC medium in the presence of 1 uM H1152 (or blebbistatin) on an amine-charged plate surface. Cultures were transferred to normoxic and normal tissue culture plates at P5. MSC purity specifications were achieved at P6. [Figure 8A-8C] (Figure 8A) Tri-lineage potential. Different steps to generate adipocytes, osteocytes and chondrocytes from MSCs. (Figure 8B) Images showing the tri-lineage potential of MSCs, Alizarin Red for osteocytes, Alcian Blue for chondrocytes and Oil Red O staining for adipocytes. (Figure 8C) MSCs were plated at 1,000 cells / cm2 in MSC differentiation medium and fed every other day for 10-14 days. The total number of plates and colonies stained using crystal violet were counted. [Figure 9A-9H]Conversion of MSCs into pericytes. (Figure 9A) Schematic of the process of converting iCell MSCs into iPSC-derived pericytes. (Figure 9B) Media formulation for generating iPSC-derived pericytes. (Figure 9C) Comparative flow cytometry of known pericyte markers PDGFRβ, NG2 and CD146 in iCell MSCs, iPSC-derived pericytes and ScienCell primary human brain vascular pericytes (HBVPs). There was no pericyte marker present in iCellMSCs upon thawing, and pericyte markers were obtained by the end of P1 in pericyte media. iPSC-derived pericytes show a higher purity of pericyte-specific markers than primary HBVPs. (Figure 9D) Morphology of iPSC-derived MSCs (P2), iPSC-derived pericytes (P1) and ScienCell primary HBVPs by bright field microscopy. (Figure 9E) Table illustrating the differences between PC1 and PC2 pericyte subtypes based on phenotype and marker expression. (FIG. 9F) iPSC-derived pericytes were stained by flow cytometry immediately after thawing and 5 days after thawing for pericyte subtype-specific markers CD274, VCAM1, desmin, DLK1, and αSMA as well as general pericyte markers PDGFRβ, NG2, CD13, and CD146. iPSC-derived pericytes reveal characteristics of contractile pericytes, subtype PC2. (FIG. 9G) IncuCyte live imaging system images of iPSC-derived pericytes in a phagocytosis assay. iPSC-derived pericytes show observable phagocytic activity of S. aureus bioparticles above control levels. (A) iPSC-derived pericytes only control. (B) iPSC-derived pericytes + NucGreen Dead 488 (NG) reagent control. (C) iPSC-derived pericytes + S. aureus pHrodo Red BioParticles (BP). (D) iPSC-derived pericytes + NucGreen Dead 488 Reagent (NG) + S. aureus pHrodo Red BioParticles (BP). All images acquired from 36 days and 16 hours after cell seeding. (Figure 9H) Quantification of phagocytic activity via total red object integrated intensity analyzed by IncuCyte software. [Figure 10A-10G]Generation of brain microvascular endothelial cells (BMECs). (Figure 10A) Schematic of the generation of brain microvascular endothelial cells. (Figure 10B) Composition of ECRA medium. (Figure 10C) Flow cytometry analysis of BMECs with co-expression of Glut1 / CD31. (Figure 10D) Immunohistochemical staining of BMECs with P-glycoprotein (green) expression on day 13. Nuclei were stained with Hoechst3342 and images were acquired at 200x magnification by ImageXpress (Molecular Devices, LLC). (Figure 10E) Functional characterization of BMECs by measuring TEER values ​​over several days after plating. (Figure 10F) Schematic of the generation of brain microvascular endothelial cells using an alternative method including plating on charged surfaces without a preconditioning step and ECM. Description of modified medium to induce generation of BMECs on charged surfaces. (FIG. 10G) Day 7 differentiating brain microvascular endothelial cells on different charged surfaces were harvested and purity quantified by staining for the presence of CD31, pGlycoprotein and Glut-1 expression and the absence of pluripotency marker (TRA-181) expression. [Figure 11] Scale-up of HPCs using a 3D differentiation process followed by purification using CD34+ magnetic beads. Schematic of scale-up and sorting of HPCs using CD34 magnetic beads. The efficiency of the sorting process by manual and CliniMAC-mediated separation is shown. The actual purity of HPCs per run (measured as the percentage of CD34 positive cells in the sorted fraction) and the efficiency of the process are outlined. [Figure 12] Media formulation for microglial differentiation. [Figure 13A-13B] (FIG. 13A) Schematic of microglial derivation from CD34+ sorted HPCs. HPCs were placed on microglial differentiation medium MDM. Cultures were fed MDM or 2xMDM every 48 hours. Cultures were split on day 12 of differentiation and 2D differentiation continued until day 23. On day 23, cells were harvested and stained for the presence of microglial culture purity markers. The remaining cultures were cryopreserved. Purity of microglial cultures was quantified before and after cryopreservation. (FIG. 13B) Microglial differentiation medium and microglial differentiation medium are shown. [Figure 14] Microglia purity assessment in the presence of MDM medium before and after cryopreservation. Day 23 differentiated microglia cultures were harvested and stained for the presence of microglia-specific markers. The remaining cells were cryopreserved using a control rate freezer. Cryopreserved cells were thawed and stained for the presence of microglia-specific markers. Cell surface expression of CD45, CD33, TREM2 and CD11b for both sets (FIG. 14A) and intracellular expression of CX3CR1 PU.1, IBA, P2RY12, TREM2 and TMEM119 by flow cytometry. [Figures 15A-15C] Microglia recovery after cryopreservation using manual vs. control rate freezer. HPCs were placed in medium and microglial differentiation was initiated in the presence of MDM. Cells were cryopreserved using the manual freezing protocol or control rate freezer (CRF) on days 20 (Figure 15B), 23 (Figure 15B) and 26 (Figure 15C) of differentiation. Cryopreserved cells were transferred to liquid nitrogen for one week. Cryopreserved microglia were thawed and placed in Microglia Maturation Medium (MMM). Cultures were fed with fresh maturation medium every 48 hours. Cells were harvested on days 3, 5, 7, 10, 12 and 14 after thawing and viable cell recovery was quantified with respect to the initial plating number. [Figure 16] Efficiency of HPCs to microglia conversion. Cryopreserved HPCs were differentiated into microglia in the presence of MDM (N=4). The total viable numbers of input HPCs and output microglia were quantified. Process efficiency was calculated based on the purity and absolute number of TREM2-positive cells present on day 23 of microglial differentiation divided by the absolute number of input live HPCs. [Figures 17A-17C]Purity analysis of microglia cryopreserved manually or in the presence of a control rate freezer at the time of thawing, 3 days post-thawing and 10 days post-thawing, at day 20 (FIG. 17A), day 23 (FIG. 17B) and day 26 (FIG. 17C). Cryopreserved microglia at days 20, 23 and 26 were thawed and plated in Microglia Maturation Medium for 3, 5, 7, 10 and 12 days. Cells were stained for the presence of Pu1, IBA, CX3CR and P2RY12 expression by flow cytometry. [Figure 18A-18B] Manual hemocytometer viable cell counts of microglia cryopreserved either manually or in the presence of a control rate freezer on days 0 (FIG. 18A) and 3 (FIG. 18B) after thawing to set up a phagocytosis assay with S. aureus bioparticles. [Figure 19] Functional characterization of microglia cryopreserved at days 20, 23, and 26 of differentiation using manual or control-rate freezer. Cryopreserved microglia were thawed and plated at 15,000 viable cells / well in 96-well plates in the presence of 200 μl of Microglia Maturation Medium per well. Cells were treated with diluted 1 μg / well of opsonized or non-opsonized pHrodo Red BioParticles (Thermo Fisher #A10010, 2 mg per vial, stored at -20°C). Plates were placed on an IncuCyte and images of phagocytosis were taken at various time points up to 5 days after thawing. Cells cryopreserved using the control-rate freezer method show stronger phagocytosis (due to higher cell viability). Manual cryopreservation revealed a slower / right-shifted rate of phagocytosis in all conditions (due to lower cell viability). [Figure 20]Functional characterization of microglia at day 14 and cryopreserved at days 20, 23, and 26 of differentiation using manual or control-rate freezers, assessed via live imaging on the IncuCyte system. Cryopreserved microglia were thawed and plated in MMM for 3 days. Viable cell counts were determined at the end of the 3 days as described in Figure 18B. 15,000 viable cells were plated in 96-well plates in the presence of 200 μl of Microglia Maturation Medium (MMM) per well. Cells were fed with fresh 50 μl of MMM medium every 48 hours. Cells were treated with diluted 1 μg / well opsonized or non-opsonized pHrodo Red BioParticles (Thermo Fisher #A10010, 2 mg per vial, stored at -20°C). Plates were placed on the IncuCyte and images of phagocytosis were taken at various time points up to 5, 7, and 14 days after thawing. Manual cryopreservation revealed a slower / right-shifted rate of phagocytosis in all conditions (due to lower cell viability). [Figure 21] The phagocytic efficiency ratio was determined by dividing the number of phagocytic red blood cells from the post-thawed samples by the total cell number. [Figure 22] Functional characterization of cryopreserved microglia using pHrodo amyloid beta. Cryopreserved day 23 microglia were thawed and plated at 15,000 viable cells / well in 96-well plates in the presence of 200 μl of Microglia Maturation Medium per well. Cells were treated with pHrodo amyloid beta. A control set included cells with medium without pHrodo amyloid beta. Plates were placed on an IncuCyte and images of phagocytosis were taken at various time points up to 24 hours after thawing. [Figure 23A-23B] Miniaturization of microglial differentiation of HPCs in the absence of ECM in a 96-well format suitable for screening applications. Schematic of microglial differentiation of HPCs (Figure 23A) and the different charged surfaces used in the experiment: ultra-low attachment (ULA), tissue culture (TC) and non-tissue culture (non-TC) vessels (Figure 23B). [Fig. 24A-24B] End-stage purity analysis of microglia on day 23 in the presence of various charged surfaces. Cryopreserved HPCs were plated at a density of 20,000–35,000 viable cells / cm2 on 96-well Primaria plates or ultra-low attachment tissue culture (TC) or non-tissue culture plates (non-TC) in the presence of 200 μl of microglial differentiation medium per well. Cells were fed every 48 h with 50 μl of MDM per well for the next 23 days of differentiation. Cells were harvested with cold PBS on day 23 and total viable cell numbers were quantified using an automated cell counter. Cells were stained for surface expression of CD11b, CD45, CD33, TREM2 and intracellular expression of TREM2, IBA, P2RY12, TMEM119. [Fig. 25A-25B] Cytokines and chemokines released by cryopreserved microglia. Day 23 cryopreserved microglia were thawed in MDM medium and plated at 50,000 cells / well in Primaria 96-well plates. Cells were plated for 3 days before initiating stimulation with 100 ng / ml LPS and 50 ng / ml interferon gamma. Stimulation was performed in triplicate over 24 hours. Supernatants were spun down to remove cells and debris and immediately placed at -20°C. Supernatants were analyzed by multiplex Luminex assay. Averages of multiple WT batches (Figure 25A) and WT, homozygous and heterozygous TREM2 knockout (KO), MECP2 HM and A53T-SNCA engineered lines (Figure 25B) are shown. [Figure 26] Cryopreserved microglia derived from multiple batches of homozygous and heterozygous TREM2 knockout (KO), MECP2 and A53T-SNCA engineered lines were stained for the presence of surface expression of CD11b, CD45, TREM2 and intracellular markers PU.1, IBA-1, CX3CR1, P2RY12 and TMEM119. Engineered iPSC lines revealed comparable expression of TREM2 by flow cytometry. [Figure 27A-27B](FIG. 27A) Release of soluble TREM2 in cryopreserved microglia derived from wild-type (WT), heterozygous (HT) and homozygous (HO) TREM2 KO engineered iPSCs. Soluble TREM2 (sTREM2) levels were quantified using Simple Step ELISA (AbCam) from conditioned media of WT and TREM2 heterozygous and homozygous KO mutants. WT and TREM2 KO microglia were thawed and plated at the same density in maturation medium in 96-well Primaria plates. Spent medium was collected on day 3 post-thaw and day 7 post-thaw. Cultures were half-fed with fresh maturation medium on days 3 and 5 post-thaw. (FIG. 27B) Release of soluble TREM2 in cryopreserved microglia derived from wild-type (WT), MECP2 HM and A53T-SNCA engineered iPSCs. Levels of soluble TREM2 (sTREM2) were quantified using Simple Step ELISA (AbCam) from conditioned medium of WT, MECP2 HM and A53T-SNCA engineered lines. Microglia were thawed and plated at equal density in maturation medium in 96-well Primaria plates. Spent medium was collected on day 3 post-thaw and day 7 post-thaw. Cultures were half-fed with fresh maturation medium on days 3 and 5 post-thaw. [Figure 28] List of media formulations to study survival kinetics of WT, HT and HO TREM2 KO engineered microglia WT, 1185 HT TREM2 KO, 1187 HO TREM2 KO. Microglia were plated at a density of 15,000 live cells per well of a 96-well plate in 250 μl of Microglia Basal Medium containing 32 different variations of cytokine formulations. Kinetics of cell survival were acquired with an IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells in the various media compositions to acquire the number of dead cells over time. Images were acquired every 8 hours and the experiment was continued for 72 hours without intermittent feeding. [Figures 29A-29C]Survival kinetics of WT (Figure 29A), 1185 HT TREM2 KO (Figure 29B), and 1187 HO TREM2 KO (Figure 29C) microglial cells. [Figures 30A-30C] Survival kinetics of WT (Figure 30A), 1185 HT TREM2 KO (Figure 30B), and 1187 HO TREM2 KO (Figure 30C) microglial cells with two cytokines. [Figure 31A-31C] Survival kinetics of WT (Figure 31A), 1185 HT TREM2 KO (Figure 31B), and 1187 HO TREM2 KO (Figure 31C) microglial cells with three cytokines. [Fig. 32A-32C] Survival kinetics of WT (Figure 32A), 1185 HT TREM2 KO (Figure 32B), and 1187 HO TREM2 KO (Figure 32C) microglial cells with four cytokines. [Fig. 33A-33E] WT, 1185 HT TREM2 KO, 1187 HO TREM2 KO microglia were plated at a density of 15,000 viable cells in 96-well plates in 250 μl of Microglia Basal Medium (Figure 33A), or Microglia Basal Medium supplemented with MMM (Figure 33B) or IL-34 (Figure 32C), or Microglia Basal Medium supplemented with IL-34 (Figure 33D), or Microglia Basal Medium supplemented with MCSF (Figure 33D), or Basal Medium supplemented with IL-34 only (Figure 33C), or MSCF or a combination of IL-34 and MCSF (Figure 33E). The kinetics of cell survival was acquired with an IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells in the various media compositions to acquire the number of dead cells over time. Images were acquired every 8 hours and the experiment was continued for 7 days without intermittent feeding. The intensity of NucGreen Dead quantifies dead cells in the culture. [Fig. 34A-34H]Functional characterization of WT, 1185 HT TREM2 KO, 1187 HO TREM2 KO microglia cryopreserved with pHrodo Red-labeled bacterial BioParticles and pHrodo Red amyloid beta on day 23. WT, 1185 HT TREM2 KO, 1187 HO TREM2 KO microglia were plated at a density of 15,000 viable cells / cm2 in 96-well plates for 3 days after thawing in 250 μl of MMM (Figures 34A-B) or MDM base (aka Microglia Basal Medium) supplemented with MSCF alone (Figures 34C-D) or IL-34 (Figures 34E-F) or a combination of IL-34 and MCSF (Figures 34G-H). Cells were treated with diluted 1 μg / well opsonized or non-opsonized pHrodo Bioparticles (Thermo Fisher #A10010, 2 mg per vial, stored at -20°C) (Figure 34A,C,E,G) or pHrodo Amyloid Beta (Figure 34B,D,F,H). Plates were placed on an IncuCyte and images of phagocytosis were taken at various time points up to 30 hours. WT and engineered microglia demonstrated phagocytic function post-thaw. Phagocytosis kinetics and efficiency differed between WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia. [Diagram 35] Purity of microglial cultures in simplified maturation medium. Post-thawing purity of cryopreserved wild-type (WT) microglia at day 23 in the presence of MMM or microglia basal medium supplemented with a combination of two key (IL-34, MSCF) cytokines in the maturation medium. Purity was quantified at days 3, 7 and 14 post-thawing by harvesting cells and purity was determined by flow cytometry by harvesting cells at the end of the differentiation process and staining cells for cell surface and intracellular staining of markers. Cryopreserved microglia retain viability and purity in maturation medium supplemented with MSCF and IL-34. This simplified medium is useful for co-culture applications of cryopreserved microglia with neurons and astrocytes to develop TREM, brain organoids. [Fig. 36A-36B] (FIG. 36A) Schematic showing a screening experiment using cryopreserved microglia. (FIG. 36B) Table of compounds tested in the screen. [Fig. 37A-37D] Results of microglial screening experiments with GW501516 (Figure 37A), leucetin L41 (Figure 37B), piceatannol (Figure 37C) and azeliragon (Figure 37D). [Fig. 38A-38D] Results of microglial screening experiments with J147 (Figure 38A), dibutyryl-cAMP (Figure 38B), isradipine (Figure 38C) and bexarotene (Figure 38D). [Fig. 39A-39E] Results of microglial screening experiments using SB-431542 (Figure 39A), SP600125 (Figure 39B), GW2580 (Figure 39C), PP2 (Figure 39D) and SB239063 (Figure 39E). [Fig. 40A-40D] Results of microglial screening experiments with GW501516 (Figure 40A), leucetin L41 (Figure 40B), piceatannol (Figure 40C) and azeliragon (Figure 40D) with LPS stimulation. [Figures 41A-41E] Results of microglial screening experiments using J147 (Figure 41A), dibutyryl-cAMP (Figure 41B), isradipine (Figure 41C), bexarotene (Figure 41D) and SB-43152 (Figure 41E) with LPS stimulation. [Fig. 42A-42D] Results of microglial screening experiments using SP600125 (Figure 41A), GW2580 (Figure 42B), PP2 (Figure 42C) and SB239063 (Figure 42D) with LPS stimulation. [Diagram 43] Summary of microglial screening experiment results. [Fig. 44A-44G](Figure 44A) Microglia - ratio with ATP / BzATP total trace. (Figure 44B) Microglia - ratio with ATP / BzATP sample trace. (Figure 44C) Response to BzATP in microglia. (Figure 44D) Differential response to ADP in microglia. (Figure 44E) Response with 100 μM BzATP in the presence of P2X7 antagonist AZ11645373. (Figure 44F) Response with 100 μM BzATP in the presence of P2X7 antagonist A438079. (Figure 44G) Dose-dependent response to show functional ADP-dependent response of microglia in the presence of AZD1283, a potent antagonist of the P2Y12 receptor. [Fig. 45A-45B] Release of soluble TREM2 in cryopreserved microglia derived from ANH- and disease-associated microglia was quantified using Simple Step ELISA (AbCam) from conditioned medium collected on day 3 (FIG. 45A) or day 7 (FIG. 45B) post-thaw. ANH- and DAM-associated microglia were thawed and plated at the same density in maturation medium in 96-well Primaria plates. Spent medium was collected on day 3 post-thaw and day 7 post-thaw. Cultures were half-fed with fresh maturation medium on days 3 and 5 post-thaw. [Fig. 46A-46J]Cytokines and chemokines released by cryopreserved microglia derived from ANH and disease-associated microglia from a panel of iPSC donors. Cryopreserved microglia were thawed in MDM medium on day 23 and plated at 50,000 cells / well in Primaria 96-well plates. Cells were plated for 3 days before initiating stimulation with 100 ng / ml LPS or IL-4+dBu-cAMP. Stimulation was performed in triplicate over 24 hours. Supernatants were spun down to remove cells and debris and immediately placed at -20°C. Supernatants were analyzed by multiplex Luminex assay. Release of M1 analytes (Figure 46A), M2 analytes (Figure 46B), interleukins (Figure 46C), chemokines (Figure 46D) and other analytes (Figure 46E) upon stimulation with LPS. Release of M1 analytes (FIG. 46F), M2 analytes (FIG. 46G), interleukins (FIG. 46H), chemokines (FIG. 46I) and other analytes (FIG. 46J) upon stimulation with IL-4+dBu-cAMP. [Fig. 47A-47J]Demonstration of phagocytic function of AHN and disease-associated microglia using pHrodo-labeled Staphylococcus aureus bioparticles and amyloid beta: Cryopreserved AHN and disease-associated microglia were plated at 5,000 cells per well of 384-well poly-D-lysine plates. S. aureus bioparticles were added to each well at 0.5 μg / mL and amyloid beta was added at 1 M / well. The kinetics of phagocytosis of S. aureus bioparticles and amyloid beta were quantified using total red object integrated intensity using an IncuCyte live cell analysis system. (Figure 47A) (ANH, S. aureus), (Figure 47B) (ANH, amyloid beta), (Figure 47C) (R47H vs. ANH, S. aureus), (Figure 47D) (R47H vs. ANH, amyloid beta), (Figure 47E) (CD33 vs. ANH, S. aureus), (Figure 47F) (CD33 vs. ANH, amyloid beta), (Figure 47G) (ABCA7 vs. ANH, S. aureus), (Figure 47H) (ABCA7 vs. ANH, amyloid beta), (Figure 47I) (APOE isoforms vs. ANH, S. aureus), (Figure 47J) (APOE isoforms vs. ANH, amyloid beta). [Fig. 48A-48D] (Figure 48A) Schematic description of the method to generate neural progenitor cells (NPCs) from iPSCs without dual SMAD inhibition. The different steps involved and the composition of the media used are described. (Figure 48B) Overview of the kinetics of NPC emergence across three iPSC lines. Decrease of pluripotency markers and emergence of NPC-specific markers at different days of the differentiation process. Quantification of purity performed by flow cytometry by cell surface and intracellular staining. (Figure 48C) Staining of astrocytes derived from NPCs across multiple passages of culture. Astrocyte purity quantified by flow cytometry by cell surface and intracellular staining. (Figure 48D) Quantification of differentiation of NPCs into neurons and purity of terminal neurons by intracellular flow cytometry. [Figure 49]Overview of surfaces suitable for derivation of iPSC-derived cell lineages. [Fig. 50A-50D] Immunohistochemical staining of 14-day (FIG. 50A) monocultures, (FIG. 50B) bi- and (FIG. 50C) tri-cultures generated from cryopreserved microglia, GABAergic or glutamatergic neurons and astrocytes in the presence of tri-culture medium. 14-day cultures were fixed using PFA and stained with Pan Neuronal Marker (1:1500; Millipore, Catalog: MAB2300), anti-Iba1 (1:500; Wako Chemicals, Catalog: 019-19741), and anti-GFAP (1:500; Abcam, Catalog: ab4674) diluted in blocking buffer. After the primary incubation, cells were stained with secondary antibody solutions, goat anti-mouse IgG1 Alexa Fluor 488 (1:1000; Invitrogen, Catalog: A21121), goat anti-rabbit IgG Alexa Fluor 568 (1:1000; Invitrogen, Catalog: A11011), goat anti-chicken IgY Alexa Fluor 647 (1:1000; Invitrogen, Catalog: A21449), and Hoechst 33342 (1:10,000; Thermo Scientific, Catalog: 62249), diluted in blocking buffer. Plates were washed and imaged with an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices). (Figure 50D) Comparison of medium supplements. [Figure 51] Tripartite culture model of microglia, neurons, and astrocytes. [Figure 52] Schematic diagram of the tripartite culture model setup for studying neuroinflammation. [Figure 53]Comparative analysis of cytokines and chemokines released in mono-, bi- and tri-cultures with GABAergic neurons, astrocytes and microglia derived from ANH, TREMHZ and TREM2HO. Analytes released by reactive and non-responsive astrocytes were quantified in mono-, bi- and tri-cultures. [Figure 54] Comparative analysis of M1 cytokines and chemokines released in mono-, bi- and tri-cultures with GABA neurons, astrocytes and microglia derived from ANH, TREMHZ and TREM2HO. [Figure 55] Comparative analysis of M2 cytokines and chemokines released in mono-, bi- and tri-cultures with GABA neurons, astrocytes and microglia derived from ANH, TREMHZ and TREM2HO. [Figure 56] Comparative analysis of C3 complement released in monocultures, bi- and tri-cultures with GABA neurons, astrocytes and microglia derived from ANH, TREMHZ and TREM2HO. [Fig. 57A-57B] Representative examples of the influence of microglia on neuronal electrophysiology when co-cultured with Gluta neurons and astrocytes and measured by MEA functional assay. (FIG. 57A) Gluta neurons and astrocytes on an MEA. (FIG. 57B) Addition of microglia to a model neural network. [Figure 58] Representative images of mono- and tripartite-cultured microglia (AHN and TREM2HZKO) phagocytosis of pHrodo Red-labeled amyloid beta fibrils obtained from corresponding kinetic phagocytosis assays with TREM2HZKO microglia. [Figure 59] Phase contrast image of a 3D tripartite culture containing cryopreserved iPSC-derived Gluta neurons, astrocytes, and microglia with cellular proportions of all three cell types. [Figure 60] Comparative analysis of calcium transients from cryopreserved 3D cultures of Gluta neurons in the presence and absence of microglia and astrocytes. [Figure 61] Schematic representation of the different steps for setting up a sandwich co-culture with iPSC-derived cryopreserved BMECs, pericytes and astrocytes. [Figure 62] TEER function of cryopreserved BMEC. iPSC-derived BMEC were cryopreserved, thawed, and seeded on FN / ColIV-coated Corning transwell inserts at 1.3×106 cells / cm2 in BMEC medium. TEER measurements were collected from day 3 to day 8 (following the day numbering scheme in sandwich transwell co-culture described in Figure 61). [Figure 63] TEER function of cryopreserved BMECs and pericytes. iPSC-cryopreserved iPSC-derived pericytes were seeded on the basolateral membrane at three different densities (666k / cm2, 333k / cm2, 500k / cm2) on day 0. iPSC-derived BMECs were cryopreserved, thawed, and seeded on the apical transwell membrane at 1.3×106 cells / cm2 or 1.0×106 cells / cm2 in BMEC medium on day 1. TEER measurements were collected from day 3 to day 8 (following the day numbering scheme in sandwich transwell co-culture described in Figure 61). [Figure 64] TEER function of cryopreserved BMECs and astrocytes. Cryopreserved astrocytes were seeded on the basolateral membrane at three different densities (666k / cm2, 333k / cm2, 500k / cm2) on day 0. iPSC-derived BMECs were cryopreserved, thawed, and seeded on the apical transwell membrane at 1.3×106 cells / cm2 or 1.0×106 cells / cm2 in BMEC medium on day 1. TEER measurements were collected from day 3 to day 8 (following the day numbering scheme in sandwich transwell co-culture described in Figure 61). [Figure 65]TEER function of cryopreserved BMEC, pericytes and astrocytes: iPSC-derived pericytes and iCell astrocytes were seeded on the basolateral membrane at three different densities (666k / cm2, 333k / cm2, 500k / cm2) on day 0. iPSC-derived BMEC were thawed and seeded on the apical transwell membrane at 1.3x106 cells / cm2 or 1.0x106 cells / cm2 in BMEC medium on day 1. TEER measurements were collected from day 3 to day 8 (following the day numbering scheme in sandwich transwell co-culture described in Figure 61). [Figure 66] Schematic diagram of an exemplary tripartite culture protocol for the culture of neurons, astrocytes, and microglia. [Figure 67] Partial loss of function of TREM2 reduces cholesterol and fatty acid biosynthesis. RNAseq analysis of isogenic edited heterozygous and homozygous TREM2KOs identified specific phenotypes resulting from partial loss that were not captured in homozygous TREM2 loss-of-function studies. As shown, partial loss of TREM2 function enhanced downregulation of SREBF2, a master regulator of cholesterol biosynthesis, resulting in reduced cholesterol and fatty acid synthomes with concomitant increased lipid efflux. [Figure 68] The Gas6 / Axl axis depends on TREM2 in a dose-dependent manner. [Figure 69] Siglec11 expression is affected by partial loss of TREM2 function. [Fig. 70A-70E] (Figure 70A) TREM2 regulates GRN expression in microglia. (Figure 70B) TREM2 regulates the expression of other ion channels in microglia. (Figure 70C) TREM2 regulates the expression of ligand-gated ion channels in microglia. (Figure 70D) TREM2 downregulates the expression of voltage-gated ion channels in microglia. (Figure 70E) TREM2 upregulates the expression of voltage-gated ion channels in microglia. [Fig. 71A-71B](FIG. 71A) TREM2 downregulates the expression of GPCRs in microglia. (FIG. 71B) TREM2 upregulates the expression of GPCRs in microglia. [Fig. 72A-72B] (FIG. 72A) TREM2 downregulates the expression of transport proteins in microglia. (FIG. 72B) TREM2 upregulates the expression of transport proteins in microglia. [Fig. 73A-73B] (FIG. 73A) TREM2 downregulates the expression of catalytic receptors in microglia. (FIG. 73B) TREM2 upregulates the expression of catalytic receptors in microglia. [Fig. 74A-74C] (Figures 74A-74B) Effect of TREM2 on controlling cellular enzyme turnover and thus affecting metabolism and function. TREM2 downregulates enzyme expression in microglia. (Figure 74C) TREM2 upregulates enzyme expression in microglia. [Figure 75] TREM2 upregulates the expression of nuclear hormone receptors in microglia. [Fig. 76A-76B] (FIG. 76A) TREM2 downregulates the expression of other proteins expressed in microglia. (FIG. 76B) TREM2 upregulates the expression of other proteins expressed in microglia. [Fig. 77A-77B] Comparison of RNAseq profiles of microglia carrying WT TREM2 with isogenic microglia carrying (Figure 77A) TREM2 HZ and (Figure 77B) TREM2 HO reveals significant downregulation of multiple transcripts. [Figure 78]Reserve respiratory capacity of TREM2 iCell microglia in monoculture. iCell microglia (MGL) were thawed and matured for 3 days before seeding for the Agilent Seahorse XFe96 Mito stress test assay. Microglia were seeded at 30,000 cells per well onto poly(ethyleneimine) and Geltrex-coated 96-well Seahorse XF Pro M cell culture plates in maintenance medium and cultured overnight. On the day of the assay, the medium was replaced with assay medium containing Seahorse XF DMEM, glucose (10 mM), sodium pyruvate (1 mM), and L-glutamic acid (2 mM). Plates were then incubated for 1 hour in a 37°C incubator with ambient CO2. Stock compounds from the Agilent Cell Mito stress test kit at 10x final concentrations of oligomycin A (10 uM), FCCP (30 uM), and rotenone / antimycin A (5 uM) were prepared in assay medium. The injection port of the XF Pro sensor cartridge was loaded with oligomycin A, FCCP, and rotenone / antimycin A according to the manufacturer's instructions. Samples were analyzed using an Agilent Seahorse XF Pro Analyzer using the Wave Controller software package. Cell numbers were determined post-assay using Hoechst nuclear dye (1:1000) and captured using an ImageXpress MetaXpress High Content Imager. Data are normalized as oxygen consumption rate (OCR) per cell. Wild-type microglia consistently responded to metabolic respiratory investigation compounds with higher spare respiratory capacity compared to TREM2 heterozygous and homozygous cell lines. [Figures 79A-79C] (Figure 79A) iCell microglia misplay uniform marker expression across the iPSC background. (Figure 79B) TREM2 knockouts show reduced soluble TREM2. (Figure 79C) TREM2 knockouts show reduced phagocytic function. [Fig. 80A-80B]Gene expression mutations in TREM2 R47H. (Figure 80A) Pathway analysis revealed upregulation of postsynaptic membrane receptor levels and downregulation of many RNA translation pathways. (Figure 80B) TREM2 R47H and TREM2 HZ reduced expression of key genes in the cholesterol biosynthesis pathway. Heterozygous mutations in TREM2 lead to increased risk of AD, whereas homozygous mutations cause a neurological condition known as Nasu-Hakola disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Description of exemplary embodiments Interactions between cells of the central nervous system, such as neurons, astrocytes, and microglia, as well as brain microvascular endothelial cells (BMECs) and pericytes, influence the neuroinflammatory response to CNS injury. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation. However, to better understand the impact of cellular crosstalk on neuroinflammation, multicellular culture models are needed. Thus, in certain embodiments, the present disclosure provides a multi-cellular in vitro culture model for the study of neuroinflammation, such as to identify novel targets, biomarkers, and therapeutics for the diagnosis, prognosis, and treatment of neurodegenerative diseases. In one embodiment, a tripartite culture of microglia, astrocytes, and neurons is provided. In another embodiment, a blood-brain barrier model is provided that includes BMECs, astrocytes, and pericytes. Additionally, assays are provided herein for studying neuroinflammation using the cell culture models of the present invention. Outcomes of the tripartite culture system or BBB model can be survival, synaptic pruning, microglial function due to Aβ aggregation, p-tau formation, neuronal MEA function, analytes released into the medium to trigger the neuroinflammatory cascade, and crosstalk between all three cell types. GWAS in AD have identified TREM2 as a key regulator of AD risk. Heterozygous mutations in TREM2 lead to increased AD risk, whereas homozygous mutations cause a neurological condition known as Nasu-Hakola disease. Thus, although the CNS is affected in both cases, the pathobiology and clinical manifestations of heterozygous and homozygous mutations are different. Recently, iPSC TREM2 KO microglia have been generated to study the effect of loss of TREM2 on microglial function and its impact on AD risk (McQuade et al., 2020; Reich et al., 2021). However, because AD results from partial loss of function, rather than complete loss of TREM2, it is unclear whether these microglial models can adequately interrogate the role of TREM2 in AD. Therefore, to better understand the role of TREM2 mutations on AD risk, we generated heterozygous TREM2 loss-of-function microglia. Validation of partial loss of TREM2 microglia identified pathways unique to HetTREM2 KO and supported the idea that heterozygous TREM2 KO microglia better model the phenotype resulting from inheritance of TREM2 mutations that lead to increased AD risk. Thus, further embodiments provide an iPSC-derived "brain-in-a-dish" tripartite culture model to identify the role of TREM2 in regulating neuroinflammatory cascades involved in neurodegeneration, as well as the role of TREM2 in microglial phagocytosis. Furthermore, heterozygous TREM2 knockout microglia can be used to identify AD molecular biomarkers. Using heterozygous TREM2 knockout microglia, alterations in the metabolome, lipidome, and secretome can be identified, including biomolecules found in microglia-derived extracellular vesicles released by microglia that reflect pathway changes identified in this study that serve as biomarkers of AD progression and phenotypic recovery following drug treatment. For example, this study identified perturbation of the cholesterol biosynthesis pathway by downregulation of TREM2, dose-dependent reduction of the Gas6-Axl axis via TREM2 perturbation, partial loss of Siglec-11 via TREM2HZ that could overcome the neuroprotective effect of CD33, upregulation of ligand-gated ion channels in microglia, changes in the expression of microglial voltage-gated ion channels, GPCRs, catalytic receptors, enzymes, and nuclear receptors, and a direct link between TREM2 downregulation associated with COMT downregulation and NRXN2 and SST expression. In further particular embodiments, the present disclosure provides methods for producing cells of multiple lineages, such as endothelial cells, mesenchymal stem cells (MSCs) and hematopoietic progenitor cells (HPCs) from induced pluripotent stem cells (iPSCs). In general, the methods include differentiating iPSCs into cells of various lineages by using a charged surface. Specifically, the differentiation methods can be in the absence of extracellular matrix (ECM) proteins, allowing for self-purification by passaging, such as for the production of MSCs and endothelial cells. In further embodiments, methods are provided for the differentiation of endothelial cells into brain microvascular endothelial cells (BMECs) or lymphatic endothelial cells, differentiation of MSCs into pericytes, and differentiation of HPCs into microglia. The process allows for efficient generation of endothelial cells and MSCs without purification such as MACS purification or the use of ECM proteins. The process can be adapted to comply with Good Manufacturing Practices (GMP). Endothelial cells constitute a network of interconnected cells in the human body that line blood vessels, lymphatic vessels and form capillaries. Endothelial cells regulate the flow of nutrients, make and respond to a variety of biologically active molecules, offering potential applications in the tool space for screening compounds and drugs for vascular toxicity, vascular permeability and therapeutic applications including treatment of tissue ischemia and bioengineering of grafts. There are many protocols used to induce endothelial cells. Nearly all processes require magnetic activated cell sorting (MAC) separation using CD31 microbeads to generate pure cultures of endothelial cells.

