Triple cell culture and uses thereof

Abstract

The present disclosure provided methods and compositions for generating a triple cell culture comprising neurons, astrocytes, and microglia. The method includes providing neurons, astrocytes, and microglial cells or precursors, and culturing them under conditions that support survival, maturation, and functional interaction. In some embodiments, the cells are derived from pluripotent stem cells and seeded in defined ratios. The cultures are suitable for modeling central nervous system function, disease processes, and for evaluating candidate therapeutic agents. Also disclosed are variations of the culture system including use of reporter genes, cryopreserved cell components, three-dimensional formats, and high-throughput assay configurations.

Description

Attorney Docket No. 01209-0020-00PCTTRIPLE CELL CULTURE AND USES THEREOFFIELD OF THE PRESENT DISCLOSURE

[0001] This disclosure relates generally to methods of generating a triple cell culture comprising neurons, astrocytes, and microglia cells, and uses thereof including as models for neurological diseases.BACKGROUND

[0002] Microglia play a key role in neurodegenerative disease. Recently, large-scale genetic association studies have identified many Alzheimer’s disease genes highly expressed in microglia in the CNS, but tools for studying human microglia function in vitro have been limited. Historically, microglia have been studied in monocultures in vitro. This has been achieved through primary mouse cultures, and, more recently, the directed differentiation of iPSCs. Unfortunately, microglia lose their identity very quickly in the absence of the brain environment. Therefore, in many in vitro assays, microglia reflect generic myeloid cell responses rather than unique microglia responses critical for understanding microglia function in the brain.

[0003] Cell culture models are valuable tools in neuroscience research for studying the interactions between different cell types within the central nervous system (CNS). Traditional co-culture systems commonly include neurons and astrocytes but often exclude microglia, limiting their ability to accurately model neuroinflammatory processes and other microglia- associated functions. Thus, there remains a need for robust triple cell culture systems involving neurons, astrocytes, and microglia to more accurately model CNS physiology and pathophysiology.SUMMARY OF THE PRESENT DISCLOSURE

[0004] The present disclosure provides a method of generating a triple cell culture comprising neurons, astrocytes, and microglia cells. The method comprises culturing neurons, astrocytes, and microglia cells together under conditions that facilitate survival, differentiation, and functional interaction of all three cell types.

[0005] The disclosure also relates to triple cell cultures themselves, comprising neurons or neuronal precursors, astrocytes, and microglia or microglial precursors. The disclosure further provides methods for using these triple cell cultures to model neurological diseases, such as neurodegenerative disorders, neuroinflammatory conditions, traumatic brain injury,Attorney Docket No. 01209-0020-00PCT and for screening therapeutic agents. Exemplary embodiments of the disclosure include, but are not limited to the following:1. A method of preparing a cell culture comprising neurons or neuronal precursors, astrocytes, and microglia cells or microglial precursors, the method comprising:(i) providing neurons or neuronal precursors;(ii) providing astrocytes;(iii) providing microglia cells or microglial precursors;(iv) seeding a cell culture vessel with the (a) neurons or neuronal precursors, (b) astrocytes, and (c) microglia cells or microglia precursor cells, wherein more neurons or neuronal precursors are seeded than astrocytes, microglia cells, or microglia precursor cells.2. The method of embodiment 1, wherein the neurons, astrocytes, and microglia cells are incubated in a triple cell culture medium comprising a serum-free basal medium that produces synaptically active neurons.3. A method of preparing a cell culture comprising neurons or neuronal precursors, astrocytes, and microglia cells or microglial precursors, the method comprising:(i) providing neurons or neuronal precursors;(ii) providing astrocytes;(iii) providing microglia cells or microglial precursors;(iv) seeding a cell culture vessel with the (a) neurons or neuronal precursors, (b) astrocytes, and (c) microglia cells or microglia precursor cells in a triple cell culture medium comprising a serum-free basal medium that produces synaptically active neurons.4. The method of embodiment 3, wherein more neurons are seeded than either astrocytes or microglia cells.5. The method of any one of embodiments 2-4, wherein the serum-free basal medium comprises BrainPhys™, or wherein the serum-free basal medium comprises inorganic salts, amino acids, vitamins, dextrose, sodium pyruvate, Hepes, cholesterol, and optionally phenol red.6. The method of any one of embodiments 2-5, wherein the triple cell culture medium comprises a serum-free basal medium supplement comprising Bottenstein’s N-l formulation, or wherein the triple cell culture medium comprises a serum-free basalAttorney Docket No. 01209-0020-00PCT medium supplemented with one or more of insulin, transferrin, selenite, putrescine, and progesterone. The method of embodiment 6, wherein the serum-free basal medium supplement is a N-2 supplement, or wherein the serum-free medium supplement comprises one or more of apo-transferrin, insulin, putrescine, sodium selenite, and progesterone. The method of any one of embodiments 2-7, wherein the triple cell culture medium comprises one or more of NeuroCult™ SMI supplement; brain-derived neurotrophic factor (BDNF); glial cell line-derived neurotrophic factor (GDNF); IL34; gmCSF; and TGFbeta. The method of any one of embodiments 1-8, wherein (iii) comprises differentiating human iPSC cells into microglial precursors. The method of embodiment 9, wherein differentiating human iPSC cells into microglial precursors comprises forming hemogenic embryoid bodies (EB) in hemogenic EB media. The method of embodiment 10, wherein the hemogenic EB media comprises a xeno-free medium capable of expanding iPSCs. The method of embodiment 11, wherein the xeno-free medium is StemFlex™, optionally further comprising StemFlex™ supplement. The method of any one of embodiments 10-12, wherein the hemogenic EB media comprises at least one of, at least two, or three of VEGF, BMP4, and SCF. The method of any one of embodiments 10-13, wherein the hemogenic EB media further comprises a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632. The method of any one of embodiments 1-13, wherein the microglia or microglial precursors in the culture express increased levels of TMEM119, CX3CR1, and / or P2RY12 relative to microglia cells maintained in a monoculture. The method of any one of embodiments 10-14, wherein EBs are formed in a cell culture vessel suitable to promote the formation of embryoid bodies; optionally wherein the cell vessel is a microwell plate such as an AggreWell™ plate. The method of any one of embodiments 10-16, wherein differentiating human iPSC cells into microglial precursors further comprises transferring EBs to a cell culture vessel coated with an extracellular matrix. The method of embodiment 17, wherein the cell culture vessel comprises a microglia precursor production medium comprising a serum-free hematopoietic basal medium.Attorney Docket No. 01209-0020-00PCT The method of embodiment 18, wherein the serum-free hematopoietic basal medium is X-VIVO®. The method of embodiment 18 or embodiment 19, wherein the microglia precursor production medium comprises at least one of, at least two of, or three of IL3, mCSF, and Glutamax™. The method of any one of embodiments 18-20, wherein the microglia precursor production media comprises 2-mercaptoethanol. The method of any one of embodiments 1-21, wherein at least 80% of the microglia or microglial precursors are positive for IBA1. The method of any one of embodiments 1-22, wherein at least 80% of the microglia or microglial precursors are positive for CX3CR1. The method of any one of embodiments 1-23, wherein microglia precursors are generated from disease-associated iPSC donors. The method of embodiment 24, wherein the disease-associated iPSC donors comprise genotypes with a mutation in one or more of TREM2, APOE, CD33, PSENJ, SNCA, or MAPT. The method of any one of embodiments 1-23, wherein microglia precursors are generated from iPSCs engineered to comprise a disease-associated genotype. The method of embodiment 26, wherein the disease-associated genotype comprises a mutation in one or more of TREM2, APOE, CD33, PSEN1, SNCA, o MAPT. The method of embodiment 26 or embodiment 27, wherein the genotype is engineered by the CRISPR / Cas9 system. The method of any one of embodiments 1-28, wherein (i) comprises differentiating a population of human iPSC cells to neuronal precursors by resuspending human iPSC cells in a neuron induction medium. The method of embodiment 29, wherein the neuron induction medium comprises DMEM as basal medium. The method of embodiment 30, wherein the neuron induction medium comprises DMEM F / 12 as basal medium. The method of any one of embodiments 29-31, wherein the neuron induction medium comprises one or more of (a) a serum-free supplement comprising Bottenstein’s N-l or N-2 supplement or comprising one or more of insulin, transferrin, selenite, putrescine, and progesterone, (b) BDNF, (c) neurotrophin-3 (NT-3), and (d) laminin.Attorney Docket No. 01209-0020-00PCT The method of embodiment 32, wherein the serum-free supplement comprises N-2 supplement, or wherein the serum-free supplement comprises apo-transferrin, insulin, putrescine, sodium selenite, and progesterone. The method of any one of embodiments 29-33, wherein the iPSC cells used to generate neuronal precursors are engineered to overexpress NGN2. The method of any one of embodiments 29-34, wherein the neuron induction media is supplemented with a ROCK inhibitor; optionally wherein the ROCK inhibitor is Y- 27632. The method of any one of embodiments 1-35, wherein at least 80% of the neurons or neuronal precursors are positive for one or more of MAP2, TUBB3, synapsin I, NeuN, and DCX. The method of any one of embodiments 1-36, wherein the microglia and neurons are derived from isogenic iPSCs. The method of any one of embodiments 1-36, wherein the microglia, astrocytes, and neurons are isogenic. The method of any one of embodiments 1-38, wherein the astrocytes are derived from human fetal astrocytes. The method of any one of embodiments 1-39, wherein (ii) comprises expanding astrocyte cells. The method of any one of embodiments 1-40, comprising forming a co-culture comprising astrocytes and neuronal precursors in a co-culture medium. The method of embodiment 41, wherein the co-culture medium is a serum-free basal medium. The method of embodiment 42, wherein the serum-free basal medium comprises BrainPhys™. The method of any one of embodiments 41-43, wherein the co-culture medium comprises one or more of (a) Bottenstein’s N-l or N-2 supplement or a supplement comprising transferrin, insulin, putrescine, selenium, and progesterone; (b) NeuroCult™ SMI supplement; (c) BDNF; and (d) GDNF. The method of any one of embodiments 41-44, wherein (iv) comprises seeding microglial precursor cells into the co-culture of neuronal precursors and astrocytes. The method of any one of embodiments 41-45, wherein the co-culture is seeded with at least a 2: 1 ratio of neuronal precursors to astrocytes.Attorney Docket No. 01209-0020-00PCT The method of embodiment 46, wherein the co-culture is seeded with at least a 3: 1 ratio of neuronal precursors to astrocytes. The method of any one of embodiments 45-47, wherein (iv) comprises seeding approximately the same number of microglial precursors as astrocytes in the co-culture. The method of any one of embodiments 1-48, wherein at least 80% of the microglia cells exhibit ramified morphology. The method of embodiment 49, wherein the microglial morphology is assessed by IBA1 immunostaining. The method of any one of embodiments 1-50, further comprising contacting the culture with an amyloid beta peptide. The method of embodiment 51, wherein the amyloid beta peptide is Api-42 or pE-Ap3- 42. The method of any one of embodiments 1-52, further comprising contacting the culture with a test agent, optionally wherein the test agent is an antibody, a soluble protein, a nucleic acid, a peptide, or small molecule drug. The method of any one of embodiments 1-53, further comprising contacting the culture with an inflammatory stimulus such as lipopolysaccharide (LPS). The method of any one of embodiments 1-54, further comprising measuring secretion of one or more cytokines, optionally wherein the cytokines are selected from IL-6, TNF- alpha, IL-lbeta, or CCL2. The method of any one of embodiments 1-55, further comprising measuring cell viability of microglia using a dead cell stain, flow cytometry, and / or imaging-based analysis. The method of any one of embodiments 1-56, wherein the method further comprises cry opreserving microglial precursors before (iv). The method of any one of embodiments 1-57, wherein the method further comprises cry opreserving neuronal precursors and / or astrocytes before (iv). A cell culture formed according to the method of any one of embodiments 1-58. A cell culture comprising neurons or neuronal precursors, astrocytes, and microglia or microglial precursors. The cell culture of embodiment 60, wherein the cell culture comprises more neurons or neuronal precursors than astrocytes or microglia or microglial precursors. The cell culture of embodiment 61, wherein the cell culture comprises at least a 2: 1 ratio of neurons or neuronal precursors to astrocytes.Attorney Docket No. 01209-0020-00PCT The cell culture of embodiment 61, wherein the cell culture comprises at least a 3: 1 ratio of neurons or neuronal precursors to astrocytes. The cell culture of any one of embodiments 60-63, wherein the cell culture comprises approximately the same number of microglia or microglial precursors as astrocytes. The cell culture of embodiment 64, wherein the cell culture comprises a 3 : 1 : 1 ratio of neurons or neuronal precursors to microglia or microglial precursors and astrocytes. The cell culture of any one of embodiments 60-65, wherein the microglia or microglial precursors express P2RY12, P2RY13, TMEM119, CX3CR1, CSF1R, AIF1, TREM2, and / or SALL1; or express increased levels of P2RY12, P2RY13, TMEM119, CX3CR1, CSF1R, AIF1, TREM2, and / or SALL1 than iPSC cells. The cell culture of any one of embodiments 60-66, wherein the microglia or microglial precursors express any one or more of cell surface markers TRA-1-60, CD34, CD45, CD43, CDl lb, P2RY12, and / or CD107a. The cell culture of any one of embodiments 60-67, wherein the microglia or microglial precursors in the culture express increased levels of TMEM119, CX3CR1, and / or P2RY12 relative to microglia cells maintained in a monoculture. The cell culture of any one of embodiments 60-68, wherein at least 80% of the microglia or microglial precursors are positive for IBA1. The cell culture of any one of embodiments 60-69, wherein at least 80% of the microglia or microglial precursors are positive for CX3CR1. The cell culture of any one of embodiments 60-70, wherein the microglia or microglial precursors do not express or show reduced expression of OCT4, SOX2, NANDG, POU5F1, and / or KLF4 compared to iPSCs. The cell culture of any one of embodiments 60-71, wherein the neurons or neuronal precursors are generated from iPSCs engineered to overexpress NGN2. The cell culture of any one of embodiments 60-72, wherein microglia precursors are generated from disease-associated iPSC donors. The cell culture of embodiment 73, wherein the disease-associated iPSC donors comprise genotypes with a mutation in one or more of TREM2, APOE, CD33, PSENJ, SNCA, or MAPT. The cell culture of any one of embodiments 60-74, wherein microglia precursors are generated from iPSCs engineered to comprise a disease-associated genotype.Attorney Docket No. 01209-0020-00PCT The cell culture of embodiment 75, wherein the disease-associated genotype comprises a mutation in one or more of TREM2, APOE, CD33, PSEN1, SNCA, or MAPT. The cell culture of embodiment 75 or embodiment 76, wherein the genotype is engineered by the CRISPR / Cas9 system. The cell culture of any one of embodiments 60-77, wherein the microglia and neurons are derived from isogenic iPSCs. The cell culture of any one of embodiments 60-78, wherein the microglia, astrocytes, and neurons are isogenic. The cell culture of any one of embodiments 60-79, wherein the astrocytes are derived from human fetal astrocytes. The cell culture of any one of embodiments 60-80, wherein the astrocytes express ALDH1L1, SLC1A2, SLC1A3, GLUL, AQP4, GJA1, SOX9, GFAP, and / or SlOObeta. The cell culture of any one of embodiments 60-81, wherein the astrocytes show reduced expression of ALPHILI, SLC1A2, SLC1A3, AQP4, GJA1, and / or SOX9; and / or show increased expression of GLUL, compared to astrocytes in a co-culture of astrocytes and neurons or neuronal precursors. The cell culture of any one of embodiments 60-82, wherein the neurons or neuronal precursors express MAP2, TUBB3, Synapsinl, NeuN, GAD1, VGLUT1, and / or DCX. The cell culture of any one of embodiments 60-83, wherein the neurons or neuronal precursors express RBFOX3, MAP2, TUBB3, SNAP25, SYT1, SYN1, DLG4, and / or NEFM. The cell culture of any one of embodiments 60-84, wherein the neurons or neuronal precursors show increased expression of MAP2, SNAP25, SYT1, SYN1, and / or NEFM; and / or wherein the neurons or neuronal precursors show reduced expression of RBFOX3, TUBB3, and / or DLG4, compared to neurons or neuronal precursors in a co-culture of astrocytes and neurons or neuronal precursors. The cell culture of any one of embodiments 60-85, wherein the cell culture is comprised within a transwell, optionally wherein neurons or neuronal precursors and astrocytes are in a bottom chamber or well of the transwell and microglia or microglial precursors are in a top chamber or well, wherein the top and bottom chambers or wells are separated by a porous membrane. The cell culture of any one of embodiments 60-86, wherein the cell culture is in a three- dimensional shape, such as a spheroid or aggregate.Attorney Docket No. 01209-0020-00PCT88. A kit comprising (a) a co-culture of astrocytes and neuronal precursors and (b) a culture of microglial precursor cells.89. A kit comprising the cell culture of any one of embodiments 59-8790. The kit of embodiment 89, further comprising instructions for use according to the method of any one of embodiments 1-58.91. A method of assessing the cell culture of any one of embodiments 59-87, comprising contacting the cell culture with an amyloid beta peptide.92. The method of embodiment 91, wherein the amyloid beta peptide is Api-42 or pE-Ap3- 42.93. A method of assessing the activity of a test agent in the cell culture of any one of embodiments 59-87, comprising contacting the culture with the test agent, optionally wherein the test agent is an antibody, a soluble protein, a nucleic acid, a peptide, or small molecule drug.94. A method of assessing the effect of an inflammatory stimulus on the cell culture of any one of embodiments 59-87, comprising contacting the cell culture with an inflammatory stimulus such as lipopolysaccharide (LPS).95. A method of assessing the cell culture of any one of embodiments 59-87, comprising measuring secretion of one or more cytokines from the cell culture, optionally wherein the cytokines are selected from IL-6, TNF-alpha, IL-lbeta, or CCL2.96. A method of assessing the efficacy of a pharmaceutical composition for the treatment of a neurodegenerative disease, the method comprising: (i) treating the cell culture of any one of embodiments 59-87 with the pharmaceutical composition, and (ii) assessing the efficacy of the pharmaceutical composition based on one or more readouts.97. The method of embodiment 96, wherein the one or more readouts comprise one or more of a change in microglial activation state, a change in cytokine secretion profile, a change in neuronal viability or morphology, a change in tau phosphorylation, a change in Dab-1 phosphorylation, a change in synaptic density, a change in phagocytic activity, or a change in expression of one or more disease-associated markers.

