Patent Application
20240417684
Described herein are embodiments of (i) human microglial-like cells (iMGLs) and (ii) methods of making iMGLs.
Microglia cells are innate immune cells of the CNS and are known to play roles in the physiological development of the CNS. In addition, microglial cells are known to play roles in neurological disorders such as Alzheimer's disease. There is a deficiency in the art of acquiring microglia cells to further investigate the roles microglia cells play in CNS development and neurological disorders.
In some embodiments, a method of producing human microglial-like (iMGLs) from pluripotent stem cells (PSCs) is provided. In some embodiments, the method comprises the steps: (i) differentiating PSCs using a media supplemented with hematopoietic differentiation factors to produce induced hematopoietic progenitor cells (iHPCs), (ii) isolating CD43+ iHPCs, (iii) differentiating the CD43+ iHPCs into iMGLs using a microglial differentiating media; and (iv) maturing the iMGLs.
In some embodiments, the method comprises the steps: (i) differentiating PSCs using a media supplemented with hematopoietic differentiation factors; and (ii) differentiating the CD43+ iHPCs into iMGLs using a microglial differentiating media.
In some embodiments, a method of producing a human microglial-like cell (iMGL) from a cell of a first type is provided. In some embodiments, the method comprises the steps of: (i) differentiating a cell of a first type into an induced hematopoietic progenitor cell (iHPC); and (ii) differentiating the iHPC to produce an iMGL.
In some embodiments, the PSCs are not derived from embryoid bodies. In some embodiments, the PSCs include single-cell PSCs.
In some embodiments, the PSCs include induced PSCs (iPSCs). In some embodiments, the PSCs include embryonic stem cells (ESCs). In some embodiments, the PSCs include mammalian PSCs. In some embodiments, the PSCs are of human origin. In some embodiments, the PSCs are mouse PSCs.
In some embodiments, a method of producing iMGLs from PSCs is provided comprising the steps: (i) differentiating PSCs into iHPCs and (ii) differentiating iHPCs into iMGLs.
In some embodiments, a composition of iMGLs is provided that comprises expression of any one, or any combination of two or more, of the following genes: RUNX1, SPI1, CSF1FR, CX3CR1, TGFBR1, RSG10, GAS6, MERTK, PSEN2, PROS1, P2RY12, P2RY13, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, TYROBP, ITGAM, APOE, SLCO2B1, SLC7A8, PPARD, TMEM119, GPR56, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1.
In some embodiments, a method of assessing chemokine, cytokine, and other inflammatory molecule secretion is provided comprising the steps: (i) treating the iMGLs with lipopolysaccharide, IFNY, or IL-1β, (ii) measuring chemokines, cytokines, and other secreted factors from iMGLs that can serve as potential biomarkers of inflammation or different neurodegenerative disease states. Some embodiments relate to a method of profiling secretion of inflammatory molecules from iMGLs comprising: (i) treating the iMGLs with lipopolysaccharide, IFNY, TNFα, or IL-1β; and (ii) measuring inflammation markers secreted by the iMGLs.
In some embodiments, a method of assessing iMGLs migration is provided comprising the steps: (i) treating the iMGLs with ADP and (ii) measuring iMGL migration. Some embodiments relate to a method of assessing iMGL migration comprising: (i) treating the iMGLs with ADP; and (ii) assessing iMGL motility and migration in response to chemical stimuli.
In some embodiments, a method of producing calcium transients in iMGLs is provided comprising the steps: (i) treating the iMGLs with ADP and (ii) producing calcium transients in iMGLs. Some embodiments relate to a method of producing calcium transients in iMGLs comprising: (i) treating the iMGLS with ADP; and (ii) interrogating calcium flux signals in the iMGLs; wherein the calcium flux signals are produced in response to electrical, biological, or chemical stimulation.
In some embodiments, a method of differentially regulating gene expression in iMGLs is provided comprising the steps: (i) co-culturing iMGLs with neurons or astrocytes and (ii) differentially regulating genes in iMGLs.
In some embodiments, a method of integrating iMGLs into the CNS/brain (e.g., neuronal) environment is provided comprising the steps: (i) co-culturing iMGLs with hiPSC 3D brain-organoids (BORGs) and (ii) invading of the iMGLs into the BORGs. Some embodiments relate to a method of integrating iMGLs into a 3D CNS environment, comprising: co-culturing iMGLs with hiPSC 3D brain-organoids (BORGs), wherein the iMGLs migrate into the BORGs and populate the BORGS, or are incorporated into the BORGs.
In some embodiments, a method of differentially regulating gene expression in iMGLs is provided comprising the steps: (i) exposing iMGLs to any of the following compounds: Aβ, Tau, fluorescently labeled Aβ, pHrodo-labeled brain-derived tau oligomers and other brain-derived proteins implicated in neurodegenerative disease i.e. synuclein, huntingtin, prion (ii) differentially regulating genes in iMGLs. Some embodiments relate to a method of establishing an iMGL gene expression profile resembling the in vivo state of the iMGLs, comprising: co-culturing iMGLs with neurons, astrocytes, or other cells of the central nervous system, thereby recapitulating a more in vivo state for the iMGLs than would otherwise be present for the iMGLs if the iMGLs were not co-cultured with the neurons, astrocytes, or other cells of the central nervous system. Some embodiments relate to a method of studying microglia dysregulation in health and disease using iMGLs, comprising: (i) exposing iMGLs to a compound selected from the group consisting of Aβ, Tau, fluorescently labeled Aβ, pHrodo-labeled brain-derived tau oligomers, and alpha-synuclein; and (ii) profiling an iMGL-omic signature selected from RNA-seq, proteomics, metabolomics, and lipidomics.
In some embodiments, a method of phagocytosing human synaptosomes (hS) in iMGLs is provided comprising the steps: (i) exposing iMGLs to hS and (ii) measuring phagocytosis of hS. Some embodiments relate to a method of studying microglia phagocytosis of compounds comprising: (i) exposing iMGLs to a compound selected from the group consisting of Aβ, Tau, fluorescently labeled Aβ, and pHrodo-labeled brain-derived tau oligomers, wherein the compound is phagocytosed, endocytosed, or ingested by the iMGLs; and (ii) measuring the phagocytosis, endocytosis, or ingestion of the compound.
Some embodiments relate to a method of investigating the role of microglia in synaptic pruning and plasticity comprising: (i) exposing iMGLs to human synaptosomes and (ii) assessing human synaptosome phagocytosis by the iMGLs.
In some embodiments, a method of determining gene regulation is provided comprising (i) exposing iMGLs to one or more of the factors CX3CL1, CD200, and TGFβ, in any combination and (ii) assessing any one or more of the differentially regulated genes in any combination: P2ry12, EGR1, TGFβ1, ETV5, CX3CR1, APOE, BIN1, CD33, GPR84, COMT, APP, PSEN1, PSEN2, HTT, GRN, FUS, TARDP, VCP, SNCA, C9ORF72, LRRK2, and SOD1.
In some embodiments, a method of assessing engraftment of iMGLs into neural tissue (e.g., cortex) is provided comprising (i) transplanting iMGLs into the neural tissue and (ii) assessing engraftment of the iMGLs into the neural tissue.
In some embodiments, a method of assessing iMGL interaction with AD neuropathy is provided comprising (i) transplanting iMGLs into hippocampi and (ii) assessing interaction of iMGLs in the hippocampi.
In some embodiments, a method of studying human microglia in a 3D neuronal environment is provided comprising transplanting iMGLs into a mammalian brain.