[0071] In certain embodiments, the present disclosure provides a method for generating a pure population of endothelial cells from iPSCs, such as episomally reprogrammed iPSCs, by a two-step process. iPSC cells are converted to hematopoietic progenitor cells (HPCs) on a positively charged amine surface, followed by further expansion and subsequent purification of endothelial cells in the presence of a negatively charged carboxyl surface. Endothelial cells can be derived from hemogenic endothelial cells without a MAC purification step. The cells express CD31 / CD144 / CD105 with high purity, can be expanded to maintain purity, and can be cryopreserved at early and late passages.

[0072] Mesenchymal stem cells (MSCs) isolated from adult human tissues can proliferate in vitro and maintain their pluripotency, making them an attractive cell source for regenerative medicine. However, current preparation methods limit their availability and ability to self-renew. iPSCs currently offer an alternative cell source similar to MSCs. Thus, certain embodiments of the present disclosure provide a method for differentiating MSCs from iPSCs, such as episomally reprogrammed iPSCs, by initiating mesodermal differentiation on a positively charged amine surface using GMP-compatible conditions. MSCs obtained by this process express all purity markers of the MSC lineage and exhibit self-renewal and pluripotency. These cells can be scaled up for clinical applications.

[0073] In a further embodiment, the present disclosure provides a method for differentiating MSCs into pericytes (PCs). Pericytes, also known as mural cells, are generally understood to line blood microvessels (i.e., capillaries, arterioles, and venules) and play an organizational or structural role in angiogenesis. Multiple criteria are used to distinguish between immature and mature pericytes, including location, morphology, gene or protein expression patterns, and perivascular density. In general, pericytes obtained according to the methods provided herein can be identified based on the expression of known pericyte molecular markers, such as, but not limited to, PDGFRβ, desmin (DES), CD13 (ANPEP; alanyl (membrane) aminopeptidase), α-SMA, RGS5 (regulator of G-protein signaling 5), NG2 (also known as CSPG4; chondroitin sulfate proteoglycan 4), CD248 (endosialin), ANG-1, CD146, CD44, CD90, and CD13.

[0074] Microglia are innate immune cells of the central nervous system that play important roles in brain development, homeostasis, and immune regulation. They are difficult to obtain from human fetal and primary tissues. Thus, further embodiments of the present disclosure provide methods for the generation, characterization, and cryopreservation of human iPSC-derived microglia (iMGL) from HPCs, such as episomally reprogrammed iCell HPCs, under defined conditions. Cryopreserved iMGL maintain purity, secrete immunomodulatory cytokines, and phagocytose pHrodo Red-labeled bacterial BioParticles and amyloid beta aggregates. The ability to produce essentially unlimited quantities of iMGL holds great promise for accelerating human neuroscience exploration into the role of microglia in normal and disease states.

[0075] Further provided herein are microglia with disruption of TREM2, MeCP2 and / or SCNA. These microglia derived from patient-derived iPSCs provide an in vitro tool to understand the complex interactions between human microglia, neurons and astrocytes in 2D or 3D organoid systems and create more accurate models to mimic neurodegenerative diseases.

[0076] Also provided herein are methods for differentiating iPSCs into neural progenitor cells (NPCs) without inhibiting SMAD signaling. These NPCs can be co-cultured with iPSC-derived microglia to generate long-term co-culture assays that mimic human brain development and the complex cell-cell interactions between neural lineages, microglia, endothelial cells, pericytes, and astrocytes in culture dishes from normal and / or disease-specific iPSC cells. iPSCs can be maintained under hypoxic conditions prior to the initiation of differentiation to generate NPCs. To initiate differentiation of neural precursors, iPSCs can be seeded in the presence of ROCK inhibitor or blebbistatin on ECM-coated surfaces. Cells can be placed in medium for the next 48 hours in the absence of ROCK inhibitor. Cells are then preconditioned in DMEMF12 medium supplemented with GSK3 inhibitor for 72 hours, with daily medium changes under normoxic conditions. At the end of the preconditioning step, the cells can be harvested and replated in 2D format on ECM-coated plates or as 3D aggregates using ultra-low attachment plates or spinner flasks in the presence of ROCK inhibitor or blebbistatin. The cultures can be fed every other day with E6 medium supplemented with N2 for the next 8 days under normoxic conditions to produce NPCs. The different steps involved in the generation of NPCs are outlined in Figure 45A.

[0077] The cells produced by the method of the present invention can be used in disease modeling, drug discovery and regenerative medicine.Also provided herein is a method of using the cells (e.g., MSCs, endothelial cells, neural progenitor cells and pericytes) for the generation of brain organoids or blood-brain barrier (BBB) ​​models.

[0078] I. Definition As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0079] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer only to alternatives or where the alternatives are mutually exclusive, but the present disclosure supports a definition that refers only to alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0080] The term "essentially" should be understood to mean that a method or composition includes only certain steps or materials that do not materially affect the basic and novel characteristics of those methods and compositions.

[0081] As used herein, a composition or medium that is "substantially free" of a particular substance or ingredient contains less than 30%, less than 20%, less than 15%, more preferably less than 10%, even more preferably less than 5% or most preferably less than 1% of the substance or ingredient.

[0082] As used herein, the terms "substantially" or "approximately" may be applied to modify quantitative comparisons, values, measurements or other expressions that may vary to an acceptable degree without resulting in a change in the basic function to which they relate.

[0083] The term "about" generally means within the standard deviation of the stated value, as determined using standard analytical techniques to measure the stated value. The term may also be used to refer to plus or minus 5% of the stated value.

[0084] As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts.Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%.Most preferred are compositions in which the amount of the particular component cannot be detected by standard analytical methods.

[0085] "Feeder-free" or "feeder-independent" is used herein to refer to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF, analogs or mimetics thereof) instead of a feeder cell layer. Thus, "feeder-free" or feeder-independent culture systems and media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state. In some cases, feeder-free cultures utilize animal-based matrices (e.g., MATRIGEL™) or are cultured on substrates such as fibronectin, collagen, or vitronectin. These approaches allow human stem cells to be kept essentially undifferentiated without the need for a "feeder layer" of mouse fibroblasts.

[0086] A "feeder layer" is defined herein as a coating layer of cells, such as the bottom of a culture dish. Feeder cells can release nutrients into the medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.

[0087] The term "defined" or "fully defined" when used in reference to a medium, extracellular matrix, or culture condition refers to a medium, extracellular matrix, or culture condition in which the chemical composition and amount of nearly all components are known. For example, a defined medium does not contain undefined factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Generally, a defined medium contains a basal medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, which contains amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources) supplemented with recombinant albumin, lipids of known composition, and recombinant insulin. An example of a fully defined medium is Essential 8™ medium.

[0088] For media, extracellular matrices or culture systems used with human cells, the term "xeno-free (XF)" refers to the condition in which the materials used are not derived from non-human animals.

[0089] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or exhibiting symptoms or symptomology of a disease (e.g., arresting further development of the symptoms and / or symptomology), (2) ameliorating a disease in a subject or patient experiencing or exhibiting symptoms or symptomology of a disease (e.g., reversing the symptoms and / or symptomology), and / or (3) causing a measurable reduction in a disease in a subject or patient experiencing or exhibiting symptoms or symptomology of a disease.

[0090] "Prophylactic treatment" includes (1) reducing or alleviating the risk of developing a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the symptoms or symptomatology of the disease, and / or (2) delaying the onset of symptoms or symptomatology of the disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the symptoms or symptomatology of the disease.

[0091] As used herein, the term "patient" or "subject" refers to a living mammal, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0092] The term "effective" as that term is used in the specification and / or claims means adequate to achieve a desired, expected or intended result. An "effective amount", "therapeutically effective amount" or "pharmacologically effective amount" when used in connection with treating a patient or subject with a compound means an amount of a compound that, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to affect the treatment or prevention of such disease.

[0093] Generally, as used herein, "pharmacologically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0094] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming somatic cells through the expression or induction of expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be generated using fetal, postnatal, neonatal, juvenile or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, Klf4, Nanog and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0095] The term "extracellular matrix protein" refers to molecules that provide structural and biochemical support to surrounding cells. Extracellular matrix proteins can be recombinant and also refer to fragments or peptides thereof. Examples include collagen and heparin sulfate.

[0096] "Three-dimensional (3D) culture" refers to an artificially created environment in which biological cells can grow or interact with their surroundings in all three dimensions. 3D cultures can be grown in a variety of cell culture vessels, such as bioreactors, small capsules in which cells can grow into spheroids, or non-adherent culture plates. In certain embodiments, the 3D culture is scaffold-free. In contrast, "two-dimensional (2-D)" culture refers to cell culture, such as a monolayer on an adherent surface.

[0097] As used herein, the "disruption" of a gene refers to the elimination or reduction of expression of one or more gene products encoded by a gene of interest in a cell, compared to the expression level of the gene product in the absence of the disruption. Exemplary gene products include the mRNA and protein products encoded by the gene. The disruption may be temporary or reversible, or may be permanent. In some cases, the disruption is the disruption of a functional or full-length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some embodiments herein, the activity or function of a gene is disrupted, as opposed to expression. Gene disruption is generally induced by artificial methods, i.e., the addition or introduction of a compound, molecule, complex or composition, and / or by disruption of the nucleic acid of the gene or the nucleic acid associated with the gene, such as at the DNA level. Exemplary methods of gene disruption include gene disruption techniques, such as gene silencing, knockdown, knockout, and / or gene editing. Examples include antisense technologies such as RNAi, siRNA, shRNA and / or ribozymes, which generally result in a transient reduction in expression and gene editing techniques that result in the inactivation or disruption of a targeted gene, for example by inducing truncation and / or homologous recombination. Examples include insertion, mutation and deletion. Disruption typically results in the suppression and / or complete absence of expression of the normal or "wild type" product encoded by the gene. Examples of such gene disruptions are insertions, frameshift and missense mutations, deletions, knock-ins and knock-outs of genes or parts of genes, including deletion of the entire gene. Such disruptions can occur in coding regions, for example in one or more exons, resulting in the inability to produce a full-length product, a functional product or any product, such as by the insertion of a stop codon. Such disruptions can also occur by disruption of promoters or enhancers or other regions that affect activation of transcription, to prevent transcription of the gene. Gene disruption includes gene targeting, including inactivation of a targeted gene by homologous recombination.

[0098] II.iPSC differentiation method A. HPC iPSCs can be differentiated into HPCs by methods known in the art, such as those described in U.S. Patent No. 8,372,642, which is incorporated herein by reference. In one method, a combination of BMP4, VEGF, Flt3 ligand, IL-3 and GM-CSF can be used to promote hematopoietic differentiation. In certain embodiments, pluripotent cells can be differentiated into HPCs and hematopoietic cells by sequential exposure of the cell culture to a first medium that prepares the iPSCs for differentiation, a second medium that contains BMP4, VEGF and FGF, followed by culture in a third medium that contains Flt3 ligand, SCF, TPO, IL-3 and IL-6. The second defined medium can also contain heparin. Additionally, inclusion of FGF-2 (50 ng / ml) in the medium containing BMP4 and VEGF can increase the efficiency of generating hematopoietic progenitor cells from pluripotent cells. Furthermore, inclusion of a glycogen synthase kinase 3 (GSK3) inhibitor (e.g., CHIR99021, BIO, SB-216763) in the first defined medium can further enhance the production of HPCs.

[0099] In general, differentiation of pluripotent cells into hematopoietic progenitor cells can be performed using defined or undefined conditions. It will be understood that in embodiments where the resulting cells are intended to be administered to a human subject, defined conditions are generally preferred. Hematopoietic stem cells can be derived from pluripotent stem cells under defined conditions (e.g., using TeSR medium), and hematopoietic cells can be generated from embryoid bodies derived from pluripotent cells. In other embodiments, pluripotent cells can be co-cultured on OP9 cells or mouse embryonic fibroblasts and subsequently differentiated.

[0100] Pluripotent cells may be able to form embryoid bodies or aggregates as part of the differentiation process. The formation of "embryoid bodies" (EBs) or clusters of growing cells to induce differentiation generally involves in vitro aggregation of human pluripotent stem cells into EBs, allowing spontaneous and random differentiation of human pluripotent stem cells into multiple tissue types representing endodermal, ectodermal and mesodermal origins. Thus, three-dimensional EBs can be used to produce some hematopoietic and endothelial cells.