[0006] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the disclosure will become apparent to one of skill in the art. These and other embodiments of the disclosure are further described by the detailed description that follows.Attorney Docket No. 01209-0020-00PCTBRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS. 1A-1C provide flow charts showing the stages of exemplary methods of generating triple cell cultures. FIG. 1A shows the overview of a method of generating a triple cell culture of the disclosure. FIG. IB shows exemplary stages 0 to 3 for generating a triple cell culture with day-to-day steps. FIG. 1C shows exemplary stages 4 and 5 for generating a triple cell culture with day-to-day steps.

[0008] FIGs. 2A-2C show images of four iPSC clones that were validated for iNeuron differentiation capacity at the iPSC (FIG. 2A), Progenitor (FIG. 2B), and mature iNeuron (FIG. 2C) stages.

[0009] FIGs. 3A-3H shows neuronal marker expression of NGN2 (FIG. 3A), NESTIN (FIG. 3B), TUBB3 (FIG. 3C), MAP2 (FIG. 3D), NEUN (FIG. 3E), SYN1 (FIG. 3F), POU5F1 (FIG. 3G), and NANOG (FIG. 3H) during iPSC differentiation in polyclonal and clonal iPSCs.

[0010] FIG. 4 shows flow cytometry graphs of benchmark pluripotency, lymphoid, myeloid, and microglia markers in microglia precursors.

[0011] FIGs. 5A-5C show graphs of neuron (FIG. 5A), astrocyte (FIG. 5B), and microglia (FIG. 5C) expression markers in a co-culture of neurons and astrocytes (“iN_Asf ’) and triple cell culture of neurons, astrocytes and microglia (“iN_Ast_iMG”) without amyloid beta (AP). FIGs. 5D-5F show graphs of neuron (FIG. 5D), astrocyte (FIG. 5E), and microglia (FIG. 5F) expression markers in a co-culture (neurons and astrocytes; “iN_Asf ’) and triple cell culture (neurons, astrocytes and microglia; “iN Ast iMG”) with the addition of Ap. Bars show mean ± SEM (z-score per gene) across biological replicates (n=4) for each marker. Z- scores were computed per gene across all samples so higher bars indicate higher relative expression of that marker in the indicated group / condition. Series labeled “iN Asf ’ (induced neurons co-cultured with astrocytes) and “iN Ast iMG” (induced neurons cultured with astrocytes and induced microglia) correspond to the two bar groups).

[0012] FIG. 6A shows images of differentiated microglia integration into the neuronalastrocyte network of the triple cell culture when compared to microglia monoculture. Microglia (shown in white) display branched or ramified morphology when in triple cell culture (top panel), but not in the microglia monoculture (bottom panel). FIG. 6B shows a comparison of expression of CD45, CD1 lb, P2RY12, and CX3CR1 in the triple cell culture compared to a microglia monoculture. Microglia show higher expression of the microgliaAttorney Docket No. 01209-0020-00PCT markers CX3CR1 and CD1 lb in the triple cell culture (top panel) relative to the microglia monoculture (bottom panel). Antibody marker signal is shifted to the right and surrounded by a rectangular gate, while isotype control antibody signal is outside of and to the left of the rectangular gate

[0013] FIGs. 7A-7F show images of triple cell cultures stained with antibodies against IBA1 and MAP2 and with DAPI (FIGs 7A and 7B), graphs of triple cell culture expression of CX3CR1 (FIG. 7C), P2RY12 (FIG. 7D), and TMEM119 (FIG. 7E), and microglia morphology comparison between different culture conditions (FIG. 7F).

[0014] FIGs. 8A-8E shows images and graphs of triple cell cultures prepared with a media with or without SMI (FIG. 8A) and comparison of the microglia morphology (FIG. 8E) and gene expression of CX3CR1 (FIG. 8B), P2RY12 (FIG. 8C), and TMEM119 (FIG. 8D). FIGs. 8F and 8G show assessment of different ratios of microglia seeded in the triple cell culture based on MAP2 network length (FIG. 8F) and percentage of IBA+ cells (FIG. 8G).

[0015] FIG. 9 shows images of microglia from triple cell cultures treated with pHrodo- labeled Api-42 (top panels) compared to triple cell cultures treated with media only (bottom panels). The top left panel and bottom left panels show microglia that constitutively express green fluorescent protein (GFP). Ap is stained in pink and microglia is stained in white. pHrodo is a pH-sensitive fluorophore used in imaging.

[0016] FIG. 10A shows graphs of the triple cell culture response to treatment with pHrodo- labeled Api-42 in comparison to media only. FIG. 10B shows the quantified uptake of pHrodo-labeled Api-42 in the microglia from the triple cell culture.

[0017] FIGs. 11A-11C show graphs of microglia in triple cell cultures responding to amyloid beta peptide Api-42) and anti-amyloid beta antibodies remternetug (FIG. 11 A), donanemab (FIG. 11B), or bapineuzumab (FIG. 11C). FIGs. 11D-11F show graphs of microglia in triple cell cultures responding to amyloid beta peptide APpE3-24 and antiamyloid beta antibodies remternetug (FIG. 11D), donanemab (FIG. HE), or bapineuzumab (FIG. HF). Isotype control antibodies (“isotype”) and an untreated sample were used in parallel triple cell cultures as controls. The uptake of amyloid beta peptide by microglia was monitored for 36 hours at 37°C. The pHrodo signal was measured and normalized to the number of microglia.

[0018] FIG. 12 shows a graph comparing the measured protein levels of pDabl in a triple cell culture after treatment with Reelin or anti-APOER2 antibody (Antibody A) relative to untreated (NT) or isotype control conditions.Attorney Docket No. 01209-0020-00PCT

[0019] FIG. 13 shows a graph of pHrodo-labeled tau uptake in triple cell cultures treated with an anti-tau antibody (E2814) relative to untreated (NT) or isotype control antibody (hlgG).

[0020] FIGs. 14A and 14B show the measured mean firing rate (FIG. 14A) and synchrony index (FIG. 14B) of induced neuron cells alone and / or in combination with induced microglia cells from the triple cell culture after treatment with amyloid beta.

[0021] FIG. 15 shows the morphology of the triple cell culture iPSC-derived microglia (1st column), triple cell culture human fetal astrocytes (2nd column), triple cell culture neurons (3rd column), and all three images merged (4th column). The first row shows healthy morphology, and the second row shows morphology after two doses of 3 uM Api-42.DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0022] Provided herein are triple cell cultures, and methods of preparing said cultures, comprising neurons, astrocytes, and microglia cells (as used herein, a “triple cell culture” refers to a cell culture comprising neurons or neuronal precursors, astrocytes, and microglia cells or microglial precursors). The triple cell cultures provided herein may be used, among other uses, for studying microglia functions in a CNS-like environment. In some embodiments of the disclosure, the triple cell culture is seeded with more neurons or neuronal precursors than microglia or microglia precursors and astrocytes. It has been discovered that seeding with more neurons (such as a 3 : 1 : 1 ratio of neurons / neuronal precursors:microglia / microglia precursors: astrocytes) yielded a more homoeostatic microglial profile when compared to standard microglial monocultures known in the art. The microglia of the triple cell cultures displayed ramified morphology, similar to microglia observed in vivo. Using live cell imaging, the present disclosure compared microglia motility in response to different CNS substates. Like microglia in the brain, microglia in the triple cell culture show increased motility of their processes in the presence of CNS substates. Like microglia monocultures, triple cell culture microglia take up pHrodo-labeled CNS substates such as amyloid beta, myelin and synaptosomes, but show less cell body movements compared to microglia monocultures, mimicking the behavior observed in mouse brains. Together, the present disclosure provides a reproducible and scalable in vitro model to study diverse microglia functions in a more brain-like environment that allows for genetic manipulation using CRISPR / Cas9 or the use of patient-derived iPSC lines and is easily accessible for evaluating drugs and antibody therapeutics.Attorney Docket No. 01209-0020-00PCT

[0023] Several in vitro models have been developed to study the central nervous system (CNS), including co-culture systems and three-dimensional (3D) cerebral organoids. While 3D brain organoids offer architectural complexity and some degree of cell-type heterogeneity, they are limited by variability between organoids, extended maturation time, and incomplete representation of key CNS-resident cell types, particularly functional microglia. In most organoid systems, microglia either fail to integrate stably or are absent altogether due to their distinct developmental origin and culture requirements. In addition, brain organoids are limited by diffusion, reducing the accessibility of therapeutic compounds to cells farther from the organoid surface and creating nutrient gradients that result in hypoxia and necrotic cores. Furthermore, light and staining compounds do not as easily penetrate brain organoids, making them less amenable to imaging assays relative to two-dimensional (2D) culture systems.

[0024] Conventional 2D co-cultures, such as neuron-astrocyte systems, permit reproducible and scalable assays but lack the immune component necessary to study neuroinflammation and microglia-mediated synaptic remodeling. Existing microglia monocultures, while useful for studying basic immunological responses, quickly lose microglial identity and fail to recapitulate CNS-specific signaling environments.

[0025] The triple cell culture system disclosed herein provides a novel and robust alternative that overcomes these limitations by incorporating neurons, such as iPSC-derived neurons, and microglia, such as iPSC-derived microglia, along with astrocytes, such as fetal- derived astrocytes, in controlled, scalable formats. This model maintains microglial identity, morphology, and function in a physiologically relevant context, enabling detailed studies of neuroinflammation, neurodegeneration, and glia-neuron interactions with greater consistency and throughput than prior models.

[0026] The triple cell culture system described herein offers multiple advantages over existing in vitro CNS models. Among the advantages: (1) microglia cultured within the triple cell culture system maintain key phenotypic and functional features observed in vivo, including ramified morphology, homeostatic gene expression (e.g., CD45, CDl lb, P2RY12, and CX3CR1) and phagocytic capacity. Unlike microglia in monoculture, which rapidly adopt a generic myeloid state, microglia in the present triple cell culture system remain transcriptionally and morphologically similar to CNS-resident microglia. (2) The use of iPSC-derived neurons and microglia enables modeling of patient-specific or genetically- engineered disease backgrounds, including knockouts or disease-associated SNPs (e.g., inAttorney Docket No. 01209-0020-00PCTTREM2 ApoE, and CZ>33). Integration with inducible expression systems (such as the NGN2 dox-inducible system) further enables rapid neuronal differentiation under controlled conditions. (3) Unlike 3D brain organoids, which suffer from batch variability and long culture timelines, the disclosed methods produce highly reproducible triple cell cultures suitable for high-throughput screening, imaging, and transcriptomic analyses within a defined and relatively short timeframe. (4) The triple cell culture system provides a tractable platform for evaluating the efficacy and toxicity of candidate compounds or biologies in a multicellular CNS-like context. Responses can be measured using imaging, flow cytometry, transcriptomics, or biochemical assays, enabling identification of disease-modifying agents that affect neuronal health, glial activation, or neuroinflammatory signaling. (5) The system is compatible with serum-free, chemically-defined media and substrate coatings (e.g., poly-L- ornithine and laminin).

[0027] Thus, provided herein are methods of generating triple cell cultures. In some embodiments, the method comprises generating neuronal precursor cells from induced pluripotent stem cells (iPSCs). In some embodiments, the method comprises obtaining astrocytes from human fetal astrocytes. In some embodiments, the method comprises generating microglial precursors from differentiated iPSC-derived microglia precursors. In some embodiments, the cells are differentiated separately under suitable conditions prior to their integration into a triple cell culture. In some embodiments, a co-culture of neuronal precursors and astrocytes is prepared before seeding with microglial precursors to form the triple cell culture.

[0028] The following protocol is referred to herein as the Exemplary Protocol. The Exemplary Protocol provides an exemplary method of generating the triple cell culture divided into five stages (FIGs. IB and 1C). The numbering of these stages from 1-5 (or stages 0-5, including stage 0 to obtain iPSCs) does not imply that the stages must be performed consecutively or that they cannot be performed in parallel. It is to be understood that the Exemplary Protocol is provided for illustrative purposes of a single working embodiment of the disclosure and is not intended to limit the scope of the present disclosure to this single working embodiment; rather, the disclosure encompasses variations, modifications, and alternative methods of generating triple cell cultures, as would be appreciated by those skilled in the art. The Exemplary Protocol is further detailed in Example 1 herein.Attorney Docket No. 01209-0020-00PCT

[0029] Stage 0: Obtaining iPSCs. In some embodiments, iPSCs of sufficient confluency may be obtained using any suitable method or source. In some embodiments, iPSCs are cultured and expanded to a desired confluency, such as at least 60% confluency, at least 70% confluency, at least 80% confluency, or at least 90% confluency. In some embodiments, iPSCs are maintained and passaged in a suitable stem cell growth medium under conditions that support pluripotency and proliferation. In some embodiments, the iPSCs from this prestage are used in subsequent stages.

[0030] Stage 1: Microglial Precursor Production. In some embodiments, embryoid bodies (EBs) are formed from iPSCs and differentiated in media conditions that promote microglial precursor production. In some embodiments, EBs are transferred to culture flasks containing microglia precursor production media to support further differentiation to microglial precursors.

[0031] Stage 2: Neural Progenitor Induction. In some embodiments, neural progenitors are induced from iPSCs under defined conditions. In some embodiments, the method involves culturing cells in neuron induction media containing suitable supplements, such as growth factors and extracellular matrix components.

[0032] Stage 3: Astrocyte Thawing and Expansion. In some embodiments, astrocyte precursors or mature astrocytes are prepared and cultured under conditions that allow for proliferation and expansion. In some embodiments, astrocytes are cultured in flasks coated with suitable substrates that support astrocyte adhesion and growth.

[0033] Stage 4: Neuron-Astrocyte Co-culture and Maturation. In some embodiments, neuronal progenitors and astrocytes are combined and co-cultured on substrate-coated plates in medium conditions optimized for their maturation and interaction.

[0034] Stage 5: Triple cell culture Induction and Maturation. In some embodiments, microglial precursors are introduced into the neuron-astrocyte co-culture under suitable conditions that support microglial integration, maturation, and function to form the triple cell culture. In some embodiments, the triple cell culture system is matured under suitable and optimized conditions until ready for experimental use.Definitions

[0035] The terms “central nervous system” or “CNS” refer to the complex of nerve tissues that control bodily function and includes the brain and spinal cord.

[0036] The terms “blood-brain barrier” or “BBB” refer to a network of brain capillary endothelial cells that are closely sealed by tight junctions.Attorney Docket No. 01209-0020-00PCT

[0037] As used herein, the term “cell culture” refers to a growth of cells in vitro in an artificial medium for research or medical treatment.

[0038] As used herein, a “medium” is a solution used to surround cells in a cell culture. While the term “media” is grammatically the plural of “medium,” the term “media” is used in both the plural and singular sense herein.

[0039] As used herein, the term “culture medium” refers to a liquid that covers cells in a culture vessel, such as a Petri plate, a multi-well plate, and the like, and contains nutrients to nourish and support the cells. Culture medium may also include growth factors added to produce desired changes in the cells.

[0040] A “serum-free” or “serum free” medium herein refers to a culture medium that does not comprise animal serum such as fetal bovine serum or any other types of animal serum. A variety of commercial “serum-free” media are available, for example.

[0041] A “basal medium” herein is a medium that comprises the essential nutrients needed for survival of the cells to be cultured, such as amino acids, vitamins, enzymatic co-factors, salts, ions, buffers, and / or carbon sources such as glucose, but that may lack other ingredients such as hormones, growth factors, or the like. A “serum-free basal medium” herein is a basal medium that is also serum-free. In some embodiments, a basal medium may be supplemented with additional factors to promote growth (aka., expansion) and / or differentiation of particular cell types.