Some embodiments of the methods, kits and compositions provided herein relate to a media for supporting generation of human iHPCs, the media comprising one or more of FGF2, BMP4, Activin A, and LiCl. Some embodiments of the methods and compositions provided herein relate to a media for supporting generation of human iHPCs, the media comprising one or more of FGF2 and VEGF. Some embodiments of the methods and compositions provided herein relate to a media for supporting generation of human iHPCs, the media comprising one or more of FGF2, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a kit for supporting generation of human iHPCs, with media that comprises one or more of FGF2, BMP4, Activin A, and LiCl. Some embodiments relate to a kit for supporting generation of iHPCs, with media that comprises one or more of FGF2 and VEGF. Some embodiments relate to a kit for supporting generation of human iHPCs, the kit including a media that comprises one or more of FGF2, VEGF, TPO, SCF, IL3, and IL6.
Some embodiments of the methods, kits and compositions provided herein relate to a media for supporting generation of human iMGLs, the media comprising one or more of CSF-1, IL-34, and TGFβ1. Some embodiments relate to a kit for supporting generation of human iMGLs, the kit including a media that comprises one or more of CSF-1, IL-34, and TGFβ1.
Some embodiments of the methods, kits and compositions provided herein relate to a media for supporting maturation or maintenance of iMGLs, the media comprising one or more of CD200 and CX3CL1. Some embodiments relate to a kit for supporting maturation or maintenance of iMGLs, the kit including a media that comprises one or more of CD200 and CX3CL1.
The compositions and related methods summarized above and set forth in further detail below describe certain actions taken by a practitioner; however, it should be understood that they can also include the instruction of those actions by another party. Thus, actions such as “transplanting iMGLs into a mammalian brain” include “instructing the transplantation iMGLs into a mammalian brain.”
Microglia are the innate immune cells of the CNS and play important roles in synaptic plasticity, neurogenesis, homeostatic functions and immune activity. Microglia also play a critical role in neurological disorders, including AD, highlighting the need to improve our understanding of their function in both health and disease. Yet, studying human microglia is challenging because of the rarity and difficulty in acquiring primary cells from human fetal or adult CNS tissue. Therefore, there is a pressing need to develop a renewable source of human microglia, such as from pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).
The challenges present in generating microglia from iPSCs are due to their unique developmental origin. Elegant lineage tracing studies show that microglia originate from yolk sac erythromyeloid progenitors (EMP) generated during primitive hematopoiesis. EMPs further develop to early primitive macrophages that migrate into the developing neural tube, and become microglial progenitors. Microglia progenitors then mature and develop ramified processes used to survey their environment, facilitate CNS development, modulate synaptic plasticity, and respond to CNS injury and pathology.
The generation of patient-derived iPSCs has facilitated new opportunities to examine the relationships between genetic risk factors and disease. Recently, genome wide association studies (GWAS) have identified several genes expressed by microglia that are associated with the risk of developing late-onset AD (LOAD). The role of these genes in microglial function and AD are just beginning to be examined in mouse models, but the generation of human microglia-like cells as described herein allows for the interrogation of human-specific genes that cannot be modeled in mice.
In AD, microglia cluster around beta-amyloid plaques highlighting their inefficacy in clearing beta-amyloid. Microglia are also implicated in the neuroinflammatory component of AD etiology, including cytokine/chemokine secretion, which exacerbate disease pathology. Furthermore, AD GWAS genes like TREM2 and CD33 are influenced by AD pathology and likely play a role in AD progression. Microglia are also the primary modulators of brain development, neuronal homeostasis, and numerous neurological disorders. Thus, there is a pressing need to further our understanding of human microglia and the influence of both pathology and disease-associated genes on microglial function.
Some of the embodiments described herein provide methods for the effective and robust generation of human iPSC microglial-like cells (IMGLs) that resemble fetal and adult microglia. These methods produce iMGLs that are useful in investigating neurological diseases like AD. In some of the embodiments described herein, microglial-like cells (iMGL) are differentiated from iPSCs to study their function in neurological diseases, such as Alzheimer's disease (AD).
The iMGLs described herein, develop in vitro similarly to microglia in vivo. Whole transcriptome analysis demonstrates that they are highly similar to adult and fetal human microglia. Functional assessment of these iMGLs, reveal that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates similar to adult/fetal microglia.
These iMGLs can be used to (i) examine the effects of fibrillar Aβ and brain-derived tau oligomers on AD-related gene expression and (ii) identify mechanisms involved in synaptic pruning, among other uses. Further, the iMGLs can be used in high-throughput studies of microglial function, providing important new insight into human neurological disease.
The following sections provide various embodiments of methods to produce iMGLs and various embodiments of the structure and function of the iMGLs. In addition, methods of using iMGLs are also provided. Also provided are non-limiting detailed explanations of the methods.
In some embodiments, methods of producing human microglial-like cells (iMGLs) from pluripotent stem cells (PSCs) are provided. In some embodiments, the method comprises the steps of: (i) differentiating PSCs using a media supplemented with hematopoietic differentiation factors to produce induced hematopoictic progenitor cells (iHPCs); (ii) isolating CD43+ iHPCs; (iii) differentiating the CD43+ iHPCs into human microglial-like cells (iMGLs) using a microglial differentiating media; and (iv) maturing the iMGLs. In some embodiments, HPC generation technology allows for collecting media enriched with precursors and carried to (iii) without isolating CD43+ iHPCs.
In some embodiments, the method comprises the steps: (i) differentiating PSCs using a media supplemented with hematopoietic differentiation factors; and (ii) differentiating the CD43+ iHPCs into iMGLs using a microglial differentiating media.
Some embodiments of the methods and compositions provided herein relate to a method of producing a human iMGL from a cell of a first type comprising the steps of: (i) differentiating a cell of a first type into an iHPC; and (ii) differentiating the iHPC to produce an iMGL. In some embodiments, the cell of a first type is not a PSC or an ESC.
In some embodiments, the PSCs are not derived from embryoid bodies. In some embodiments, the PSCs include single-cell PSCs. In some embodiments, the PSCs are not CD43+ before differentiation. In some embodiments, the PSCs are not CD34+ before differentiation. In some embodiments, the PSCs are not CD314. In some embodiments, the PSCs are not CD45+ before differentiation.
In some embodiments, the PSCs are or include induced PSCs (iPSCs). In some embodiments, the PSCs are or include embryonic stem cells (ESCs). In some embodiments, the PSCs are mammalian PSCs. In some embodiments, the PSCs are human PSCs. In some embodiments, the PSCs are mouse PSCs.
In some embodiments, differentiating PSCs to produce iHPCs comprises an incubation period that is between 5 and 15 days. For example, the incubation period is 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days. In some embodiments, the incubation period is 10 days. In some embodiments, the oxygen percentage that the PSCs are exposed to varies the 10-day period. In some embodiments, during the incubation period the iPSCs are incubated in a hypoxic or normoxic environment. In some embodiments, during days 1 through 10 of the incubation period the PSCs are incubated in a hypoxic or normoxic environment. In some embodiments, during the first part of the 10 day period, the PSCs will be exposed to an oxygen environment between 3% and 7%. In some embodiments, the first part of the 10 day period is 4 days (days 1-4) and the oxygen environment is 5%. In some embodiments, during the second part of the 10 day period, the PSCs will be exposed to an oxygen environment between 15% and 25%. In some embodiments, the second part of the 10 day period is 6 days (days 5-10) and the oxygen environment to which the PSCs are exposed is 20%. In some embodiments, differentiating PSCs to produce iHPCs comprises an incubation period that is between 3 and 21 days. In some embodiments, the incubation period is up to 28 days. In some embodiments, the incubation period is over 28 days. In some embodiments, the incubation period is less than 3 days.