[0101] To promote the formation of aggregates, cells were transferred to low-attachment plates and cultured in 75% IMDM (Gibco), 0.05% N2, and 1% B-27 supplemented with 25% Ham's Modified F12 (Cellgro) without RA, 200 mM l -glutamine, 0.05 mg / ml ascorbic acid-2-phosphate magnesium salt (Asc 2-P) (WAKO), and 4.5 × 10 -4The wells may be incubated overnight in serum-free differentiation (SFD) medium consisting of MTG. The next day, cells may be collected from each well and centrifuged. The cells may then be resuspended in "EB differentiation medium" consisting of SFD basal medium supplemented with approximately 50 ng / ml bone morphogenetic protein (BMP4), approximately 50 ng / ml vascular endothelial growth factor (VEGF) and 50 ng / ml zb FGF for the first four days of differentiation. The cells are half-fed every 48 hours. On the fifth day of differentiation, the medium is replaced with a second medium consisting of SFD medium supplemented with 50 ng / ml stem cell factor (SCF), approximately 50 ng / ml Flt-3 ligand (Flt-3L), 50 ng / ml interleukin-6 (IL-6), 50 ng / ml interleukin-3 (IL-3), 50 ng / ml thrombopoietin (TPO). The cells are half-fed with fresh differentiation medium every 48 hours. Medium exchange is performed by spinning down the differentiation culture at 300g for 5 minutes, aspirating half the volume from the differentiation culture, and replenishing with fresh medium. In certain embodiments, the EB differentiation medium may contain about BMP4 (e.g., about 50ng / ml), VEGF (e.g., about 50ng / ml), and optionally FGF-2 (e.g., about 25-75ng / ml or about 50ng / ml). The supernatant may be aspirated and replaced with fresh differentiation medium. Alternatively, the cells may be fed half of the fresh medium every two days. Cells may be harvested at various time points during the differentiation process.

[0102] HPCs can be cultured from pluripotent stem cells using defined media. + Methods for differentiation into stem cells are described, for example, in U.S. Patent Application Publication No. 12 / 715,136, which is incorporated by reference in its entirety. It is anticipated that these methods may be used in the present disclosure.

[0103] For example, using a defined medium, +The pluripotent cells may be cultured in a first defined medium containing the growth factors BMP4, VEGF, Flt3 Ligand, IL-3 and / or GMCSF. The pluripotent cells may be cultured in a second medium containing either (Flt3 Ligand, IL-3 and GMCSF) or (Flt3 Ligand, IL-3, IL-6 and TPO). The first and second medium may also contain one or more of SCF, IL-6, G-CSF, EPO, FGF-2 and / or TPO. Substantially hypoxic conditions (e.g., less than 20% O2) may further promote hematopoietic or endothelial differentiation.

[0104] Cells may be subsequently individualized via mechanical or enzymatic means (e.g., using trypsin or TrypLE™). A ROCK inhibitor (e.g., H1152 or Y-27632) may also be included in the medium. It is anticipated that these approaches may be automated, for example, using robotic automation.

[0105] In certain embodiments, substantially hypoxic conditions may be used to promote differentiation of pluripotent cells into hematopoietic progenitor cells. As will be appreciated by those skilled in the art, atmospheric oxygen content less than about 20.8% would be considered hypoxic. Human cells in culture can be grown in atmospheric conditions with reduced oxygen content compared to ambient air. This relative hypoxia may be achieved by reducing the atmospheric oxygen exposed to the culture medium. Embryonic cells typically develop in vivo under conditions of reduced oxygen, generally about 1% to about 6% atmospheric oxygen and ambient levels of carbon dioxide. Without wishing to be bound by theory, it is anticipated that hypoxic conditions may mimic aspects of certain embryonic developmental conditions. As shown in the examples below, in certain embodiments, hypoxic conditions may be used to promote further differentiation of induced pluripotent cells into more differentiated cell types, such as HPCs.

[0106] The following hypoxic conditions may be used to promote differentiation of pluripotent cells into hematopoietic progenitor cells. In certain embodiments, atmospheric oxygen content of less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, about 5%, about 4%, about 3%, about 2%, or about 1% may be used to promote differentiation into hematopoietic progenitor cells. In certain embodiments, the hypoxic atmosphere comprises about 5% oxygen gas.

[0107] Regardless of the particular medium used for the expansion of a given hematopoietic progenitor cell, the medium used is preferably supplemented with at least one cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more usually 10 ng / mL to 100 ng / mL. Suitable cytokines include, but are not limited to, c-kit ligand (KL) (also called hematopoietic stem cell factor (StI), mast cell growth factor (MGF) and stem cell factor (SCF)), IL-6, G-CSF, IL-3, GM-CSF, IL-1α, IL-11 MIP-1α, LIF, c-mpl ligand / TPO and flk2 / flk3 ligand (Flt2L or Flt3L). In particular, the culture includes at least one of SCF, Flt3L and TPO. More particularly, the culture includes SCF, Flt3L and TPO.

[0108] In one embodiment, the cytokines are included in the medium and replenished by medium perfusion. Alternatively, when using a bioreactor system, the cytokines can be added separately as a concentrated solution through a separate inlet port without medium perfusion. When cytokines are added without perfusion, they are typically added as a 10-100x solution in an amount equal to 1 / 10-1 / 100 of the volume of the bioreactor, with fresh cytokines added approximately every 2-4 days. Furthermore, fresh concentrated cytokines can also be added separately in addition to the cytokines in the perfusion medium.

[0109] Exemplary HPC Differentiation Methods Differentiation of 2D HPCs: iPSCs maintained on MATRIGEL™ or vitronectin in the presence of E8 can adapt to hypoxia for at least 5-10 passages. Cells are split from subconfluent iPSCs and plated on amine culture dishes at a density of 250,000 cells / well in the presence of serum-free defined (SFD) medium supplemented with 5uM blebbistatin. 24 hours after plating, SFD medium supplemented with 50ng / ml BMP4, VEGF and FGF2 is added to the cultures. The next day, fresh medium is replaced to remove blebbistatin. On day 5 of the differentiation process, cells are placed in medium containing 50ng / ml Flt-3 ligand, SCF, TP0, IL3 and IL6 with 5U / ml heparin. Cells are fed every 48 hours throughout the differentiation process. The entire process is carried out on charged amine plates under hypoxic conditions. HPCs are quantified by the presence of CD43 / CD34 cells and CFUs.

[0110] Differentiation of 3D HPCs: Cells were split from subconfluent iPSCs and plated in spinner flasks at a density of 250,000–500,000 cells per ml in the presence of serum-free defined (SFD) medium supplemented with 5 μM blebbistatin or 1 μM H1152. 24 hours after plating, the SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 was replaced. On day 5 of the differentiation process, cells were placed in medium containing 50 ng / ml Flt-3 ligand, SCF, TP0, IL3, and IL6, along with 5–10 U / ml heparin. Cells were fed every 48 hours throughout the differentiation process. The entire process was performed under hypoxic conditions. HPCs were quantified by the presence of CD43 / CD34. HPCs were MACS sorted using CD34 beads.

[0111] B. Gene Disruption In certain aspects, the expression, activity or function of the TREM2, MeCP2 and / or SCNA genes is disrupted in cells such as PSCs (e.g., ESCs or iPSCs). In some embodiments, gene disruption is carried out by causing disruption of the gene, such as knockout, insertion, frameshift mutation such as missense or biallelic frameshift mutation, deletion and / or knock-in of all or part of the gene, such as one or more exons or parts thereof. For example, disruption can be performed by sequence-specific or targeted nucleases, including DNA-binding targeted nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases such as CRISPR-associated nucleases (Cas) that are specifically designed to target the sequence of a gene or part thereof.

[0112] In some embodiments, disruption of gene expression, activity and / or function is carried out by disrupting the gene. In some embodiments, the gene is disrupted such that its expression is reduced by at least or about 20, 30 or 40%, typically at least or about 50, 60, 70, 80, 90 or 95%, compared to its expression in the absence of the gene disruption or in the absence of components introduced to effect the disruption.

[0113] In some embodiments, the disruption is transient or reversible, such that expression of the gene is subsequently restored, in other embodiments, the disruption is not reversible or transient, e.g., permanent.

[0114] In some embodiments, gene disruption is typically performed by inducing one or more double-strand breaks and / or one or more single-strand breaks in a gene in a targeted manner.In some embodiments, double-strand or single-strand breaks are performed by a nuclease, for example an endonuclease, such as a gene-targeting nuclease.In some aspects, the break is induced in the coding region of a gene, for example an exon.For example, in some embodiments, the induction occurs near the N-terminal part of the coding region, for example the first exon, the second exon or a subsequent exon.

[0115] In some embodiments, double-stranded or single-stranded breaks undergo repair through cellular repair processes, such as by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the repair process is error-prone, resulting in disruption of the gene, such as a frameshift mutation, e.g., a biallelic frameshift mutation, which may result in a complete knockout of the gene. For example, in some embodiments, the disruption includes inducing a deletion, mutation, and / or insertion. In some embodiments, the disruption results in the presence of a premature stop codon. In some embodiments, the insertion, deletion, translocation, frameshift mutation, and / or the presence of a premature stop codon results in disruption of the expression, activity, and / or function of the gene.

[0116] In some embodiments, gene disruption is achieved using antisense technology, such as RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA) and / or ribozymes, to selectively suppress or block the expression of a gene. siRNA technology is RNAi using a double-stranded RNA molecule that has a sequence that is homologous to the nucleotide sequence of the mRNA transcribed from the gene and a sequence that is complementary to the nucleotide sequence. siRNAs can generally be siRNAs that contain multiple RNA molecules that are homologous / complementary to one region of the mRNA transcribed from the gene or homologous / complementary to different regions. In some embodiments, siRNAs are included in polycistronic constructs. In certain embodiments, siRNAs suppress both wild-type and mutant protein translation from endogenous mRNA.

[0117] In some embodiments, the disruption is achieved using a DNA targeting molecule, such as a DNA binding protein or a DNA binding nucleic acid, or a complex, compound, or composition comprising the same, that specifically binds or hybridizes to the gene. In some embodiments, the DNA targeting molecule comprises a DNA binding domain, such as a zinc finger protein (ZFP) DNA binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA binding domain, or a DNA binding domain from a meganuclease. Zinc finger, TALE, and CRISPR system binding domains can be engineered to bind to a given nucleotide sequence, for example, through engineering (changing one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Engineered DNA binding proteins (zinc finger or TALE) are non-naturally occurring proteins. Rational criteria for design include the application of substitution rules and computerized algorithms to process information in databases that store information and binding data of existing ZFP and / or TALE designs. See, e.g., U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261. See also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496, and U.S. Patent Application Publication No. 2011 / 0301073.

[0118] In some embodiments, the DNA targeting molecule, complex or combination comprises a DNA binding molecule and one or more additional domains, such as an effector domain for promoting gene suppression or destruction.For example, in some embodiments, gene destruction is carried out by a fusion protein comprising a DNA binding protein and a heterologous regulatory domain or a functional fragment thereof.In some embodiments, the domain comprises a transcription factor domain, such as activator, repressor, coactivator, corepressor, silencer, oncogene, DNA repair enzymes and their associated factors and modifiers, DNA rearrangement enzymes and their associated factors and modifiers, chromatin-associated proteins and their modifiers, such as kinases, acetylases and deacetylases, and DNA modifying enzymes, such as methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases and their associated factors and modifiers. For details on the fusion of DNA binding domain and nuclease cleavage domain, see, for example, US Patent Application Publication Nos. 2005 / 0064474; 2006 / 0188987 and 2007 / 0218528, which are incorporated herein by reference in their entirety.In some embodiments, the additional domain is a nuclease domain.Thus, in some embodiments, gene disruption is facilitated by gene or genome editing using engineered proteins such as nucleases and nuclease-containing complexes or fusion proteins, which are composed of sequence-specific DNA binding domains fused or complexed with non-specific DNA cleavage molecules such as nucleases.

[0119] In some embodiments, these targeted chimeric nucleases or nuclease-containing complexes induce targeted double-strand or single-strand breaks and perform precise genetic modification by stimulating cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR). In some embodiments, the nuclease is an endonucleases such as zinc finger nucleases (ZFNs), TALE nucleases (TALENs), and RNA-guided endonucleases (RGENs) or meganucleases such as CRISPR-associated (Cas) proteins.

[0120] In some embodiments, a donor nucleic acid, such as a donor plasmid or a nucleic acid encoding an engineered antigen receptor, is provided and inserted by HDR at the site of gene editing following introduction of a DSB. Thus, in some embodiments, gene disruption and introduction of an antigen receptor, such as a CAR, are performed simultaneously, whereby the gene is partially disrupted by knock-in or insertion of a nucleic acid encoding a CAR.

[0121] In some embodiments, no donor nucleic acid is provided. In some embodiments, NHEJ-mediated repair following introduction of a DSB results in an insertion or deletion mutation that can cause gene disruption, for example, by creating a missense mutation or a frameshift.

[0122] 1. ZFPs and ZFNs In some embodiments, the DNA targeting molecule comprises a DNA binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease. Examples include ZFNs, TALEs, and TALENs.

[0123] In some embodiments, the DNA targeting molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, a region of amino acid sequence within the binding domain, the structure of which is stabilized by the coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial ZFP domains that target specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers.

[0124] ZFPs include those in which the single finger domain is approximately 30 amino acids long, contains two invariant histidine residues coordinated through zinc to two cysteines in a single beta turn, and contains an alpha helix with two, three, four, five, or six fingers. In general, the sequence specificity of ZFPs can be altered by making amino acid substitutions at the four helical positions (-1, 2, 3, and 6) of the zinc finger recognition helix. Thus, in some embodiments, ZFPs or ZFP-containing molecules are engineered to bind to non-natural, e.g., selected, target sites.

[0125] In some embodiments, disruption of MeCP2 is carried out by contacting a first target site of a gene with a first ZFP, thereby disrupting the gene, hi some embodiments, the target site of the gene is contacted with a fusion ZFP comprising six fingers and a regulatory domain, thereby inhibiting expression of the gene.

[0126] In some embodiments, the contacting step further comprises contacting a second target site in the gene with a second ZFP. In some embodiments, the first and second target sites are adjacent. In some embodiments, the first and second ZFPs are covalently linked. In some embodiments, the first ZFP is a fusion protein comprising a regulatory domain or at least two regulatory domains.

[0127] In some embodiments, the first and second ZFPs each comprise a regulatory domain or are fusion proteins each comprising at least two regulatory domains, hi some embodiments, the regulatory domains are transcriptional repressors, transcriptional activators, endonucleases, methyltransferases, histone acetyltransferases, or histone deacetylases.

[0128] In some embodiments, the ZFP is encoded by a ZFP nucleic acid operably linked to a promoter. In some aspects, the method further comprises initially administering the nucleic acid to the cell in a lipid:nucleic acid complex or as a naked nucleic acid. In some embodiments, the ZFP is encoded by an expression vector comprising a ZFP nucleic acid operably linked to a promoter. In some embodiments, the ZFP is encoded by a nucleic acid operably linked to an inducible promoter. In some aspects, the ZFP is encoded by a nucleic acid operably linked to a weak promoter.

[0129] In some embodiments, the target site is upstream of the transcription start site of the gene. In some embodiments, the target site is adjacent to the transcription start site of the gene. In some embodiments, the target site is adjacent to an RNA polymerase pause site downstream of the transcription start site of the gene.

[0130] In some embodiments, the DNA targeting molecule is or comprises a zinc finger DNA binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one type liS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is derived from the type liS restriction endonuclease Fok I. Fok I catalyzes double-stranded cleavage of DNA, typically 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand.

[0131] In some embodiments, ZFN targets genes present in engineered cells. In some aspects, ZFN efficiently generates double-strand breaks (DSBs) at predetermined sites in the coding region of genes, for example. Typical regions targeted include exons, regions encoding N-terminal regions, first exons, second exons, and promoter or enhancer regions. In some embodiments, transient expression of ZFNs promotes highly efficient and permanent destruction of target genes in engineered cells. Notably, in some embodiments, delivery of ZFNs results in permanent destruction of genes with efficiency of over 50%.

[0132] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) in collaboration with Sigma-Aldrich (St. Louis, MO, USA) has developed a platform for zinc finger construction (CompoZr) that allows researchers to bypass zinc finger construction and validation altogether and provides zinc fingers specifically targeted to thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or custom designed.

[0133] 2. TAL, TALE and TALEN In some embodiments, the DNA targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as a transcription activator-like protein effector (TALE) protein, see, e.g., U.S. Patent Application Publication No. 2011 / 0301073, which is incorporated herein by reference in its entirety.

[0134] A TALE DNA binding domain or TALE is a polypeptide that contains one or more TALE repeat domains / units. The repeat domains are responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also called "repeat") is typically 33-35 amino acids long and exhibits at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. Each TALE repeat unit typically contains one or two DNA binding residues at positions 12 and / or 13 of the repeat that constitute a repeat variable dipeptide (RVD). The natural (canonical) code for DNA recognition of these TALEs has been determined such that the HD sequence at positions 12 and 13 binds to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NO binds to T, and non-canonical (non-classical) RVDs are also known. See US Patent Application Publication No. 2011 / 0301073. In some embodiments, TALEs can target any gene by designing the TAL array with specificity for the target DNA sequence. The target sequence generally begins with a thymidine.

[0135] In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some embodiments, a TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain for cleaving a nucleic acid target sequence.

[0136] In some embodiments, TALENs recognize and cleave target sequences in genes. In some embodiments, DNA cleavage results in double-strand breaks. In some embodiments, cleavage stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). In general, NHEJ is an imperfect repair process that often leads to changes in DNA sequence at the cleavage site. In some embodiments, the repair mechanism involves rejoining the remains of the two DNA ends via direct religation (Critchlow and Jackson, 1998) or so-called microhomology-mediated end joining. In some embodiments, NHEJ-mediated repair results in small insertions or deletions that can be used to disrupt and thereby silence genes. In some embodiments, the modification can be a substitution, deletion or addition of at least one nucleotide. In some embodiments, cells in which a cleavage-induced mutagenesis event, i.e., a mutagenesis event subsequent to an NHEJ event, has occurred can be identified and / or selected by methods well known in the art.

[0137] In some embodiments, TALE repeats are assembled to specifically target genes. A library of TALENs targeting 18,740 human protein-coding genes has been constructed. Custom-designed TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA) and Life Technologies (Grand Island, NY, USA).

[0138] In some embodiments, the TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some embodiments, the plasmid vectors may contain a selection marker that provides for identification and / or selection of cells that have received the vector.

[0139] 3. RGEN (CRISPR / Cas system) In some embodiments, the disruption is carried out using one or more DNA-binding nucleic acids, such as RNA-guided endonuclease (RGEN)-mediated disruption. For example, the disruption can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In general, "CRISPR system" refers collectively to the transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0140] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) with nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system, and can be derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0141] In some embodiments, a Cas nuclease and a gRNA (comprising a fusion of a crRNA and an immobilized tracrRNA specific for a target sequence) are introduced into a cell. Generally, a target site at the 5' end of the gRNA allows the Cas nuclease to target a target site, e.g., a gene, using complementary base pairing. The target site can typically be selected based on the location directly 5' of a protospacer adjacent motif (PAM) sequence, such as NGG or NAG. In this regard, the gRNA targets a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence to which the guide sequence is designed to have complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0142] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by disruption as described herein. In other embodiments, a Cas9 variant considered a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each directed by a pair of different gRNAs targeting sequences such that simultaneous introduction of a nick introduces a 5' overhang. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0143] The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within an organelle of a cell. In general, a sequence or template that can be used for recombination into a target locus that comprises a target sequence is referred to as an "editing template", or an "editing polynucleotide", or an "editing sequence". In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is a homologous recombination.

[0144] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs therefrom). A tracr sequence that may include or consist of all or a portion of a wild-type tracr sequence (e.g., about 20 or more, about 26 or more, about 32 or more, about 45 or more, about 48 or more, about 54 or more, about 63 or more, about 67 or more, about 85 or more or more nucleotides of a wild-type tracr sequence) may form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex (such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned).

[0145] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. The components can also be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined into a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector can include one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of the one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0146] The vector can include regulatory elements operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0147] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is mutated with respect to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide that contains the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of S. pyogenes Cas9 converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination nicks both strands and can be used to induce NHEJ or HDR.

[0148] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species show a particular bias for a particular codon of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the properties of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of the selected tRNA in a cell generally reflects the codon that is most frequently used in peptide synthesis. Thus, a gene can be tuned for optimal gene expression in a given organism based on codon optimization.

[0149] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50% or more, about 60% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97.5% or more, about 99% or more, or more, when optimally aligned using a suitable alignment algorithm.

[0150] Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0151] CRISPR enzymes can be part of fusion proteins that contain one or more heterologous protein domains. CRISPR enzyme fusion proteins can contain any additional protein sequences and optionally linker sequences between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP). CRISPR enzymes may be fused to genetic sequences that encode proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tags, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions and herpes simplex virus (HSV) BP16 protein fusions.

[0152] C. Charged cell surfaces In some embodiments, the present disclosure relates to charged surfaces for cell culture. The charged surfaces can be positively charged, such as amine surfaces or nitrogen-containing functional groups, or negatively charged, such as carboxyl surfaces or oxygen-containing functional groups. The cell surface can be treated to change the surface charge of the culture vessel.

[0153] In some embodiments, the surface is neutrally charged, such as a surface that includes both negatively and positively charged functional groups. For example, the CORNING PRIMARIA® surface features a unique mixture of oxygen-containing (negatively charged) and nitrogen-containing (positively charged) functional groups on a polystyrene surface. The surface supports the growth of cells that may attach poorly or have limited differentiation potential when cultured on traditional TC surfaces. In some embodiments, the surface includes a ULA surface coating. For example, the Corning Ultra-Low Attachment surface is a covalently bonded hydrogel layer that is hydrophilic and neutrally charged. Because proteins and other biomolecules passively adsorb to polystyrene surfaces via hydrophobic or ionic interactions, the hydrogel naturally inhibits nonspecific immobilization by these forces and inhibits subsequent cell adhesion. The surface is highly stable, non-cytotoxic, biologically inert and non-degradable. Other examples that can support the generation of microglia from HPCs include: Corning CellBIND Culture (U.S. Patent No. 6,617,152) uses a higher energy microwave plasma to incorporate more oxygen into polystyrene surfaces, making them more hydrophilic (wettable) while increasing surface stability compared to traditional plasma or corona discharge treated surfaces. Corning Synthemax self-coating substrates are unique, animal-free, synthetic vitronectin-based peptides that contain an RGD motif and adjacent sequences. The synthetic peptides are covalently attached to the polymer backbone for a passive coating, orientation, and presentation of the peptide for optimal cell binding and signaling.

[0154] The cell culture surface may be coated with a plasma polymerized film. The source of the plasma polymerization is one or more monomers. Useful polymerizable monomers may include unsaturated organic compounds such as olefinic amines, halogenated olefins, olefinic carboxylic acids and carboxylates, olefinic nitrile compounds, oxygenated olefins and olefinic hydrocarbons. In some embodiments, the olefins may include vinyl and allylic types. In other embodiments, cyclic compounds such as cyclohexane, cyclopentane, and cyclopropane may be used.

[0155] As will be appreciated by those skilled in the art, various plasma polymerization techniques may be utilized to deposit one or more monomers onto the cell culture surface. Preferably, a positively charged polymerized film is deposited on the surface. As will be appreciated by those skilled in the art, the plasma polymerized surface may have a negative charge depending on the protein used therewith. Amines are preferably used as the monomer source for the polymer. In some embodiments, plasma polymerized monomers are made using a plasma source to generate a gas discharge that provides energy to initiate polymerization of the gaseous monomers and deposits a thin polymer film onto the culture vessel. Cyclic compounds may be utilized that may include gas plasma by glow discharge methods. Derivatives of these cyclic compounds, such as 1,2-diaminocyclohexane, are also generally polymerizable in gas plasma.

[0156] Mixtures of polymerizable monomers may be used. Additionally, the polymerizable monomers may be blended with other gases that are not generally considered to be polymerizable themselves, such as argon, nitrogen, and hydrogen.

[0157] It is contemplated that any culture vessel useful for adherent culture may be used. Preferred cell culture vessel configurations contemplated by the present disclosure include multi-well plates (such as 6-well, 12-well and 24-well plates), dishes (such as Petri dishes), test tubes, culture flasks, roller bottles, tubes or shaker flasks, and the like.