[0042] As used herein, the term “differentiation” refers to a process whereby an unspecialized cell acquires the features of a specialized cell. Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

[0043] The term “expression system” refers to one or more nucleic acid molecules comprising coding sequence and control sequence(s) in operable linkage, along with a host cell and / or other in vitro transcription and translation machinery, such that one or more proteins encoded by the nucleic acid molecule(s) are capable of being produced.

[0044] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a type of pluripotent stem cell, similar to an embryonic stem cell, formed by the introduction of certain embryonic genes (such as a OCT4, SOX2, NANOG, POU5F1, and KLF4 transgenes) (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), herein incorporated by reference for all purposes) into a somatic cell. An induced pluripotent stem cell may be prepared from any fully (e.g., mature or adult) or partially differentiated cell using methodsAttorney Docket No. 01209-0020-00PCT known in the art. For example, but not by way of limitation, an induced pluripotent stem cell may be prepared from a fibroblast, such as a human fibroblast; an epithelial cell, such as a human epithelial cell; a blood cell such as a lymphocyte or hematopoietic cell, or cell precursor or myeloid cell, such as a human lymphocyte, hematopoietic cell or cell precursor or human myeloid cell; or a renal epithelial cell, such as a human renal epithelial cell. In certain non-limiting embodiments, an induced pluripotent stem cell contains one or more introduced reprogramming factors associated with producing pluripotency. In some embodiments, a human induced pluripotent stem cell is not identical to a human embryonic pluripotent stem cell.

[0045] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.

[0046] “Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0047] As used herein, the term “a population of cells” or “a cell population” refers to a group of at least two cells. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at leastAttorney Docket No. 01209-0020-00PCT about 1000 cells. The population may be a pure population comprising one cell type, such as a population of proprioceptors, or a population of undifferentiated stem cells. Alternatively, the population may comprise more than one cell type, for example a mixed cell population.

[0048] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of up to 10% above and down to 10% below the value or range remain within the intended meaning of the recited value or range. It is understood that wherever aspects are described herein with the language “about” or “approximately” a numeric value or range, otherwise analogous aspects referring to the specific numeric value or range are also provided.

[0049] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise. For example, reference to an “antibody” is a reference to from one to many antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

[0050] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially of’ are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0051] As used herein the terms “hemogenic embryoid bodies (hemogenic EBs)” or “embryoid bodies (EBs)” refer to three-dimensional aggregates of human induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) cultured under conditions that promote primitive myeloid and microglial precursor formation. EBs are characterized by spheroidal morphology, presence of organized outer epithelial-like layers, and the emergence of distinct germ layer derivatives within the aggregate. EBs also typically form spontaneously when pluripotent stem cells are cultured in suspension or on low-attachment surfaces, in contrast to adherent monolayer cultures.

[0052] Additional terms and phrases are defined in sections that follow or elsewhere in this disclosure.

[0053] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety for all purposes.Attorney Docket No. 01209-0020-00PCTI. Methods of Preparing Triple Cell Cultures

[0054] In some embodiments, the triple cell culture system is prepared using a multi-stage protocol involving the sequential and / or parallel preparation of neurons or neuronal precursors, microglia or microglial precursors, and astrocytes. For example, in some embodiments, a triple cell culture system is prepared by a method comprising (i) providing neurons or neuronal precursors; (ii) providing astrocytes; (iii) providing microglia cells or microglial precursors; and (iv) seeding a cell culture vessel with those three cell types. In some embodiments, one or more of the above cells may be derived from iPSC cells prior to preparation of the triple cell culture. In some embodiments, the triple cell culture system is prepared by first co-culturing neuronal precursors and astrocytes. In some embodiments, the protocol is according to the Exemplary Protocol, above, or Example 1 herein. For example, in some embodiments, triple cell cultures may be prepared by (i) iPSC expansion (stage 0 of Example 1 herein), (ii) microglia precursor production (stage 1), (iii) neural progenitor induction (stage 2), (iv) astrocyte expansion (stage 3), (v) iPSC-neuron and astrocyte coculture plating and maturation (stage 4), and (vi) triple cell culture induction and maturation (stage 5). See FIGs. 1A-1C, for example. However, numerous aspects of the protocol may be adjusted.

[0055] In some embodiments, the method of preparing the triple cell culture comprises obtaining iPSC cells (Stage 0 according to the Exemplary Protocol). In some embodiments, Stage 0 involves expanding iPSC cells to obtain a sufficient number of iPSC cells at a sufficient confluency for downstream differentiation. In some embodiments, iPSC cells are thawed and seeded onto an appropriate surface, such as an extracellular matrix-coated culture vessel, such as plates coated with Geltrex™ or other suitable basement membrane substitutes. In some embodiments, the iPSCs are cultured in a defined stem cell medium, such as a feeder-free, defined culture medium suitable for the maintenance and expansion of undifferentiated human iPSCs, such as StemFlex™. In some embodiments, the iPSC medium is supplemented with ROCK inhibitor (such as Y-27632). In some embodiments, the iPSCs are expanded until reaching an appropriate confluency, such as 60-90% confluency.Microglia and Microglia Precursors and their Formation from iPSCs

[0056] “Microglial cells” or “microglia” are resident immune cells of the CNS. Microglia can elicit overt degeneration of neurons and glia and are normally involved in the maintenance of brain homeostasis, the clearing of cellular debris, pruning of synapses, and eliminating aggregations. Microglia become activated in response to injury, infection, orAttorney Docket No. 01209-0020-00PCT neurodegeneration. Genome-wide association studies have linked microglia innate immunity with Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (Lou Gherig’s disease), autism, and tumorogenesis. Microglia have been implicated as primary agents in certain developmental and age-related disorders. Accurate representation of microglial responses is important for modeling CNS disease.

[0057] As used herein the term “microglia precursors” refers to cells derived from iPSCs that exhibit phenotypic and functional characteristics of progenitor cells committed to the microglial lineage. In some embodiments, microglia precursors can be distinguished from undifferentiated iPSCs and other hematopoietic progenitors by one or more of the following features: expression of lineage-specific markers associated with early microglial development, including, for example, P2RY12, P2RY13, TMEM119, CX3CR1, CSF1R, AIF1, TREM2, and / or SALL1; no expression or reduced expression of pluripotency markers including, for example, OCT4, SOX2, NANOG, POU5F1, and / or KLF4; and the ability to migrate, survive, and further mature into functional microglia when co-cultured with neurons, astrocytes; and respond to inflammatory events (e.g., lipopolysaccharide, ATP, cytokines) by modulating cytokine secretion, phagocytosis, or calcium flux.

[0058] In some embodiments, microglia precursor cells can be differentiated from human pluripotent stem cells (such as human induced pluripotent stem cells or embryonic stem cells). In some embodiments, the iPSCs express pluripotency markers, including, for example, OCT4, SOX2, NANOG, POU5F1, and / or KLF4, which are not expressed by microglial precursors or which are expressed to a reduced degree in microglial precursors. In some embodiments, expression of such markers may be assessed at the RNA level; in other embodiments, it may be assessed at the protein level.

[0059] Some embodiments of the methods herein include a method of preparing the triple cell culture comprising generating microglial precursors from iPSCs (Stage 1 according to the Exemplary Protocol). In some embodiments, the iPSCs are prepared according to the method of Stage 0 herein. In some embodiments, single-cell suspensions of iPSCs are seeded into microwell plates (such as AggreWell™ 800 plates) in a “hemogenic EB medium.” In some embodiments, the microwell plates comprise conical microwells designed to promote the formation of size-controlled, homogeneous cell aggregates or spheroids (i.e., embryoid bodies). In some embodiments, the microwell plates comprise a hydrophilic surface to prevent nonspecific cell adhesion and to facilitate formation of embryoid bodies.Attorney Docket No. 01209-0020-00PCT

[0060] A “hemogenic EB medium” herein is a medium that is conducive to embryoid body (EB) formation (such as hemogenic EB media described herein). In some embodiments, the hemogenic EB medium is a serum-free or low serum basal medium, that may be supplemented with one or more growth factors such as vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP4), and / or stem cell factor (SCF), and that may or may not also be xeno-free. As used herein, “xeno-free” means that the media is free of nonhuman animal-derived components such as fetal bovine serum or other sera. A “xeno-free” medium, however, can in some embodiments include human-derived components such as human serum, human cell lysates, or human-derived proteins including recombinant proteins. Thus, in some embodiments, the hemogenic EB medium comprises a defined xeno-free low- serum or serum-free basal media, such as a StemFlex™ base. In some embodiments, the hemogenic EB media comprises growth factors such as vascular endothelial growth factor (VEGF). In some embodiments, the hemogenic EB media comprises growth factors such as bone morphogenetic protein 4 (BMP4). In some embodiments, the hemogenic EB media comprises growth factors such as stem cell factor (SCF).

[0061] In some embodiments, once formed, hemogenic embryoid bodies (EBs) are transferred to adherent flasks pre-coated with extracellular matrix components (such as gelatin) and, in some embodiments, cultured in “microglial precursor production media.” As used herein, “microglial precursor production media” comprise “serum-free hematopoietic basal media” that may be supplemented with ingredients such as, for example, one or more cytokines, growth factors, and / or hormones, such as one or more of IL-3, mCSF, 2- mercaptoethanol, and / or L-glutamine (e.g., Glutamax™). As used herein, a “serum-free hematopoietic basal medium” includes a serum-free basal medium designed for growth of hematopoietic stem and progenitor cells without the need for animal serum. In some embodiments, the medium may also be xeno-free. In some embodiments, the microglial precursor production media comprises a serum-free hematopoietic cell medium, such as X- VIVO®, or any other medium suitable for proliferation of this cell type. In some embodiments, the microglial precursor production media comprises IL-3. In some embodiments, the microglial precursor production media comprises mCSF. In some embodiments, the microglial precursor production media comprises 2-mercaptoethanol. In some embodiments, the microglial precursor production media comprises a stabilized form of L-glutamine, such as Glutamax™. Through Stage 1, EBs adhere and differentiate into microglial precursors over a period of several weeks, with media changes performed atAttorney Docket No. 01209-0020-00PCT intervals to support precursor production. In some embodiments, microglial precursors are harvested when they reach sufficient density, as determined by suitable methods such as microscopy or turbidity.Neural Precursors or Neurons and their Production from iPSCs

[0062] “Neurons” are electrically excitable cells responsible for transmitting information through electrochemical signaling. They are the primary functional cells of the CNS, enabling essential processes such as cognition, sensory perception, motor coordination, and memory formation. Dysfunction and loss of neurons are central features of many neurological diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In culture, neurons typically require specific growth factors and extracellular matrix components to support their differentiation, maturation, and synapse formation. In cell culture models, neurons are commonly obtained from primary cultures derived from embryonic or postnatal animal tissues or from differentiated pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). In the triple cell culture system described herein, it has been discovered that neuronal media, such as BrainPhys™ supplemented with SMI, is capable of supporting functionalized microglia while promoting neuronal integrity and identity.

[0063] As used herein the term “neuronal precursors” (also called “neural progenitors” and “neuronal precursors” or like terms) refers to cells derived from iPSCs that exhibit properties of early neural lineage commitment and that give rise to “neurons” also known as “mature neurons.” In some embodiments, neuronal precursors are distinguished from iPSCs by the expression of neural stem and early neuronal lineage markers including TUBB3; and / or electrophysiological capabilities consistent with neuronal maturation upon further differentiation, including acquisition of voltage-gated sodium and potassium currents and the ability to generate action potentials.

[0064] In some embodiments, the method of preparing the triple cell culture comprises inducing neural precursors from iPSCs (Stage 2 in the Exemplary Protocol). In some embodiments, iPSCs are dissociated into single cells and seeded onto a suitable basement membrane, such as on a Geltrex™-coated plate in neuron induction media. A “neuron induction media” or “neural induction media” as used herein refers to media used to induce iPSCs to differentiate into neural precursors or neurons. Thus, such media may include small molecules and growth factors that promote such differentiation of the cells. In some embodiments, the neuron induction media is based on a basal medium comprising aminoAttorney Docket No. 01209-0020-00PCT acids, vitamins, glucose, and inorganic salts, such as a Dulbecco’s Modified Eagle Medium (DMEM)-based media. “DMEM” refers to a widely used basal cell culture medium formulated to support the growth of a variety of mammalian cells. DMEM generally comprises relatively high concentrations of amino acids, vitamins, and glucose to promote enhanced cell growth and viability. DMEM also generally comprises essential and non- essential amino acids, inorganic salts, glucose, and buffering agents, and may be supplemented with serum, antibiotics, and / or other additives as required for specific cell types or experimental conditions. In some embodiments, the neuron induction media is a DMEM F / 12-based media (Dulbecco’s modified Eagle medium supplemented with nutrient mixture F-12) or suitable alternative, such as DMEM media supplemented with other nutrient mixtures. In some embodiments, the nutrient mixtures comprise of sugar, vitamins, amino acids, and / or growth factors suitable to support cell proliferation and / or differentiation. In some cases, the media may further comprise one or more of a serum-free supplement derived from Bottenstein’s N-l formulation such as N-2 media supplement, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), laminin or another extracellular matrix protein, and / or doxycycline. In some cases, the neural induction medium may also be supplemented with a ROCK inhibitor such as Y-27632. In some embodiments, the iPSCs may be engineered to express neurogenic transcription factors, such as NGN2, under inducible promoters. In some embodiments, the neuron induction media comprises a serum- free supplement derived from Bottenstein’s N-l formulation, a defined supplement originally developed for serum-free culture of CNS cells. In some embodiments, Bottenstein’s N-l formulation comprises one or more of insulin, transferrin, selenite, putrescine, and progesterone. In some embodiments, the serum-free supplement derived from the N-l formulation is N-2 media supplement. In some embodiments, the N-2 supplement comprises one or more of insulin, apo-transferrin, progesterone, putrescine, and sodium selenite. In some embodiments, the neuron induction media comprises BDNF. In some embodiments, the neuron induction media comprises NT-3. In some embodiments, the neuron induction media comprises extracellular matrix proteins, such as laminin. In some embodiments, the neuron induction media comprises doxycycline to activate transgene expression (such as with the NGN2 dox-inducible system). Neurons are maintained in induction media for several days, with media changes. In some embodiments, the cells are monitored for neuronal character during and / or after the production process, such as for neurite outgrowth and / or neuronal marker expression. In some embodiments, neurons or neuronal precursors prepared in thisAttorney Docket No. 01209-0020-00PCT step are positive for one or more of MAP2, beta-III-tubulin, synapsin 1, NeuN, and DCX. In some embodiments, expression of those markers may be detected at the RNA level while in other embodiments, expression of those markers may be detected at the protein level.Provision of Astrocytes for Triple Cell Cultures

[0065] “Astrocytes” are glial cells critical for maintaining homeostasis within the CNS. They provide structural support, regulate neurotransmitter uptake, maintain the blood-brain barrier, and support neuronal survival and synapse formation. Astrocytes are also implicated in the pathology of several neurological disorders, including Alzheimer's disease, Huntington's disease, and multiple sclerosis, through mechanisms such as impaired glutamate clearance and neuroinflammatory signaling. Astrocytes used in cell culture are typically derived from primary animal tissues, human fetal sources, or pluripotent stem cells, depending on the experimental requirements. In some embodiments, astrocytes are obtained from human fetal sources or other suitable precursor populations. In some embodiments, astrocytes present in triple cell cultures herein are characterized by expression of ALDH1L1, SLC1A2, SLC1A3, GLUL, AQP4, GJA1, and / or SOX9. In some embodiments, astrocytes present in triple cell cultures herein are characterized by decreased expression of ALDH1L1, SLC1A2, SLC1A3, AQP4, GJA1, and / or SOX9 compared to astrocytes in a co-culture with neurons in the absence of microglia. In some embodiments, astrocytes present in triple cell cultures herein are characterized by increased marker expression of GLUL compared to astrocytes in a co-culture with neurons in the absence of microglia. See, e.g., Figs. 5B and 5E.