In some embodiments, hematopoietic differentiation factors used to differentiate PSCs comprise FGF2, BMP4, Activin A, LiCl, VEGF, TPO, SCF, IL3, and IL6. The media will comprise any one or more of these factors in any combination. In some embodiments, the PSCs are incubated in different media throughout the incubation period of the PSC differentiation step. In some embodiments, a 10 day incubation period is provided wherein, during day 1 the media comprises FGF2, BMP4, Activin A, and LiCl, during days 3 and 4 the media comprises FGF2 and VEGF, and during days 5 through 10 the media comprises FGF2, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a medium comprising any one or a combination of the factors FGF2, BMP4, Activin A, LiCl, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a medium comprising any one or a combination of the factors FGF2, BMP4, Activin A, and LiCl. Some embodiments relate to a medium comprising any one or a combination of the factors FGF2 and VEGF. Some embodiments relate to a medium comprising any one or a combination of the factors FGF2, VEGF, TPO, SCF, IL3, and IL6.
In some embodiments, the concentration of each of the factors FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL6 in the media is between 5 ng/ml and 100 ng/ml. In some embodiments, the concentration of each of the factors FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL6 in the media is between 30 ng/ml and 70 ng/ml or between 40 ng/ml and 60 ng/ml. In some embodiments, the concentration of each of the factors FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL6 in the media is 50 ng/ml. In some embodiments, the concentration of Activin A in the media is between 9 ng/ml and 16 ng/ml or between 11 ng/ml and 14 ng/ml. In some embodiments, the concentration of Activin A in the media is 12.5 ng/ml. In some embodiments, the concentration of LiCl in the media is between 1 nM and 3 nM. In some embodiments, the concentration of LiCl in the media is between 1 mM and 3 mM. In some embodiments, the concentration of LiCL in the media is 2 mM.
Isolation of iHPCs
Any method known in the art is used to isolate iHPCs or CD43+ iHPCs. In some embodiments, the method used to isolate iHPCs or CD43+ iHPCs is FACS. In some embodiments, the isolation step comprises selecting for the CD43+ marker. In some embodiments, a marker other than CD43+ is used to isolate HPCs. In some embodiments, the isolation step comprises selecting for CD34+ cells. In some embodiments, the isolation step comprises selecting for CD31+ cells or CD45+ cells. In some embodiments, the isolation step comprises selecting for another marker known to identify iHPCs.
In some embodiments, isolating iHPCs results in isolation of iHPCs that are greater than 80% pure, for example, greater than 90%. In some embodiments, isolating CD43+ iHPCs results in isolation of CD43+ iHPCs that are greater than 80% pure, for example, greater than 90%.
Differentiating iHPCs into iMGLs
Any method known in the art to mature microglia cells may be used to mature iMGLs.
In some embodiments, differentiating CD43+ iHPCs into iMGLS comprises an incubation period of between 20 and 30 days. In some embodiments, the incubation period is 25 days.
In some embodiments, the media used to differentiate the iHPCs into iMGLs comprises any one or combination of the factors CSF-1, IL-34, and TGFβ1. In some embodiments, the media comprises all of the factors CSF-1, IL-34, and TGFβ1. In some embodiments, the concentration of the CSF-1 in the media is between 5 ng/ml and 50 ng/ml. In some embodiments, the concentration of the CSF-1 in the media is between 15 ng/ml and 35 ng/ml or between 20 ng/ml and 30 ng/ml. In some embodiments, the concentration of CSF-1 in the media is 25 ng/ml. In some embodiments, the concentration of the IL-34 in the media is between 25 ng/ml and 125 ng/ml. In some embodiments, the concentration of the IL-34 in the media is between 80 ng/ml and 120 ng/ml or between 90 ng/ml and 110 ng/ml. In some embodiments, the concentration of IL-34 in the media is 100 ng/ml. In some embodiments, the concentration of the TFGβ-1 in the media is between 2.5 ng/ml and 100 ng/ml. In some embodiments, the concentration of the TFGβ-1 in the media is between 30 ng/ml and 70 ng/ml or between 40 ng/ml and 60 ng/ml. In some embodiments, the concentration of TGFβ-1 in the media is 50 ng/ml. Some embodiments relate to a medium comprising any one or a combination of the factors CSF-1, IL-34, and TGFβ1.
In some embodiments, the media used to differentiate the iHPCs into iMGLs comprises TFGβ-2. In some embodiments, the concentration of the TFGβ-2 in the media is between 2.5 ng/ml and 100 ng/ml. In some embodiments, the concentration of the TFGβ-2 in the media is between 30 ng/ml and 70 ng/ml or between 40 ng/ml and 60 ng/ml. In some embodiments, the concentration of TGFβ-2 in the media is 50 ng/ml.
In some embodiments, the media used to differentiate the iHPCs into iMGLs comprises a TFGβ mimetic. Examples of TGFβ mimetics include IDE-1 and IDE-2. In some embodiments, the TFGβ mimetic has one or more off-target effects and/or affects a SOX signaling pathway. In some embodiments, the concentration of the TFGβ mimetic in the media is between 2.5 ng/ml and 100 ng/ml. In some embodiments, the concentration of the TFGβ mimetic in the media is between 30 ng/ml and 70 ng/ml or between 40 ng/ml and 60 ng/ml. In some embodiments, the TGFβ mimetic activates a TGFβ signaling pathway.
In some embodiments, the media used to differentiate iHPCs into iMGLs is serum-free media.
Maturation of iMGLs
In some embodiments, maturing the iMGLs comprises an incubation period between 1 and 5 days. In some embodiments, the incubation period for maturing the iMGLs is 3 days.
In some embodiments, maturation step comprises incubating the iMGLs in media comprising either or both of CD200 and CX3CL1. In some embodiments, the CD200 is human recombinant CD200 and the CX3CL1 is human recombinant CX3CL1.
In some embodiments, the concentration of each of CD200 and CX3CL1 in the media is between 1 ng/ml and lug/ml In some embodiments, the concentration of each of CD200 and CX3CL1 in the media is between 80 ng/ml and 120 ng/ml, or between 90 ng/ml and 110 ng/ml. In some embodiments, the concentration of each of CD200 and CX3CL1 is 100 ng/ml.
Characteristics of the iMGLs Produced
In some embodiments, the iMGLs produced using the methods described herein results in a pure population of iMGLs that is between 70% pure and 100% pure. In some embodiments, the iMGLs produced using the methods described herein results in a pure population of iMGLs that is between 80% pure and 100% pure. For example, the population of iMGLs will be 80% pure, 81% pure, 82% pure, 83% pure, 84% pure, 85% pure, 86% pure, 87% pure, 88% pure, 89% pure, 90% pure, 91% pure, 92% pure, 93% pure, 94% pure, 95% pure, or 96% pure, 97%, 98%, 99%, 99%, or 100%. In some embodiments, the population of iMGLs produced is greater than 96%.
Assessing the purity of the iMGLs is accomplished through utilization of any method known in the art of determining the purity of microglial cells. In some embodiments, the purity levels are assessed by the expression and/or co-localization of the factors P2RY12 and TREM12. In some embodiments, the purity levels are assessed by the expression and/or co-localization of Trem2, Iba1, and/or Pu1.
The iMGLs produced by any of the methods described herein will express any factor or any combination of factors that a typical microglial cell expresses. In some embodiments, the iMGLS produced are c-kit/CD45+. In some embodiments, the c-kit/CD45+ iMGLs are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteinomics. In some embodiments, other cell types are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteinomics. In some embodiments, the iMGLs produced comprise two separate populations of iMGLs: (1) CD45+/CX3CR1− and (2) CD45+/CX3CR1+. In some embodiments, the iMGLs produced are CD43+, CD235a+, or CD41+. In some embodiments, the iMGLs produced are CD43+/CD235a+/CD41+.