[0158] Materials for cell culture surfaces can include plastics (e.g., polystyrene, acrylonitrile butadiene styrene, polycarbonate); glass; microporous filters (e.g., cellulose, nylon, fiberglass, polyester, and polycarbonate); materials for bioreactors used in batch or continuous cell culture or genetic engineering (e.g., bioreactors), which may include hollow fiber tubing or microcarrier beads; polytetrafluoroethylene (Teflon), ceramics, and related polymeric materials.

[0159] In certain embodiments, the cell cultures are free or essentially free of any extracellular matrix proteins, such as laminin, fibronectin, vitronectin, MATRIGEL™, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin.

[0160] D. Differentiation of HPCs into microglia Microglia are innate immune cells of the central nervous system that play a key role in brain development, homeostasis and immune regulation. They are difficult to obtain from human fetal and primary tissues. In certain embodiments, the methods of the present invention describe the generation, characterization and cryopreservation of human iPSC-derived microglia (iMGL) from episomally reprogrammed HPCs under defined conditions. Cryopreserved iMGL maintain purity, secrete immunomodulatory cytokines, and phagocytose pHrodo Red-labeled bacterial BioParticles and amyloid beta aggregates. The ability to produce essentially unlimited quantities of iMGL holds great promise for accelerating human neuroscience research into the role of microglia in normal and disease states.

[0161] In an exemplary method, fresh or cryopreserved HPCs are thawed and plated in microglia differentiation medium containing FLT-3 ligand and IL-3. Cells may be plated at a density of 10-50K / cm2, such as 20-35K / cm2. Microglia differentiation medium may contain IL-34, TGFβ1 or M-CSF (MDM) or analogs or mimetics of each. Culturing may be performed on a charged surface such as MATRIGEL™ coated plates, or Primaria plates, or ultra-low attachment plates, or tissue culture plates (TC), or non-tissue culture plates (Non-TC), and may be high throughput such as 96-well plates (e.g., 200 μl of microglia differentiation medium per well). For the next 23 days of differentiation, cells may be half-fed every 48 hours with 2× Microglia Differentiation Medium (MDM), 50 μl of medium per well. In certain embodiments, differentiation is performed in the absence of ECM proteins such as MATRIGEL®. Cells are harvested with cold PBS on day 23 and total viable cell numbers are quantified using an automated cell counter. Cells are stained for surface expression of CD11b, CD11c, CD45, CD33, TREM-2 and intracellular expression of TREM-2, IBA, CX3CR1, P2RY12, TMEM119.

[0162] E. Endothelial cells iPSCs maintained on MATRIGEL™ or vitronectin in the presence of E8 can adapt to hypoxia for at least 5-10 passages. Cells can be split from subconfluent iPSCs and plated in amine culture dishes at a density of 250,000 cells / well in the presence of serum-free defined (SFD) medium supplemented with 5uM blebbistatin or 1uM H1152. 24 hours after plating, SFD medium supplemented with 50ng / ml BMP4, VEGF and FGF2 can be added to the cultures. Cells can be fed every 48 hours throughout the differentiation process. The entire process can be performed under hypoxic conditions. Cells harvested at the end of differentiation can be cryopreserved or replated on carboxyl surfaces at a density of 25k / cm2 to initiate endothelial differentiation in the presence of VascuLife VEGF Endothelial Medium or SFD Endothelial Medium.

[0163] In an exemplary method, cryopreserved day 6 HPCs or viable cultures are plated at 25k / cm on a carboxyl surface in the presence of VascuLife VEGF Endothelial Medium or SFD Endothelial Medium and hypoxic conditions. 2 Cells are given a fresh feed of endothelial medium 24 hours after plating and cultures are fed every 48 hours until they reach confluence. It may take 5-6 days for cells to reach confluence. Cells are harvested using TrypLE Select, stained for surface endothelial markers CD31, CD105 and CD144 and plated at 25k / cm using endothelial medium. 2 The cells are then replated on a carboxyl surface at 4°C and placed in a hypoxic incubator. The cells are fed the full volume of endothelial medium on days 2, 4, and 6 after splitting. On day 7, the cells are harvested, stained, and replated three more times in the same manner.

[0164] In some embodiments, endothelial cells are converted to brain microvascular endothelial cells. In an exemplary method, live or cryopreserved HPCs (e.g., day 7 HPCs obtained on amine surfaces in the presence of SFD supplemented with BMP4, VEGF, and FGF2) are plated on ECM containing fibronectin (e.g., 50-200 μg / mL, particularly 100 μg / mL) and collagen I (e.g., 100-500 μg / mL, particularly 400 μg / mL) with ECRA medium (human endothelial SFM [Gibco], 1% platelet-poor plasma-derived bovine serum [Fisher], 20 ng / mL bFGF [Promega], 10 uM retinoic acid). Cells are plated at 50-100 k / cm. 2 , especially 75k / cm 2 The cells may be plated at a density of 100-1500 nm. The cultures may be maintained under low oxygen incubator conditions. The cultures may be fed with ECRA medium every other day until confluent. The confluent cultures are then harvested, such as by using TrypLE. Staining may be performed on the harvested cells to detect PECAM-1 (CD31) and GLUT-1. The harvested cells may be replated, such as onto Transwell inserts, with ECRA medium and placed in low oxygen incubator conditions. The cultures may be fed with ECRA medium every other day until confluent. The confluent cultures may be tested for transendothelial electrical resistance (TEER).

[0165] F. Mesenchymal cells In some embodiments, iPSCs are differentiated into MSCs. For example, FIG. 5C shows a schematic of the 2D HPC differentiation process to generate MSCs. iPSCs maintained on MATRIGEL™ or vitronectin in the presence of E8 are adapted to hypoxia for at least 5-10 passages. Cells are split from subconfluent iPSCs and plated in amine culture dishes at a density of 250,000 cells / well in the presence of serum-free defined (SFD) medium supplemented with 5uM blebbistatin or 10uM H1152. 24 hours after plating, SFD medium supplemented with 50ng / ml BMP4, VEGF and FGF2 is added to the culture. Cells are fed every 48 hours throughout the differentiation process. The entire process is carried out under hypoxic conditions. At the end of the 6th or 7th day of differentiation, the cells are placed in GMP-MSC medium. Cells were grown to confluence, harvested at the end of each passage, and then plated on the amine surface at 50K / cm in GMP-MSC medium supplemented with 5 uM blebbistatin or 10 uM H1152. 2 The cells are replated at a density of 100-200 μg / ml to selectively allow growth and proliferation of the MSCs.

[0166] In some embodiments, cryopreserved day 6 HPCs or live cultures at the end of differentiation on day 6 are placed in the presence of MSC medium in the presence of 10 uM H1152 on amine-charged plate surfaces. Cells are given a fresh feed of MSC medium 24 hours after plating and cultures were fed every 48 hours until they reached confluence. It took 5-6 days for cells to reach confluence. Cells are harvested using TrypLE and stained for the absence of surface MSC markers CD73, CD44, CD105, CD49d and endothelial markers CD31 and CD144. New cultures are passaged 3 times using the process above under hypoxic conditions and amine surfaces. Cultures are transferred to normoxic and normal tissue culture plates at P4.

[0167] In some embodiments, MSCs can be further differentiated into pericytes. In an exemplary method, MSCs are seeded (e.g., 1-20k / cm) in ScienCell Pericyte Medium (catalog: 1201). 2 Cell density of 10k / cm on tissue culture plastic (TCP) 6-well plates, specifically 2 The cells are then cultured at 4°C (at 4°C) and placed in normoxic incubator conditions. Cultures may be fed every other day with ScienCell Pericyte Medium until confluent. Confluent cultures may then be harvested, such as by using TrypLE. Staining may be performed on the harvested cells to detect neuroglial antigen 2 / chondroitin sulfate proteoglycan (NG2) and PDGFR-beta (CD140b). Harvested cells may be replated in ScienCell Pericyte Medium (e.g., at a cell density of 1-20k / cm). 2 , especially 10k / cm for TCP 6-well plates 2 ). Cultures may be fed with ScienCell Pericyte Media every other day until confluent, and harvested and stained in the same manner as above. Cells are then replated until expanded and culture purity is maintained. Additionally, cells may stain positive for the presence of CD146, CD49a, CD166, CD54, CD73, CD105, CD13, CD56, CD49d, and / or CD44.

[0168] G. Differentiation medium The cells can be cultured with nutrients necessary to support the growth of each particular population of cells. Generally, the cells are cultured in a growth medium that includes a carbon source, a nitrogen source, and a buffer to maintain pH. The medium can also include fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffering agents, pH indicators, and inorganic salts. Exemplary growth media include minimal essential media, such as Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance the growth of stem cells. Examples of minimal essential media include, but are not limited to, Minimum Essential Medium Eagle (MEM) Alpha Medium, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. Additionally, the minimal essential medium can be supplemented with additives such as horse, calf, or fetal bovine serum. Alternatively, the medium can be serum-free. In other cases, the growth medium may include "Knockout serum replacement," referred to herein as a serum-free formulation optimized to grow and maintain undifferentiated cells, such as stem cells, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application Publication No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a feeder-free, completely defined medium.

[0169] In some embodiments, the medium may or may not contain any substitute for serum. Serum substitutes may include materials that suitably contain albumin (lipid-rich albumin, albumin substitutes such as recombinant albumin, vegetable starch, dextran and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol or equivalents. Serum substitutes may be prepared, for example, by the methods disclosed in WO 98 / 30679. Alternatively, and more conveniently, commercially available materials may be used. Commercially available materials include KNOCKOUT™ serum substitute (KSR), lipid concentrates of known composition (Gibco) and GLUTAMAX™ (Gibco).

[0170] Other culture conditions can be appropriately defined. For example, the culture temperature can be about 30 to 40°C, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39°C, but is not particularly limited thereto. In one embodiment, the cells are cultured at 37°C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen partial pressure can be at least up to or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.

[0171] H. Multicellular Cell Culture In certain embodiments, the present disclosure provides multi-cell culture models for the study of neuroinflammation, such as to identify novel targets, biomarkers, and therapeutics for the diagnosis, prognosis, and treatment of neurodegenerative diseases. In one embodiment, tripartite cultures of microglia, astrocytes, and neurons are provided.

[0172] The neurons may be excitatory and / or inhibitory, increasing or decreasing the activity of target neurons, respectively. Cortical (GABAergic / inhibitory), dopaminergic, cholinergic, serotonergic, glutamatergic, strychnine-sensitive glycine receptor expressing neurons, acetylcholine, epinephrine or norepinephrine, or histamine-responsive neurons may be used.

[0173] Glutamatergic neurons produce glutamate, one of the most common excitatory neurotransmitters in the central nervous system (CNS), which plays a role in fundamental processes such as learning, cognition, and memory, and dysregulation of glutamatergic transmission can lead to several neurological conditions.

[0174] GABAergic neurons produce gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). GABA is synthesized primarily from glutamate, catalyzed by glutamic acid decarboxylase (GAD), and is present in 30-40% of synapses. GABA induces Cl- influx or K+ efflux, resulting in neuronal hyperpolarization and a decrease in action potentials. Dysfunction of GABA neurotransmission may cause several disorders, including schizophrenia and epilepsy.

[0175] Dopaminergic neurons in the midbrain are the main source of dopamine (DA) in the mammalian central nervous system. Their loss is associated with Parkinson's disease (PD), one of the most prominent human neurological disorders. In some embodiments, neuronal types typically found in the hippocampus, amygdala, periphery (peripheral neurons), motor neurons, or cortical neurons (e.g., glutamatergic neurons or excitatory neurons) may be used in various embodiments. For example, in some embodiments, cholinergic neurons may be generated, for example, as described in U.S. Pat. No. 8,513,017 or U.S. Pat. No. 8,796,022. In some embodiments, motor neurons may be generated, for example, as described in U.S. Pat. No. 8,735,149. In some embodiments, dopaminergic neurons can be generated as described in WO2013067362; WO2013163228; WO2012080248; or WO2011130675.

[0176] Glutamatergic and GABAergic neurons may be positive for SCL1, BCL11B, Calb2, CD24, CDH1, CUX1Cux2, DCX, DLG4, Dlx, Dlx2, Emx1, Emx2, eomes, ETV1, FOXG1, FOXP2, Fut4, GABRA2, GAD1, GAD2, GAPDH, GFAP, GRIN2B, HoxB4, HTR2C, ISL1, ITGB1, LHX2, Neurog1, NKX2-1, Nos1, NPY, NR4A2, PAX6, POU3F2, PVALB, RELN, SATB2 SLC17A6, SLC17A7, SLC17A8, SLC32A1, SOX1, Sox10, SST, SYN1, and Tbr1. GABAergic neurons can be characterized by expression of SL3A1, GAD2, and DlX2. Glutamatergic neurons can be characterized by expression of Emx2, SLC17A6, and Tbr1.

[0177] In some embodiments, GABAergic, glutamatergic, dopaminergic, or cholinergic neurons can be generated from pluripotent stem cells, such as embryonic stem cells or iPS cells, using the following methods. For example, in some embodiments, neurons can be generated from pluripotent stem cells using the methods of US Patent Application Publication No. 2012 / 0276063. For example, in some embodiments, bFGF and TGFβ can be omitted from the medium used to culture pluripotent cells, such as iPS cells, before the onset of aggregate formation (while the cells are still in adherent culture), (e.g., omitted from defined media such as TeSR or Essential8 medium), which can be used to promote neural differentiation of pluripotent cells. In some embodiments, if iPS cells are "primed" in the absence of TeSR growth factors for several days before aggregate formation, i.e., cultured in any medium without basic fibroblast growth factor (bFGF) and transforming growth factor β (TGFβ), the cells can develop into neural lineages with purity, rapidity, and consistency. Other methods of generating neurons include Zhang et al. (2013), U.S. Patent No. 7,820,439, WO 2011 / 091048, U.S. Patent No. 8,153,428, U.S. Patent No. 8,252,586, and U.S. Patent No. 8,426,200.

[0178] Cultures of neural cell types derived from pluripotent cells, including iPS cells, are also commercially available and may be purchased. For example, iCell® Neurons, iCell® Dopa Neurons, and iCell® Astrocytes are derived from human iPS cells and are available from Cellular Dynamics International (Madison, Wisconsin). iCell® Neurons are human induced pluripotent stem cell (iPSC) derived neurons that exhibit biochemical, electrophysiological, and pathophysiological properties characteristic of native human neurons. Due to their high purity, functional relevance, and ease of use, iCell® Neurons represent a highly useful in vitro test system for investigating neurobiology in many areas of basic research and drug development.

[0179] In some embodiments, defined media (i.e., media that does not contain tissue, feeder cells, or cell-conditioned media) may be used to produce neurons or astrocytes from pluripotent cells such as iPS cells.

[0180] The medium used to produce neurons or astrocytes from iPS cells may be essentially free of serum and / or serum-derived growth factors. In further embodiments, the medium may have or be essentially free of exogenously added TGFβ superfamily signaling regulators, including positive regulators or inhibitors of BMP signaling and / or Activin / Nodal / TGFβ / GDF signaling. For example, the BMP signaling inhibitor may be Dorsomorphin and the Activin / Nodal / TGFβ / GDF signaling inhibitor may be SB431542. In still further embodiments, the medium may have or be essentially free of exogenously added other FGF signaling regulators, particularly FGF inhibitors.

[0181] In certain embodiments, mature neural cells comprising cultured cells may be identified as mature neural cells by expression of one or more of Dcx, MAP-2, synapsin 1, TuJ1, NSE, Map2a, Gap43, NF, CD24, CDH2 / CD325, synaptophysin, and CD56 / NCAM. Such cell cultures may be produced by the methods described herein or other methods, including those later developed.

[0182] Neuronal cells can be characterized according to a number of phenotypic criteria, including but not limited to, microscopic observation of morphological features, detection or quantification of expressed cellular markers, enzymatic activity, neurotransmitters and their receptors, and electrophysiological function.

[0183] Certain cells that may be used in various embodiments have morphological features characteristic of neural cells, which are recognized by those of skill in the art, such as neurons that include a small cell body and multiple processes reminiscent of axons and dendrites.

[0184] Neurons may also be characterized by whether they express phenotypic markers characteristic of a particular type of neuronal cell, including, but not limited to, dopaminergic neurons (markers include TH, AaDC, Dat, Otx-2, FoxA2, ​​LMX1A, and VMAT2), cholinergic neurons (markers include NGF, ChAT), GABAergic neurons (markers include GAD67 and vGAT), glutamatergic neurons (markers include vGLUT1), serotonergic neurons, motor neurons (markers include HB9, SMN, ChAT, NKX6), sensory neurons (markers include POU4F1 and peripherin), astrocytes (markers include GFAP and Tapal), oligodendrocytes (markers include O1, O4, CNPase, and MBP). Neurons may express one, two, three, four, five, or more markers of a particular type of neuronal cell type.

[0185] Astrocytes are a subtype of glial cells in the central nervous system. They are also known as astrocytes. They are generally star-shaped and their many processes typically encase synapses made by neurons in vivo. Astrocytes are classically identified using histological analysis. Many of these cells express the intermediate filament glial fibrillary acidic protein (GFAP). Three forms of astrocytes exist in the CNS: fibrous, protoplasmic, and radial. Fibrous glia are typically located within white matter, have relatively few organelles, and exhibit long, unbranched cell processes. This type often has "vascular feet" that physically connect the cells to the outside of the capillary wall when the cells are in close proximity to the capillary wall. Protoplasmic glia are found in gray matter tissue, have a greater amount of organelles, and exhibit highly branched, short tertiary processes. Radial glia are arranged in a plane perpendicular to the axis of the ventricle. Radial glia are present primarily during development and may play a role in neuronal migration in vivo. The exceptions are Müller cells in the retina and Bergmann glial cells in the cerebellar cortex, which persist into adulthood. A variety of methods can be used to generate astrocytes from pluripotent stem cells, such as embryonic stem cells or iPS cells. These methods include, for example, U.S. Patent Application Publication No. 2012 / 0276063, which is incorporated herein by reference in its entirety without disclaimer.

[0186] In some embodiments, the cell culture may further include exogenous Aβ oligomers, such as synthetic oligomers.

[0187] The cell culture may be a 3D cell culture. The 3D cell culture may be a brain organoid. The term "organoid" as used in the context of this application refers to a three-dimensional cellular structure that mimics the tissue and function of an organ. Organoids are composed of tissue-specific cell types that self-organize through cell sorting and spatially restricted lineage commitment. Organoids may be derived from stem cells, such as embryonic stem cells or induced pluripotent stem cells. The term "brain organoid" refers to an organoid that has anatomical features that resemble those of the brain. It is generally understood that brain organoids are composed of various cell types of the brain. These cell types may have different developmental capabilities, with some cell types being less differentiated than others. In some embodiments, brain organoids may include structures and cell types of the retina, cortex, midbrain, hindbrain, brainstem, and / or spinal cord.

[0188] In another embodiment, a blood-brain barrier model is provided that includes BMECs, astrocytes, and pericytes. The model may be in a sandwich format that includes BMECs on the apical (i.e., blood) side, a layer of ECM proteins (e.g., collagen IV and fibronectin), and astrocytes and pericytes on the basolateral (i.e., brain) side. The sandwich format may further include a permeable membrane insert between the apical and basolateral sides. The ECM proteins may be present on the apical side of the membrane. In some embodiments, the membrane may be coated with collagen, laminin, proteoglycan, vitronectin, fibronectin, poly-D-lysine, and / or polysaccharides. The basolateral side of the membrane may further include a layer of gelatin. Alternatively, the basolateral side may be coated with a polyalkylene oxide, a poloxamine, a cellulose, a hydroxyalkylated cellulose, a polypeptide, a polysaccharide, a carbohydrate, a protein, a copolymer thereof, or a combination thereof, more specifically composed of or derived from poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-polypropylene oxide) block copolymers, carboxymethylcellulose, hydroxyethylcellulose, methylhydroxypropylcellulose, polysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin, alginate, collagen, albumin, ovalbumin, a copolymer thereof, or a combination thereof.

[0189] In some embodiments, the model may be provided in a microfluidic device. Various microfluidic device configurations useful for supporting cells are known in the art, including in the form of an in vitro vascular model. See, for example, US Patent Application Publication No. 2011 / 0053207 and US Patent Application Publication No. 2014 / 0038279, which are incorporated herein by reference. In general, a microfluidic device including a blood-brain barrier model as taught herein may include a chamber dimensioned to receive the blood-brain barrier model therein, such that the endothelial cell layer and the neuronal cell layer define a boundary between a first chamber or opening in fluid contact with the endothelial cell layer of the model and a second chamber or opening in fluid contact with the neuronal cell layer of the model. The fluid may be a liquid, such as a medium or buffer. The device may further include a fluid inlet and a fluid outlet for each chamber, a fluid reservoir (e.g., a medium reservoir) connected thereto, and the like.

[0190] In some embodiments, the cells used in the cell culture are generated from iPS cells generated from cells obtained from a healthy donor. In other embodiments, the donor has a disease. For example, in some embodiments, the donor has a disease, such as a neurological or neurodegenerative disease, such as epilepsy, autism, attention deficit hyperactivity disorder (ADHD), amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth (CMT), Huntington's disease, familial epilepsy, schizophrenia, familial Alzheimer's disease, Friedreich's ataxia, spinocerebellar ataxia, spinal muscular atrophy, hereditary spastic paraparesis, leukodystrophy, phenylketonuria, Tay-Sachs disease, Wilson's disease, addiction disorder, depression, or mood disorder. The disease can be a genetic disease or an increased genetic susceptibility to a particular neurological disease.

[0191] Additionally, assays are provided herein for studying neuroinflammation using the cell culture models of the present invention. Outcomes of the tripartite culture system or BBB model can be survival, synaptic pruning, microglial function due to Aβ aggregation, p-tau formation, neuronal MEA function, analytes released into the medium to trigger the neuroinflammatory cascade, and crosstalk between all three cell types.

[0192] Cells are generally seeded in suitable culture vessels, such as flasks, 6-well, 24-well, or 96-well plates, or other tissue culture plates. Culture vessels used to culture cells may include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, as long as they can culture stem cells. Cells may be cultured in volumes of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range of volumes derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any ex vivo device or system that supports a biologically active environment for cells to grow in. A bioreactor has a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0193] I. Cryopreservation The cells produced by the methods disclosed herein can be cryopreserved at any stage of the process, such as stage I, stage II, or stage III, see, for example, PCT Publication WO 2012 / 149484 A2, which is incorporated herein by reference. The cells can be cryopreserved with or without a substrate. In some embodiments, the storage temperature is in the range of about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, about -90°C to about -100°C, and overlapping ranges thereof. In some embodiments, lower temperatures are used for storage (e.g., maintenance) of the cryopreserved cells. In some embodiments, liquid nitrogen (or other similar liquid coolant) is used to preserve the cells. In further embodiments, the cells are preserved for more than about 6 hours. In further embodiments, the cells are preserved for about 72 hours. In some embodiments, the cells are preserved for 48 hours to about 1 week. In yet other embodiments, the cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, the cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The cells can also be stored for longer periods of time. The cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.

[0194] In some embodiments, an additional cryoprotectant can be used. For example, cells can be cryopreserved in a cryopreservation solution that includes one or more cryoprotectants, such as DM80, and serum albumin, such as human or bovine serum albumin. In certain embodiments, the solution includes about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In other embodiments, the solution includes about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% dimethyl sulfoxide (DMSO) or albumin. In certain embodiments, the solution includes 2.5% DMSO. In another specific embodiment, the solution includes 10% DMSO.

[0195] The cells may be cooled, for example, at about 1° C. / min during cryopreservation. In some embodiments, the cryopreservation temperature is about −80° C. to about −180° C. or about −125° C. to about −140° C. In some embodiments, the cells are cooled to 4° C. before cooling at about 1° C. / min. The cryopreserved cells may be transferred to the vapor phase of liquid nitrogen before thawing for use. In some embodiments, for example, once the cells reach about −80° C., they are transferred to a liquid nitrogen storage area. Cryopreservation may also be performed using a controlled rate freezer. The cryopreserved cells may be thawed, for example, at a temperature of about 25° C. to about 40° C., typically at a temperature of about 37° C.