[0066] In some embodiments, the method of preparing the triple cell culture comprises thawing and expansion of astrocytes (Stage 3 in the Exemplary Protocol). In some embodiments, cryopreserved human fetal astrocytes are thawed and seeded onto suitable culture flasks. In some embodiments, the human fetal astrocytes are seeded onto culture surfaces pre-coated with a treatment to enhance cell attachment and adhesion, such as poly-L- ornithine (PLO) and rinsed to remove residual coating. Astrocytes are expanded in suitable complete astrocyte media under standard culture conditions. Complete astrocyte media components were purchased from Sciencell. Complete astrocyte media typically contains basal media DMEM, 5 to 10% fetal bovine serum, and astrocyte growth supplement, which typically includes BSA, Insulin, IGF-1, and FGF-2. Media changes are performed at regular intervals, and astrocytes are cultured until reaching suitable confluency for downstream coculture. Co-culture media contains either neurobasal or Brainphys™ basal media, N2 andAttorney Docket No. 01209-0020-00PCTSMI supplements, BDNF, and GDNF. Triple cell culture media contains the same components with additional factors, such as IL34, GMCSF, and TGFB, which are important for microglia maintenance and maturation.Co-culturing of neuronal precursors and astrocytes

[0067] In some embodiments, the method of preparing the triple cell culture comprises coculturing the induced neuronal precursors and astrocytes (Stage 4 in the Exemplary Protocol). In some embodiments, neuronal precursors and astrocytes are sequentially seeded into multiwell plates (e.g., 96-well plates). In some embodiments, the plates are pre-coated with PLO and / or laminin. In some embodiments, neurons are seeded first and allowed to adhere, followed by the addition of astrocytes. The co-culture is maintained in a suitable co-culture medium to support the growth and maturation of neurons. In some embodiments, the coculture medium is a serum-free basal medium such as BrainPhys™, or a neuronal medium comprising inorganic salts, amino acids, vitamins, dextrose, sodium pyruvate, Hepes, cholesterol, and optionally phenol red. The serum-free basal medium may in some cases be supplemented with one or more of Bottenstein’s N-l supplement, N-2 supplement, NeuroCult™ SMI neuronal supplement (“SMI”), BDNF, GDNF, and / or doxycycline. In some embodiments, the co-culture media comprises N-2 media supplement or suitable alternative such as other N-l -derived supplements. In some embodiments, the co-culture media comprises SMI. In some embodiments, the co-culture media comprises BDNF. In some embodiments, the co-culture media comprises GDNF. In some embodiments, the coculture media comprises doxycycline (such as for NGN2-expressing cells). Media is replaced as needed while avoiding disrupting the forming neural network. The neuron-astrocyte coculture in some embodiments is allowed to mature for several days to achieve physiological relevance and to promote astrocyte-neuron interaction.

[0068] In some embodiments, the cells may be assayed for gene expression or morphology before proceeding to formation of a triple cell culture.Preparation of Triple Cell Cultures

[0069] In some embodiments, the method of preparing the triple cell culture comprises integrating microglial precursors into a mature neuron-astrocyte co-culture such as that described above to form the triple cell culture (Stage 5 in the Exemplary Protocol). In some embodiments, microglial precursors harvested as described in Stage 1 above are counted and suspended in a triple cell culture medium. In some embodiments, the triple cell culture medium comprises serum free basal medium suitable for neuronal growth, such asAttorney Docket No. 01209-0020-00PCTBrainPhys™ or a neuronal medium comprising inorganic salts, amino acids, vitamins, dextrose, sodium pyruvate, Hepes, cholesterol, and optionally phenol red. In some embodiments, the triple cell culture medium comprises one or more of Bottenstein’s N-l supplement, N-2 supplement, SMI, BDNF, GDNF, IL34, gmCSF, TGFbeta, and / or doxycycline. For example, in some embodiments, BrainPhys™ may be supplemented with one nor more of those supplements to form the triple cell culture medium. In some embodiments, the triple cell culture medium comprises SMI. In some embodiments, the triple cell culture medium comprises BDNF. In some embodiments, the triple cell culture medium comprises GDNF. In some embodiments, the triple cell culture medium comprises IL34. In some embodiments, the triple cell culture medium comprises gmCSF. In some embodiments, the triple cell culture medium comprises TGFp. The addition of certain supplements can promote microglial maturation and functionality. In some embodiments, the microglial precursors are added to the co-culture at a defined ratio. In some embodiments, the neurons outnumber either the astrocytes or the microglia precursors. For example, in some embodiments, about 1.5, about 2, about 2.5, about 3, about 3.5, or about 4 neurons are present per microglia or microglial precursor. In some embodiments, about 3 neurons are present per microglia or microglial precursor. In some embodiments, about 1.5, about 2, about 2.5, about 3, about 3.5, or about 4 neurons are present per astrocyte. In some embodiments, about 3 neurons are present per astrocyte. In some embodiments, about 3 neurons are present to about 1 astrocyte and about 1 microglia or microglial precursor (i.e., about a 3: 1 : 1 ratio). For example, in a 96-well format, about 20,000 neuronal precursors, about 6,500 microglia precursors, and about 6,500 astrocytes may be seeded. It has been discovered herein that seeding a greater number of neuronal precursors and an intermediate number of microglia precursors promotes MAP2 network length of the neurons while producing a triple cell culture with highly ramified microglia. The triple cell culture may then be maintained in media comprising, for example, BrainPhys™, and further comprising, for example, 2% SMI, 1% N2-A, 20 ng / ml BDNF, 20 ng / ml GDNF, 100 ng / ml IL34, 25 ng / ml TGFb, and 10 ng / ml gmCSF. The resulting triple cell culture in some embodiments is matured with periodic feeding and is typically ready for experimental use within 1-2 weeks following microglial seeding.

[0070] In some embodiments, the ratio of neurons or neuronal precursors to astrocytes to microglia or microglial precursors seeded in the triple cell culture may be defined such that more neurons (or neuronal precursors) are introduced to the triple cell culture than eitherAttorney Docket No. 01209-0020-00PCT microglia (or microglia precursors) or astrocytes. In some embodiments, a higher ratio of neurons (or neuronal precursors) relative to astrocytes and microglia allows the triple cell culture to more closely resemble the physiological CNS environment. For example, neuron:microglia:astrocyte ratios may range from about 10: 1 : 1 to about 2: 1 : 1. In an exemplary embodiment, the ratio of neuron:microglia:astrocyte is about 3: 1 : 1.

[0071] In some embodiments, the methods of preparing the triple cell culture comprise the use of particular supplements. In some embodiments, SMI neuronal supplement, alternative neuronal supplements, or additional growth factors and cytokines, such as BDNF, GDNF, IL- 34, GM-CSF, and TGF-beta, at varying concentrations based on experimental needs and desired outcomes are used.

[0072] The triple cell culture preparation methods disclosed herein provide a reproducible and modular platform for assembling a human CNS-like in vitro model comprising three major central nervous system cell types. The protocols disclosed herein support genetic manipulation, cryopreservation, and adaptation to high-throughput screening formats.Cell sources

[0073] The triple cell culture system described herein can be constructed using a variety of cell sources depending on the intended application, including, but not limited to, disease modeling, drug screening, and mechanistic studies.

[0074] Induced pluripotent stem cells (iPSCs). In some embodiments, neurons and / or microglia are derived from human induced pluripotent stem cells (iPSCs). iPSCs may be obtained from healthy donors or from individuals carrying genetic mutations or polymorphisms associated with neurological diseases. These patient-derived iPSCs provide a foundation for personalized or precision modeling of neurodegenerative or neurodevel opmental conditions, such as Alzheimer’s disease, Parkinson’s disease, autism spectrum disorder, or Rett syndrome.

[0075] Genome-edited iPSCs. In some embodiments, the iPSCs used to generate neurons or microglia are genetically modified using genome-editing technologies such as the CRISPR / Cas9 system, TALENs, zinc finger nucleases, or any other suitable method. Such editing can introduce or correct specific disease-associated variants (such as TREM2, ApoE, or CD33 variants), disrupt gene expression (such as TREM2 knockout), or introduce reporter constructs (such as CX3CR1-GFP) for functional assays or imaging studies. These engineered lines enable the study of causal relationships between genotype and phenotype in a controlled background.Attorney Docket No. 01209-0020-00PCT

[0076] Isogenic iPSCs. In some embodiments, neurons and microlia are derived from a single iPSC line. In some embodiments, all three cell types — neurons, astrocytes, and microglia — are derived from a single iPSC line, providing an isogenic system that minimizes genetic variability and facilitates interpretation of functional outcomes. In some embodiments, cell types may be non-isogenic, such as when fetal-derived astrocytes from one source are used alongside iPSC-derived neurons and microglia from other sources. Non- isogenic systems may be desirable when a specific cell source (e.g., human fetal astrocytes) provides superior functional performance or is more readily available for specific experimental needs.

[0077] Additional sources. In some embodiments, astrocytes may be obtained from primary fetal brain tissue, from commercially available human astrocyte cell lines, or from differentiated pluripotent stem cells. In some embodiments, microglia may be obtained from primary sources (e.g., fetal or neonatal brain). In some embodiments, microglial precursors are generated from iPSCs.

[0078] Cells used in the present disclosure may be assessed for identity by conventional methods, including immunocytochemistry, flow cytometry, and transcriptomic analysis, to confirm expression of lineage-specific markers (e.g., MAP2 and synapsins for neurons; GFAP and S100P for astrocytes; TMEM119, Ibal, and P2RY12 for microglia). Additional markers are known in the art, including cell state markers.

[0079] This flexibility in sourcing and customization makes the disclosed triple cell culture system a broadly applicable platform for modeling a wide range of genetic backgrounds and disease conditions relevant to the central nervous system.Neurons

[0080] In some embodiments, the neurons of the triple cell culture herein are synaptically active. As used herein the term “synoptically active neurons” refers to neurons within the triple cell culture system that are capable of forming and / or participating in functional synaptic connections with other neurons. Such neurons of the triple cell culture exhibit one or more of the following characteristics: increased expression of pre- and post-synaptic markers (e.g., MAP2, SNAP25, SYT1, SYN1, and / or NEFM) or decreased expression of pre- and post-synaptic markers (e.g., RBFOX3, TUBB3, and / or DLG4) compared to neuronal precursors or iPSCs; measurable electrophysiological activity indicative of synaptic transmission, including spontaneous or evoked postsynaptic currents, action potential propagation, or network oscillations; functional calcium flux or neurotransmitter release inAttomey Docket No. 01209-0020-00PCT response to stimulation; morphological features consistent with synapse formation, including dendritic spines and axonal boutons; and / or an electrophysiological profile characteristic of neuronal maturation, including higher firing rates and increased network synchrony (e.g., increased Synchrony Index), consistent with synapse formation and integration into the neuronal network. Accordingly, in some embodiments, the triple cell culture media may be assessed for one or more of these activities. In some embodiments, the pre- and post-synaptic markers are measured though quantitative RT-PCR or RNA-sequencing. In some embodiments electrophysiological activity is measured by microelectrode array. In some embodiments morphological features are measured with immunocytochemistry.Microglial cells

[0081] The general features of microglia and microglial precursor cells are described above. In some embodiments, as part of the process of producing the triple cell culture, tests are used to distinguish microglial precursors from mature microglia, such as in some embodiments by differences in gene expression, cellular maturity, and / or functional activity between iPSC-derived cells and primary cells. See FIG. 4 (which shows a flow cytometry assay showing benchmark pluripotency, lymphoid, myeloid, and microglia markers in microglia precursors). In some embodiments, such tests are performed at key differentiation stages to control for factors such as batch-to-batch variability from iPSCs. In some embodiments, an antibody panel, such as comprising antibodies against TRA-1-60, CD34, CD45, CD43, CD1 lb, P2RY12, and / or CD107a may be used to assess expected positive and negative markers characteristic of microglia precursors which would indicate if a particular batch of differentiated cells is appropriate to use in the triple culture system (see FIG. 4). Thus, in some embodiments, benchmarking with antibody panels provides a reference standard that facilitates evaluation of differentiation and maturity of the iPSC-derived microglia precursors prior to subsequent differentiation stages such as maturation in the monoculture or triple culture system.

[0082] In certain embodiments, the microglia or microglial precursor cells incorporated into the triple cell culture system are genetically engineered to express reporter genes, enabling real-time tracking, quantification, and functional analysis of microglial behavior. In some embodiments, microglia are engineered to express green fluorescent protein (GFP) or other suitable fluorescent reporter. In some embodiments, the microglia are engineered to express a reporter under the control of the CX3CR1 promoter, a microglia-specific and homeostatic transcriptional driver. Use of the CX3CR1 promoter, such as for a CX3CR1-Attorney Docket No. 01209-0020-00PCTGFP reporter, would allow visualization of microglial morphology, migration, and process motility within the triple cell culture, and facilitate live-cell imaging and automated morphological analysis (e.g., branching complexity, surveillance behavior). Such reporter constructs may be introduced by conventional means, including, but not limited to, lentiviral or retroviral transduction, CRISPR / Cas9-mediated knock-in, or any other suitable method of gene editing. The resulting cells may be clonally selected or pooled, and validated by flow cytometry, fluorescence microscopy, and comparison with endogenous CX3CR1 expression.

[0083] In some embodiments, microglia are engineered with alternative or dual reporters, such as a fluorescence-tagged lineage marker (e.g., Ibal-RFP or TMEM119-mCherry), NF- KB or IRF reporters (e.g., GFP under inflammatory response promoters) for quantifying activation status, calcium indicators (e.g., GCaMP) for monitoring functional signaling, or fluorescent phagocytosis probes (e.g., pH-sensitive fluorophores linked to amyloid beta or synaptosomes). These reporters may be constitutive or inducible, and enable multiplexed assays assessing microglial reactivity, inflammatory signaling, and interactions with other CNS cell types.

[0084] In some embodiments, microglia engineered with reporter genes allow for live-cell imaging of dynamic microglial behavior in response to stimuli (e.g., treatment with ATP, LPS, or Ap peptides), quantitative readouts of morphology and activation in high-content imaging or flow cytometry, automated image analysis of ramification, clustering, and migration, tracking of microglia in long-term experiments and lineage studies, and / or screening of compounds that modulate microglial activation, phagocytosis, or cytokine production.Astrocytes

[0085] As noted above, astrocytes are glial cells critical for maintaining homeostasis within the CNS. They provide structural support, regulate neurotransmitter uptake, maintain the blood-brain barrier, and support neuronal survival and synapse formation. Astrocytes may be derived from primary animal tissues, human fetal sources, or pluripotent stem cells, depending on the experimental requirements. In some embodiments, astrocytes are obtained from human fetal sources or other suitable precursor populations, i.e., astrocytes derived from “human fetal astrocytes.” In some embodiments, astrocytes within the triple cell cultures herein are characterized by expression of ALDH1L1, SLC1A2, SLC1 A3, GLUL, AQP4, GJA1, and / or SOX9. In some embodiments, the expression of ALDH1L1, SLC1A2, SLC1 A3, AQP4, GJA1, and / or SOX9 is decreased compared to that of astrocytes within aAttorney Docket No. 01209-0020-00PCT neuron / astrocyte co-culture. In some embodiments, the expression of GLUL is increased in astrocytes of the triple cell culture compared to astrocytes within a neuron / astrocyte coculture.Quality control markers

[0086] To ensure reproducibility, functional integrity, and lineage fidelity of the triple cell culture system, cells may be validated using established molecular and phenotypic markers. Such quality control (QC) assays may be performed at key stages during or after differentiation and / or after introduction of each cell type to a co-culture or to the triple cell culture.

[0087] Neuronal and neuronal precursor markers. Neuronal cells or neuronal precursor cells of the triple cell culture can be validated using a combination of structural, synaptic, and identity-specific markers. In certain embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of neuronal cells or neuronal precursor cells are assessed for expression of one or more of the following: MAP2 (microtubule-associated protein 2; a neuronal dendritic marker), piII-Tubulin (TUBB3; an early neuronal marker), Synapsin I (a presynaptic vesicle marker), NeuN (Rbfox3; a mature neuronal nuclear marker), VGLUT1 (a glutamatergic neuron identity marker), GAD1 (also known as GAD67; a GABAergic neuron identity marker), and / or DCX (doublecortin; a neuroblast or immature neuron marker). Some of these neuronal markers may be mutually exclusive, such as VGLUT1 and GAD1. In some embodiments, neuronal cells or neuronal precursor cells are assessed for expression of one or more of the following RBFOX3, MAP2, TUBB3, SNAP25, SYT1, SYN1, DLG4, and NEFM (see FIG. 5A and 5D). The selection of appropriate quality control markers depends upon the desired identity of the cell, and those skilled in the art can readily select appropriate quality control markers. In some embodiments, subtype or region-specific markers (e.g., CTIP2, TBR1, SATB2) may be used depending on desired neuronal identity. The foregoing lists are representative and do not limit the scope; additional markers known to those skilled in the art may be substituted or added.

[0088] Astrocyte markers. In some embodiments, human fetal astrocytes or differentiated astrocyte-like cells can be validated through the expression of canonical astrocytic markers, including: GFAP (glial fibrillary acidic protein; an intermediate filament protein), S100P (a calcium-binding protein specific to mature astrocytes), AQP4 (aquaporin-4; an astrocyte endfoot marker), ALDH1L1 (a pan-astrocyte marker), and / or GLAST (EAAT1) or GLT-1Attorney Docket No. 01209-0020-00PCT(EAAT2) (glutamate transporters indicating functional uptake). In some embodiments, additional markers such as CD44 and vimentin may also be used to evaluate astrocyte maturation state (to identify immature or reactive astrocytes).