Any of the methods for producing iMGLs described herein will result in a differentiation step of the CD43+ iHPCs in which there is a commitment of cells to a microglial lineage early during the differentiation process. In some embodiments, iMGLs that are c-kit-/CD45+ are detected on day 14 of the incubation period used for differentiating CD43+ iHPCs into iMGLs. Determining whether there is a commitment to an iMGL lineage is done through testing for expression of any factors that are known to be markers for cells that are committed to a microglia fate. In some embodiments, determining whether the cells are committed to an iMGL lineage is determined through assessing expression of the transcription factor PU.1 and/or the microglia-enriched protein Trem2. In some embodiments, the cell markers are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteinomics.
In some embodiments, a method of producing iMGLs from induced PSCS is provided that comprises the steps: (i) differentiating PSCs into induced hematopoietic progenitor cells (iHPCs) and (ii) differentiating iHPCs to produce iMGLs. In some embodiments, this method further comprises step (iii) of maturing the iMGLS produced from step (ii). In some embodiments, the PSCs include induced PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the PSCs are mammalian PSCs, such as from a human or a mouse.
In some embodiments, TRIM14, CABLES1, MMP2, SIGLEC 11 and 12, MITF, and/or SLC2A5 mRNA and/or protein expression is enriched in the produced iMGLs. In some embodiments, COMT, EGR2, EGR3, and/or FFAR2 mRNA and/or protein expression is enriched in the produced iMGLs.
Gene Expression of iMGLs
In some embodiments, iMGLs are provided that express a specific gene profile. Any of the iMGLs described herein will comprise a gene expression profile similar to microglia cells. In some embodiments, any of the compositions of iMGLs described herein comprise expression of any of the following genes: RUNX1, PU.1, CSF1FR, CX3CR1, TGFBR1, RSG10, GAS6, PROS1, P2RY12, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, ITAM, APOE, SLCO2B1, SLC7A8, PPARD, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1. Any of the iMGLs disclosed herein will comprise expression of any of these genes in any combination.
RUNX1, SPI1, CSF1FR, CX3CR1, TGFBR1, RSG10, GAS6, MERTK, PSEN2, PROS1, P2RY12, P2RY13, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, TYROBP, ITGAM, APOE, SLCO2B1, SLC7A8, PPARD, TMEM119, GPR56, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1
In some embodiments, in any of the compositions of iMGLs described herein TREM2 and P2RY12 are co-expressed. In some embodiments, any of the compositions of iMGLs described herein do not express any one or more of the genes KLF2, TREM1, MPT, ITGAL, and ADGRE5.
Any of the iMGLs described herein will secrete a chemokine profile similar to microglia cells in response to any stimuli known in the art to stimulate chemokines in microglia cells. In some embodiments, the chemokines secreted are any one or more of TNFα, CCL2, CCL4, and CXCL10, in any combination, and are secreted in response to stimulation by lipopolysaccharide, IFNY, or IL-1β.
In some embodiments, a method of stimulating chemokine secretion from iMGLs is provided. The method comprises (i) treating iMGLs with any factor known in the art to stimulate cytokine secretion in microglia cells and (ii) secreting chemokines from the microglia cells. In some embodiments, the factor used to treat the iMGLs is lipopolysaccharide, INFY, or IL-1β. The chemokines secreted may comprise any chemokines known to be secreted by microglia cells. In some embodiments, the chemokines secreted comprise any one or more of the following: TNFα, CCL2, CCL4, and CXCL10.
Any of the iMGLS described herein will migrate in response to ADP treatment and/or ADP treatment will trigger calcium transients. In some embodiments, inhibition of P2ry12 negates ADP mediated migration of iMGLs and/or ADP mediated calcium transients. In some embodiments, the inhibition of P2ry 12 occurs through the inhibitor PSB0739.
In some embodiments, a method of migrating iMGLs is provided. The method comprises (i) treating iMGLs with ADP and (ii) migrating the iMGLs. In some embodiments, a method of producing calcium transients is provided. The method comprises (i) treating the iMGLs with ADP and (ii) producing calcium transients in iMGLs.
Phagocytoses by iMGLs
Any of the iMGLs described herein are capable of phagocytosis. Any of the iMGLs described herein are able to phagocytose any factor known in the art that microglia can phagocytose. In some embodiments, the factor(s) that iMGLs phagocytose comprise any one or more of the following: Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers.
In some embodiments, a method of iMGLs phagocytoses is provided. The method comprises (i) exposing iMGLs to any one or more of the compounds: Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers and (ii) phagocytosing the compound.
Any of the iMGLs provided herein are capable of phagocytosing human synaptosomes (hS). In some embodiments, a method of iMGLs phagocytosing hS is provided. The method comprises (i) exposing the iMGLS to hS and (ii) phagocytosing hS. In some embodiments, hS are fluorescently labeled.
Utility of iMGLS in Studying Alzheimer's Disease
Any of the iMGLs described herein are capable of regulating gene expression in response to different stimuli. In some embodiments, the stimuli comprise neurons, for example, rat-hippocampal neurons. In some embodiments, any of the iMGLs described herein are capable of differentially regulating any one or more the genes: CABLES, TRIM4, MITF, MMP2, and SLCA25. In some embodiments, the iMGLs upregulate any one or more the genes: TYROPB, CD33, and PICALM.
In some embodiments, methods of regulating gene expression in iMGLs are provided. One of the methods comprises (i) co-culturing iMGLs with neurons and (ii) differentially regulating genes in iMGLs. The neurons co-cultured with iMGLs will comprise be any neurons from any species. In some embodiments, the neurons are rat-hippocampal neurons.
Another method comprises (i) exposing iMGLs to any one or more the compounds: Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers and (ii) differentially regulating genes. The differentially regulated genes will comprise any combination of genes that would be differentially regulated in microglia in response to Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers. In some embodiments, the differentially regulated genes are upregulated genes comprising any one or more of CD33, TYROPB, and PICALM, in any combination.
Methods of Using iMGLs
In some embodiments, a method of assessing gene expression in iMGLs in response to neuronal cues is provided. The method comprises, according to several embodiments, (i) exposing iMGLs to one or more of the factors CX3CL1, CD200, and TGFβ, in any combination and (ii) assessing one or more the of the differentially regulated genes in any combination: P2ry12, EGR1, TGFβ1, ETV5, CX3CR1, APOE, BIN1, CD33, GPR84, COMT, APP, PSEN1, PSEN2, HTT, GRN, FUS, TARDP, VCP, SNCA, C9ORF72, LRRK2, and SOD1.
In some embodiments, a method of assessing engraftment of iMGLs into a cortex is provided. The method comprises, according to several embodiments, (i) transplanting iMGLs into a cortex and (ii) assessing engraftment of the iMGLs into the cortex. In some embodiments, step (ii) occurs at least 2 weeks after step (i), for example, at least 3 weeks after step (i), at least 4 weeks after step (i), at least 5 weeks after step (i), at least 6 weeks after step (i), at least 7 weeks after step (i), at least 8 weeks after step (i), at least 9 weeks after step (i), at least 10 weeks after step (i), at least 11 weeks after step (i), at least 12 weeks after step (i), at least 13 weeks after step (i), at least 14 weeks after step (i), at least 15 weeks after step (i), at least 16 weeks after step (i), at least 17 weeks after (i), at least 18 weeks after step (i), at least 19 weeks after step (i), or at least 20 weeks after step (i). In some embodiments, step (ii) occurs 2 months after step (i). In some embodiments, the method further comprises transplanting the iMGLS into the cortext of a mouse. In some embodiments, the mouse is a MITRG mouse.