[0196] III.How to use The present disclosure provides methods that can produce large numbers of cells of multiple lineages, as well as compositions that include cells of the CNS. These cell populations can be used for many important research, development and commercial purposes, including, but not limited to, in vivo cell transplantation or implantation; in vitro screening of antivirals, cytotoxic compounds, carcinogens, mutagens, growth / regulatory factors, pharmaceutical compounds, and the like; elucidation of mechanisms of liver disease and infectious diseases; studying the mechanisms by which drugs and / or growth factors act; diagnosis and monitoring of cancer in patients; gene therapy; and production of biologically active products, to name just a few.

[0197] The multicellular cultures provided herein can be used, for example, to test the effect of molecules on neuronal differentiation or survival, or for toxicity testing, or for testing the effect of molecules on neuronal or neuronal function. This can include screening to identify compounds that affect neuronal activity, plasticity (e.g., long-term potentiation), or function. The cell cultures can be used to discover, develop, and test new drugs and compounds that interact with and affect the biology of neural stem cells, neural progenitor cells, or differentiated neural cells or neuronal cell types. Neuronal cells are also highly useful in studies designed to identify the cellular and molecular basis of neural development and dysfunction, including, but not limited to, axon guidance, neurodegenerative diseases, neuroplasticity, and learning and memory. Such neurobiology studies can be used to identify novel molecular components of these processes, provide new uses for existing drugs and compounds, and identify new drug targets or drug candidates.

[0198] In some embodiments, one or more specific compounds may be tested to determine whether the compound has an effect that may be beneficial in treating the disease. Based on the effect of the compound on functional activity, it may be possible to determine whether the compound may be useful in treating the disease. In some embodiments, the cells are derived from iPS cells from a subject with a disease (e.g., a genetic disease or a disease with a genetic component or risk factor), such as a neurological or neurodegenerative disease (e.g., autism, epilepsy, ADHD, schizophrenia, bipolar disorder, etc.). In some embodiments, the cells may be cultured in the presence of a first compound or toxin, such that the neuronal culture exhibits similar characteristics to the disease state. In these embodiments, a second compound may be provided to the cell culture to determine whether the second compound can alleviate or reduce the effects of the first compound or toxin. In other embodiments, the cell culture may be used to determine whether the compound produces a toxic or adverse effect on the cell culture.

[0199] For example, one or more candidate drugs can be added to the culture medium at various concentrations. Drugs that promote the expression of the polypeptide of interest expressed in the cells are considered to be useful. Such drugs can be used as therapeutic agents to prevent, delay, ameliorate, stabilize, or treat injuries, diseases, or disorders characterized by defects in neurodevelopment or neurofunction. The identified drugs can be used to treat or prevent neurological conditions. In another embodiment, the activity or function of cells of the organoid is compared in the presence and absence of the candidate compound. Compounds that desirably change the activity or function of cells are selected as useful in the method.

[0200] Agents useful in the present method can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts, or chemical libraries, or polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the art of drug discovery and development will understand that the exact source of the test extract or compound is not critical to the screening procedure of the method of the present invention. Agents used for screening can include known agents known as therapeutic agents for the treatment of neurological conditions. Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant, fungal, prokaryotic or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing polypeptides.

[0201] Assays for determining the functional activity of cells may include viability assays, microglial phagocytosis assays, calcium assays, MEA assays, synaptic pruning by microscopy assays, signal transduction tracking phosphorylated intermediates of various pathways, analysis of analytes released into the culture medium alone, two-way or three-way, including normal and disease-specific cell types. For example, for disease modeling applications comparing isogenic or patient-specific cells to AHN controls, treatment or exposure to neurogenerative proteins such as amyloid beta, myelin, synaptosomes or tau will decrease calcium signaling and electrical activity and increase neuroinflammatory cytokines. In some embodiments, measuring functional activity includes measuring dendritic area (e.g., MAP2), synapse number (e.g., synapsin 1 / 2), cell number (e.g., CUX2), or axon area (e.g., beta III tubulin). For example, an increase in any of these functional activity measures (eg, greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90%) may be indicative of a candidate agent.

[0202] In some embodiments, early pathogenic changes can be quantified by observing changes or primarily downregulation of gene expression profiles associated with the onset of neurodegeneration. In certain embodiments, upregulation of immune-related genes associated with the release of neuroinflammatory cytokines can be measured as associated with neurodegeneration. In some embodiments, the method can include detecting the levels of Gas6-Axl, Siglec-11, ligand-gated ion channels, voltage-gated ion channels, GPCRs, catalytic receptors, enzymes, nuclear receptors, COMT, NRXN2, and / or SST in microglia. In some embodiments, the GPCR comprises ADGRD1, ADGRE3, ADGRE5, ADGRG1, ADGRG3, ADORA2B, ADRB1, ADRB2, C5AR1, C5AR2, CCR2, CXCR2, CXCR4, EDNRA, FPR3, FZD1, GPBAR1, GPR157, LTB4R, LTB4R2, P2RX1, P2RY1, P2RY12, PTGER4, and / or SUCNR1. In certain embodiments, the GPCR comprises AVPR2, CNR2, GPR18, GPR84, and / or LPAR6. In certain embodiments, the ligand-gated ion channel comprises P2XRX1, P2RY12, and / or P2RY10.

[0203] The assay may be performed in a high-throughput manner. For example, cell cultures can be placed or placed on culture dishes, flasks, roller bottles or plates (e.g., single multi-well dishes or dishes such as 8, 16, 32, 64, 96, 384 and 1536 multi-well plates or dishes), optionally in defined locations, for the identification of potential therapeutic molecules. Screenable libraries include, for example, small molecule libraries, siRNA libraries, adenovirus transfection vector libraries. The screening platform can be automated, such as robotic automation. The culture platform can include an automated cell washer and a high content imager.

[0204] In some embodiments, the assay can quantify the response of the cell cultures to different neuroinflammatory stimuli that mimic sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch wound), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profiles of control and LPS-exposed cultures can be measured.

[0205] The blood-brain barrier model described herein can be used for compound or treatment screening or testing (e.g., for efficacy, toxicity, or other metabolic or physiological activity), such as for pharmacodynamic or pharmacokinetic testing of drug crossing the blood-brain barrier. Such testing can be performed by providing the in vitro blood-brain barrier model described herein under conditions that maintain the constituent cells of the product in a viable state (e.g., in an oxygenated medium); applying the compound to be tested (e.g., a drug candidate) to the cells (e.g., by administration to the endothelial layer); and then detecting the penetration of the compound through the endothelial layer and / or other physiological responses (e.g., injury, scar tissue formation, infection, cell proliferation, bum, cell death, release of markers such as histamine release, cytokine release, genetic changes, etc.), which can indicate whether the compound can cross the blood-brain barrier and / or has a therapeutic effect, toxicity, or other metabolic or physiological activity in the brain when delivered systemically (e.g., intravascularly) to a mammalian subject. A control sample of the blood-brain barrier may be maintained under similar conditions and a control compound (e.g., saline, complex vehicle or carrier) applied thereto to obtain comparative results, or damage may be determined based on comparison with historical data or data obtained by application of dilutions of test compounds, etc.

[0206] Methods for determining whether a test compound has immune activity may include testing for immunoglobulin production, chemokine production, and cytokine production by assessing the migration of innate immune cells, such as microglia or astrocytes in a blood-brain barrier model, or neutrophils and macrophages, into the neuronal layer.

[0207] Methods of crossing the blood-brain barrier (e.g., the human blood-brain barrier) that can be tested using the models taught herein include, but are not limited to, differential paracellular tight junction permeability, passive diffusion through cell layers, receptor-mediated transcytosis, and / or cellular efflux inhibition.

[0208] In some embodiments, the model can be used for subject-specific testing (e.g., for efficacy, toxicity, or other metabolic or physiological activity), where at least some of the cells in the model are from the subject, such as for pharmacodynamic or pharmacokinetic testing of drug blood-brain barrier crossing. For example, the subject's fibroblasts can be directed into induced pluripotent stem cells (e.g., induced pluripotent neural stem cells), which can then be directed into one or more cell types for the model, such as neurons, oligodendrocytes, endothelial cells, astrocytes, or microglia.

[0209] Further provided herein are methods of treating neurodegenerative diseases by administering a TREM2 agonist, which may be an antibody that induces TREM2 function, or an activator of downstream signaling pathways induced by TREM2, such as spleen tyrosine kinase (pSyk), PI3K, TYRO protein tyrosine kinase binding protein (TYROBP), DNAX-activating protein of 12 kDa (DAP12), AKT kinase, or the PLCγ pathway.

[0210] The terms "neurodegenerative disease or disorder" and "neurological disorder" include diseases or disorders that primarily involve the peripheral or central nervous system. The compounds, compositions, and methods provided herein can be used to treat neurological or neurodegenerative diseases and disorders. As used herein, the terms "neurodegenerative disease", "neurodegenerative disorder", "neurological disease", and "neurological disorder" are used interchangeably.

[0211] Examples of neurological disorders or diseases include diabetic peripheral neuropathy (including third nerve palsy, mononeuropathy, multiple mononeuropathy, diabetic amyotrophy, autonomic neuropathy, and thoracic and abdominal neuropathy), Alzheimer's disease, age-related memory decline, senility, age-related dementia, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multiple system degeneration (Shy-Drager syndrome), motor neuron diseases including amyotrophic lateral sclerosis ("ALS"), degenerative ataxias, corticobasal degeneration, Guam ALS-Parkinson's disease-dementia complex, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, multiple sclerosis ("MS"), synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Jackson syndrome ... These include, but are not limited to, chronic neurological disorders such as Joseph's disease / Spinocerebellar ataxia type 3 and olivopontocerebellar degeneration, Gilles de la Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinal muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Wernicke-Korsakoff related dementia (alcohol-induced dementia), Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff disease, familial spastic disorders, Wohifart-Kugelberg-Welander disease, spastic paraparesis, progressive multifocal leukoencephalopathy, and prion diseases (including Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, kuru, and fatal familial insomnia). Other conditions further included in the method of the present disclosure include age-related dementia and other dementias, as well as conditions involving memory loss, including vascular dementia, diffuse white matter disease (Binswanger's disease), dementia of endocrine or metabolic origin, dementia due to head trauma and diffuse brain injury, dementia pugilistica, and frontal lobe dementia. Also included are cerebral ischemia or infarction, including embolic and thrombotic occlusion, and other neurodegenerative diseases resulting from any type of intracranial hemorrhage (including but not limited to epidural, subdural, subarachnoid, and intracerebral), and intracranial and intravertebral lesions (including but not limited to contusion, penetration, shear, compression, and laceration). Thus, the term also encompasses acute neurodegenerative disorders, such as stroke, traumatic brain injury, schizophrenia, peripheral nerve injury, hypoglycemia, spinal cord injury, epilepsy, and those involving anoxia and hypoxia.

[0212] A. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising the cells and a pharma- ceutically acceptable carrier.

[0213] Thus, the cell composition for administration to a subject according to the present invention can be formulated in any conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate processing of the compound into a medicament that can be used. The appropriate formulation will vary depending on the selected route of administration.

[0214] The pharmaceutical compositions and formulations described herein comprise an active ingredient (such as cells) having a desired degree of purity in the form of a lyophilized formulation or an aqueous solution, optionally in one or more pharma- ceutical acceptable carriers (see Remington's Pharmaceutical Sciences 22). ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmacopoeitic carriers include, but are not limited to, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include interstitial drug dispersion agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0215] B. Distribution for Commercial, Therapeutic and Research Purposes In some embodiments, a reagent system is provided that includes cells present at any time during manufacture, distribution, or use. Kits may include any combination of cells described in this disclosure in combination with undifferentiated pluripotent stem cells or other differentiated cell types, often sharing the same genome. Each cell type may be packaged together, in separate containers in the same facility, or at different locations at the same or different times, under the control of the same or different entities that share a business relationship. Pharmaceutical compositions may be packaged in appropriate containers, optionally with written instructions for the desired purpose, such as mechanistic toxicity.

[0216] In some embodiments, kits are provided that may include, for example, one or more media and components for producing cells. The reagent system may be packaged in either aqueous media or lyophilized form, as appropriate. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed and preferably appropriately divided. If the kit includes multiple components, the kit will generally also include a second, third, or other additional container into which the additional components may be separately placed. However, various combinations of components may be included in the vials. The components of the kits may be provided as dry powders. If the reagents and / or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits of the present disclosure will also typically include a means for containing the components of the kit in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. The kits may also include instructions for use, such as in electronic, such as printed or digital, format. EXAMPLES

[0217] The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many modifications can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0218] Example 1 – Generation of endothelial cells iPSCs maintained on MATRIGEL™ or vitronectin in the presence of E8 were adapted to hypoxia for at least 5-10 passages. Cells were split from subconfluent iPSCs and plated in amine culture dishes at a density of 250,000 cells / well in the presence of serum-free defined (SFD) medium supplemented with 5uM blebbistatin or 1uM H1152. 24 hours after plating, SFD medium supplemented with 50ng / ml BMP4, VEGF and FGF2 is added to the cultures. Cells are fed every 48 hours throughout the differentiation process. The entire process is performed under hypoxic conditions. Cells are harvested at the end of differentiation and either cryopreserved or cultured at 25k / cm to initiate endothelial differentiation in the presence of VascuLife VEGF Endothelial Medium or SFD Endothelial Medium. 2 The carboxyl surface can be replated at a density of 100 μg / cm2 (Figure 2).

[0219] Cryopreserved day 6 HPCs or live cultures were cultured in the presence of VascuLife VEGF Endothelial Medium, with 25k / cm2 of carboxyl surface in the presence of 1 μM H1152 and hypoxic conditions. 2Cells were plated at 250 nmol / cm2. Cells were given a fresh feed of VascuLife 24 hours after plating and cultures were fed every 48 hours until they reached confluence. It took 5-6 days for cells to reach confluence. Cells were harvested using Accumax with minimal agitation or pipetting, stained for surface endothelial markers CD31, CD105 and CD144, and plated at 25 nmol / cm2 using VascuLife+H1152. 2 The cells were replated on a carboxyl surface at 20°C and placed in hypoxic incubator conditions. On days 2, 4 and 6 after splitting, the cells were fed the full amount of VascuLife. On day 7, the cells were harvested, stained and replated three more times in the same manner. The histograms show the increasing purity of endothelial cells at each replating step. Pure endothelial cells were generated using serial passage purification without CD31+MACS. Endothelial cells can be cryopreserved at the end of replating passage 3 (Figure 3).

[0220] Example 2 – Generation of mesenchymal stem cells Figure 5C shows a schematic of the 2D HPC differentiation process to generate MSCs. iPSCs maintained on MATRIGEL™ or vitronectin in the presence of E8 were adapted to hypoxia for at least 5-10 passages. Cells were split from subconfluent iPSCs and plated in amine culture dishes at a density of 250,000 cells / well in the presence of serum-free defined (SFD) medium supplemented with 5uM blebbistatin or 1uM H1152. 24 hours after plating, SFD medium supplemented with 50ng / ml BMP4, VEGF and FGF2 was added to the cultures. Cells were fed every 48 hours throughout the differentiation process. The entire process was performed under hypoxic conditions. At the end of day 6 / 7 of differentiation, cells were placed in GMP-MSC medium. The phenotype of the precursor population was analyzed after recovery (Figure 5C). Cells were grown to confluence, harvested at the end of each passage, and then plated on the amine surface at 50K / cm in GMP-MSC medium supplemented with 5uM blebbistatin or 1uM H1152. 2The cells were replated at a density of 100x to selectively allow the growth and proliferation of MSCs.

[0221] Cryopreserved day 6 HPCs from 3D / 2D HPC differentiation or live cultures at the end of day 6 differentiation resulting from 2D HPC differentiation were placed in the presence of MSC medium in the presence of 1 uM H1152 on amine charged plate surfaces. Cells were given a fresh feed of MSC medium 24 hours after plating and cultures were fed every 48 hours until they reached confluence. It took 5-6 days for cells to reach confluence. Cells were harvested using TrypLE and stained for lack of surface MSC markers CD73, CD44, CD105, CD49d as well as endothelial markers CD31 and CD144. New cultures were passaged 3 times using the process above under hypoxic conditions and amine surfaces. Cultures were transitioned to normoxic and normal tissue culture plates at P4. MSC purity specifications were achieved at P6 (Figures 6, 7). MSCs cryopreserved at P3 were thawed and placed in lineage-specific differentiation matrices as described in Figure 8A to demonstrate the tri-lineage potential to generate osteocytes, chondrocytes, and adipocytes (Figure 8B). The clonal expansion potential of cryopreserved MSCs was demonstrated at 1000 cells / cm in 10 cm tissue culture plates. 2 This was demonstrated by plating MSCs at a density of 100-200 nm. Cells were fed with MSC medium for 2 weeks, with medium changed every other day. Emergent colonies were stained with crystal violet and scored (Figure 8C).

[0222] Example 3 – Generation of pericytes from MSCs iCell MSC and iPSC-derived pericytes were sampled at 50% confluency and analyzed by flow cytometry for known pericyte markers PDGFRβ, NG2 and CD146. Cryopreserved MSCs were thawed and plated at 35,000 cells / cm in 6-well plates in MSC maintenance medium without extracellular matrix (ECM). 2Cells were allowed to reach confluence and replated at 15,000 cells / cm2 in 6-well plates without extracellular matrix (ECM) in SFD pericyte medium (SPM) (Figure 9A; Figure 9B).

[0223] Primary human brain vascular pericytes (HBVP) (ScienCell#1200) were thawed and plated at 5,000 cells / cm in pericyte medium (ScienCell#1201) on poly-L-ornithine-coated 6-well plates. 2 These cells were used as a positive control for the differentiation process. ScienCell HBVP, iCell MSC and iPSC derived pericytes were analyzed by flow cytometry for known pericyte markers PDGFRβ, NG2 and CD146 (Figure 9C). Pericyte markers of iCell MSC were absent upon thawing. HBVP and iPSC derived pericytes showed expression of known pericyte markers PDGFRβ, NG2 and CD146, with iPSC derived pericytes having higher purity than ScienCell HBVP (Figure 9C). iPSC derived pericytes show similar morphology to ScienCell HBVP (Figure 9D).

[0224] Based on their function, pericytes can be phenotypically classified as PC1 (pro-inflammatory) or PC2 (contractile) (Rustenhoven et al., 2017). The characteristics of both subtypes are described in Figure 9E. After thawing, iPSC-derived pericytes were subtyped by flow cytometry for PC1 and PC2 markers CD274, VCAM1, calponin, desmin, DLK1 and αSMA (Figure 9F). iPSC-derived pericytes reveal the characteristics of contractile pericytes, subtype PC2.

[0225] In addition to the non-specific phagocytic uptake seen in chronic and acute BBB models, pericytes specifically regulate the neuronal microenvironment by handling the clearance of specific macromolecules in both physiological and pathological conditions (Winkler et al., 2014). iPSC-derived pericytes were cultured at 15,000 cells / cm in 96-well plates with PDL coating (Greiner #655946) in SPM. 2 iPSC-derived pericytes were seeded at 100x the normal concentration for 10 min at 20°C. Cells were left undisturbed for 3 days after plating, after which the dead indicator NucGreen Dead 488 (Invitrogen #R37109) and S. aureus pHrodo Red BioParticles (Invitrogen #A10010) were added to the cells. Plates were placed in an IncuCyte live imaging system for over a month with weekly feedings (containing the same concentrations of live / dead and bioparticle reagents). iPSC-derived pericytes show observable phagocytic activity of S. aureus bioparticles over controls.

[0226] Example 4 – Generation of Brain Microvascular Endothelial Cells (BMEC) iPSCs maintained on MATRIGEL™ or vitronectin in the presence of E8 were adapted to hypoxia for at least 5-10 passages to generate brain microvascular endothelial cells. Live HPCs or cryopreserved HPCs (e.g., day 6 HPCs obtained on amine surfaces in the presence of SFD supplemented with BMP4, VEGF and / or FGF2, such as BMP4 and FGF2) were plated on ECM containing fibronectin (e.g., 50-200 μg / mL, particularly 100 μg / mL) and collagen IV (e.g., 100-500 μg / mL, particularly 400 μg / mL) with ECRA medium (human endothelial SFM (Gibco), 1% platelet-poor plasma-derived bovine serum (Fisher), 20 ng / mL bFGF (Promega), 10 uM retinoic acid). Cells were plated at 50-100 k / cm. 2 , especially 75k / cm 2The cells were plated at a density of 100-150 μg / ml. Cultures were fed daily and maintained under low oxygen incubator conditions. Cultures are fed with ECRA medium every other day until confluent. Confluent cultures are then harvested, such as by using TrypLE. The harvested cells are stained to detect PECAM-1 (CD31) and GLUT-1 to confirm their identity as BMECs (Figure 10B). The harvested cells are replated, such as onto Transwell inserts, using ECRA Medium and placed in low oxygen culture conditions (Figure 10A). Cultures may be fed with ECRA medium every other day until confluent. Confluent cultures may be tested for the presence of P-gp, CD105, Glu-1 and CD31 expression by flow cytometry (Figure 10C) and immunocytochemistry (Figure 10D) as well as transendothelial electrical resistance (TEER) and compared to blank medium (Figure 10E). For immunohistochemistry, cells were washed three times with 200 μl DPBS and then incubated with rabbit anti-P-gp antibody (1:50 in blocking buffer (10% FBS, 0.01% TritonX in DPBS)) overnight at 4°C. After washing three times with 200 μl DPBS, P-gp was stained with secondary antibody (1:1000, donkey anti-rabbit IgG Alexa Fluor 488 (Invitrogen)). Nuclei were stained with Hoechst3342 (Thermo Fisher) and images were acquired at 200× magnification by ImageXpress (Molecular Devices, LLC).

[0227] Example 5 – Generation of Microglia iPSCs maintained on MATRIGEL™ or vitronectin in the presence of E8 were adapted to hypoxia for at least 5-10 passages. Differentiation of 2D HPCs: Cells were split from subconfluent iPSCs and plated in amine culture dishes at a density of 250,000-500,000 cells / well in the presence of serum-free defined (SFD) medium supplemented with 5uM blebbistatin or 1uM H1152. 24 hours after plating, SFD medium supplemented with 50ng / ml BMP4, VEGF and FGF2 was added to the cultures. A complete medium change was performed the next day.

[0228] On day 5 of the differentiation process, cells were placed in medium containing 50 ng / ml Flt-3 Ligand, SCF, TP0, IL3 and IL6 with 5 U / ml heparin. Cells were fed every 48 hours throughout the differentiation process. The entire process was performed under hypoxic conditions. HPCs were quantified by the presence of CD43 / CD34 cells.

[0229] Differentiation of 3D HPCs: Cells were split from sub-confluent iPSCs and plated in spinner flasks at a density of 250,000-500,000 cells per ml in the presence of serum-free defined (SFD) medium supplemented with 5uM blebbistatin or 1uM H1152. 24 hours after plating, the SFD medium supplemented with 50ng / ml BMP4, VEGF and FGF2 was replaced. On day 5 of the differentiation process, cells were placed in medium containing 50ng / ml Flt-3 ligand, SCF, TP0, IL3 and IL6, along with 5U / ml heparin. Cells were fed every 48 hours throughout the differentiation process. The entire process was performed under hypoxic conditions. HPCs were quantified by the presence of CD43 / CD34. An overview and efficiency of the process is shown in (Figure 11) and the composition of the medium is shown in (Figure 12).

[0230] HPCs were placed on microglial differentiation medium MDM or 2xMDM (Figure 11). Cultures were fed every 48 hours. Purity markers of microglial cultures on day 23 of differentiation were quantified before and after cryopreservation (Figure 13A,B).

[0231] The purity of live and cryopreserved microglial cultures was assessed on day 23. Day 23 differentiated microglial cultures were harvested and stained for the presence of microglia-specific markers. The remaining cells were cryopreserved using a control rate freezer. Cryopreserved cells were thawed and stained for the presence of microglia-specific markers. Cell surface expression of CD45, CD33, TREM2, and CD11b (Figure 14A) and intracellular expression of PU.1, IBA, P2RY12, TREM2, and TMEM119 by flow cytometry (Figure 14B) for both sets. The results revealed that cryopreserved microglia retained their purity after cryopreservation.