[0089] In some embodiments, astrocytes are characterized by expression of ALDH1L1, SLC1A2 (EAAT2), SLC1A3 (EAAT1), GLUL, AQP4, GJA1 (Connexin-43), and / or SOX9 (transport, channels, canonical astrocyte identity) (see FIG. 5B and 5E). In some embodiments, astrocytes within the triple cell culture may also show decreased expression of one or more of ALDH1L1, SLC1A2 (EAAT2), SLC1A3 (EAAT1), GLUL, AQP4, GJA1 (Connexin-43), and / or SOX9 compared to that of astrocytes within a neuron / astrocyte coculture. (See, e.g., Fig. 5B.) In addition, in the presence of Abeta, astrocytes within the triple cel culture may also show increased expression of one or more of ALDH1L1, SLC1 A2 (EAAT2), SLC1A3 (EAAT1), GLUL, AQP4, GJA1 (Connexin-43), and / or SOX9, compared to expression in the absence of Abeta but otherwise identical conditions. In some embodiments, astrocytes are characterized by expression of one or more cell surface markers chosen from TRA-1-60, CD34, CD45, CD43, CD1 lb, P2RY12, and / or CD107a (see FIG. 4). The foregoing lists are representative and do not limit the scope; additional markers known to those skilled in the art may be substituted or added.

[0090] Microglia and microglial precursor markers. iPSC-derived microglia or microglial precursors may be evaluated for expression of known microglial markers. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of microglia cells or microglia precursor cells in the triple cell culture are positive for one or more of the following: TMEM119 (a homeostatic microglia-specific marker), P2RY12 (a purinergic receptor associated with CNS- resident microglia), CX3CR1 (a fractalkine receptor involved in neuron-microglia signaling), Ibal (AIF1; a general microglial / macrophage marker), and / or TREM2 (an immune signaling receptor implicated in neurodegeneration). In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of microglia cells or microglia precursor cells in the triple cell culture express a low level of CD1 lb and / or CD45, confirming the microglia are not yet activated in the triple cell culture. In contrast, in some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of microglia cells or microglia precursor cells in the triple cell culture express an increased level of CD1 lb and / or CD45, indicating the microglia cells are activated, such as in response to stimuli, aAttorney Docket No. 01209-0020-00PCT pathogen, or injury. Low to intermediate CD45 and / or CD1 lb expression is consistent with homeostatic, non-activated microglia, and may be desirable, in some embodiments, for the triple cell culture system before experimental use (wherein said experimental use may be designed to activate microglia cells).

[0091] In some embodiments, microglia are characterized by expression of one or more markers chosen from P2RY12, P2RY13, TMEM119, CX3CR1, CSF1R, AIF1 (IBA1), TREM2, and / or SALL1, and optionally also by homeostatic surveillance, and presence of CSF1R / TREM2 signaling. (See FIGs. 5C and 5F). The foregoing lists are representative and do not limit the scope; additional markers known to those skilled in the art may be substituted or added.

[0092] Flow cytometry, immunocytochemistry, transcriptomic profiling (e.g., single-cell RNA-seq), or any other suitable method may be used to assess expression of the preceding neuronal, microglial, and / or astrocyte markers. In some embodiments, functional assays such as phagocytosis of pHrodo-labeled particles, motility response, and ramified morphology scoring are used to assess microglial maturity and CNS relevance.

[0093] In some embodiments the preceding validations may be carried out at different times points during generation of the triple cell culture, including after seeding of the coculture, after full maturation of the triple cell culture, and following exposure of the triple cell culture to test agents.Structural embodiments

[0094] In addition to two-dimensional (2D) systems, the triple cell culture systems disclosed herein may, in some embodiments, incorporate structural modifications to more closely mimic the physical and spatial properties of the central nervous system (CNS). These embodiments may further support enhanced physiological relevance, experimental flexibility, and mechanistic interrogation of neurobiological processes.

[0095] In some embodiments, the triple cell culture is cultured in spatially separated but diffusively connected compartments using transwell systems. As used herein, a “transwell system” is a type of cell culture system comprising multiple chambers including a bottom chamber or well comprising cultured cells, for example, of one type and one or more upper chambers or wells comprising cultured cells, for example, of a different type that are separated from the bottom chamber or well by a porous membrane that may allow exchange of soluble proteins or other molecules to and / or from the different chambers or wells. The system may also allow for migration of the cells within their chambers or wells such as in theAttorney Docket No. 01209-0020-00PCT presence of a chemoattractant. For example, in some embodiments, neurons and astrocytes may be co-cultured on the bottom surface of a culture well, while the microglia cells are seeded on the upper surface of a porous transwell insert. The insert allows for exchange of soluble factors (e.g., cytokines or neurotransmitters) without direct physical contact between cell types. This format facilitates studies of chemotaxis, paracrine signaling, and barrier function, and permits selective sampling or manipulation of specific compartments. Transwell formats are especially useful for modeling blood-brain barrier interactions, microglial migration, or astrocyte-mediated modulation of immune responses.

[0096] In some embodiments, one or more cell types in the triple cell culture system are embedded within or cultured on top of soft hydrogels or extracellular matrix (ECM) mimetics. For example, in some embodiments, neurons are cultured on laminin-rich substrates to promote synapse formation. In some embodiments, microglia may be seeded on a solubilized basement membrane preparation, such as Matrigel™, collagen I, hyaluronic acid, or synthetic hydrogels engineered with brain-like stiffness to maintain homeostatic morphology and motility. In some embodiments, ECM components may be used to differentially modulate astrocyte reactivity or glial scar formation. These embodiments provide improved biomechanical signaling and structural support, enabling analysis of mechanotransduction, ECM-mediated signaling, and cell-matrix interactions.

[0097] In some embodiments, the triple cell culture system has a three-dimensional structure, for example, with more than one layer of cells arranged in a particular shape or as an aggregate in space. In other embodiments, it may have a two-dimensional shape such as on a flat plate or similar surface. For instance, in some embodiments it is configured as a three-dimensional (3D) spheroid or organoid-like aggregate, for example, as opposed to a flat, 2D, linearly arranged culture system. Such aggregates may be generated by aggregating pre-differentiated neurons, astrocytes, and microglia into low-attachment wells or microwell arrays and embedding mixed or sequentially assembled cell populations within hydrogel droplets or scaffold-free hanging drop cultures. Although such formats significantly increase complexity, the use of 3D formats allow for multilayered cell-cell interactions, recapitulation of spatial organization and cytoarchitecture, and gradient-based signaling and diffusion limitations, similar to the in vivo brain environment. 3D triple cell cultures may be used for long-term maturation, modeling of focal injury or hypoxia, or screening for drugs affecting structural network integrity, migration, and long-range signaling.Attorney Docket No. 01209-0020-00PCT

[0098] In some embodiments, the selection of a 2D, 3D, transwell, or ECM-based format may be based at least in part on the intended experimental use of the triple cell culture system. For example, high-throughput screening may favor 2D plate-based formats. Mechanistic studies of microglia migration or neurovascular crosstalk may favor transwell inserts. Morphogenesis or chronic modeling of disease progression may favor 3D aggregates or soft hydrogel systems.

[0099] Each format described herein may be combined with any suitable ratio of neurons, astrocytes, and microglia, and all media, supplements, and validation techniques described in the present disclosure are contemplated with these alternative structural embodiments.Methods of preserving and expanding triple cell cultures

[0100] The triple cell culture system disclosed herein is compatible with workflows that support cry opreservation and scalable production. For example, “cry opreservation” as used herein when referring to a cell culture or to a triple cell culture may involve flash freezing the cell culture, optionally in the presence of cryoprotectants such as DMSO. Preservation of the triple cell culture or intermediate stages used to generate the triple cell culture can allow for incorporating the system into high-throughput workflows such as drug screening or toxicology assays.

[0101] In some embodiments, individual cell types (neurons, astrocytes, or microglia) or precursor populations may be cryopreserved at defined stages of differentiation, enabling modular assembly of the triple cell culture on demand. Cryopreservation techniques of these cell types are well-known in the art.

[0102] For example, in some embodiments, microglia precursors may be harvested from iPSC differentiation cultures and cryopreserved using serum-free cryoprotectant media such as media containing DMSO (e.g., 90% culture media + 10% DMSO) under controlled-rate freezing conditions. Upon thawing, microglial precursors may be re-plated in appropriate media (e.g., iMg precursor production media or triple cell culture media as discussed in Example 1 of the present disclosure) and retain high viability and differentiation potential.

[0103] In some embodiments, iPSC-derived neurons, such as NGN2-induced or early-stage precursors, may be frozen prior to final maturation and thawed for co-culture seeding with astrocytes with minimal loss of function.

[0104] In some embodiments, human fetal astrocytes, commonly obtained from commercial sources, are frequently shipped cryopreserved and may be banked and expanded prior to integration into the co-culture with the neuronal precursors.Attorney Docket No. 01209-0020-00PCT

[0105] In some embodiments, a co-culture comprising iPSC-derived neurons, such as NGN2-induced or early-stage precursors, and astrocytes may be frozen prior to forming the triple cell culture.

[0106] These intermediate cryopreservation steps allow for batch-standardization across experiments, scheduling flexibility, and / or long-term storage of validated cell lines or donorspecific models. In some embodiments, entire co-cultures (e.g., neuron-astrocyte co-cultures or the triple cell culture system) may be cryopreserved as monolayers or in suspension. Techniques for optimizing post-thaw viability and functionality are known in the art.

[0107] In some embodiments, the triple cell culture system may be adapted for use in multiwell plates, including but not limited to, 96-well plates, 384-well plates, 24-well plates, or 6-well plates, depending upon the number of cells desired. The triple cell culture system may be adapted to different size formats by standardizing the seeding density (e.g., the number of cells seeded per cm2). Use of automated liquid handling systems for reproducible plating, feeding, and compound addition are contemplated for use with the methods of preparing the triple cell culture. In some embodiments, substrates such as poly-L-omithine and laminin can be pre-applied to plates at scale, and media can be batch-prepared to ensure consistency across experimental runs. In some embodiments, barcoded microplates and automation-compatible culture vessels are used for sample tracking and integration into screening pipelines.

[0108] In some embodiments, large-scale production of individual cell types (e.g., iPSC- derived microglia or NGN2-induced neurons) may be performed in flasks or bioreactor systems. In some embodiments, these cells can be aliquoted and cryopreserved as working cell banks, seeded into multiwell formats for downstream co-culture or triple-culture preparation, and / or distributed, such as for collaborative, therapeutic, diagnostic, or other commercial purposes. This scalability enables efficient generation of disease models from patient lines or genome-edited iPSCs across multiple replicates, time points, or therapeutic conditionsII. Disease Models using Triple Cell Cultures

[0109] The triple cell culture system provided herein provides a physiologically relevant and scalable platform for modeling neurological disease states and evaluating the effect of therapeutic agents in a multicellular human CNS-like environment. In particular, the presence of neurons, astrocytes, and microglia enables the interrogation of both cell-autonomous andAttorney Docket No. 01209-0020-00PCT non-cell autonomous responses to disease-associated stimuli, genetic perturbations, or pharmacological interventions.

[0110] In some embodiments, the triple cell culture system is used to model a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia (FTD). In some embodiments, disease modeling is achieved by using iPSC-derived neurons and / or iPSC-derived microglia generated from patient donors harboring disease-associated mutations or single nucleotide polymorphisms (SNPs), such as variants in TREM2, ApoE, PSEN1, SNCA, or MAP T genes. In some embodiments, gene-editing techniques, such as the CRISPR / Cas9 system, are used to generate isogenic iPSC lines with targeted disruptions or knock-ins to model specific genetic disorders or molecular pathways seen in patients, such as specific disease-associated mutations or SNPs.[OHl] In some embodiments, the triple cell culture system is used to evaluate cellular responses to exogenous disease-relevant insults. In some embodiments, the exogenous disease-relevant insult is an amyloid beta peptide, alpha-synuclein aggregate, a tau species, oxidative stress, a cytokine, or a pathogen-associated molecular pattern (PAMP) such as lipopolysaccharide (LPS) or poly(I:C). Microglial responses to these insults may be assessed via changes in morphology. For example, the microglia of the triple cell culture systems disclosed herein were found to exhibit a ramified morphology. A switch to a more ameboid morphology indicates an activated state and can also indicate phagocytosis of a target. Microglial responses may also be assessed via changes in motility, other measures of phagocytic activity, cytokine secretion, and expression of activation markers (such as CD1 lb, CD45, IL-6, and TNF-alpha). Neuronal health may be evaluated by various assessments, including synapse density, MAP2 network integrity, or apoptosis markers. Astrocyte responses may include GFAP upregulation or polarization toward reactive states.

[0112] Further provided herein are methods of screening therapeutic agents using the triple cell culture system. In some embodiments, a method comprises contacting the triple cell culture with one or more candidate therapeutic agents and measuring one or more cellular, molecular, or functional responses. In some embodiments, the therapeutic agent is selected from the group consisting of a small molecule, a peptide, a polypeptide, an enzyme, an antibody, an antibody fragment, a nucleic acid-based agent (such as an siRNA or antisense oligonucleotide), a viral vector, or a gene-editing component.Attorney Docket No. 01209-0020-00PCT

[0113] In some embodiments, the therapeutic agent is an antibody or antibody derivative, including monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, singledomain antibodies (such as nanobodies), or fusion proteins. In some embodiments, the antibody targets a disease-associated protein such as amyloid beta, phosphorylated tau, a- synuclein, TDP-43, ApoER2, complement proteins, or inflammatory cytokines. The agent may be tested for its ability to modulate microglial uptake of pathogenic species, reduce inflammatory signaling, or protect neuronal structure and viability. In some embodiments, the triple cell culture may be used in a high-throughput screening format, such as to assess a panel of therapeutic antibody candidates.

[0114] In some embodiments, the therapeutic agent is an enzyme, such as a protease, lipase, glycosidase, or other catalytic protein capable of degrading extracellular aggregates, modifying post-translational modifications, or reshaping the extracellular matrix. In some embodiments, the enzyme is delivered in recombinant form or via gene therapy vectors (such as AAV-based delivery). The triple cell culture system enables real-time assessment of enzyme activity and downstream cellular effects in a CNS-like context.

[0115] In some embodiments, the therapeutic agent is assessed for its ability to reduce microglial activation or inflammatory cytokine production. In some embodiments, the therapeutic agent is assessed for its ability to promote neuronal survival or neurite outgrowth. In some embodiments, the therapeutic agent is assessed for its ability to modulate astrocyte reactivity. In some embodiments, the therapeutic agent is assessed for its ability to restore homeostatic intercellular signaling. Quantitative readouts may include, but are not limited to, immunocytochemistry, ELISA, multiplex cytokine assays, qPCR, RNA sequencing, phagocytosis assays, flow cytometry, calcium imaging, and high-content image analysis.

[0116] In some embodiments, the triple cell culture system is used to assess the specificity and efficacy of therapeutic antibodies targeting distinct conformers or isoforms of pathological proteins. For example, antibodies may be conjugated to pH-sensitive fluorophores (e.g., pHrodo) and incubated with labeled amyloid beta or tau peptides to quantify microglial uptake. Differential responses to various antibody-aggregate combinations may reveal selective engagement of toxic species, enabling optimization of therapeutic candidates.

[0117] In some embodiments, the triple cell culture is configured for high-throughput drug screening by culturing cells in multiwell plates (such as 96- or 384-well formats) and automated compound addition, imaging, and data acquisition. In some embodiments, culturesAttorney Docket No. 01209-0020-00PCT are exposed to compound libraries, including FDA-approved drugs, biologic candidates, or novel small molecules, to identify agents that modulate neuroinflammatory pathways or protect against disease-relevant stressors.

[0118] The triple cell culture system disclosed herein thus offers a versatile and scalable platform for preclinical testing of therapeutic candidates in a humanized CNS environment, with applications in drug discovery, precision medicine, and mechanistic studies of neurodegeneration and neuroinflammation.

[0119] Also provided herein are methods for screening therapeutic agents, comprising contacting the triple cell culture provided herein with one or more test agents, measuring one or more parameters, and optionally selecting one or more test agents based on the measured one or more parameters.III. Kits

[0120] Further provided herein are kits for preparing or using the triple cell cultures disclosed herein. The kits may be configured to support research, diagnostic, drug discovery, toxicology, or therapeutic screening applications, including applications involving human CNS models, such as CNS disease models.

[0121] In some embodiments, the kit comprises a triple cell culture provided in live or cryopreserved form. In some embodiments, the kit comprises only a portion of the triple cell culture components, such as a pre-assembled neuron-astrocyte co-culture. In some embodiments, the kit comprises separate components, such as a pre-assembled neuronastrocyte co-culture and a separate culture of microglia precursors. In some embodiments, the kit comprises differentiated, cryopreserved cells ready for thawing and plating. In some embodiments, the kit comprises pre-plated cultures, such as pre-plated cultures in multi-well formats (e.g., 96-well plates) prepared and optionally matured to a defined developmental stage.

[0122] In some embodiments, the kit comprises a container comprising cryopreserved iPSC-derived neurons or neuronal precursors. In some embodiments, the kit comprises a container comprising cryopreserved fetal-derived or iPSC-derived astrocytes. In some embodiments, the kit comprises a container comprising cryopreserved iPSC-derived microglial precursors or mature microglia. In some embodiments, the kit comprises a hemogenic EB media as defined herein, or component ingredients thereof. In some embodiments, the kit comprises iMg precursor production media as defined herein, or component ingredients thereof. In some embodiments, the kit comprises neuron inductionAttorney Docket No. 01209-0020-00PCT media, or component ingredients thereof. In some embodiments, the kit comprises co-culture media as defined herein, or component ingredients thereof. In some embodiments, the kit comprises triple cell culture media as defined herein, or component ingredients thereof.