In some embodiments, a method of assessing iMGL interaction with AD neuropathy is provided. The method comprises, in several embodiments, (i) transplanting iMGLs into hippocampi and (ii) assessing interaction of the iMGLs in the hippocampi. In some embodiments, the method comprises assessing migration of iMGLs towards plaques. In some embodiments, the method comprises assessing iMGL phagocytosis of fibrillary Aβ.
In some embodiments, a method of studying human microglia in a 3D neuronal environment is provided comprising transplanting iMGLs into a mammalian brain. In some embodiments, the mammalian brain is a mouse brain. In some embodiments, the iMGLs are transplanted into the hippocampi of the mouse brain. In some embodiments, the mouse is a wild-type mouse. In some embodiments, the mouse is an AD mouse strain.
Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure.
A two-step fully-defined protocol was developed to successfully generate microglia-like cells (iMGLs) from iPSCs in just over five weeks (
Second, CD43+ iHPCs were grown in serum-free differentiation medium (formulated in house) containing CSF-1, IL-34, and TGFβ1. By day 14, cells expressed the myeloid-associated transcription factor PU.1 and the microglia-enriched protein TREM2 (
By day 38, iMGLs exhibited high purity as assessed by purinergic receptor P2RY12 and TREM2 co-localization and quantification (>96%) (
The transcriptome of the iMGLs was profiled in comparison to human primary fetal microglia (Fetal MG) and adult microglia (Adult MG). The CD14+/CD16− monocytes (CD14 M), CD14+/CD16+ inflammatory monocytes (CD16 M), myeloid dendritic cells (Blood DCs), iHPCs, and iPSCs were also examined, in order to compare them to stem cells and other myeloid molecular signatures. Correlational analysis and Principal Component Analysis (PCA) revealed striking similarity of iMGLs to Fetal MG and Adult MG (Fetal MG and Adult MG are located in the same circled cluster in
Biclustering analysis using 300 microglial, macrophage, and other immune related genes adapted from previous studies identified similarities between groups and highlighted common gene clusters. This analysis again showed that iMGLs cluster with microglia but are distinct from other myeloid cells, iHPCs and iPSCs (
iMGLs were validated as surrogates of microglia using both functional and physiological assays. Cytokine/chemokine secretion by iMGLs stimulated by Lipopolysaccharide (LPS), and by IL-1β and IFNγ (two cytokines that are elevated in AD patients and mouse models) were measured. Results shows that iMGLs secreted 10 of the examined cytokines at low but detectable levels (Table 5). However, in response to IFNγ or IL-1β, iMGLs secreted 8 different chemokines including TNFα, CCL2, CCL4, and CXCL10. As expected, iMGLs robustly responded to LPS with induction of all measured cytokines except for CCL3 (see, Table 5 for values). Collectively, this data shows that iMGLs differentially release cytokines/chemokines based on their cell-surface receptor stimuli, a finding that closely aligns with the responses observed in acutely isolated primary microglia (Rustenhoven et al., 2016).
Because iMGLs express the microglial-enriched purinergic receptor P2ry12, which can sense extracellular nucleotides leaked from degenerating neurons and has been shown to be critical for microglial homeostatic function (
Microglia along with astrocytes, also play a critical role in synaptic pruning. Because in vitro synaptosome phagocytosis assays are an established surrogate to study pruning, the ability of iMGLs to phagocytose human synaptosomes (hS) was quantitatively assessed. In comparison to MD-Mφ, iMGL phagocytosis of pHrodo-labeled hS was less robust (
Because microglia (and iMGLs) express both C1q and CR3 (CD11b/CD18 dimer), iMGLs were used to assess whether synaptic pruning in human microglia primarily involves this pathway. Using an additive-free CD11b antibody, iMGL phagocytosis of hS was significantly reduced (−40.0%, ***p<0.0001) (
Previous reports have shown that impaired microglia clearance of beta-amyloid (Aβ) is implicated in the pathophysiology of AD. Therefore, iMGLs were examined to determine with they can phagocytose Aβ or tau, two hallmark AD pathologies. Similar to primary microglia, iMGLs internalized fluorescently labeled fibrillar Aβ (
Microglia genes are implicated in late onset AD, yet how they modify disease risk remains largely unknown. Thus, iMGLs were investigated to determine how these genes might influence microglia function and AD risk. Hierarchical clustering using just these 25 AD-GWAS genes demonstrated that iMGLs resemble microglia and not peripheral myeloid cells (
1TREATMENTS: FAB (5 MG/ML) OR BDTO (5 MG/ML) 24 H.
In the brain, neurons and glia interact with microglia and influence function and gene expression. Therefore, iMGLs were cultured with rat-hippocampal neurons (21 div) to assess how iMGLs respond to neuronal cues (
A fundamental characteristic of microglia is the surveillance of the CNS environment with their highly ramified processes. To investigate how iMGLs might interact within a CNS environment, iMGLs were cultured with human iPSC (hiPSC) 3D brain-organoids (BORGs). BORGs include neurons and astrocytes that self-organize into a cortical-like network, but lack microglia (
Neurons, astrocytes, and endothelial cells in the brain interact with microglia to influence gene expression and function. The differentiation protocol attempted to recapitulate CNS cues present in the brain by including signals derived from these other cell types including CX3CL1, CD200, and TGFβ. Whole transcriptome RNA-seq analysis confirmed the importance of these factors for establishing microglia in vitro (
CX3CL1 and CD200 are both neuronal-and endothelial-derived cues that can further educate iMGLs toward an endogenous microglia phenotype. CX3CL1 and CD200 were tested to determine how inclusion or exclusion of these factors modulates iMGL phenotype. The addition of CD200 and CX3CL1 to iMGLs increased the expression of select genes like COMT (
Next, it was examined whether iMGL maturation can be achieved with direct contact with the CNS environment. iMGLs were cultured with rat-hippocampal neurons (21 DIV) to assess how iMGLs respond to neuronal surface cues (
A fundamental characteristic of microglia is the surveillance of the CNS environment with their highly ramified processes. To investigate how iMGLs might interact within a human brain environment, iMGLs were cultured with hiPSC 3D brain-organoids (BORGs). BORGs include neurons, astrocytes, and oligodendrocytes that self-organize into a cortical-like network, but lack microglia (
iMGLs were examined within the context of a CNS environment in vivo. iMGLs (day 38) were transplanted into the cortex of MITRG mice that are Rag2-deficient and IL2rγ-deficient mice and also express the human forms of four cytokines knocked-in (M-CSFh;IL-3/GM-CSFh;TPOh), allowing for xenotransplantation and survival of myeloid and other leukocytes (
iMGLs were transplanted into the hippocampi of xenotransplantation-compatible AD mice, previously generated and characterized, to examine how iMGLs interact with AD neuropathology in vivo (
All cell culture flasks, reagents, supplements, cytokines, and general reagents were purchased from ThermoFisher (Carlsbad, CA) unless otherwise noted.
Maintenance and Culture of Human Pluripotent Stem Cells (hPSCs)
All stem cell work was performed with approval from UC Irvine Human Stem Cell Research Oversight (hSCRO) and IBC committees. Use of human tissue was performed in accordance and approval of Institutional Review Board (IRB). Human iPSC cell lines ADRC F5 and ADRC F14 (control subjects) were generated by the UCI ADRC Induced Pluripotent Stem Cell Core using non-integrating Sendai virus (Cytotune). iPSCs were confirmed to be karyotype normal by G-banding, sterile, and pluripotent via Pluritest (UCLA) Analysis. iPSCs were maintained feeder-free on matrigel (MTG) in complete TeSR-E8 medium (Stemcell Technologies) in a humidified incubator (5% CO2, 37° C.).