[0232] HPCs were placed in culture medium to initiate microglial differentiation in the presence of MDM and intermittent feeding was performed with 2xMDM. Cells were cryopreserved on days 20, 23 and 26 of differentiation using a manual freezing protocol or a control rate freezer (CRF). Cryopreserved cells were transferred to liquid nitrogen for 1 week. Cryopreserved microglia were thawed and placed in Microglia Maturation Medium (MMM). Cultures were fed with fresh Microglia Maturation Medium every 48 hours. Cells were harvested on days 3, 5, 7, 10, 12 and 14 after thawing and the recovery of viable cells was quantified with respect to the initial plating number (Figures 15A-15C).

[0233] Cryopreserved HPCs were differentiated into microglia in the presence of MDM. The total viable numbers of input HPCs and output microglia were quantified. Process efficiency was calculated based on the purity and absolute number of TREM2-positive cells present at day 23 of microglial differentiation divided by the absolute number of input live HPCs (Figure 16).

[0234] Cryopreserved microglia at day 20 (Figure 17A), day 23 (Figure 17B) or day 26 (Figure 17C) of differentiation were thawed in Microglia Maturation Medium (MMM) and fed fresh medium every 48 h. Total viability and absolute cell numbers were quantified 3, 7 and 10 days after thawing. Data revealed a higher post-thaw recovery rate for day 23 microglia than day 26 microglia (Figures 17A-17C).

[0235] Next, functional assessment of cryopreserved microglia was performed on days 20, 23, and 26 of the differentiation process. Cells were thawed and plated at 15,000 viable cells / well in 96-well plates in the presence of 200 μl of Microglia Maturation Medium per well. Cells were treated with 1 μg / well of diluted opsonized or non-opsonized pHrodo Red BioParticles (Thermo Fisher #A10010, 2 mg per vial, stored at -20°C). Plates were placed on an IncuCyte and images of phagocytosis were taken at various time points up to 5 days after thawing. Cells cryopreserved using the control rate freezer method show stronger phagocytosis (due to higher cell viability (Figure 19)).

[0236] Functional assessment was extended to later time points post-thaw. Phagocytic potential was assessed on days 5, 7 and 14 post-thaw for cryopreserved microglia at days 20, 23 and 26 of differentiation using manual or control rate freezers assessed via live imaging on the IncuCyte system. Cryopreserved microglia were thawed and plated in MMM for 3 days. Viable cell counts were measured at the end of the 3 days as described in Figure 18B. 15,000 live cells were plated in a 96-well plate with diluted 1 μg / well opsonized or non-opsonized pHrodo Red BioParticles in the presence of 200 μl per well of Microglia Maturation Medium (MMM), plates were placed on an IncuCyte and images of phagocytosis were taken at various time points up to 5, 7 and 14 days post-thaw. Manual cryopreservation revealed a slower / right-shifted rate of phagocytosis in all conditions (due to lower cell viability).

[0237] The phagocytic index is a measure of phagocytic activity determined by counting the number of bacteria ingested per phagocyte during a limited period of incubation of a suspension of bacteria and phagocytes. The ability of cryopreserved microglia to phagocytose labeled bacterial particles was quantified by the ratio of number of phagocytic red objects / total live cells. This ratio was determined as the phagocytic index (Figure 21).

[0238] Cryopreserved microglia were thawed and plated at 15,000 viable cells / well in 96-well plates in the presence of 200 μl of Microglia Maturation Medium per well. Cells were treated with 1 μg / well of diluted opsonized or non-opsonized pHrodo Red BioParticles (Thermo Fisher #A10010, 2 mg per vial, stored at -20°C). Plates were placed on an IncuCyte and images of phagocytosis were taken at various time points up to 5 days after thawing. Cells cryopreserved using the control rate freezer method show stronger phagocytosis (due to higher cell viability (Figure 22)).

[0239] Next, differentiation of HPCs into microglia was further developed in a 96-well format suitable for screening applications in the absence of ECM. Differentiation was performed in ultra-low attachment (ULA), tissue culture (TC) and non-tissue culture (non-TC) vessels (Figure 23A). Cryopreserved HPCs were plated at 20,000–35,000 viable cells / cm on 96-well Primaria plates or ultra-low attachment tissue culture (TC) or non-tissue culture plates (non-TC) in the presence of 200 ul of microglial differentiation medium per well. 2 Cells were plated at a density of 100-200 μl per well (Figure 23B). For the next 23 days of differentiation, cells were fed with 50 μl of MDM medium per well every 48 hours. On day 23, cells were harvested with cold PBS and total viable cell numbers were quantified using an automated cell counter. Cells were stained for surface expression of CD11b, CD45, CD33, TREM2 and intracellular expression of TREM2, IBA, P2RY12, TMEM119 (Figures 24A-24B).

[0240] [Table 1]

[0241] Cytokines and chemokines released by cryopreserved microglia. Day 23 cryopreserved microglia were thawed in MDM medium and plated at 50,000 cells / well in Primaria 96-well plates. Cells were plated for 3 days before initiating stimulation with 100 ng / ml LPS and 50 ng / ml interferon gamma. Stimulation was performed in triplicate over a 24-hour period. Supernatants were spun down to remove cells and debris and immediately placed at -20°C. Supernatants were analyzed by multiplex Luminex assay.

[0242] Example 6 – Engineering iPSCs to generate variants that mimic neurodegenerative diseases The function of TREM2 was disrupted by introducing an indel into exon 2, leading to a frameshift and premature translational stop. A TAL-nuclease (hereafter Pair TREM2) was designed to bind to a DNA sequence centered at amino acid 58 within exon 2. The cell line used for the engineering was FCDI iPSC line 01279.107. TAL-nuclease mRNA and a co-selection plasmid expressing blasticidin resistance under the control of an SV40 promoter were electroporated into cells using a BioRad Gene Pulser Xcell system at settings of 125V / 950uF. Cells were plated and a short blasticidin selection was applied on days 1 and 2 after electroporation. Surviving cells were expanded and single cells were sorted into 96-well plates on day 7 after electroporation. After approximately 2 weeks, 81 clones were selected and genotyped by PCR and sequencing.

[0243] Seven of the 81 clones sequenced showed sequence alterations. Three clones contained one allele with a one base pair insertion, three clones contained one allele with a one base pair deletion, and one clone was a compound heterozygote with one allele containing a one base pair insertion and one allele containing a four base pair deletion. The seventh clone contained a 24 base pair deletion that was not expected to introduce a frameshift. Clones were expanded, cryopreserved, and subjected to sequence confirmation and karyotype analysis. After differentiation into microglia, two major clones were selected as examples of heterozygous or homozygous disruptions. The heterozygous clone 01279.1185 contained an allele with a 1 bp insertion, resulting in a frameshift at position 60 of TREM2, which then terminated 45 amino acids later. Homozygous clone 01279.1187 contained one allele with a 1-bp frameshift insertion at position 59, terminating 16 amino acids later, and a second allele with a 4-bp deletion causing a frameshift at position 59, terminating 46 amino acids later.

[0244] [Table 2]

[0245] [Table 3]

[0246] Example 7 - Generation of additional isogenic lines that mimic neurodegeneration: A Parkinson's disease model was produced by genetically engineering episomally reprogrammed iPSC01279 by nuclease-mediated homologous recombination and donor oligo SJD14-133. The resulting iPSC contained the SNP rs104893877, which changed amino acid 53 from alanine to threonine, resulting in the A53T variant of the alpha-synuclein gene (SNCA) and two silent mutations resulting in the SNCA A53T iPSC line.

[0247] An isogenic model to study Rett syndrome was generated using nuclease-mediated homologous recombination and the donor plasmid p1553, which inserted a series of stop codons before the methyl-CpG binding domain followed by a PGKp-puromycinR-SV40pA selection cassette flanked by LoxP sites. The MECP2 HM line, derived from the parent line 01279, provided a disease model for Rett syndrome.

[0248] Generation of HPCs and microglia from isogenically engineered iPSCs: Homozygous and heterozygous TREM2 KO iPSCs derived from 01279 iPSCs and SNCA A53T and MECP2 HM engineered lines derived from 01279 were acclimated and maintained to hypoxia by passaging for 10 passages in the presence of E8 and MATRIGEL™. Cells were karyotyped, iPSC banks were created, and HPC differentiation was initiated via the 3D HPC differentiation protocol. Cells were split from subconfluent iPSCs and plated in spinner flasks at a density of 250,000-500,000 cells per ml in the presence of serum-free defined (SFD) medium supplemented with 5 μM blebbistatin or 1 μM H1152. 24 hours after plating, SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 was replaced. On day 5 of the differentiation process, cells are placed in medium containing 50 ng / ml Flt-3 Ligand, SCF, TP0, IL3 and IL6, along with 5-10 U / ml heparin. Cells are fed every 48 hours throughout the 13-day differentiation process. The entire process is carried out under hypoxic conditions. HPCs were quantified by the presence of CD43 / CD34. HPCs are cryopreserved after MAC sorting using CD34 beads. Microglia were generated by thawing cryopreserved HPCs and placing the cells into a 23-day differentiation process as described in Example 5.

[0249] Cryopreserved microglia from day 23 wild type and TREM-engineered clones were thawed and the presence of TREM-2 expression along with CD45 was quantified by flow cytometry (Figure 26).

[0250] Cryopreserved day 23 microglia derived from isogenically engineered lines were thawed and stained for the presence of microglia-specific markers. Cells were stained to quantify cell surface expression of CD45, CD33, TREM2 and CD11b, as well as intracellular expression of PU.1, IBA, P2RY12, TREM2 and TMEM119 proteins by flow cytometry. Figure 26 summarizes the purity obtained with all four isogenically engineered iPSCs. The results demonstrate the generation of highly pure microglia from isogenically engineered iPSCs without any modification to the differentiation protocol.

[0251] The levels of soluble TREM2 (sTREM2) protein secreted by microglia after thawing were quantified using Simple Step ELISA (AbCam) from conditioned media collected from WT and TREM2 heterozygous and homozygous KO mutants (Figure 27A). WT and TREM2 KO microglia were thawed and plated at the same density in maturation medium in 96-well Primaria plates. Spent medium was collected on day 3 post-thaw and day 7 post-thaw. Cultures were half-fed with fresh maturation medium on days 3 and 5 post-thaw. The data revealed differences in the levels of soluble TREM2 between WT, heterozygous TREM2 KO and homozygous KO microglia. This assay can be used as a functional assay to distinguish between WT and TREM2 engineered iPSCs.

[0252] The levels of soluble TREM2 (sTREM2) protein secreted by microglia after thawing were quantified using Simple Step ELISA (AbCam) from conditioned media collected from WT and TREM2 heterozygous and homozygous KO mutants, MECP2HM and SNCA-A53T (Figure 27A). WT and TREM2 KO microglia were thawed and plated at the same density in maturation medium in 96-well Primaria plates. Spent medium was collected on day 3 post-thaw and day 7 post-thaw. Cultures were half-fed with fresh maturation medium on days 3 and 5 post-thaw. The data revealed differences in the levels of soluble TREM2 between WT, heterozygous TREM2 KO and homozygous KO microglia. This assay can be used as a functional assay to distinguish between WT and TREM2 engineered iPSCs. The release of soluble TREM2 was impaired in A53T-SNCA microglia, whereas MECP2HM microglia did not reveal any changes in the levels of sTREM2 released in the medium (Figure 27B).

[0253] Cytokines and chemokines released by isogenically engineered cryopreserved microglia. Day 23 cryopreserved microglia derived from WT, 1185 HT TREM2 KO, 1187 HO TREM2 KO A53T-SNCA and MeCP2HM microglia were thawed in MDM medium and plated at 50,000 cells / well in Primaria 96-well plates. To confirm M1-mediated responses, cells were plated for 3 days before starting stimulation with 100 ng / ml LPS. Stimulation was performed in triplicate over a 24-hour period. Supernatants were spun down to remove cells and debris and immediately placed at -20°C. Supernatants were analyzed by multiplex Luminex assay. The results of this multiplex Luminex assay are captured as a heatmap in Figure 27C. Engineered strains secreted higher levels of IL-6 compared to ANH controls. TREM2 HZ and TREM2 HO and MeCP2HM microglia released less TNF-alpha but increased levels of IL6 compared to ANH. A53T-SNCA microglia released similar levels of IL-6 and TNF-alpha compared to AHN control microglia.

[0254] All engineered strains released the M2 cytokine IL-10 when treated with an M1 stimulus (LPS). MECP2HM microglia released less IL-10 compared to AHN control microglia (Figure 27E). AHN and engineered microglia were able to release CCL2 / MCP-1, CCL20 / MIP-3 alpha, CCL4 / MIP-1 beta, CCL5 / RANTES, CX3CL1 / fractalkine, CXCL1 / GRO alpha, CXCL10 / IP-10, CXCL2 / GRO beta, IL-8 / CXCL8 in response to LPS stimulation. There were some inherent differences in the levels of cytokine release. TREM2HO revealed the highest levels of CCL4, the main analyte released during the development of Alzheimer's disease (AD). MECP2HM, TREM2HZ and TREM2HO microglia released high levels of CXCL1 / GRO. This represents an attempt to recruit the accessory cell type granulocytes that promote microbial killing and trigger inflammatory responses during phagocytosis. MECPHM microglia revealed spontaneous secretion of IL-8 / CXC18. This analyte is elevated in brain injury and induces the expression of proinflammatory proteases and MMP-2 and MMP-9. MECPHM microglia secreted high levels of IL-6. These results suggest that MECPHM are primed for proinflammatory responses. Engineered and AHN microglia released similar levels of PDL-1, CD40, FLT-3 and PDGFAA in the medium in response to LPS.

[0255] Cryopreserved microglia were thawed in maturation medium and allowed to recover for 48 hours before performing screening experiments (Figure 36). 5,000 microglia from TREM2 WT and TREM2 HOKO were plated in 40uL medium per well of a 384-well plate for 24 hours. In the first set (Plate 1), cells were pre-treated with compounds at a final concentration of 1uM. 24 hours after cells were treated with compounds, pHrodo-labeled amyloid beta was added to Plate 1 at a final concentration of 1uM and phagocytosis was acquired in IncuCyte S3 for 96 hours (Figures 37-39). In the second set (Plate 2), cells were plated for 24 hours and then treated with 1ug / ml LPS at a final concentration of 1ug / mL (Figures 40-42). 24 hours after exposure to LPS and 48 hours after initial plating, pHrodo-labeled amyloid beta was added to Plate 2 at a final concentration of 1uM. Cells were imaged using an IncuCyte once per hour for up to 96 hours. Phagocytosis data was acquired as total red object integrated intensity x μM2 / image. Final volumes for all treatments remained constant. The results of the screen are summarized in Figure 43.

[0256] To understand the cytokines required for microglia survival after thawing in maturation medium, a schematic matrix was designed with 32 different medium formulations (Figure 28). WT, 1185 HT TREM2 KO, 1187 HO TREM2 KO microglia were placed at a density of 15,000 live cells in 96-well plates in 250 μl of Microglia Basal Medium or MMM or Maturation Medium supplemented with a single cytokine (Figure 29), two cytokines (Figure 30), three cytokines (Figure 31) or four cytokines (Figure 32). The kinetics of cell survival was acquired with an IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells in the various medium compositions to acquire the number of dead cells over time. Images were acquired every 8 hours and the experiment was continued for 72 hours without intermittent feeding. The intensity of NucGreen Dead quantifies the dead cells in the culture.

[0257] WT, 1185 HT TREM2 KO, 1187 HO TREM2 KO microglia were plated at a density of 15,000 viable cells in 96-well plates in 250 μl of Microglia Basal Medium (Figure 33A), MMM (Figure 33B), Microglia Basal Medium supplemented with IL-34 (26C), Microglia Basal Medium supplemented with IL-34 (Figure 33D), Microglia Basal Medium supplemented with MCSF (Figure 33D) or Basal Medium supplemented with IL-34 only (Figure 33C) or a combination of MSCF or IL-34 and MCSF (Figure 33E). The kinetics of cell survival was acquired with an IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells in the various media compositions to acquire the number of dead cells over time. Images were acquired every 8 hours and the experiment was continued for 7 days without intermittent feeding. The intensity of NucGreen Dead quantifies dead cells in the culture.

[0258] Functional properties of WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia cryopreserved with pHrodo Red-labeled bacterial BioParticles and pHrodo Red amyloid beta were assessed on day 23. WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia were cultured in 96-well plates at 15,000–30,000 viable cells / cm in 250 μl of MMM (Figures 33A–B) or MDM-based (also known as Microglia Basal Medium) supplemented with MSCF alone (Figures 33C–D) or IL-34 (Figures 33E–F) or a combination of IL-34 and MCSF (Figures 33G–H) for 3 days after thawing. 2Cells were plated at a density of 1 μg / well. Cells were treated with diluted opsonized or non-opsonized pHrodo Bioparticles (Thermo Fisher #A10010, 2 mg per vial, stored at -20°C) (Figure 33A,C,E,G) or pHrodo Amyloid Beta (Figure 33B,D,F,H). Plates were placed on an IncuCyte and images of phagocytosis were taken at various time points up to 30 hours. WT and engineered microglia demonstrated phagocytic function post-thaw. Phagocytosis kinetics and efficiency differed between WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia.

[0259] The post-thaw purity of cryopreserved wild type (WT) microglia at day 23 in the presence of MMM or microglia basal medium supplemented with a combination of two key (IL-34, MSCF) cytokines in the maturation medium was determined (Figure 35). Purity was quantified at days 3, 7 and 14 post-thaw by harvesting cells and the purity of CD45, CD33, TREM2, CD11b, CX3CR1, P2RY12, TMEM119, IBA was determined by flow cytometry by harvesting cells at the end of the differentiation process and staining cells for cell surface and intracellular staining of markers. Cryopreserved microglia retain viability and purity in maturation medium supplemented with MSCF and IL-34. This simplified medium lends itself to the application of co-culture of cryopreserved microglia with neurons and astrocytes to develop brain organoid models to study the contribution of many SNPs and mutations associated with neurodegeneration.

[0260] Example 8 –Generation of disease-associated microglia from patient-derived iPSCs Recent genetic studies have shown that polymorphisms in several microglia-enriched genes are associated with altered risk of developing Alzheimer's disease (AD), Parkinson's disease (PD) and several neurodegenerative diseases. Summarizing the list of risk-associated SNPs from GWAS studies, a panel of end-stage microglia was generated from donors exhibiting mutations in TREM2, CD33 and ABCA7 along with APOE isoforms. Cryopreserved microglia from patients derived from iPSCs provide an in vitro tool to understand the complex interactions between human microglia, neurons and astrocytes in 2D or 3D organoid systems and create more accurate models to mimic neurodegenerative diseases (McQuade et al., 2019).

[0261] Generation of HPCs from episome-reprogrammed AHN and disease-specific iPSCs: Episome-reprogrammed iPSCs generated from normal and disease-specific donors were acclimated to hypoxia for at least 5-10 passages using E8 / MATRIGEL™ prior to banking source material for differentiation into hematopoietic cells and subsequent differentiation into microglia. Genotypes of the panel of iPSCs are shown in Table 4. iPSCs derived from all donors were karyotyped and iPSC banks were created to initiate HPC differentiation via the 3D HPC differentiation protocol, performed as described in Example 5. Microglia were generated by thawing cryopreserved HPCs and placing the cells in a 23-day differentiation process as described in Example 5. Cryopreserved day 23 microglia derived from various donors were thawed and stained for the presence of microglia-specific markers. Cells were stained for cell surface expression of CD45, CD33, TREM2 and CD11b and intracellular expression of PU.1, IBA, P2RY12, TREM2 and TMEM119 proteins by flow cytometry. Table 5 summarizes the purity obtained across all AHN and disease-associated microglia (DAM). Results show that highly pure microglia are generated from a panel of healthy and disease-specific donors with no modifications to the differentiation protocol.

[0262] [Table 4]

[0263] [Table 5]

[0264] Levels of soluble TREM2 (sTREM2) protein secreted by microglia after thawing were quantified from conditioned media collected from microglia generated from a panel of iPSC donors using Simple Step ELISA (AbCam) (Figure 45). Microglia generated from apparently healthy normal and disease-specific donors were thawed and plated at equal density in maturation medium in 96-well Primaria plates. Spent media was collected on day 3 post-thaw and day 7 post-thaw. Cultures were half-fed with fresh maturation medium on days 3 and 5 post-thaw.

[0265] The data revealed differences in the levels of soluble TREM2 between various samples of microglia derived from donors exhibiting the R47H genotype, reaching the highest soluble TREM levels at 3 days post-thawing and remaining high at 7 days post-thawing. Although this donor was asymptomatic and therefore classified as AHN, iPSC-derived microglia secreted high levels of sTREM. This data is consistent with the high levels of sTREM2 observed in the cerebrospinal fluid of Alzheimer's disease patients and associated with this mutational status (Cheng et al., 2016). This data is a strong example for the use of iPSC-derived microglia in designing predictive kits to screen for the onset of neurodegenerative diseases. Younger donors with the APOE4 / 4 / genotype, indicative of the onset of AD, showed higher levels of soluble TREM compared to older donors with the same genotype. The presence of SNPs in the CD33 or ABCA7 genes did not appear to enhance the release of soluble TREM in the supernatant medium. AHN donor 12068 showed high levels of soluble TREM at 3 and 7 days after thawing.

[0266] Neuroinflammation contributes to the progression and pathogenesis of many neurodegenerative diseases. Microglia and astrocytes present in the brain release cytokines and can play both pro- and anti-inflammatory roles in the brain depending on the stimuli and microenvironment. This variation between pro- and anti-inflammatory profiles has been associated with the development of AD and other neurodegenerative diseases. To quantify the levels of cytokines and chemokines released by disease-associated microglia (DAM), cryopreserved AHN and DAM microglia were thawed in MDM medium and plated at 50,000 cells / well in Primaria 96-well plates. After plating the cells for 3 days, stimulation was initiated with 100ng / ml LPS to confirm M1-mediated responses or 10ng / ml IL-4 with 10uM dBu-cAMP to trigger M2-specific responses. Stimulation was performed in triplicate over a 24-hour period. The supernatant was spun down to remove cells and debris and immediately placed at -20°C. Supernatants were analyzed in a multiplex Luminex assay, the results of which are captured as a heat map in Figure 27C.

[0267] The chemokines CCL1, CCL2, CCL3, CCL4, CCL8, CCL11, CCL13, CCL17, CCL18, CCL20, CCL22, and CCL24 function as chemoattractants, mediate the recruitment of myeloid cells, granulocytes, lymphoid cells, or neural progenitor cells to areas of inflammation, enhance phagocytic responses, and are commonly upregulated in Alzheimer's disease (AD) or multiple sclerosis. In response to LPS or dBu-cAMP, microglia from APOE E4 / E4, AP0E E2 / E4, TREM2 R47H, ABCA7 G1527A, and CD33 (harboring the rs429358 SNP) strains released higher levels of all these analytes compared to the AHN strain. The fold increase varied between the various microglial genotypes from 0.1-fold to 7-fold. This data from disease-associated microglia confirms previous findings showing increased CCL2 and CCL5 expression in AD brain samples. Expression of CCL2 in the brain and cerebrospinal fluid (CSF) has been reported as a reliable predictor of AD severity by Westin et al.

[0268] Microglia derived from APOE E4 / E4, AP0E E2 / E4, TREM2 R47H, ABCA7 G1527A and CD33 (harboring rs429358 SNP) had slightly elevated levels of soluble CD163, a marker of inflammation and inflammatory diseases associated with M2 polarization. Release of sCD163 prevents monocyte hyperactivation and reduces secretion of proinflammatory cytokines TNF-alpha, IL-1beta, IL-6 and IL-8. A similar trend was observed with chitinase-3, which plays a role in tissue remodeling during neuroinflammation (Melief et al., 2012; Minett et al., 2016).

[0269] PD-L1 and its receptor PD-1 induce inhibitory signals that regulate the balance between T cell activation, tolerance, and immune-mediated tissue damage. In response to LPS, APOE E4 / E4-derived microglia were not increased compared to AHN-derived microglia. TREM2 R47H, APOE E2 / E4, and CD33 (harboring the rs429358 SNP)-derived microglia showed an increase compared to the AHN lineage. In response to IL-4 + dBu-cAMP, APOE E4 / E4, TREM2 R47H, and ABCA7 G1527A showed a slight increase over the AHN lineage, but APOE E2 / E4-derived microglia showed a 7-fold increase compared to AHN-derived microglia.