[0123] In some embodiments, the kit comprises instructions for preparing the triple cell culture. In some embodiments, the instructions comprise at least a portion of the Exemplary Protocol disclosed herein. In some embodiments, the instructions are provided as software instructions.

[0124] In some embodiments, the kit may be configured for a particular use, such as for drug discovery screening, validation for testing biologies, such as antibodies or enzymes, against known CNS-relevant targets, or a disease modeling kit, such as wherein the cells in the kit are derived from a donor line harboring a neurodegenerative mutation.EXAMPLES

[0125] The present disclosure may be better understood by reference to the following nonlimiting examples. The following examples are presented in order to more fully illustrate certain embodiments and should not be construed as limiting the broad scope of the present disclosure. While certain embodiments of the present disclosure have been shown as described herein, it will be apparent that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the embodiments. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the methods described herein.Example 1. Preparation of Neuron- Astrocyte-Microglia Triple Cell Culture

[0126] The following describes the discovery and development of a genetically and functionally relevant human CNS triple cell culture cell model comprising neurons, astrocytes, and microglia cells. Briefly, differentiated iPSC-derived microglia precursors are seeded onto an iPSC-derived neuron and human fetal astrocyte co-culture which then forms the mature triple cell culture.

[0127] In detail, a five-stage protocol (stages 1-5) was developed starting with iPSC cells (prepared in stage 0) (see FIGs. 1A-1C).

[0128] Media recipes. The following media were used.

[0129] Hemogenic EB media. Base of StemFlex™ with StemFlex™ supplement (Gibco™, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA, cat. No. A3349401) with 100Attorney Docket No. 01209-0020-00PCT ng / mL HumanKine® recombinant human VEGF165 protein (Proteintech®, Rosemont, Illinois, USA, cat. No. HZ-1038), 100 ng / mL human BMP-4 recombinant protein (PeproTech®, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA, cat. No. 120- 05), and 40 ng / mL human SCF recombinant protein (PeproTech® cat. No. 300-07).

[0130] iMg precursor production media. Base of X- VIVO® serum-free hematopoietic cell medium (Lonza, Basel, Switzerland, cat. No. 02-053Q) with 25 ng / mL human IL-3 recombinant protein (PeproTech® cat. No. 200-03), 100 ng / mL human M-CSF recombinant protein (PeproTech® cat. No. 300-25), 1 / 500 volume 2-mercaptoethanol (Gibco™ cat. No. 21985023), and 1 / 100 volume GlutaMAX™ (Gibco™ cat. No. 35050061).

[0131] Neuron induction media. Base of DMEM / F- 12 (Gibco™ cat. No. 11320082) with 1 / 100 volume N-2 supplement (Gibco™ cat. No. 17502048), 1 / 100 volume MEM non- essential amino acid solution (Gibco™ cat. No. 11140050), 10 ng / mL BDNF recombinant protein (PeproTech® cat. No. 450-02), 10 ng / mL human NT-3 recombinant protein (PeproTech® cat. No. 450-03), and 0.2 pg / mL mouse laminin (Sigma-Aldrich, Inc., St. Louis, Missouri, cat. No. L2020).

[0132] Co-culture media. Base of BrainPhys™ neuronal medium (StemCell™ Technologies, Inc., Cambridge, Massachussetts, cat. No. 05790) with 1 / 100 volume N2 Supplement-A (StemCell™ cat. No. 7152), 1 / 50 volume NeuroCult™ SMI Neuronal Supplement (StemCell™ cat. No. 5711), 20 ng / mL BDNF (PeproTech® cat. No. 450-02), and 20 ng / mL GDNF (PeproTech® cat. No. 450-10).

[0133] Triple cell culture media. Base of Brainphys™ media (StemCell™ cat. No. 05790), 1 / 100 volume N2 (StemCell™ cat. No. 7152), 1 / 50 volume SMI (StemCell™ cat. No. 5711), 20 ng / mL BDNF (StemCell™ cat. No. 78005), 20 ng / mL GDNF (StemCell™ cat. No. 78058), 100 ng / mL IL34 (Proteintech® cat. No. HZ1316), 10 ng / mL gmCSF (PeproTech® cat. No. 300-03), and 25 ng / mL TGFbeta (PeproTech® cat. No. 100-21).

[0134] Stage 0. iPSC expansion. This preliminary stage prepares iPSC cells for use in subsequent stages. Any suitable means of obtaining confluent iPSC cells is contemplated.

[0135] Day 1. iPSC thawing and plating. 1 mL of 1% Geltrex™ solution (Gibco cat. No. A14133-02) was added to two wells of a 6-well plate and incubated at 37 °C for at least one hour. The Geltrex™ was always kept cold before adding to a 6-well plate. A cryopreserved vial of iPSCs was thawed by swirling in a water bath or bead bath until a small amount of ice remained. The vial of iPSCs originated from a 70-90% confluent well of a 6-well plate. The cell suspension in the cryovial was transferred to 10 mL of cold StemFlex™ media in a 15Attorney Docket No. 01209-0020-00PCT mL conical tube. The suspension was aspirated slowly to avoid breaking up aggregates. The 15 mL conical was spun down at 300 x g for 5 minutes. The resulting supernatant was aspirated and the cell pellet was resuspended in 4 mL StemFlex™ supplemented with 10 pM Y-27632 ROCK inhibitor. The 1% Geltrex™ solution was aspirated from each well of the 6- well plate and 2 mL per well of the StemFlex™ cell suspension was immediately added to each of two wells. The 6-well plate was transferred to an incubator at 37 °C with 5% CO2. Gentle shifting of the plate was used to distribute iPSCs evenly.

[0136] Day 2. iPSC feeding. The media was aspirated from both wells and 2 mL of fresh StemFlex™ was added to each well.

[0137] Day 3. iPSC feeding. If the culture was 50-90% confluent, the protocol was skipped to the Day 4 passaging protocol. If the culture was <50% confluent, the media was aspirated from both wells and 2 mL of fresh StemFlex™ was added per well.

[0138] Day 4. iPSC passaging. 1 mL of Geltrex™ solution was added to each of two wells of a 6-well plate and incubated at 37 °C for at least one hour. The media was aspirated from the iPSC plate and each well was washed with 1 mL DPBS. 1 mL of VERSENE™ (or any suitable chelating agent) was added to each well and incubated at 37 °C for 4 minutes. Once the edges of the colonies peeled up with visible spaces between cells, the VERSENE™ was aspirated and 3 mL StemFlex™ was ejected onto bottom of wells to lift iPSC layer into iPSC aggregates. The Geltrex™ was aspirated from the Geltrex™-coated 6-well plate and 2 mL StemFlex™ was added to each well. 0.5 mL of the iPSC aggregate suspension was added to each well of the Geltrex™-coated 6-well plate. The plates were then incubated at 37 °C with 5% CO2. Gentle shifting of the plate was used to distribute iPSCs evenly.

[0139] Days 5-21. iPSC feeding and passaging. The media was aspirated from each well and 2 mL of fresh StemFlex™ was added per well. iPSC feeding continued until iPSCs were 60-90% confluent before moving to Stage 1 and / or Stage 2. iPSCs were passaged and maintained at 10-80% confluency before moving to Stage 1 and / or Stage 2.

[0140] Stage 1. Microglia precursor production. The following procedure begins with a full 6-well plate of 60-90% confluent iPSCs from Stage 0, above. Any suitable methods of generating confluent iPSCs are contemplated.

[0141] Day 1. Hemogenic embryoid body (EB) induction. 300 pl of anti-adherent solution was added to each well of an AggreWell™ 800 plate. Plates were spun at 1000 x g for 2 minutes. StemFlex™ media was aspirarated from iPSC wells and the wells were washed with 1 mL DPBS each. 1 mL Accutase® was added to each iPSC well and incubated at 37 °C / 5%Attorney Docket No. 01209-0020-00PCTC02 for 3 minutes. Once the cells could be lifted by swirling of the plate, 1 mL of StemFlex™ media was added to each well and the resulting cell suspension was added to a 50 mL conical tube. The cell suspension was triturated with a 5 mL serological to form a single cell suspension. The conical was then spun at 300 x g for 5 minutes. The resulting supernatant was aspirated and resuspending in 3 mL hemogenic EB media with 10 pM Y- 27632 ROCK inhibitor. The cells were counted and additional EB media with 10 pM Y- 27632 ROCK inhibitor was added to bring the suspension to 750,000 cells / mL. The antiadherent solution was aspirated from the AggreWell™ 800 plate and each well was washed with 2 mL PBS. 2 mL of the iPSC suspension was added to each well of the AggreWell™ 800 plate and the plate was then spun at 400 x g for 2 minutes. The plates were moved to a 37 °C / 5% CO2 incubator.

[0142] Day 2 and Day 3. EB Feeding. On each day, aspirate 1 mL of media was aspirated from each well of the AggreWell™ 800 plate. 1 mL of hemogenic EB media was added per well without disturbing EBs.

[0143] Day 4. EB Feeding and Precursor Production Flask Coating. 1 mL media was aspirated from each well of AggreWell™ 800 plate. 1 mL hemogenic EB media was added to each well of the AggreWell™ 800 plate without disturbing EBs. Separately, 20 mL of 0.5% gelatin solution was added to each of three T150 flasks and incubated at 37 °C overnight.

[0144] Day 5. Precursor Production Flask Seeding. Gelatin solution was aspirated from each T150 flask and 20 mL iMg precursor production media was added to each flask. 1.5 mL was aspirated from each well of the AggreWell™ 800 plate without disturbing EBs. Using a 5mL pipette, 2 mL media was transferred from a T150 flask and ejected into a well of the AggreWell™ to suspend the EBs. The EB suspension from was transferred to a second AggreWell™ well so that the EBs from two AggreWell™ wells were in the same approximately 2 mL suspension. The 2 mL EB suspension was transferred to the T150 flask from which the 2mL was taken from previously. The process is repeated until each T150 flask has been seeded with EBs from two AggreWells™. The flasks were then incubated at 37 °C / 5% CO2 and the flasks were shifted along the x and y axis to distribute EBs evenly. The flasks were left undisturbed for at least four days to allow for adherence.

[0145] Days 12 and 23 (days can be adjusted to avoid weekend feeding). Precursor Production Flask Feeding. 10 mL iMg precursor production media was added to each flask.

[0146] Days 23-40+. iMicroglia Precursor Production Flask Feeding. 10 mL iMg precursor production media was added to each flask twice a week. iPSC microglia precursors wereAttorney Docket No. 01209-0020-00PCT harvested at any point after day 40 (iMicroglia Precursor Production Flask day 35, up to protocol day 82, iMicroglia Precursor Production Flask day 77) if precursors could be detected in the supernatant via phase microscopy and the supernatant was cloudy.

[0147] Stage 2, Neural Progenitor Induction.

[0148] This stage began with a full 6-well plate of 60-90% confluent iPSCs.

[0149] Day 21. iNeuron induction plate seeding. 1 mL 1% Geltrex™ solution was added to each well of a 6-well plate and incubated at 37 °C for at least one hour. GelTrex™ was kept cool at all times before cooling. Stemflex™ was aspirated from iPSCs and the cells are washed with 1 mL DPBS per well. 1 mL Accutase® was added to each well and incubated at 37 °C with 5% CO2 for 3 minutes. Once cells could be lifted by swirling of the plate, 1 mL Stemflex™ was added to each well and the cell suspension was transferred to a 50 mL conical tube. The suspension was triturated with a 5 mL serological pipette and then spun at 300 x g for 5 minutes. The supernatant was aspirated and resuspended in 3 mL neuron induction media supplemented with 2 pg I mL doxycycline. The cells were counted and enough Stemflex™ (supplemented with 10 pM ROCK inhibitor, 2 pg / mL doxycycline) media was added to bring the cell suspension to about 150,000 cells per mL. The 1% GelTrex™ solution was aspirated from wells of the 6-well plate and 2 mL cell suspension was added to each well. The plates were incubated at 37 °C with 5% CO2 with gentle rocking to distribute the iPSCs evenly.

[0150] Day 22. iNeuron induction plate feeding. Media was aspirated from the iNeuron induction plate and 2 mL Stemflex™ supplemented with 2 pg / mL doxycycline was added per well.

[0151] Days 23-25. iNeuron induction plate feeding. The plates were fed daily by aspirating media from the plates and adding 2 mL neuron induction media supplemented with 2 pg / mL doxycycline per well.

[0152] Stage 3, Human fetal astrocyte thawing and expansion.

[0153] This stage began with a cryopreserved vial of 2-3 million astrocytes. Astrocytes were used which had not undergone more than 10 population doublings since purchase.

[0154] Day 14. 20 mL of 15% PLO solution (poly-L-omithine; Sigma cat. No. P4957) is diluted in PBS in a T150 flask and incubated at 37 °C for at least two hours. The PLO solution was aspirated from the T150 flask and washed three times with 40 mL PBS. A cryopreserved vial of human fetal astrocytes was thawed by swirling in a water bath or bead bath until a small amount of ice remained. Immediately, the cryovial was transferred to a cellAttorney Docket No. 01209-0020-00PCT culture hood and a pipette was used to transfer the cell suspension to 20 mL of cold astrocyte media in a 50 mL conical. The suspension was aspirated and ejected slowly to avoid shear stress. PBS was aspirated from the T150 flask and 20 mL of the astrocyte suspension was added. The T150 flask was incubated at 37 °C / 5% CO2 with shifting along x and y axis to distribute astrocytes evenly.

[0155] Day 15. Media was aspirated from the flask and 20 mL of complete astrocyte media was added.

[0156] Day 18 (day can be adjusted to avoid weekend feeding). The media was aspirated from the flask and 20 mL of complete astrocyte media was added.

[0157] Day 22 (day can be adjusted to avoid weekend feeding). The media was aspirated from the flask and 20 mL of complete astrocyte media was added.

[0158] Stage 4, iPSC-neuron and human fetal astrocyte co-culture plating and maturation

[0159] This stage began with a 70-95% confluent T150 flask of astrocytes from previous stage 3 and NGN2 neuron precursors from stage 2.

[0160] Day 25. 96-well plate coating. 50 pL of 15% PLO solution diluted in DPBS was added to the center 60 wells of five 96-well plates and 200 mL PBS was added to the border wells to mitigate evaporation. The plates were incubated at 37 °C overnight.

[0161] Day 26. The PLO solution was aspirated from 96-well plates and they were washed three times with 200 pL PBS per well. 50 pL of 10 pg / mL laminin solution in DPBS was added to the center 60 wells of the PLO-coated 96-well plates and incubated at 37 °C for at least two hours. Media was aspirated from the iNeuron induction plate and 1 mL DPBS was added per well. The DPBS is aspirated from the iNeuron induction plate and 1 mL Accutase® was added per well and incubated for 37 °C / 5% CO2 for 3 minutes.

[0162] Once the cells could be lifted by swirling the plate, 1 mL StemFlex™ was added to each well and the cell suspension was transferred to a 15 mL conical. The suspension was triturated with a serological pipette to form a single cell suspension. The suspension was spun at 300 x g for 5 minutes. The supernatant was aspirated and the cells were resuspended in 3 mL co-culture media supplemented with 2 pg / mL doxycycline. The cells were counted and enough co-culture media supplemented with 2 pg / mL doxycycline was added to bring the cell suspension to 400,000 viable cells per mL.

[0163] The laminin solution was aspirated from the center 60 wells of two 96-well plates and immediately a 50 pL of cell suspension was added to each well. The 96-well plates wereAttorney Docket No. 01209-0020-00PCT incubated at 37 °C / 5% C02. The process was repeated until all 96-well plates have been seeded.

[0164] After 45 minutes, the media was aspirated from the astrocyte T150 flask and 20 mL of DPBS was immediately added. The DPBS was aspirated from the T150 flask and 15 mL trypsin was added. The flask was incubated at 37 °C / 5% CO2 for 5 minutes. 15 mL astrocyte media was added to the T150 flask and the cell suspension was transferred to a 50 mL conical and spun at 300 x g for 5 minutes. The supernatant was aspirated and the astrocytes were resuspended in 5 mL co-culture media supplemented with 2 pg / mL doxycycline. The astrocytes were counted and enough co-culture media + doxycycline was added to obtain about 133,000 viable astrocytes per mL. 50 pL of the astrocyte suspension was added to the center 60 wells of the five 96-well plates seeded above and the plates were incubated at 37 °C / 5% CO2.

[0165] Day 30. iNeuron-astrocyte co-culture feeding (day can be adjusted to avoid weekend feeding). From this day on, media was replaced carefully to avoid disturbing cell layer. An aliquot of co-culture media supplemented with 2 pg / mL doxycycline was warmed to 37 °C. 50 pL was aspirated from each well of the co-culture plates and 50 pL of the 37 °C co-culture media supplemented with 2 pg / mL doxycycline was added.