Differentiation of iPSCs to Hematopoietic Progenitor Cells (iHPCs)
Human iPSC derived hematopoietic progenitors were generated using defined conditions with several modifications to previously published protocols (Kennedy et al., 2007, Sturgeon et al., 2014). Briefly, iPSCs were triturated to generate a single-cell suspension and seeded in 6-well plates at 1-6×105 cells per well in E8 medium+Y-27632 ROCK Inhibitor (10 μM; R&D Systems). In some embodiments, Y-27632 is substituted with Thiazovivin (R&D systems). Cells were cultured for 24 hours under normoxic (20% O2) conditions after which the E8 media was changed to differentiation media composed of a base media and cytokines: IMDM/F12 (50:50), insulin (0.02 mg/ml), holo-transferrin (0.011 mg/ml), sodium selenite (0.0134 mg/ml), L-ascorbic acid 2-Phosphate magnesium (64 μg/ml; Sigma), monothioglycerol (400 μM), PVA (5 mg/ml; Sigma), L-alanyl-L-glutamine (2 mM), chemically-defined lipid concentrate (1×), non-essential amino acids (NEAA; 1×), FGF2 (50 ng/ml), BMP4 (50 ng/ml), Activin-A (12.5 ng/ml), and LiCl (2 mM) in hypoxia (5% O2). After two days, media was changed to base media supplemented with FGF2 (50 ng/ml) and VEGF (50 ng/ml). On day 4, media was changed to media containing FGF2 (50 ng/ml), VEGF (50 ng/ml), TPO (50 ng/ml), SCF (50 ng/ml), IL-6 (50 ng/ml), and IL-3 (50 ng/ml). On Day 6, media was supplemented with aforementioned medium. Cells were cultured for an additional 4 days (10 days total), after which, CD43+ cells were isolated by FACS for iMGL differentiation. Additionally, iPSC-derived HPCs (Cellular Dynamics) were identified as a commercial source of CD43+ progenitors.
Generation of Microglia-Like Cells From iHPCs
CD43+ iHPCs were plated in Matrigel-coated 6-well plates (BD Biosciences) with serum-free complete differentiation media at a density of 1-2×105 cells per well. Differentiation media consists of M-CSF (25 ng/ml), IL-34 (100 ng/ml; Peprotech), and TGFβ-1 (50 ng/ml; Militenyi) added to a base media (phenol-free DMEM/F12 (1:1), insulin (0.2 mg/ml), holo-transferrin (0.011 mg/ml), sodium selenite (0.0134 mg/ml), Penicillin/streptomycin (1% v/v), B27 (1% v/v), N2 (0.5%, v/v), monothioglycerol (200 μM), and additional insulin (4 μg/ml) just before addition to cells). Cells were supplemented with complete differentiation media every two days. At day 12, early iMGLs were collected (300×g for 5 mins at 25° C.) and a 50% media change was performed. After 25 days of microglial differentiation (35 days from iPSC), iMGLs were cultured in complete differentiation media supplemented with CD200 (100 ng/ml, Novoprotein) and CX3CL1 (100 ng/ml; Peprotech) for an additional three days, cultured with hippocampal neurons, or cultured with human brain-organoids.
Isolation of PBMCs from Human Blood
Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll-paque (GE Healthcare) gradient separation. In brief, blood was layered on top of Ficoll-Paque and centrifuged in swinging bucket rotator without brake (400×g, 40 minutes, 18° C.). After centrifugation, plasma and upper layers were removed and PBMCs isolated from the interphase. Cells were then washed once with ice-cold PBS and used immediately.
CD14 and CD16 monocytes were isolated via negative selection from PBMCs using the EasySep™ Monocyte Enrichment Kit (Stemcell Technologies) according to manufacturer's instructions. Isolated cells were washed three times with PBS and sorted by FACs for either RNA-sequence analysis or used for further macrophage differentiation.
Isolated monocytes were plated onto tissue culture treated 6-wells at 2×106 cells/ml in RPMI-1640 media at 37° C. 5% CO2 incubator. After two hours, media was aspirated to waste and adherent monocytes washed three times with DPBS and replaced with complete media composed of RPMI-1640, FBS (10% v/v), Penicillin/streptomycin (1% v/v), L-alanyl-L-glutamine (2 mM). To generate MD-MD, M-CSF (25 ng/ml) was added to wells and cells differentiated for 5 days.
Cells were harvested and washed three times with DPBS and stored in RNAlater, RNA preservation solution. RNA was extracted from all cell types using using RNeasy Mini Kit (Qiagen) following manufacturer's guidelines. RNA integrity (RIN) was measured for all samples using the Bioanalzyer Agilent 2100 series. All sequencing libraries analyzed were generated from RNA samples measuring a RIN score≥9. The Illumina TruSeq mRNA stranded protocol was used to obtain poly-A mRNA from all samples. 200 ng of isolated mRNA was used to construct RNA-seq libraries. Libraries were quantified and normalized using the Library Quantification Kit from Kapa Biosystems and sequenced as paired-end 100 bp reads on the Illumina HiSeq 2500 platform.
RNA-seq reads were mapped to the hg38 reference genome using STAR aligner and mapped to Gencode version 24 gene annotations using RSEM. Genes with expression (<1 FPKM) across all samples were filtered from all subsequent analysis. Differential gene expression analysis was performed on TMM normalized counts with EdgeR (Robinson et al., 2010). Multiple biological replicates were used for all comparative analysis. A p-value≤0.001 and a 2-fold change in expression were used in determining significant differentially expressed genes for respective comparisons. PCA analysis was performed using the R package rgl and plotted using plot3d. Clustering was performed using R hclust2 and visualized using Java Tree View 3.0.
Trans-well migration assays to ADP was performed as previously described (De Simone et al., 2010; Moore et al., 2015). iMGLs (5.5×104 cells/well) were cultured in serum-free basal media without cytokines for 1hour. Next, iMGLS were pre-exposed to DMSO or PSB0739 (50 μM, Tocris) for 1 hr at 37° C. in 5% CO2 cell culture incubator. Cells were then washed three times with basal medium and plated in trans-well migration chambers (5 μm polycarbonate inserts in 24 wells; Corning) containing Adenosine 5′-phosphate (ADP, 100 μM; Sigma) in the bottom chamber in 37° C. in 5% CO2. After 4 hours, cells were washed three times and fixed in PFA (4%) for 15 minutes at room temperature. Cells were stained with Hoechst stain for 10 mins to visualize nuclei of cells. A blinded observer counted total cells per slide and then scrubbed cells off top surface, washed with PBS, and recounted to record migrated cells. Migration was reported as migrated over total cells per well. Fluorescent images of cells were captured using Olympus IX71 inverted microscope.
For calcium imaging, iMGLs were plated on poly-L-lysine-coated coverslips and 1 hour later were incubated with Fura-2-AM (Molecular Probes) calcium dye diluted in Ringer solution containing (in mM): NaCl 140, KCl 4.5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, sucrose 5, pH=7.4. After a 1-hour incubation, the dye was washed out 3 times using Ringer solution and treated for 1 hour with either P2RY12 inhibitor PSB0739 (50 μM, Tocris) or Vehicle (DMSO) and used for experiments. Baseline Ca2+ signal (I340/I380) were measured for more than 100 s and then ADP (10 μM) was introduced under stead flow after baseline measurement. Ca2+ recordings were performed on Zeiss (Axiovert 35)-based imaging setup and data acquisition was conducted with Metafluor software (Molecular Devices). Data analysis was performed using Metafluor, Origin Pro, and Prism 6.0.