[0270] APOE E4 / E4, AP0E E2 / E4, TREM2 R47H, ABCA7 G1527A and CD33 (containing rs429358 SNP) derived microglia released slightly elevated levels of soluble fractalkine, a soluble chemokine that promotes chemotaxis, survival and enhances neuroprotection by reducing levels of TNF-α and nitric oxide during neuroinflammation.

[0271] Microglia from APOE E4 / E4, AP0E E2 / E4 and ABCA7 G1527A released high levels of CXCL1 / GROalpha in response to both LPS and dBu-cAMP, whereas microglia from TREM2 R47H and CD33 showed only a small increase in the levels of this cytokine released, implying a correlation of this analyte with APOE and ABCA7 genotypes. Recent studies have shown that CXCL1 may contribute to the inflammatory response in the development of AD, but not as a potential genetic factor predisposing to AD in the pathogenesis of this disease. Under physiological conditions, CX3CR1 maintains microglial homeostasis by limiting their activation. The high levels of this cytokine released after stimulation indicate the occurrence of a rescue mechanism by CX3CR1 to maintain disease-related microglial homeostatic function associated with APOE or ABCA7 G1527A genotypes. Instead, high levels of CX3CR1 secreted by APOE E4 / E4 and ABCA7 G1527A-activated microglia may be a signal that promotes neuronal degeneration ( Atagi et al., 2015 ; Wolfe et al., 2018 ).

[0272] Microglia from APOE E4 / E4 and AP0EE2 / E4 released high levels of IL-6 in response to both LPS and IL4 / dBu-cAMP, while all other genotypes secreted similar levels of IL-6 as AHN. IL-6 secretion attracts granulocytes and promotes a cell-mediated humoral Th2 response, leading to inflammation. This mechanism also supports the greater neuroinflammation associated with the APOE E4 / E4 genotype.

[0273] Microglia from ABCA7 G1527A released high levels of IL-1 beta and IL-1 alpha in response to LPS, whereas the other genotypes secreted levels comparable to AHN microglia.

[0274] APOE E4 / E4, AP0E E2 / E4 and ABCA7 G1527A derived microglia also released higher levels of IL-8 / CXCL8 in response to LPS than dBu-cAMP. ABCA7 G1527A derived microglia released higher levels of IL-8, suggesting the development of a proinflammatory response that contributes to brain injury.

[0275] Microglia also secrete proteolytic enzymes and matrix metalloproteinases that may clear Aβ deposits and limit the AD process, playing a neuroprotective role in AD. APOE E4 / E4, AP0E E2 / E4, TREM2 R47H, ABCA7 G1527A and CD33 (harboring rs429358 SNP)-derived microglia showed slightly elevated levels of MMP-9 and MMP-12 compared to AHN-derived microglia.

[0276] R47H TREM2-derived microglia released 7-fold more IL-12 p70 compared to AHN-derived microglia when stimulated by LPS or M1 stimulation. On the other hand, AP0E E2 / E4 released 7-fold more IL-12 p70 compared to AHN-derived microglia in response to IL4+dBu-cAMP or M2 stimulation. The other genotypes revealed a slight increase in IL-12 secretion levels. Primary microglia produce IL-12 in the brain and control immune responses during infection or in Th1 cell-mediated autoimmune diseases of the CNS. The increased levels of IL-12 by R47H TREM2-derived microglia imply a strong activation of NK and T cell cytotoxic activity.

[0277] Finally, APOE E4 / E4, AP0E E2 / E4, ABCA7 G1527A-derived microglia released similar or slightly higher levels of IL-13, IL-18, IL-23 and alpha-synuclein, indicating a lack of correlation between these analytes and the above genotypes.

[0278] Neuroinflammation is an important contributor to the pathogenesis and progression of Alzheimer's disease (AD). The combination of several inflammatory mediators creates unique signatures associated with specific SNP mutations. Using iPSC-derived microglia from disease-specific donors, we can determine the key signature cytokines associated with different microglial SNP and mutation-associated genotypes.

[0279] Microglial phagocytic function is important to maintain neuroprotective effects. Microglial-mediated phagocytosis can be impaired by disease-specific SNPs or mutations, which may affect important homeostatic mechanisms in the brain. To compare the role of disease-associated SNPs on microglial phagocytic function, we assessed the phagocytic function of disease-associated microglia (DAM) in the presence of pHrodo-labeled bacteria Staphylococcus aureus and amyloid beta. This function can be used for high-throughput screening applications.

[0280] Among these microglia-expressed disease-related genes, sequence variants in genes encoding triggering receptor expressed in myeloid cells 2 (TREM2) and APO E isoforms are associated with an abnormally increased risk of AD. APOE is the major cholesterol carrier in the brain and plays a key role in lipid transport, cholesterol homeostasis and synaptic stability. Anti-ApoE immunotherapy inhibits amyloid accumulation and deposition, further supporting the role of ApoE in Aβ aggregation and clearance. ApoE expression has been shown to be significantly upregulated in disease-associated microglia. ApoE4 / E4 isoforms have been shown to essentially affect microglial physiology by upregulating motility and phagocytosis in vitro. Overexpression of ApoE4 / E4 has been shown to decrease amyloid beta (Abeta) uptake in contrast to other isoforms. The role of ApoE2, the third most common major ApoE isoform, in neurodegeneration has been shown to delay the onset of familial AD. ApoE isotype-specific effects on iPSC-derived microglial function have not been thoroughly investigated so far.

[0281] TREM2 senses lipids and mediates phagocytosis of myelin. Loss-of-function (LOF) variants of TREM2 are associated with increased amyloid plaque seeding, reduced amyloid clusters, and additional interactions with the ApoE-triggered signaling cascade, leading to reduced microglial clusters and ApoE accumulation in amyloid plaques with dysfunction.

[0282] To compare the role of disease-associated SNPs on microglial phagocytic function, we assessed the phagocytic function of disease-associated microglia (DAM) in the presence of the bacteria Staphylococcus aureus and amyloid beta.

[0283] On the same lineage, ATP-binding cassette transporter A7 (ABCA7) has been identified as a susceptibility factor for late-onset Alzheimer's disease in genome-wide association studies. ABCA7 has been shown to mediate phagocytosis and affect membrane trafficking. ABCA7 is strongly associated with AD. Phagocytic clearance of amyloid beta is impaired in Abca7- / - mice. iPSCs derived from patients carrying a missense variant associated with the G1527A substitution in ABCA7 provide BCA7, which plays a role in regulating brain amyloid beta homeostasis in response to altered phagocyte function.

[0284] CD33 is an immunomodulatory receptor associated with Alzheimer's disease (AD) susceptibility through regulation of microglial phagocytosis. Because TREM2 interacts downstream of CD33 in regulating microglial physiology and metabolism, we could use WT and iPSC-derived microglia expressing the CD33 rs3865444 SNP to validate the role of CD33 in association with impaired phagocytic function (Caldeira et al., 2017).

[0285] Cryopreserved AHN and DAM microglia were thawed, cultured in maturation medium for 3 days, and exposed to pHrodo-labeled amyloid beta and pHrodo S. aureus. Phagocytosis kinetics were measured using an IncuCyte live cell analysis system. Total red object integrated intensity was used to quantify functional responses.

[0286] TREM2 R47H and ABCA7-G1527A microglia revealed a stronger phagocytic capacity against the bacterium Staphylococcus aureus (S. Aureus) compared to AHN microglia. CD33 (harboring the rs429358 SNP) microglia revealed a comparable phagocytic capacity against the bacterium Staphylococcus aureus (S. Aureus) compared to AHN microglia. TREM2 R47H, ABCA7-G1527A and CD33 (harboring the rs429358 SNP) microglia revealed a reduced strength of phagocytosis in the presence of amyloid beta compared to AHN microglia.

[0287] CW13030EE1 APOE 4 / 4 showed higher intensity of phagocytosis by S. aureus and amyloid beta compared to AHN microglia. CW13098AA1 APOE 4 / 4 showed lower intensity of phagocytosis by both S. aureus and amyloid beta compared to AHN microglia. CW13005AA1 APOE 2 / 4 microglia revealed stronger phagocytic ability for amyloid beta and reduced phagocytosis for S. aureus compared to AHN-derived microglia. CW13074AA1 APOE 4 / 4 microglia showed similar phagocytic tendency as AHN microglia for S. aureus and slightly enhanced phagocytosis from amyloid beta to AHN microglial cell lines.

[0288] A comprehensive understanding of genetic alterations targeting homeostatic, pro- and anti-inflammatory microglial subtypes can provide novel biological insights and facilitate target prioritization for immune-modulatory therapeutic approaches in neurodegenerative diseases.

[0289] Example 9 – Further characterization of microglia Extracellular nucleotides such as ATP and ADP are known to induce receptor-mediated pathways called the "purinergic signaling" pathway. Physiological processes such as tissue homeostasis, wound healing, neurodegeneration, immunity, inflammation, and cancer are regulated by purinergic signaling. Extracellular ATP and P2 receptors are important for the activation mechanism of microglia. P2X receptors are ionotropic receptors that bind ATP or its derivatives. One of the P2Y receptors is a G protein-coupled receptor that responds to ADP. Under pathological conditions, nucleotides such as ATP are released or leaked from damaged cells and function as "find me" or "eat me" signals, triggering process elongation, chemotaxis, and phagocytosis by microglia. Activation of P2 receptors also induces cytokine production from microglia, including interleukin 1b (IL-1b), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNFα). Such proinflammatory mediators have been shown to dynamically alter the expression and function of G protein-coupled receptors (GPCRs) in astrocytes.

[0290] The microglial purinergic receptor responses were characterized. Microglia were thawed and reconstituted in microglial differentiation medium to a cell suspension of 200,000 cells / well. 15 μl of the microglial cell suspension was added per well of a 384-well plate. Cells were treated with different doses (0–1000 nM) of BzATP and ADP. The responses to BzATP and ADP were characterized by the induction of AZ11645373 (P2X7 antagonist) and AZD1283 (P2Y 12 The activity of FLPR Calcium was also measured in the presence of 10 μl of 4X stock of compound or inhibitor for all treatments. Cells were exposed to compound for 30 minutes in the presence or absence of inhibitor prior to assay. One bottle of FLPR Calcium 6 was reconstituted to 11 ml in Assay Buffer B and 15 μl of dye solution was added to the cell suspension in the presence of compound. Cells were incubated at 37°C for 1.5 hours and imaged on the FDSS μCELL system.

[0291] Figure 44A shows microglia with ATP / BzATP total traces and Figure 44B shows microglia with ATP / BzATP sample traces. Figures 44C-F show microglial responses to BzATP in the presence of P2X7 antagonist AZ11645373, ADP, BzATP and BzATP in the presence of P2X7 antagonist A438079. Figure 44G shows dose-dependent responses to demonstrate functional ADP-dependent responses of microglia in the presence of AZD1283.

[0292] Example 10 –Generation of neural progenitor cells from iPSCs Successful development of in vitro disease models depends on the availability of large amounts of end-stage lineages derived from patient-derived iPSCs. Neural progenitor cells (NPCs) are self-renewing precursor cells with the capacity to generate neurons and glia (Breunig et al., 2011). There are many established protocols with varying efficiency for generating NPCs from primary neural cells and iPSCs (Shi et al., 2012a, Shi et al., 2012b). The majority of recent protocols rely on inhibition of SMAD signaling pathways. This application describes a simple protocol to generate NPCs across different iPSC lineages, taking advantage of the spontaneous drift of iPSCs towards the ectoderm, without using a dual SMAD inhibition pathway. The rationale for generating this cell type is to generate long-term co-culture assays that, in combination with iPSC-derived microglia, mimic human brain development and the complex cell-cell interactions between neural lineages, microglia, endothelial cells, pericytes, and astrocytes in culture dishes from normal and disease-specific iPSC cells.

[0293] In this study, multiple episome-reprogrammed iPSC lines were maintained on MATRIGEL™, laminin or vitronectin coated plates and E8 medium. iPSCs were maintained under hypoxic conditions prior to the initiation of differentiation to generate neural progenitor cells. To initiate neural precursor differentiation, iPSCs were harvested and plated at 15K / cm on MATRIGEL™, laminin or vitronectin plates using E8 medium in the presence of rock inhibitors. 2 iPSC cultures were seeded at 100°C for 1 h. Cells were placed in fresh E8 medium in the absence of rock inhibitors for the next 48 h. The next step included a preconditioning step, which involved placing iPSC cultures in DMEMF12 medium supplemented with 3 uM CHIR for 72 h and changing the medium daily under normoxic conditions. At the end of the preconditioning step, cells were harvested and re-plated in 2D format on MATRIGEL™, laminin or vitronectin plates at 30K / cm2, or 3D aggregates were generated using ultra-low attachment plates or spinner flasks at a density of 300,000 cells per ml in the presence of rock inhibitors. Cultures were fed every other day with E6 medium supplemented with N2 for the next 8 days under normoxic conditions. At day 14 of differentiation, cultures were harvested and individualized using TrypLE. Cells were stained for the presence of Tra-162, CD56, CD15 by cell surface staining for flow cytometry, and for the presence of Sox1, Nestin, β3 microglobulin and Pax-6 expression by intracellular staining for flow cytometry. The different steps involved in the generation of NPCs are outlined in FIG. 45A. The appearance of NPC markers at different days of differentiation across the three iPSC lines is summarized in FIG. 45B. CD56 was used as a marker for NPCs obtained in this manner. Cells were cryopreserved using CS10 and retained purity and proliferation potential after thawing. NPCs were placed into downstream differentiation protocols to generate astrocytes and Pan neurons.

[0294] Astrocytes were differentiated from NPCs following the protocol outlined by Julia et al. On day 14, NPC cells were plated at 15k / cm2 onto MATRIGEL™ coated 6-well plates in Science Cell astrocyte medium. Plates were given a complete medium change every 2 days. Every 6 days or when cultures were approximately 90% confluent, plates were harvested using Accumax and plated at 15k / cm2 onto MATRIGEL™ coated 6-well plates. 2 Cultures were fed and replated as above for four passages. At the end of the fourth passage, cultures were stained for surface markers, CD44 and glutamate aspartate transporter (GLAST) and intracellular markers, glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1), glutamine synthetase (GS), aquaporin 4 (AQP4), and S100 calcium binding protein B (S100β) (Figure 45C).

[0295] Cortical glutamatergic neurons were generated from NPCs using the protocol developed by Slosarek et al. Day 14 NPCs were plated in E6 medium supplemented with 1 μM cyclic AMP, 10 ng / ml brain-derived neurotrophic factor (BDNF) and 10 ng / ml glial-derived neurotrophic factor (GDNF) for 30 days. The medium was then changed to cortical neuronal differentiation medium (E6 medium, supplemented with 1 μM cyclic AMP, 10 ng / ml BDNF, 10 ng / ml GDNF, 100 ng / ml insulin-like growth factor-I and 2% B27) for an additional 30 days (Brennand et al., 2011). Cortical glutamatergic neurons were observed from days 14 to 36 in different iPSC lines. Purity of the neuronal cultures was confirmed by staining for the presence of β3 tubulin, MAP2 expression.

[0296] Example 11 - Tripartite culture containing neurons, microglia and astrocytes Tripartite cultures were set up by first maturing iCell microglia. Cryopreserved microglia were thawed in ultra-low attachment (ULA) 6-well plates for 3 days prior to tripartite culture. Cells were placed in the presence of microglia maturation medium containing IL-34 (100ng / mL; Peprotech / 200-34), TGFβ1 (50ng / mL; R&D Systems / 240-B), M-CSF (25ng / mL; Peprotech / 300-25), CD200 (100ng / mL; Acro Biosystems / OX2-H5228), and fractalkine (100ng / mL; Peprotech / 300-31).

[0297] [ka]

[0298] CD200, also known as OX-2 membrane glycoprotein (OX-2), is a type 1 membrane glycoprotein that contains two immunoglobulin domains, one Ig-like C2-type (immunoglobulin-like) domain and one Ig-like V-type (immunoglobulin-like) domain, and thus belongs to the immunoglobulin superfamily. CD200 / OX-2 is widely expressed on multiple cell types. CD200 interacts with a structurally related receptor (CD200R) expressed primarily on myeloid cells and is involved in the regulation of macrophage and mast cell function. CD200 also plays a role in the prevention of graft rejection, autoimmune diseases, and spontaneous abortion.

[0299] Fractalkine is a CX3CL chemokine that signals through the CX3CR1 receptor. It has been shown to chemoattract monocytes, microglial cells, and NK cells. Fractalkine is the only CXC3C chemokine to date that contains three amino acid residues between the first and second cysteine ​​residues of the chemokine domain.

[0300] 3 days after thawing, microglial cultures were harvested, washed, and the cell pellet was diluted with triplicate culture medium (Table 6) to obtain 7,500 cells per 70 µL well of a 96-well plate. Cells may be seeded at a density of 7,500 cells per well of a 96-well plate.

[0301] [Table 6]

[0302] Cryopreserved GABA neurons were thawed and resuspended to obtain 50,000 cells per 70 μL well of a 96-well plate in triplicate culture medium. Neurons may be seeded at a density of 50,000 cells per well of a 96-well plate. Similarly, cryopreserved astrocytes were thawed and diluted in triplicate culture medium to a density of 8,000 cells per 70 μL well of a 96-well plate. Astrocytes may be seeded at a density of 8,000 cells per 70 μL well of a 96-well plate.

[0303] The day before setting up the co-cultures, plates were coated with PEI / Geltrex solution (PEI at a concentration of 0.07% and Geltrex at concentrations of 12–18 ng / mL) and rinsed before plating the cells of interest. All cell types were plated to generate mono-, bi-, or tri-partite cultures. On the day of plating, 70uL of microglia, 70uL of GABA neurons, and 70uL of iCell astrocytes were each adjusted to the densities listed above and plated into each well. Cells were spread by gently shaking the plate on a flat surface and cultured at 37°C and 5% CO2. Cells were fed with half the tri-partite culture medium from the plate every 4 days. Combinations of mono-cultures of neurons, astrocytes, and microglia and bi-partite cultures of neurons and astrocytes, or astrocytes and microglia, or neurons and microglia were also generated at the same densities. Mono-, bi-, and tri-cultures were maintained in tri-culture medium and the correct ratios of cell types for 14 days before end-stage staining or assays were performed. A schematic of this set-up is shown in (Figure 66).

[0304] The 14-day cultures were stimulated with LPS for 24 hours, and cultures with and without stimulation, as well as combined mono- and bipartite cultures, were fixed and stained using Pan Neuronal Marker (1:1500; Millipore, Cat: MAB2300), anti-Iba1 (1:500; Wako Chemicals, Cat: 019-19741), and anti-GFAP (1:500; Abcam, Cat: ab4674) to detect the presence of the solution. Images of mono- (Figure 50A), bi- (Figure 50B) and tripartite cultures (Figure 50C), a collage of images in tripartite culture medium with and without microglial factors (TGFβ, IL-34, and MSCF) are shown (Figure 50D), and finally a high-resolution image of the tripartite culture is shown (Figure 50E). All images were acquired using an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices). Quantification of viable cells in triplicate cultures was performed by FUJIFILM AI-based image analysis software (Figures 51A-51B).

[0305] Disease-related differences were examined in tripartite cultures of microglia, astrocytes and neurons by replacing apparently healthy microglia with TREM2HZ and TREM2HO microglia. Comparative analysis of various cytokines and chemokines released by monocultures, bicultures and combined tripartite cultures was assessed in the presence and absence of LPS stimulation. A schematic of the experimental design is shown in (Figure 52).

[0306] Co-cultures were maintained for 14 days before initiating stimulation with 1 μg / mL LPS. Stimulation was performed twice over 24 hours. Supernatants were spun down to remove cells and debris and immediately placed at -20°C. Supernatants were analyzed in triplicate by multiplex Luminex assay.

[0307] The results of the Luminex assay are presented as heat maps in Figures 53-56. A compilation of cytokines released by reactive (CX3CL1) and non-responsive (FGF2) astrocytes is summarized in Figure 53. A compilation of all M1 factors released into the medium (TNF alpha, IL-6, CCL2, CCL3, CCL4, IL-1 beta, IL-12, IL-13, IL-8, Interferon gamma, IL1-Alpham FAS Ligand, IL-2, GMCSF, Granzyme B, ICAM-1, CXCL11) is summarized in Figure 54. A compilation of M2 (anti-inflammatory) factors (IL-4, IL-10, IL-21, VEGF, CCL5, IL-17, IL1-RII, GCSG, CXCL5) is summarized in Figure 55. The release of the C3 complement system is summarized in Figure 56.

[0308] Microglia influence neuronal network activity on microelectrode arrays (MEAs). Synchronized burst co-cultures of Gluta neurons and astrocytes (cell ratios ranging from 4:1 to 6:1) were established after 2 weeks in BrainPhys medium supplemented with B27, NSS, N2, and laminin. Subsequent addition of microglia to existing cultures at similar cell numbers as astrocytes (MGL:ASC ratios ranging from 0.25:1 to 1:1) at weeks 2, 3, 4, or beyond resulted in changes in network burst architecture that were visualized and quantified on MEAs. Microglial influences can be short-lived or long-lasting and can be strongly influenced by medium and supplements, thereby offering various strategies to improve neuronal networks (Figure 57).

[0309] 3D or 2D cultures of microglia, astrocytes and glutan neurons can be grown in BrainPhys Neural Medium supplemented with B27 containing vitamin A, 0.5 mM Glutamax N2 Plus medium supplement, 460 μM thioglycerol, 1X insulin-transferrin-selenium, 5.4 μg / ml human insulin solution, 10-25 ng / ml BDNF, 10-25 ng / ml GDNF, 1 mM creatine, 200 nM L-ascorbic acid, 1 μg / ml laminin or fibronectin, 5-15 ng / ml TGF-b, 100 ng / ml human IL-34, 100 ng / ml human M-CSF, 1.5 μg / ml cholesterol, 1 ng / ml 9 or 11 eicosenoic acid, and 100 ng / ml oleic or linoleic acid.

[0310] Microglia exhibit robust phagocytosis of various substrates (bioparticles, amyloid beta, tau, synaptosomes, etc.) when cultured alone under standard conditions. A panel of iPSC-derived microglia (including TREM2 HZ and TREM2 HO) for disease modeling yielded distinct characteristics and kinetics in phagocytosis assays. Creation of more complex bi- and tri-partite cultures with GABA or gluta neurons and astrocytes resulted in enhanced phagocytic activity (Figure 58). This platform approach can be used to investigate the distinct mechanisms of TREM2 variants on microglial biology, phenotype, and function.

[0311] To study the role of microglia and astrocytes in neuronal MEA function, 3D tripartite culture spheroid formation with astrocytes, microglia, and Gluta Neurons was performed. Cryopreserved microglia, astrocytes, and Gluta Neurons were thawed in NB complete medium supplemented with IL-34 and MCSF (NB+Glutamax+B27-VitA+NSS). A wide range of ratios was tested between the three cell types, and the representative images shown in Figure 59 were generated using a ratio of 5k:5k:30k (Microglia:Astrocytes:Gluta Neuron). Spheroids were formed in S-Bio V-bottom 96-well plates in NB complete medium supplemented with IL-34 and MCSF. After 48 hours, the medium was switched to Brainphys complete medium (Brainphys+N2+B27-vitA+NSS) and the cultures were maintained for 2 weeks.

[0312] Functional assessment of calcium transients was performed in tripartite spheroids at days 7 and 15 in the presence of all three cell types. Calcium transients were measured using EarlyTox calcium dye (Molecular Devices) and recorded with a CLARIOstar instrument. Robust spontaneous calcium oscillations were detected in tripartite spheroids as early as day 7 after assembly, revealing the presence of a fully functional neuronal network as shown in Figure 60. Calcium oscillations can be detected by fluorescent dyes that change emission with the voltage of calcium concentration. Instruments that can detect this are FDSS, Flipper, or time-lapse confocal imaging.