[0166] Stage 5, Triple cell culture induction and maturation (days 38-52),

[0167] Day 33. iMicroglia precursor seeding. 45 mL supernatant from the iMicroglia precursor production flasks was strained through a 40 pm cell strainer into 50 mL conicals. If the supernatant cells were clumped, they were triturated 4 times with a serological pipette to disperse. The adherent cell layer was not disturbed. The 50 mL conicals were spun at 300 x g for 5 minutes. The supernatant was aspirated and the pellets resuspended in 3 mL triple cell culture media supplemented with 2 pg / mL doxycycline. The cells were counted and enough triple cell culture media was added to bring the iMicroglia precursor suspension to about 133,000 viable cells per mL. The iMicroglia precursor suspension was warmed to 37 °C. 50 pL media from each well of the co-culture plates was aspirated and 50 pL of pre-warmed microglia precursor suspension was added to each well.

[0168] Days 37-47+. Triple cell culture feeding. The triple cell cultures were fed twice a week. An aliquot of triple cell culture media was warmed to 37 °C. 50 pL media was aspirated from each well and 50 pL of pre-warmed triple cell culture media was added to each well. The triple cell cultures were ready for experimental use around day 47.Attorney Docket No. 01209-0020-00PCT

[0169] The exemplary triple cell culture protocol is shown in the flowchart of FIGs. 1A-1C and microglia, astrocyte, and neuron staining of GFAF, MAP2, and IBA1 is shown in the right panel of FIG. 1C. The staining shows the neutrite projections from neurons and ramified morphology of the microglia cells in the triple cell culture.Example 2. Triple cell culture microglia are ramified and express microglia marker proteins

[0170] Triple cell cultures prepared according to the protocol of Example 1, above, were assembled. Immunocytochemistry (ICC) and confocal imaging were used to analyze microglial morphology and marker expression. The triple cell cultures were stained with anti- IBA1 (ionized calcium-binding adapter molecule 1), a cytoplasmic protein expressed in microglia, and an established marker for microglial identity and morphology. The triple cell cultures were also stained with DAPI nuclear stain and anti-MAP2. The IBA1 branch length per cell is a quantitative measure of microglial ramification and was calculated by segmenting IBA1+ processes and quantifying total length per microglial cell. Ramified morphology, characterized by long, thin, branched processes, is associated with resting, homeostatic microglia in vivo. In contrast, ameboid (rounded) morphology typically indicates activated or inflamed microglia. Microglia in the triple cell culture were compared to a monoculture of microglia.

[0171] As shown in FIG. 6A, microglia in the triple cell culture exhibited extensive ramification and integration into the neuronal-astrocyte network, with no disruption of MAP2+ neuronal processes as shown by the MAP2 morphology. In contrast, microglial cells in the monoculture were more ameboid.

[0172] In addition, as shown in FIG. 6B, flow cytometry was performed against CD45, CD1 lb, P2RY12, and CX3CR1. Microglia cells in the triple cell culture expressed CX3CR1 and showed heterogenous CD1 lb expression compared to monoculture. CD1 lb is an integrin highly expressed on microglia and other myeloid cells, and is a general marker for this cell type. CX3CR1 is highly expressed in microglia.

[0173] These results confirm that the method described in Example 1 supports microglial maturation, functionalization, and functional morphology in the context of neuronal network formation.Example 3. Culture conditions

[0174] To assess the media conditions capable of promoting and maintaining microglial identity and function in the triple cell culture, triple cell cultures were prepared according toAttorney Docket No. 01209-0020-00PCT(a) the method of Example 1 using DMEM-based media with minimal neuronal supplements instead of the BrainPhys™ media supplemented with SMI and N2 or (b) the method of Example 1 (using the BrainPhys™ media supplemented with SMI and N2). The BrainPhys™ media was desirable for use in the triple cell culture system to allow for the measurement of neuron function.

[0175] Triple cell cultures were matured in each condition then analyzed for expression of microglial markers and morphological features. Cells were stained with antibodies against IBA1 and MAP2 and with DAPI. As shown in FIGs. 7A and 7B, IBA1 branch length showed microglia cultured in the presence of the BrainPhys™ media optimized for neuronal cells exhibited ramified morphology and expressed high levels of CX3CR1 (a chemokine receptor involved in neuron-microglia signaling) and TMEM119 (a highly specific homeostatic microglial surface protein), similar to DMEM-based media which was suitable for microglial monoculture.

[0176] Triple cell cultures were also prepared with BrainPhys™ media with or without SMI. As shown in FIG. 8A, the neuronal supplement SMI promoted ramified microglia morphology and gene expression (CX3CR1, P2RY12, and TMEM119).

[0177] The ratio of cells seeded in the triple cell culture were also assessed. In the 96-well format, triple cell cultures were prepared according to Example 1, including with about 20,000 neuronal precursors per well and about 6,500 astrocytes per well. However, the number of microglial precursors varied. About 1,000, 5,000, or 10,000 microglial precursors were seeded into the triple cell culture and the MAP2 network length and the percent cells positive for IBA1 were assessed as compared to seeding with 0 microglia precursors. As shown in FIGs. 8F and 8G, in a 96-well plate, seeding densities of 20,000 neuronal precursors, 5,000 microglia precursors, and 6,500 astrocytes per well (about a 3 : 1 : 1 ratio) resulted in microglia integration without compromising network complexity, as assessed by MAP2 network length and percent cells positive for IB Al.Example 4. Microglia of the triple cell culture are sensitive to activating insults

[0178] The triple cell culture prepared according to Example 1 were treated with 1 pM pHrodo-labeled Api-42. As shown in FIG. 9, compared to triple cell cultures treated with media only, significant pHrodo signal was detected in the triple cell cultures treated with pHrodo-labeled Api-42, indicating the microglia cells were phagocytosing the amyloid beta peptide. As shown in FIGs. 10A and 10B, the microglia became transiently ameboid (FIG.Attorney Docket No. 01209-0020-00PCT10A) in response to the amyloid beta peptide uptake (FIG. 10B). These results suggest the microglia of the triple cell culture exhibit responses similar to those observed in vivo.Example 5. Functional microglial response to amyloid beta exposure

[0179] To assess whether microglia in the triple cell culture prepared according to the method of Example 1 could respond dynamically to insulting stimuli, cultures were exposed to amyloid beta peptide (Api-42 or APpE3-24), which are associated with Alzheimer’s disease. 1 pg / mL of anti-amyloid beta antibodies (remternetug, donanemab, or bapineuzumab, each at 1 ug / mL) and pHrodo-labeled amyloid beta peptide (Api-42 or APpE3-24, 1 pM) were added to the triple cell cultures. Isotype control antibodies (“isotype”) and an untreated sample (no amyloid beta peptide and no antibody) were used in parallel triple cell cultures as controls. The uptake of pHrodo-labeled amyloid beta peptide by microglia was monitored for 36 hours at 37 °C. The pHrodo (a pH-sensitive fluorophore that fluoresces in acidic phagolysosomes) signal was measured and normalized to the number of microglia. Microglia monocultures did not respond to anti-amyloid beta antibodies; however, the microglia in the triple cell culture responded in an amyloid beta species-specific manner. As shown in FIGs. 11A-11F, the anti-amyloid beta antibodies donanemab and remternetug, which target the pyroglutamate-modified Ap (APpE3-24) significantly increased microglia phagocytosis of APpE3-42 (FIGs. 11D and HE) but not Api-42 (FIGs. HA and 11B). Bapineuzumab, which targets unmodified Api-42, significantly drove phagocytosis of Api- 42 (FIG. 11C) and less significantly for APpE3-24 (FIG. HF).

[0180] Further, as shown in FIG. 15, treatment with Api-42 resulted in marked disruption of the morphology of all three cell types when compared to untreated controls. Specifically, microglia exhibited a more activated and amoeboid morphology, astrocytes showed hypertrophic changes, and neuronal processes appeared reduced and disorganized, indicative of neurotoxic effects associated with Ap exposure. Moreover, as illustrated in FIGs. 5D-5F, treatment with Api-42 caused opposite results in the expression of cell-type specific markers in neurons, astrocytes and microglia of the triple cell culture relative to the untreated condition shown in FIGs. 5A-5C. Thus, the triple cell culture system provides a relevant in vitro model for studying neurodegenerative mechanisms and evaluating potential therapeutic interventions, and for studying Alzheimer’s disease and potential therapeutic treatments.Example 6. Neuronal maturation in triple cell culture

[0181] Induced neuronal (iNeuron) cultures were generated from induced pluripotent stem cells (iPSCs) via NGN2 induction. During initial differentiation experiments, the iNeuronAttorney Docket No. 01209-0020-00PCT cultures exhibited a subpopulation of seemingly non-neuronal cells. It was hypothesized that this non-neuronal growth was attributable to incomplete drug selection and / or copy number variation in the polyclonal iPSC population, which permitted spontaneous differentiation of non-neuronal lineages. To address this, clonal iPSC cell lines were derived from the polyclonal iPSC population and validated, expanded, and differentiated under optimized neuronal differentiation media conditions (N2-supplemented Neurobasal medium; N2 / NB). Longitudinal imaging was used to monitor cellular morphology throughout the differentiation process, while RT-qPCR was performed to assess temporal changes in gene expression. As shown in FIGs. 2A-2C, four representative clones (Al, A3, DI, and D6) exhibited the best iPSC (FIG. 2A) and iNeuron morphology (FIGs. 2B and 2C). FIGs. 3A-2H show the lineage-specific expression profiles of the clones (Al, A3, DI, and D6). Specifically, neuronal lineage markers including NGN2, TUBB3, MAP2, NEUN, and SYN1 were upregulated during the transition from iPSCs (day 0) to induced neuronal progenitor cells (iNPCs; day 5) and mature iNeurons (day 27). Conversely, pluripotency-associated genes including POU5F1 (OCT4) and NANOG were progressively downregulated across the differentiation time course. The transient upregulation of NESTIN at day 5 reflected the intermediate progenitor state.

[0182] To assess the effect of the triple cell culture system on neuronal maturation, electrophysiological activity was measured. Neurons were cultured either alone or in triple cell culture. As shown in FIGs. 14A and 14B, neurons in the triple cell culture exhibited electrophysiological profiles characteristic of enhanced maturation. Specifically, spike firing rates increased significantly over time relative to neuron-only and dual coculture conditions. See FIG. 14A. Moreover, the triple cell culture condition displayed greater network synchrony even after Ap challenge, compared to other culture conditions. See FIG. 14B. These results indicate that the triple cell culture promotes mature synapse formation and neuronal activity.

[0183] Together, the results from Examples 1-6 confirm that microglia within the triple cell culture system retain functional responsiveness to disease-relevant protein aggregates, mimicking key aspects of innate immune activation in the CNS. These results suggest the triple cell culture system is a functional screening platform for therapeutic antibody candidates, capable of resolving species-specific effects on microglial uptake behavior.Example 7. Triple cell culture system as a model system for the Reelin signaling cascade pathwayAttorney Docket No. 01209-0020-00PCT

[0184] The triple cell culture system prepared according to the method of Example 1 was used to assess the effect of agents affecting the Reelin signaling cascade pathway. The Reelin signaling pathway regulates neuronal positioning and tau phosphorylation through engagement of the ApoER2 receptor and activation of the intracellular adaptor protein Disabled-1 (Dabl), which is phosphorylated upon pathway activation (to give pDabl). Downstream, this cascade influences kinase activity affecting the phosphorylation state of tau, a microtubule-associated protein implicated in neurodegenerative diseases.

[0185] After maturation of the triple cell culture, the culture was treated with one or more test agents, such as an anti-ApoER2 antibody. In some conditions, recombinant human Reelin protein was used as a positive control. The cultures were incubated with the test agent for a defined period, such as between 1 hour and 24 hours, under standard culture conditions. Following treatment, cell lysates and / or culture supernatants were collected for analysis.

[0186] The level of phosphorylated Dabl (pDabl) was measured as a proximal and early readout of ApoER2 activation. pDabl was quantified by ELISA using phospho-specific antibodies. Additionally, downstream effects on tau phosphorylation were assessed by Western blot using phospho-tau-specific antibodies, such as AT8 (recognizing phosphorylated serine 202 and threonine 205) and S396 (recognizing phosphorylated serine 396).

[0187] An increase in pDabl levels relative to untreated or isotype control conditions indicated that the test agent activates or potentiates ApoER2 signaling, either directly (such as in the case of a receptor agonist, or Reelin protein itself) or indirectly (such as by enhancing endogenous Reelin activity). As shown in FIG. 12, an anti-APOER2 antibody (labeled “Antibody A”) at concentrations of 0.1 nM to 10 nM showed an increase in pDabl levels relative to an isotype antibody and an untreated control (“NT”). Reelin protein itself also showed an increase in pDabl levels in the triple cell culture system.

[0188] Changes in phospho-tau levels, while more downstream, can provide additional insight into the functional consequences of receptor activation.Example 8. Plasma cell model

[0189] The triple cell culture system can be used to evaluate microglia-mediated plasma cell / plasmablast phagocytosis. In one example, peripheral blood mononuclear cells (PBMCs) are isolated from healthy human donor blood samples by Ficoll-Paque density gradient centrifugation. Isolated PBMCs are resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin at a concentration of 2 x 106Attorney Docket No. 01209-0020-00PCT cells / mL. A 2x B cell activation cocktail is prepared as follows: 10 mL RPMI 1640 medium supplemented with 10% FBS, 2 pg / mL Resiquimod / R848 (a TLR7 / 8 agonist; Sigma cat. No. SML0196-10mg; dissolved in DMSO), 20 ng / mL IL-2 (ThermoFisher Scientific cat. No. PHC0021; dissolved in 100 mM acetic acid), 50 ng / mL IL- 10 (StemCell™ Technologies cat. No. 78024; dissolved in water) and 18.2 pL 2-mercaptoethanol. B cells are activated by mixing 0.5 mL of PBMC suspension (the final concentration of 1 x 106cells / mL) with 0.5 mL of 2x B cell stimulation cocktail in wells of a 24-well plate and incubated at 37 °C / 5% CO2 for 6 days. Plasma cell differentiation is analyzed by flow cytometry by staining for CD19+ / CD38+ / BCMA+ cells (consistent with plasma cell phenotype). B cells are purified from this mixture by immunomagnetic positive selection, with the Easy Sep™ Human CD 19 Positive Selection Kit II (StemCell™ Technologies cat. No. 17854) according to manufacturer’s instructions. Purified B cells are resuspended in BrainPhys™ media and added to 96-well plates containing triple cell cultures at a seeding density of 6,500-20,000 B cells / well.

[0190] To induce antibody-dependent phagocytosis, triple cell cultures may then be treated with candidate anti-BCMA antibodies or isotype control antibodies and incubated overnight at 37 °C. B cell phagocytosis is quantified by counting the number of CD19+ / CD38+ doublepositive cells per well through flow cytometry. Alternatively, B cell / plasma cell phagocytosis is quantified by co-localization of GFP+ microglia in the triple cell culture and fluorescently- labeled, purified CD 19+ B cells through live cell imaging. Candidate anti-BCMA antibodies that significantly decrease total count of CD19+ / CD38+ compared to isotype control may indicate the antibodies activated microglia-mediated engulfment of plasma cells.Example 9. Tau uptake by triple cell culture system

[0191] The triple cell culture system can be used to model tau protein intake. In one study, the triple cell culture system was prepared according to the method of Example 1 to maturation with EGFP-expressing microglia. Labeled tau, such as 75 nM pHrodo-labeled tau, was diluted to concentration in triple cell culture media and then added to the cells. The pHrodo signal was monitored using live cell imaging, such as an Incucyte®, for 24 hours in a 37 °C / 5% CO2 incubator. The pHrodo intensity was normalized to the number of EGFP- positive microglia. The area under the curve was calculated.

[0192] To assess the ability of candidate therapeutics to increase tau uptake, the 75 nM pHrodo-labeled tau was mixed in media with 75 nM of an anti -tau antibody (labeled as “E2814”), for 1 hour at room temperature. See FIG. 13. The 75 nM tau protein and 75 nMAttorney Docket No. 01209-0020-00PCT anti-tau antibody mixture in triple cell culture media was then added to the triple cell culture. The 75 nM tau protein and 75 nM anti-tau antibody (“E2814”) mixture was compared to a mixture of the tau protein with an isotype control antibody (labeled as “hlgG”) and a pHrodo- labeled tau in media alone (labeled as “NT” for not treated). The results are shown in FIG. 13 and were assessed by comparing the area under the curve of the anti-tau antibody experiment with, for example, an isotype control.Example 10. Cytokine secretion following inflammatory stimulation

[0193] The triple cell culture system prepared according to the method of Example 1 is used to model neuroinflammatory activation by treating the cultures with an inflammatory stimulant, such as lipopolysaccharide (LPS), for a defined period, such as 1 hour to 24 hours. Following incubation, cell culture supernatants are collected and analyzed using a multiplex cytokine assay to quantify the levels of key inflammatory mediators, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a), C-C motif chemokine ligand 2 (CCL2, also known as MCP-1), interleukin- 1 beta (IL-ip), and interleukin- 10 (IL- 10).