Cells were washed with cold PBS and fixed with cold PFA (4%) for 20 min at 25° C. followed by three washes with PBS. Cells were blocked with PBS with either 0.05% goat or donkey serum, and with Triton X100 (0.01%) for 1 hour at 25° C. Primary antibodies (1:500) were added in blocking solution overnight at 4° C. Cells were then washed three times with PBS and stained with Alexa Fluor® conjugated secondary antibodies at 1:400 for 1 hour at 25° C. After secondary staining, cells were washed three times followed by coverslip with DAPI-counterstain mounting media (Fluoromount, southern Biotech). Primary antibodies used for immunocytochemistry analysis include: β-3Tubulin (Biolegend), GFAP (Abcam), Iba1 (Wako), ITGB5 (Abcam), MMP-9 (Novus), MerTK (Biolegend), P2RY12 (Sigma), PROS1 (Abcam), PU.1 (Cell Signaling Technology) hCytoplasm (SC121; Takara Bio Inc.), TREM2 (R&D Systems), TGFβR1 (Abcam)
For ICC, cells were washed three times with DPBS (1×) and fixed with cold PFA (4% w/v) for 20 min at room temperature followed by three washes with PBS (1×). Cells were blocked with blocking solution (1×PBS, 5% goat or donkey serum, 0.2% Triton X-100) for 1 h at room temperature. ICC primary antibodies were added at respective dilutions (see below) in blocking solution and placed at 4° C. overnight. The next day, cells were washed 3 times with PBS for 5 min and stained with Alexa Fluor® conjugated secondary antibodies at 1:400 for 1 h at room temperature in the dark.
After secondary staining, cells were washed 3 times with PBS and coverslipped with DAPI-counterstain mounting media (Fluoromount, southern Biotech). For BORG IHC, tissue were collected and dropped-fixed in PFA (4% w/v) for 30 min at room temperature and then washed three times with PBS. BORGs were then placed in sucrose solution (30% w/v) overnight before being embedded in O.C.T (Tissue-Tek). Embedded tissue was sectioned at 20 μm using a cryostat and mounted slides were stored at −20° C. until staining. For BORG staining, mounted tissue was removed from storage and warmed by placing at room temperature for 30 min. Tissue were rehydrated and washed with room temperature PBS (1×) 3 times for 5 min.
Heat-mediated antigen retrieval was performed by using Citrate Buffer (10 mM Citrate, 0.05% Tween 20, pH=6.0) at 97° C. for 20 min and then allowed to cool to room temperature. After antigen retrieval, slides were washed three times with PBS. Slides were then washed once in PBS-A solution (1×PBS with 0.1% Triton X-100) for 15 min. Tissue was blocked using PBS-B solution (PBS-A, 0.2% BSA, and 1.5% goat or donkey serum) for 1 h at room temperature. After block, primary antibodies were added to PBS-B solution (250-350 μl/slide) at appropriate dilutions (see below) and incubated overnight at room temperature. The next day, slides were washed with PBS-A solution 3 times for 5 min each. Tissue were blocked for 1 h using PBS-B solution at room temperature. After block, slides were incubated with Alexa Fluor® conjugated secondary antibodies (all at 1:500) and Hoechst stain (1×) in PBS-B (for 250-300 μl/slide) for 2 h at room temperature in the dark.
After secondary staining, slides were washed 5 times with PBS for 5 min. Slides were cover slipped using fluoromount (Southern Biotech). For mouse brain IHC, brains were collected, fixed, and processed as mentioned above. Free-floating sections were blocked in blocking solution (1×PBS, 0.2% Triton X-100, and 10% goat serum) for 1 h at room temperature with gentle shaking. For human TMEM119 staining, heat mediated antigen retrieval was performed prior to blocking, as performed previously (Bennett et al., 2016). Free-floating tissue antigen retrieval was performed by placing floating sections in a 1.5 ml micro centrifuge tube containing 1 ml of Citrate Buffer solution and placing in a pre-heated temperature block set at 100° C. Tissue was heated for 10 min at 100° C. then removed and allowed to come to room temperature for 20 min before washing with PBS 3 times for 5 min and then proceeding with blocking step. For AD mouse brain staining of amyloid plaques, floating sections were placed in 1× Amylo-Glo® RTD™ (Biosensis) staining solution for 10 min at room temperature without shaking.
After staining, sections were washed in PBS 3 times for 5 minutes each and briefly rinsed in MiliQ DI water before being placed back in to PBS followed by blocking. Primary antibodies were added to staining solution (1×PBS, 0.2% Triton X-100, and 1% goat scrum) at appropriate dilutions (see below) and incubated overnight at 4° C. with slight shaking. The next day, sections were washed 3 times with PBS and stained with Alexa Fluor® conjugated secondary antibodies at 1:400 for 1 h at room temperature with slight shaking in the dark. After secondary staining, sections were washed in PBS 3 times for 5 min and mounted on glass slides. After mounting, slides were cover slipped with DAPI-counterstain mounting media (Fluoromount, southern Biotech). Primary antibodies:
Immunofluorescent sections were visualized and images captured using an Olympus FX1200 confocal microscope. To avoid non-specific bleed-through each laser line was excited and detected independently. All images shown represent either a single confocal z-slice or z-stack. Bright field images of cell cultures were captured on an Evos XL Cell Imaging microscope.
Cells were suspended in FACs buffer (DPBS, 2% BSA, and 0.05 mM EDTA) and incubated with human Fc block (BD Bioscience) for 15 min at 4° C. For detection of microglial surface markers, cells were stained with anti CD11b-FITC clone ICRF44, anti CD45-APC/Cy7 clone HI30, anti CX3CR1− APC clone 2A9-1, anti CD115-PE clone 9-4D, and anti CD117-PerCP-Cy5.5 clone 104D2. Live/dead cells were gated using Zombie Violet™ live/dead stain, all from Biolegend (San Diego, CA). Cells were run on FACs Aria II (BD Biosciences) and analyzed with FlowJo software (FlowJo).
1×105 cells were suspended in 100 μl of FACs buffer and added to Shandon glass slides (Biomedical Polymers) and assembled in a cytology funnel apparatus. Assembled slides containing cells were loaded in a cytospin instrument and centrifuged (500 rpm, 5 min). Slides were allowed to air-dry for two minutes and immediately stained in 100% May-Grunwald stain (Sigma) for 5 min. Next, slides were washed in PBS for 1.5 min and immediately placed in 4% Giemsa stain (Sigma) for 20 min at room temperature. Slides were washed in double-distilled H2O 6 times and allowed to air-dry for 10 min. Slides were preserved using glass coverslips and permount (Sigma).
RNA Isolation and qPCR Analysis
Cells were stored in RNAlater stabilizing reagent and RNA was isolated using Qiagen RNeasy Mini Kit (Valencia, CA) following manufacturer's guidelines. qPCR analysis was performed using a ViiA™ 7 Real-Time PCR System and using Taqman qPCR primers. Analysis of AD-GWAS genes utilized a custom Taqman Low Density Array card using the primers described below.
All procedures were performed under an IUCAC approved protocol. Primary cortical and hippocampal neuron cultures were derived from embryonic rat (E18). Briefly, dissected tissue was dissociated with trypsin, triturated, and plated on 6-well plates coated with poly-L-lysine coated in serum-free Neurobasal supplemented with B27 (1% v/v) (NB medium). Cells were plated at a density of 5×106 cells/ml and maintained in culture until used.
iMGL Co-Culture With Rat Neurons
Rat hippocampal or cortical neurons were cultured for 21 days with 50% media change every 3-4 days. iMGLs were cultured with neurons at a 1:5 ratio (1×106 iMGL to 5×106 neurons) in 50% iMGL and 50% NB medium. After 3 days, iMGLs were collected for RNA isolation.
iMGLs culture media was replaced with basal media for 2 hours prior to stimulation with IFNγ (20 ng/ml), IL1β (20 ng/ml), and LPS (100 ng/ml) for 24 hours, after which cells were collected for RNA and conditioned media assessed for cytokine secretion. To simultaneously assess multiple cytokine and chemokine analytes from iMGL conditioned media, conditioned media from each treatment group was processed and analyzed using the V-PLEX human cytokine 30-plex kit (Mesoscale) according to the manufacturer protocol.