[0313] Example 12 - Setting up a blood-brain barrier model using cryopreserved BMECs, pericytes and astrocytes A blood-brain barrier (BBB) ​​model was generated using cryopreserved iPSC-derived BMECs, pericytes, and astrocytes. The sequence of steps involved in setting up a sandwich BBB with a specific ratio of all three cell types is shown in Figure 61. The details of the setup are described as follows: The apical end (blood side) of the transwell was coated with collagen IV (200-400 μg / mL) / fibronectin (50-100 μg / mL) and left overnight at 4 °C or incubated at 37 °C for 4 h. The entire plate with the lid was inverted and the base of the plate was removed without disturbing the transwell. The basolateral side (brain side) of the inverted transwell was coated using 0.1% gelatin for 30-60 min at room temperature. The base of the plate was replaced using a spacer to ensure that the coated basolateral side of the transwell was not disturbed. The transwell was covered to reduce evaporation of the coated apical and basolateral sides. Astrocytes and pericytes were thawed in IMDM medium containing 10% FBS and spun at 600g for 8 minutes. Cells were then counted and mixed at a 1:2 ratio in astrocyte:pericyte medium (A:P medium) containing Y-27632 (Table 7). Astrocytes were cultured at 333,333 cells / cm. 2 and pericytes were plated at 666,666 cells / cm 2The 0.1% gelatin was removed from the inverted basolateral side of the transwells and the combined cell suspension was plated on the inverted basolateral side of the transwells at 100-170 μL per transwell. The base of the plate was replaced on the spacer on the inverted transwells and the plate was incubated in a normoxic atmosphere (5% O2) for 4 hours. The plate was then placed in a BSC and the plate base and spacer were removed. The plate base was then replaced and the plate was inverted back to its original orientation. The collagen / fibronectin was removed from the apical side of the transwells and each well was added 300 μL to the apical side of the transwell and 1 mL of A:P medium to the well. The plate was then incubated overnight at 37°C in a normoxic atmosphere. BMECs were thawed and placed in EFRA2+Y-27632 at a 1:1 ratio. A: P medium was removed from the apical side of the transwell and BMECs were placed at 1.3 × 10 in 300 μL EFRA2 + Y-27632 onto the apical side of the transwell. 6 / cm 2 The medium in the wells was aspirated, and 1 mL of EFRA2+Y-27632 was added. The plate was placed in a normoxic atmosphere at 37°C.

[0314] Figure 62 shows thawed and cultured 1.3 × 10 cells in BMEC medium on days 3 to 8 after thawing. 6 cells / cm 2 Figure 63 shows the TEER function of cryopreserved BMECs seeded on Corning transwell inserts coated with FN / ColIV at 20 °C. Figure 63 shows the TEER function of cryopreserved BMECs and pericytes. Figure 64 shows the TEER function of cryopreserved BMECs and astrocytes and finally, Figure 65 shows the TEER function of cryopreserved BMECs, pericytes and astrocytes. The ratios of all cell types used in the triplicate culture setup are shown in Table 8.

[0315] [Table 7]

[0316] [Table 8]

[0317] Example 13 - Characterization of the role of TREM2 in microglia GWAS in AD have identified TREM2 as a major regulator of AD risk. Heterozygous mutations in TREM2 lead to increased AD risk, whereas homozygous mutations cause a neurological condition known as Nasu-Hakola disease. Thus, although the CNS is affected in both cases, the pathobiology and clinical manifestations of heterozygous and homozygous mutations are distinct. To better understand the role of TREM2 mutations on AD risk, we generated heterozygous TREM2 loss-of-function microglia. Validation of partial loss of TREM2 microglia identified pathways unique to HetTREM2 KO, supporting the idea that heterozygous TREM2 KO microglia better model the phenotype resulting from inheritance of TREM2 mutations leading to increased AD risk.

[0318] Use of heterozygous TREM2 LOF microglia to identify druggable pathways in AD. Heterozygous mutations in TREM2 cause AD, whereas homozygosity causes Nasu-Hakola disease. Examination of HZ and HO TREM2 KO lines compared to AHN isogenic microglia identified unique pathways altered by partial loss of TREM2 function in microglial mutants where complete loss of TREM2 function was not evident or absent. For example, gene expression of SREBF2, the master transcriptional regulator of cholesterol biosynthesis, was significantly decreased with a concomitant decrease in cholesterol biosynthetic enzyme genes, suggesting downregulation of cholesterol synthesis due to partial, but not complete, loss of TREM2 function (Figure 67). Furthermore, the HZ TREM2 phenotype identified reprogramming of fatty acid metabolism / catabolism, indicating that TREM2 signaling plays a critical role in controlling lipid homeostasis in microglia. Thus, a unique pathway was discovered only in the context of partial loss of function of TREM2 that may serve as a novel therapeutic target for restoring microglial function and homeostasis in AD and other neurological diseases.

[0319] Targeting identified pathways through heterozygous loss of TREM2 function to improve liver function, lung function, vision loss, and reduce atherosclerosis. Given that TREM2 is also expressed in other tissue-resident macrophages (alveolar macrophages, Kupffer cells, subretinal microglia), the generation of hetTREM2 KO identified that TREM2 signaling regulates lipid homeostasis and that TREM2 can serve as a therapeutic target to restore lipid dysfunction in different tissue-resident macrophages that play a role in the pathogenesis of numerous chronic diseases.

[0320] Targeting TREM2 function enhances interferon signaling without downregulating cholesterol biosynthesis. Furthermore, interferon properties were identified. Recent studies have shown a link between interferon signaling and cholesterol biosynthesis. Therefore, the interferon axis can also be targeted to regulate cholesterol biosynthesis associated with tissue-resident macrophages to restore homeostatic function.

[0321] Targeting the Gas6-Axl axis via TREM2. Examination of HZ and HO TREM2 microglia identified a dose response for gene expression of the Gas6 / Axl axis (Figure 68). The Axl signaling axis is associated with disease-associated microglia that play a role in responding to neurodegenerative cascades in AD and other neurological diseases (Krasemann et al., 2018). Thus, activation of TREM2 enhances Gas6 / Axl signaling and enhances the DAM phenotype. However, the Gas6 / Axl pathway also plays a role in supporting an immunosuppressive environment in the tumor microenvironment. Thus, antagonizing TREM2 and downregulating Gas6 / Axl can enhance immune cell recruitment (Tanaka and Siemann, 2020).

[0322] Activation of Siglec-11 via TREM2 is neuroprotective. Using HZ TREM2 microglia, we identified downregulation of Siglec11 (Wang and Neumann, 2010), a CD33-associated protein that prevents microglial neurotoxicity, as a specific gene with partial, but not complete, TREM2 loss (Figure 69). The TREM2-Siglec11 axis was only evident in HZ TREM2 microglia. Thus, activation of TREM2 may be neuroprotective via upregulation of microglial Siglec-11.

[0323] Targeting TREM2 to restore GRN function in FTD. HZ and HO LOF microglia demonstrated downregulation of GRN due to loss of TREM2 signaling (Figure 70). Thus, activation of TREM2 can enhance GRN levels and restore the GRN loss-of-function phenotype in FTD.

[0324] Effect of TREM2 on ion channels. A dose-dependent increase in gamma-aminobutyric acid receptor subunit epsilon (GABRE) by TREM2HZ and TREM2HO can activate microglia to release interleukin-6 and interleukin-12p40.

[0325] Enhancement of expression of zinc-activated ligand-gated ion channels of the cysteine ​​loop superfamily of ligand-gated ion channels by TREM2 suggests a direct link between TREM2 and ion channel expression in generating a relevant functional model of neuroinflammation.

[0326] G protein-coupled receptors (GPCRs) are among the most frequently targeted receptors in the development of novel therapeutic agents for central nervous system (CNS) disorders. As new functions for GPCRs are discovered, the number of drugs targeting GPCRs is expected to increase, especially in the case of the 100 orphan GPCRs that currently have no known endogenous ligands or clearly defined functions.

[0327] Microglia lacking TREM2 undergo global changes in their metabolism, resulting in reduced ATP levels and signs of stress and death. Given that the majority of today's pharmaceutical drugs target GPCRs, and TREM2 induces changes in many GPCRs, it is suggested that TREM2 is both a ligand and a regulator of microglial survival and function. GPCRs were found to be regulated by TREM2, which can be used for drug targeting applications. Perturbation of TREM2 expression downregulated ADGRD1, ADGRE3, ADGRE5, ADGRG1, ADGRG3, ADORA2B, ADRB1, ADRB2, C5AR1, C5AR2, CCR2, CXCR2, CXCR4, EDNRA, FPR3, FZD1, GPBAR1, GPR157, LTB4R, LTB4R2, P2RX1, P2RY1, P2RY12, PTGER4, and SUCNR1. Expression of TREM2 downregulated the expression of AVPR2, CNR2, GPR18, GPR84, and LPAR6.

[0328] Downregulation of TREM2 dose-dependently decreased the expression of P2XRX1, P2RY12, and P2RY10, which function as highly calcium-permeable ligand-gated ion channels. The TREM2-induced decrease in P2RX and P2RY levels supports a role for TREM2 in age-related changes in human microglia, including cell adhesion and axon guidance, which are regulated by calcium signaling.

[0329] Effect of TREM2 on transport proteins. ATP-binding cassette (ABC) transporters are membrane-bound proteins that actively move endogenous and xenobiotic solutes across cell membranes, often against a concentration gradient, at the expense of energy via ATP hydrolysis. ABC transporters were discovered as proteins in the process of Aβ protein production, degradation, and clearance. Certain transport proteins were found to be regulated by TREM2, which controls metabolism, phagocytic function, and may play a role in the generation of DAMs (disease-associated microglia).

[0330] ATP6ViG2 is a multisubunit enzyme that mediates acidification of intracellular compartments in eukaryotic cells to perform import and sorting functions. Downregulation of ATP6ViG2 by TREM2 HZ and TREM2 HO strains supports a role for TREM2 in protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation.

[0331] ATP8A1 catalyzes ATP hydrolysis coupled to the transport of aminophospholipids from the outside to the inside of various membranes, ensuring the maintenance of asymmetric distribution of phospholipids. Downregulation of ATP8A1 in TREM2 HZ and TREM2 HO lines supports a role for TREM2 in microglial function metabolism and migration.

[0332] The solute carrier (SLC) family of membrane transport proteins includes over 400 members organized into 66 families located in the cell membrane. In addition to their role in providing essential nutrients and osmolytes to neurons and glial cells, SLC transporters also play a role in the termination of synaptic transmission of amino acid neurotransmitters. SLC family members found to be affected by TREM2 perturbation include ABCA3, ABCA7, ABCG1, ATP10A, ATP13A1, ATP13A2, ATP2A3, ATP6V1E2, ATP8A1, EPB41, SLC10A3, SLC15A3, SLC16A1, SLC16A3, SLC18A2, SLC19A1, SLC22A23, SLC25A1, SLC25A23, These include SLC25A4, SLC25A45, SLC26A11, SLC29A2, SLC29A3, SLC2A1, SLC2A5, SLC37A1, SLC37A2, SLC38A1, SLC38A5, SLC40A1, SLC41A2, SLC43A3, SLC44A2, SLC45A3, SLC47A1, SLC6A12, SLC6A8, SLC7A1, SLC7A5, SLCO4C1, and SPNS3. Perturbation of TREM2 also upregulated the expression of transporters SLC11A1, ABCC5, SLC25A20, SLC35E3, ABCA1, SLC25A27, SLC15A2, and SLC25A42.

[0333] Many of these transported proteins control pH, acidify intracellular compartments, facilitate transport of monocarboxylic acids, enhance transport of drugs, toxins and hormones, and increase mitochondrial permeability. Data confirm the expression of a wide variety of transporters on microglia that shape physiological and survival functions. The effect of TREM2 on down- or up-regulation of a subset of transporters reveals their cooperative role in survival, phagocyte function and the development of neurodegenerative diseases.

[0334] TREM2 downregulated the expression of many important proteins that control the development of neuroinflammation. TREM2 downregulated ACVR1B, ACVRL1.AXL.CIITACSF2RBCSF3R, EPHB6, IL11RA, IL17RA, IL21R, IL27RA, IL6R, IL6ST, IL7R, ITGAM, ITGAX, ITGB3, LMTK3, NLRP1, TLR3, TLR5, TNFRSF11A, TNFRSF12A, TNFRSF21, TNFRSF25, and TNFRSF9. Downregulation of TREM2 enhanced the expression of IL21R, ITGA2B, ITGA7, RYK, TLR2, TNFRSF10C, and FAS. Many of these molecules can be used in diagnostic kits to detect neurodegeneration.

[0335] TREM2 downregulated the expression of the following enzymes expressed in microglia: LIPG, INPP5A, PADI4, GRK5.PCSK5, NEK2, ACE, PTK2, TSSK6, MMP19, SMPD3, PDE3B, and PDE6G. PRSS8, TPSAB1, PRKACB, DAGLA, QPCT, CDK14, ACE2, CPA3, NT5M, PIK3R6, RPS6KA5, PCSK4, TTK, HDC, CAMKK1, INPP1, MAPK12, LPIN1, IRAK2, PLK2, PDE6B, NAT8L, PROC, SPHK1, HP GD, CIT, INPP5E, CDK18, PTGS2,CASK, PLK1, FAAH,BCR, HDAC4, DMPK, ITPKB, ADA, CES1, MVK, TESK2, PIK3CA,TGM2, CFD, BUB1B, DPEP2, ENPP2, CYP27A1, ULK2, PIK3CB.SQLEKMT5C , AURKB, MAPK13, MAP3K12, TOP2A, PKMYT1, HMGCS1, ACAT2, DNMT3A, RRM2, CYP51A1, MGLL, KMT5A, BACE1, PIK3CG, TRIB1.PIK3R2, FASN, MMP2, ADCK2, LSS, GRK6, HMGCR, SRC, ACSS 2, STK38L, FGR, IDI1, KDM5C, ABHD2, KSR1, LTC4S, FURIN, MVD, PRKAR2A, RPS6KA4, DAPK1, MPST, FDFT1, ADAM10, DDAH2, LGMN, PKN1, FDPS, SGK1, GAA, CPM, ALDH2, ASAH1, and CTSD.

[0336] TREM2 upregulated the expression of the following enzymes expressed in microglia: MMP9, CASP5, LIMK2, PLA2G4C, RAB27A, CFB, CYP2R1, CTSK, NUDT7, PRKY, ANPEP, SMPDL3A, PLA2G4B, STK32B, and PDK4.

[0337] TREM2 downregulated the expression of the following proteins expressed in microglia: BCL2, BIRC5, BIRC7, BRPF3, CD1D, CD22, CD276, CD37, CD6, CD74, CRY1, EPAS1, FABP3, FCGR3A, FCMR, HSPA1B, ILDR2, KIF11, LILRA4, NOTCH4, RBP1, RBP4, RBP7, RGS1, RGS2, RGS3, TACSTD2, TUBA1A, and XIAP. TREM2 upregulated the expression of the following proteins expressed in microglia: CD14, CD36, CD80, CLEC4E, FCER1G, LAG3, LY96, PVRIG, RBP5, and SLAMF7. This study is the first to show a direct link between perturbation / downregulation of TREM2 associated with downregulation of COMT, NRXN2, and SST expression.

[0338] The COMT gene encodes an enzyme called catechol-O-methyltransferase, which controls personality, planning, behavioral inhibition, abstract thinking, emotions, and working (short-term) memory. COMT catalyzes O-methylation, inactivating catecholamine neurotransmitters, catechol hormones, and shortening the biological half-life of neuroactive drugs, such as L-DOPA, alpha-methylDOPA, and isoproterenol. There is a wide variability in the levels of COMT in sporadic AD. A genetic risk score based on the accumulation of multiple risk alleles in BDNF, COMT, and APOE for AD has been used to predict late-life cognitive impairment in AD (Wollam et al., 2015). NRXN2 belongs to the family of Nurexins, which function as cell adhesion molecules and receptors. NRXN2 deletion in mice causes autism-related behaviors. The hormone somatostatin (SST) controls the rate of neurotransmission and cell proliferation in the CNS.

[0339] In addition to COMT, NRXN2, and SST, TREM2HZ microglia further downregulated aldehyde dehydrogenase (ALDH1A2), which catalyzes the synthesis of retinoic acid (RA) from retinaldehyde. Homeobox 3 (HOXB3) is a nuclear protein with a homeobox DNA-binding domain that is involved in development. Insulin-like growth factor binding protein 2 (IGFBP2) controls neuronal plasticity to regulate high-level cognitive behaviors such as spatial learning and memory and information processing, and the serine protease proteinase 3 (PRTN3) degrades elastin, fibronectin, laminin, vitronectin, and collagen types I, III, and IV, promoting transmigration.

[0340] In addition to COMT, NRXN2, and SST, TREM2HO microglia further downregulate ankyrin (ANK1), which links integral membrane proteins to the underlying spectrin-actin cytoskeleton and plays a key role in activities such as cell motility, activation, proliferation, and maintenance of specialized membrane domains. ANKI interacts with the interferon signaling pathway. Non-ubiquitous CaM kinase (PNCK), upregulated by pregnancy, is a unique member of the calmodulin kinase family. PNCK is primarily expressed in the central nervous system. It phosphorylates CREB1 and SYN1 / synapsin, triggering signaling pathways that activate microglia. CaMKII is dysregulated in AD, and this dysregulation is a major cause of synaptic degeneration, NFT formation, and memory deficits. Downregulation of PNCK by TREM2 suggests a role for PNCK in microglial proliferation and activation. Other genes that are downregulated include tubulin beta 4a chain binding to GFP and nucleotides (TUBB4), sterile alpha motif domain containing 11 (SAMD11), and B9 domain containing protein (B9D1). The functions of these genes are cell adhesion, proliferation, and migration functions of microglia. Downregulation of genes listed with TREM2 may be a contributing factor to the development of AD.

[0341] All methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications may be applied to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Atagi et al., J Biol Chem.290(43):26043-50,2015. Brennand et al., 2011 Caldeira et al.,Front Aging Neurosci.9:277,2017. Cheng et al.,Clin Chim Acta.463:88-95,2016. International Publication No. 02 / 016536 Brochure International Publication No. 03 / 016496 Brochure International Publication No. 98 / 30679 Brochure International Publication No. 98 / 53058 Brochure International Publication No. 98 / 53059 Brochure International Publication No. 98 / 53060 Brochure Julia et al.Stem cell reports.9(2):600-614,2017. McQuade et al., J Mol Biol.431(9):1805-17,2019. Melief et al.,Glia.60(10):1506-17,2012. Minett et al., J Neuroinflammation.13(1):135,2016. PCT Publication International Publication No. 2012 / 149484 Pamphlet Rustenhoven et al.,Trends In Pharmacological Sciences,38(3),291-304,2017. Slosarek et al.Cell Rep.24(9):2248-2260,2018. U.S. Patent Application Publication No. 12 / 715,136 U.S. Patent No. 6,140,081 U.S. Patent No. 6,453,242 U.S. Patent No. 6,534,261 U.S. Patent No. 6,617,152 U.S. Pat. No. 8,372,642 US Patent Application Publication No. 2002 / 0076747 US Patent Application Publication No. 2005 / 0064474 US Patent Application Publication No. 2005 / 0260186 US Patent Application Publication No. 2006 / 0104968 US Patent Application Publication No. 2006 / 0188987 US Patent Application Publication No. 2007 / 0218528 US Patent Application Publication No. 2011 / 0301073 US Patent Application Publication No. 2011 / 0301073 US Patent Application Publication No. 2011 / 0301073 Westin et al.PLoS ONE.7:e30525,2012. Winkler et al., Brain pathology (Zurich, Switzerland), 24(4), 371-386, 2014. Wolfe et al.,Int J Mol Sci.20(1),2018.

Claims

1. 1. A cell culture comprising induced pluripotent stem cell (iPSC)-derived microglia, astrocytes, and / or neurons in a medium, wherein the neurons are GABAergic neurons, dopaminergic neurons, or glutamatergic neurons.

2. The culture of claim 1 , wherein the cell culture is further defined as a tripartite culture.

3. The culture of claim 1 , wherein the neurons are excitatory or inhibitory neurons.

4. (a) the microglia are at least 90% positive for TREM2, P2RY12, TMEM119, IBA-1, and / or CX3CR1, and / or are mature microglia; (b) the astrocytes are positive for S100beta, GFAP, and CD44; and / or The neuron is a member of the IL-16 receptor agonist family, and is a member of the IL-16 receptor agonist family. 1, positive for at least two of the markers selected from the group consisting of LHX2, Neurog1, NKX2-1, Nos1, NPY, NR4A2, PAX6, POU3F2, PVALB, RELN, SATB2, SLC17A6, SLC17A7, SLC17A8, SLC32A1, SOX1, Sox10, SST, SYN1, and Tbr1; The culture of claim 1.

5. 5. The culture of any of claims 1-4, wherein the microglia are derived from an isogenically engineered iPSC line, from a donor expressing a disease-associated SNP, generated from a disease-associated iPSC donor with a genotype associated with TREM2, APOE, CD33, BIN, ABCA7, SNPS, or neurodegeneration, or comprise disruptions in TREM2, methyl-CpG binding protein 2 (MeCP2), and / or alpha-synuclein (SCNA).

6. The culture of any one of claims 1 to 4, wherein the cell culture is a two-dimensional (2D) culture or a three-dimensional (3D) culture.

7. The culture of any one of claims 1 to 4, wherein the medium further comprises IL-34, M-CSF, or TGFβ.

8. The culture of any one of claims 1 to 4, wherein the cells are cultured on a surface coated with polyethyleneimine (PEI) or an extracellular matrix protein.

9. 9. The culture of claim 8, wherein the extracellular matrix is ​​basement membrane extract (BME) purified from mouse Engelbreth-Holm-Swarm tumor, collagen, or laminin.

10. The culture of claim 6, wherein the 3D culture is a cerebral organoid culture and comprises a functional neuronal network comprising calcium oscillations.

11. The culture of any one of claims 1 to 4, wherein the microglia, astrocytes and neurons are isogenic.

12. 5. The culture of any of claims 1 to 4, wherein the culture comprises microglia and astrocytes in a ratio of 1:1, or wherein the culture comprises microglia, astrocytes, and neurons in a ratio of 1:1:5 or 2:6:

1.

13. The culture has 15,000 cells / cm 2 ~25,000 cells / cm 2 or the culture contains microglia at a cell density of 125,000 cells / cm 2 ~160,000 cells / cm 2 or the culture contains neurons at a cell density of 25,000 cells / cm 2 ~35,000 cells / cm 2 5. The culture of claim 1, comprising astrocytes at a cell density of 0.1 to 0.

25.

14. The culture of any one of claims 1 to 4, wherein the culture is xeno-free, feeder-free, and / or conditioned medium-free, or the medium is a defined medium.

15. A cell culture comprising brain microvascular endothelial cells (BMEC), pericytes, and astrocytes in a sandwich format.

16. The culture of claim 15, further defined as a blood-brain barrier model.

17. The culture of claim 15 or 16, wherein the sandwich format comprises an extracellular matrix layer comprising at least two extracellular matrix proteins between two cell layers, optionally wherein the at least two extracellular matrix proteins are collagen IV and fibronectin.

18. 17. The culture of claim 15 or 16, wherein the sandwich format comprises BMECs on the apical side, the extracellular matrix layer in the middle, and astrocytes and pericytes on the basolateral side.

19. 16. The culture of claim 15, wherein the extracellular matrix layer further comprises gelatin, the astrocytes and pericytes, and / or the sandwich format further comprises a permeable membrane insert comprising a polytetrafluoroethylene (PFTE), polycarbonate, or polyethylene terephthalate (PFTE) insert.

20. (a) the permeable membrane insert is coated on the apical side with human collagen IV and human fibronectin; (b) the basolateral side of the permeable membrane insert is coated with gelatin; (c) the astrocytes and pericytes are present in a ratio of 1:2; (d) the astrocytes and pericytes are on the basolateral side of the permeable membrane insert and the BMECs are on the apical side of the permeable membrane insert; (e) the BMECs, astrocytes, and pericytes are in a medium containing a ROCK inhibitor; 20. A culture according to any one of claims 15, 16 and 19.

21. the BMECs, astrocytes, and pericytes are present in a ratio of 4:1:2, 4:1:1, 4:2:1, or 2:1:1; The BMEC is 1×10 6 cells / cm 2 ~1.5 x 10 6 cells / cm 2 are seeded at a cell density of The astrocytes are 300,000 cells / cm 2 ~700,000 cells / cm 2 and / or The pericytes are present at a density of 300,000 cells / cm 2 ~700,000 cells / cm 2 are seeded at a cell density of 20. A culture according to any one of claims 15, 16 and 19.

22. (a) contacting a test compound with a culture according to any one of claims 1 to 4, 15, 16 and 19; and (b) Measuring the functional activity of the cells A method for screening a therapeutic compound for treating a neurodegenerative disease, comprising:

23. 20. Use of a culture according to any one of claims 1 to 4, 15, 16 and 19 as a model for a neurodegenerative disease.

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