[0194] Cytokine levels from LPS-treated cultures are compared to untreated control triple cell cultures. A significant increase in pro-inflammatory cytokines, such as IL-6, TNF-a, and CCL2, indicates activation of microglia and engagement of neuroimmune signaling pathways.

[0195] The triple cell culture system may then be used to evaluate the ability of test agents to modulate or ameliorate inflammation induced by this exogenous stimulation. Triple cell cultures will be pre-treated, co-treated, or post-treated with a test agent, such as a small molecule or biologic, prior to or following exposure to inflammatory stimulants such as LPS. The effect of the test agent is assessed by measuring changes in cytokine secretion profiles relative to stimulated but untreated control cultures. A reduction in one or more pro- inflammatory cytokines in the presence of the test agent indicates an anti-inflammatory or immunomodulatory activity.

Claims

Attorney Docket No. 01209-0020-00PCTWHAT IS CLAIMED IS:

1. A method of preparing a cell culture comprising neurons or neuronal precursors, astrocytes, and microglia cells or microglial precursors, the method comprising:(i) providing neurons or neuronal precursors;(ii) providing astrocytes;(iii) providing microglia cells or microglial precursors;(iv) seeding a cell culture vessel with the (a) neurons or neuronal precursors, (b) astrocytes, and (c) microglia cells or microglia precursor cells, wherein more neurons or neuronal precursors are seeded than astrocytes, microglia cells, or microglia precursor cells.

2. The method of claim 1, wherein the neurons, astrocytes, and microglia cells are incubated in a triple cell culture medium comprising a serum-free basal medium that produces synaptically active neurons.

3. A method of preparing a cell culture comprising neurons or neuronal precursors, astrocytes, and microglia cells or microglial precursors, the method comprising:(i) providing neurons or neuronal precursors;(ii) providing astrocytes;(iii) providing microglia cells or microglial precursors;(iv) seeding a cell culture vessel with the (a) neurons or neuronal precursors, (b) astrocytes, and (c) microglia cells or microglia precursor cells in a triple cell culture medium comprising a serum-free basal medium that produces synaptically active neurons.

4. The method of claim 3, wherein more neurons are seeded than either astrocytes or microglia cells.

5. The method of any one of claims 2-4, wherein the serum-free basal medium comprises BrainPhys™, or wherein the serum-free basal medium comprises inorganic salts, amino acids, vitamins, dextrose, sodium pyruvate, Hepes, cholesterol, and optionally phenol red.Attorney Docket No. 01209-0020-00PCT6. The method of any one of claims 2-5, wherein the triple cell culture medium comprises a serum-free basal medium supplement comprising Bottenstein’s N-l formulation, or wherein the triple cell culture medium comprises a serum-free basal medium supplemented with one or more of insulin, transferrin, selenite, putrescine, and progesterone.

7. The method of claim 6, wherein the serum-free basal medium supplement is a N-2 supplement, or wherein the serum-free medium supplement comprises one or more of apo-transferrin, insulin, putrescine, sodium selenite, and progesterone.

8. The method of any one of claims 2-7, wherein the triple cell culture medium comprises one or more of NeuroCult™ SMI supplement; brain-derived neurotrophic factor (BDNF); glial cell line-derived neurotrophic factor (GDNF); IL34; gmCSF; and TGFbeta.

9. The method of any one of claims 1-8, wherein (iii) comprises differentiating human iPSC cells into microglial precursors.

10. The method of claim 9, wherein differentiating human iPSC cells into microglial precursors comprises forming hemogenic embryoid bodies (EB) in hemogenic EB media.

11. The method of claim 10, wherein the hemogenic EB media comprises a xeno-free medium capable of expanding iPSCs.

12. The method of claim 11, wherein the xeno-free medium is StemFlex™, optionally further comprising StemFlex™ supplement.

13. The method of any one of claims 10-12, wherein the hemogenic EB media comprises at least one of, at least two, or three of VEGF, BMP4, and SCF.

14. The method of any one of claims 10-13, wherein the hemogenic EB media further comprises a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632.

15. The method of any one of claims 1-13, wherein the microglia or microglial precursors in the culture express increased levels of TMEM119, CX3CR1, and / or P2RY12 relative to microglia cells maintained in a monoculture.Attorney Docket No. 01209-0020-00PCT16. The method of any one of claims 10-14, wherein EBs are formed in a cell culture vessel suitable to promote the formation of embryoid bodies; optionally wherein the cell vessel is a microwell plate such as an AggreWell™ plate.

17. The method of any one of claims 10-16, wherein differentiating human iPSC cells into microglial precursors further comprises transferring EBs to a cell culture vessel coated with an extracellular matrix.

18. The method of claim 17, wherein the cell culture vessel comprises a microglia precursor production medium comprising a serum-free hematopoietic basal medium.

19. The method of claim 18, wherein the serum-free hematopoietic basal medium is X- VIVO®.

20. The method of claim 18 or claim 19, wherein the microglia precursor production medium comprises at least one of, at least two of, or three of IL3, mCSF, and Glutamax™.

21. The method of any one of claims 18-20, wherein the microglia precursor production media comprises 2-mercaptoethanol.

22. The method of any one of claims 1-21, wherein at least 80% of the microglia or microglial precursors are positive for IBA1.

23. The method of any one of claims 1-22, wherein at least 80% of the microglia or microglial precursors are positive for CX3CR1.

24. The method of any one of claims 1-23, wherein microglia precursors are generated from disease-associated iPSC donors.

25. The method of claim 24, wherein the disease-associated iPSC donors comprise genotypes with a mutation in one or more of TREM2, APOE, CD33, PSEN1, SNCA, or MAPT.

26. The method of any one of claims 1-23, wherein microglia precursors are generated from iPSCs engineered to comprise a disease-associated genotype.Attorney Docket No. 01209-0020-00PCT27. The method of claim 26, wherein the disease-associated genotype comprises a mutation in one or more of TREM2, APOE, CD33, PSEN1, SNCA, oxMAPT.

28. The method of claim 26 or claim 27, wherein the genotype is engineered by the CRISPR / Cas9 system.

29. The method of any one of claims 1-28, wherein (i) comprises differentiating a population of human iPSC cells to neuronal precursors by resuspending human iPSC cells in a neuron induction medium.

30. The method of claim 29, wherein the neuron induction medium comprises DMEM as basal medium.

31. The method of claim 30, wherein the neuron induction medium comprises DMEM F / 12 as basal medium.

32. The method of any one of claims 29-31, wherein the neuron induction medium comprises one or more of (a) a serum-free supplement comprising Bottenstein’s N-l or N-2 supplement or comprising one or more of insulin, transferrin, selenite, putrescine, and progesterone, (b) BDNF, (c) neurotrophin-3 (NT-3), and (d) laminin.

33. The method of claim 32, wherein the serum-free supplement comprises N-2 supplement, or wherein the serum-free supplement comprises apo-transferrin, insulin, putrescine, sodium selenite, and progesterone.

34. The method of any one of claims 29-33, wherein the iPSC cells used to generate neuronal precursors are engineered to overexpress NGN2.

35. The method of any one of claims 29-34, wherein the neuron induction media is supplemented with a ROCK inhibitor; optionally wherein the ROCK inhibitor is Y- 27632.

36. The method of any one of claims 1-35, wherein at least 80% of the neurons or neuronal precursors are positive for one or more of MAP2, TUBB3, synapsin I, NeuN, and DCX.

37. The method of any one of claims 1-36, wherein the microglia and neurons are derived from isogenic iPSCs.Attorney Docket No. 01209-0020-00PCT38. The method of any one of claims 1-36, wherein the microglia, astrocytes, and neurons are isogenic.

39. The method of any one of claims 1-38, wherein the astrocytes are derived from human fetal astrocytes.

40. The method of any one of claims 1-39, wherein (ii) comprises expanding astrocyte cells.

41. The method of any one of claims 1-40, comprising forming a co-culture comprising astrocytes and neuronal precursors in a co-culture medium.

42. The method of claim 41, wherein the co-culture medium is a serum-free basal medium.

43. The method of claim 42, wherein the serum-free basal medium comprises BrainPhys™.

44. The method of any one of claims 41-43, wherein the co-culture medium comprises one or more of (a) Bottenstein’s N-l or N-2 supplement or a supplement comprising transferrin, insulin, putrescine, selenium, and progesterone; (b) NeuroCult™ SMI supplement; (c) BDNF; and (d) GDNF.

45. The method of any one of claims 41-44, wherein (iv) comprises seeding microglial precursor cells into the co-culture of neuronal precursors and astrocytes.

46. The method of any one of claims 41-45, wherein the co-culture is seeded with at least a 2: 1 ratio of neuronal precursors to astrocytes.

47. The method of claim 46, wherein the co-culture is seeded with at least a 3 : 1 ratio of neuronal precursors to astrocytes.

48. The method of any one of claims 45-47, wherein (iv) comprises seeding approximately the same number of microglial precursors as astrocytes in the co-culture.

49. The method of any one of claims 1-48, wherein at least 80% of the microglia cells exhibit ramified morphology.

50. The method of claim 49, wherein the microglial morphology is assessed by IBA1 immunostaining.Attorney Docket No. 01209-0020-00PCT51. The method of any one of claims 1-50, further comprising contacting the culture with an amyloid beta peptide.

52. The method of claim 51, wherein the amyloid beta peptide is Api-42 or pE-Ap3-42.

53. The method of any one of claims 1-52, further comprising contacting the culture with a test agent, optionally wherein the test agent is an antibody, a soluble protein, a nucleic acid, a peptide, or small molecule drug.

54. The method of any one of claims 1-53, further comprising contacting the culture with an inflammatory stimulus such as lipopolysaccharide (LPS).

55. The method of any one of claims 1-54, further comprising measuring secretion of one or more cytokines, optionally wherein the cytokines are selected from IL-6, TNF-alpha, IL- Ibeta, or CCL2.

56. The method of any one of claims 1-55, further comprising measuring cell viability of microglia using a dead cell stain, flow cytometry, and / or imaging-based analysis.

57. The method of any one of claims 1-56, wherein the method further comprises cry opreserving microglial precursors before (iv).

58. The method of any one of claims 1-57, wherein the method further comprises cry opreserving neuronal precursors and / or astrocytes before (iv).

59. A cell culture formed according to the method of any one of claims 1-58.

60. A cell culture comprising neurons or neuronal precursors, astrocytes, and microglia or microglial precursors.

61. The cell culture of claim 60, wherein the cell culture comprises more neurons or neuronal precursors than astrocytes or microglia or microglial precursors.

62. The cell culture of claim 61, wherein the cell culture comprises at least a 2: 1 ratio of neurons or neuronal precursors to astrocytes.Attorney Docket No. 01209-0020-00PCT63. The cell culture of claim 61, wherein the cell culture comprises at least a 3: 1 ratio of neurons or neuronal precursors to astrocytes.

64. The cell culture of any one of claims 60-63, wherein the cell culture comprises approximately the same number of microglia or microglial precursors as astrocytes.

65. The cell culture of claim 64, wherein the cell culture comprises a 3: 1 : 1 ratio of neurons or neuronal precursors to microglia or microglial precursors and astrocytes.

66. The cell culture of any one of claims 60-65, wherein the microglia or microglial precursors express P2RY12, P2RY13, TMEM119, CX3CR1, CSF1R, AIF1, TREM2, and / or SALL1; or express increased levels of P2RY12, P2RY13, TMEM119, CX3CR1, CSF1R, AIF1, TREM2, and / or SALL1 than iPSC cells.

67. The cell culture of any one of claims 60-66, wherein the microglia or microglial precursors express any one or more of cell surface markers TRA-1-60, CD34, CD45, CD43, CDl lb, P2RY12, and / or CD107a.

68. The cell culture of any one of claims 60-67, wherein the microglia or microglial precursors in the culture express increased levels of TMEM119, CX3CR1, and / or P2RY12 relative to microglia cells maintained in a monoculture.

69. The cell culture of any one of claims 60-68, wherein at least 80% of the microglia or microglial precursors are positive for IBA1.

70. The cell culture of any one of claims 60-69, wherein at least 80% of the microglia or microglial precursors are positive for CX3CR1.

71. The cell culture of any one of claims 60-70, wherein the microglia or microglial precursors do not express or show reduced expression of OCT4, SOX2, NANDG, POU5F1, and / or KLF4 compared to iPSCs.

72. The cell culture of any one of claims 60-71, wherein the neurons or neuronal precursors are generated from iPSCs engineered to overexpress NGN2.Attorney Docket No. 01209-0020-00PCT73. The cell culture of any one of claims 60-72, wherein microglia precursors are generated from disease-associated iPSC donors.

74. The cell culture of claim 73, wherein the disease-associated iPSC donors comprise genotypes with a mutation in one or more of TREM2, APOE, CD33, PSENJ, SNCA, or MAPT.

75. The cell culture of any one of claims 60-74, wherein microglia precursors are generated from iPSCs engineered to comprise a disease-associated genotype.

76. The cell culture of claim 75, wherein the disease-associated genotype comprises a mutation in one or more of TREM2, APOE, CD33, PSEN1, SNCA, o MAPT.

77. The cell culture of claim 75 or claim 76, wherein the genotype is engineered by the CRISPR / Cas9 system.

78. The cell culture of any one of claims 60-77, wherein the microglia and neurons are derived from isogenic iPSCs.

79. The cell culture of any one of claims 60-78, wherein the microglia, astrocytes, and neurons are isogenic.

80. The cell culture of any one of claims 60-79, wherein the astrocytes are derived from human fetal astrocytes.

81. The cell culture of any one of claims 60-80, wherein the astrocytes express ALDH1L1, SLC1A2, SLC1A3, GLUL, AQP4, GJA1, SOX9, GFAP, and / or SlOObeta.

82. The cell culture of any one of claims 60-81, wherein the astrocytes show reduced expression of ALPHILI, SLC1A2, SLC1A3, AQP4, GJA1, and / or SOX9; and / or show increased expression of GLUL, compared to astrocytes in a co-culture of astrocytes and neurons or neuronal precursors.

83. The cell culture of any one of claims 60-82, wherein the neurons or neuronal precursors express MAP2, TUBB3, Synapsinl, NeuN, GAD1, VGLUT1, and / or DCX.Attorney Docket No. 01209-0020-00PCT84. The cell culture of any one of claims 60-83, wherein the neurons or neuronal precursors express RBFOX3, MAP2, TUBB3, SNAP25, SYT1, SYN1, DLG4, and / or NEFM.

85. The cell culture of any one of claims 60-84, wherein the neurons or neuronal precursors show increased expression ofMAP2, SNAP25, SYT1, SYN1, and / or NEFM; and / or wherein the neurons or neuronal precursors show reduced expression of RBFOX3, TUBB3, and / or DLG4, compared to neurons or neuronal precursors in a co-culture of astrocytes and neurons or neuronal precursors.

86. The cell culture of any one of claims 60-85, wherein the cell culture is comprised within a transwell, optionally wherein neurons or neuronal precursors and astrocytes are in a bottom chamber or well of the transwell and microglia or microglial precursors are in a top chamber or well, wherein the top and bottom chambers or wells are separated by a porous membrane.

87. The cell culture of any one of claims 60-86, wherein the cell culture is in a three- dimensional shape, such as a spheroid or aggregate.

88. A kit comprising (a) a co-culture of astrocytes and neuronal precursors and (b) a culture of microglial precursor cells.

89. A kit comprising the cell culture of any one of claims 59-8790. The kit of claim 89, further comprising instructions for use according to the method of any one of claims 1-58.

91. A method of assessing the cell culture of any one of claims 59-87, comprising contacting the cell culture with an amyloid beta peptide.

92. The method of claim 91, wherein the amyloid beta peptide is Api-42 or pE-Ap3-42.

93. A method of assessing the activity of a test agent in the cell culture of any one of claims 59-87, comprising contacting the culture with the test agent, optionally wherein the test agent is an antibody, a soluble protein, a nucleic acid, a peptide, or small molecule drug.Attorney Docket No. 01209-0020-00PCT94. A method of assessing the effect of an inflammatory stimulus on the cell culture of any one of claims 59-87, comprising contacting the cell culture with an inflammatory stimulus such as lipopolysaccharide (LPS).

95. A method of assessing the cell culture of any one of claims 59-87, comprising measuring secretion of one or more cytokines from the cell culture, optionally wherein the cytokines are selected from IL-6, TNF-alpha, IL-lbeta, or CCL2.

96. A method of assessing the efficacy of a pharmaceutical composition for the treatment of a neurodegenerative disease, the method comprising: (i) treating the cell culture of any one of claims 59-87 with the pharmaceutical composition, and (ii) assessing the efficacy of the pharmaceutical composition based on one or more readouts.

97. The method of claim 96, wherein the one or more readouts comprise one or more of a change in microglial activation state, a change in cytokine secretion profile, a change in neuronal viability or morphology, a change in tau phosphorylation, a change in Dab-1 phosphorylation, a change in synaptic density, a change in phagocytic activity, or a change in expression of one or more disease-associated markers.

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