Human 3D brain organoids were generated as previously described with some modifications (Lancaster et al., 2013) with modifications detailed in Supplemental Information.
Fibrillar fluorescent amyloid-beta (fAβ1-42) was generated. Briefly, fluorescently labeled Aβ peptide (Anaspec; Fremont, CA) was first dissolved in 0.1% NH4OH to 1 mg/ml, then further diluted in sterile endotoxin-free water and incubated for 7 days at 37° C. fAβ was thoroughly mixed prior to cell exposure.
Tau oligomers were isolated by immunoprecipitation with the T22 antibody using PBS-soluble fractions of homogenates prepared from AD brain. These were then purified by fast protein liquid chromatography (FPLC) using PBS (pH 7.4). Additional analyses include Western blots to detect contamination with monomeric tau or large tau aggregates (tau-5, normally appear on top of the stacking gel) and using a mouse anti-IgG to identify non-specific bands. BDTOs were subsequently conjugated to pHrodo-Red according to the manufacturer's protocol.
Human tissue samples were obtained at autopsy and minced, slowly frozen in 0.32 M sucrose with 10% DMSO and stored at −80° C. To obtain a crude synaptosome fraction, tissue was thawed in a 37° C. water bath and homogenized in 10 mm Tris buffer (pH 7.4) with proteinase inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich) using a glass/Teflon homogenizer (clearance 0.1-0.15 mm). The homogenate was centrifuged at 1000 g at 4° C. for 10 min, the supernatant was removed and centrifuged again at 10 000 g at 4° C. for 20 min. Resulting pellets were resuspended in sucrose/Tris solution and stored at −80° C. Synaptosomes were conjugated to pHrodo-Red according to the manufacturer protocol.
iMGLs and MD-M□, were incubated with mouse anti CD16/32 Fc-receptor block (2 mg/ml; BD Biosciences) for 15 minutes at 4° C. Cells were then stained with anti CD45-APC clone (mouse cells; Tonbo Biosciences; San Diego, CA) at 1:200 in flow cytometer buffer. Samples were then analyzed using Amnis Imagestreamerx Mark II Imaging Flow Cytometer (Millipore). E. coli, human synaptosome, fAβ, and BDTO phagocytosis was analyzed using the IDEAS software onboard Internalization Wizard algorithm. Additive free Anti-CD11b antibody (Biolegend) was used for CD11b blockade.
Statistical analysis was performed using Graphpad Prism 6 software. Comparisons involving more than two groups utilized 1-way ANOVA followed by Tukey's post hoc test and corrected p-values for multiple comparisons were reported. Comparisons of two groups utilized two-tailed Students t-test. All differences were considered significantly different when p<0.05. Statistical analysis for RNA-sequencing is detailed above and all other statistical analysis are reported in the figure legends.
iMGLs (5.5×104 cells/well) were cultured in serum-free basal media without cytokines for 1 hour. Next, iMGLS were pre-exposed to DMSO or PSB0739 (50 μM, Tocris) for 1 hr at 37° C. in 5% CO2 cell culture incubator. Cells were then washed three times with basal medium and plated in trans-well migration chambers (5 μm polycarbonate inserts in 24 wells; Corning) containing Adenosine 5′-phosphate (ADP, 100 μM; Sigma) in the bottom chamber in 37° C. in 5% CO2. After 4 hours, cells were washed three times and fixed in PFA (4%) for 15 minutes at room temperature. Cells were stained with Hoechst stain for 10 mins to visualize nuclei of cells. A blinded observer counted total cells per slide and then scrubbed cells off top surface, washed with PBS, and recounted to record migrated cells. Migration was reported as migrated over total cells per well. Fluorescent images of cells were captured using Olympus IX71 inverted microscope.
iPSCs were cultured and maintained on Vitronectin XF (Stem Cell Technologies) in 6-well tissue culture treated plates (BD Falcon) and maintained with TeSR-E8 media (Stem Cell Technologies) daily, at 37° C. with 5% CO2. At approximately 80% confluency, iPSCs were detached from the Vitronectin XF substrate using the standard ReLeSR protocol (Stem Cell Technologies) and centrifuged, pelleted, and suspended in embryoid body (EB) media, which consists of KO DMEM/F12 (Invitrogen), KOSR (20% v/v) (v/v), L-alanyl-L-glutamine (2 mM), NEAA (1×), 2-Mercaptoethanol (0.1 mM), rhubFGF (4 μg/ml), and HSA (0.1% v/v) and ROCK inhibitor (50 μM), to form EBs. Approximately 1×104 cells were plated per well of a standard V-bottom 96-well plate coated with Lipidure (1% v/v; AMSBio) to avoid having the EBs attach to the 96-well plate. After 4 days in EB media with bFGF (4 ng/ml) and ROCK inhibitor (50 μM), both the bFGF and ROCK inhibitor were discontinued leaving the brain organoids in basic EB media for an additional 3 days (7 days total). After the EB media phase, the EB media is replaced with neural epithelium (NE) media which consists of DMEM/F12, N2 supplement (0.1% v/v), L-alanyl-L-glutamine (2 mM), MEM-NEAA (0.1% v/v), Heparin solution (0.2 mg/ml; Sigma), and filtered using 0.22 μm PES filter (EMD Milipore). The brain organoids were transferred to an ultra-low attachment 24-well plate (Corning) using cut P200 pipette tips, with 1-2 EBs per well in 1 ml NE media. The EBs were neutralized in the NE media for five days, after which they were transferred into Matrigel (Corning) using a mold created from siloconized parafilm and a sterile empty P200 box. The brain organoids were kept in a 6 cm suspension petri dish with differentiation media consisting of KO DMEM/F12 (50%), Neurobasal medium (50%), N2 supplement (0.1% v/v), B27 without vitamin A supplement (0.1% v/v), Insulin solution (0.1% v/v; Sigma), 2-Mercaptoethanol (0.1 mM), L-alanyl-L-glutamine (2 mM), MEM-NEAA (1×), and Penicillin/Streptomycin (0.1% v/v). After five days of being exposed to differentiation media containing B27 without vitamin A, the differentiation media was replaced by a formulation that is identical except for the replacement of B27 without vitamin A to B27 with vitamin A; at this time point, the brain organoids are also transferred to a 125 ml spinning flask bioreactor (Corning) siliconized with Sigmacote (Sigma), where they were fed differentiation media with vitamin A weekly for 8 weeks. After 12 weeks, Borgs were utilized for iMGL co-culture studies.
Briefly, normal appearing cortical tissue was resected from pharmacologically intractable non-malignant cases of temporal lobe epilepsy. Tissue was cleaned extensively and mechanically dissociated. A single cell suspension was generated following gentle enzymatic digestion using trypsin and DNAse prior to passing through a nylon mesh filter. The single cell suspension underwent a fickle ultracentrifugation step to remove myelin. Dissociated cells were centrifuged, counted, and plated at 2×106 cells/mL in MEM supplemented with 5% FBS, 0.1% P/S and 0.1% glutamine. Microglia were grown for 3 days, collected and plated at 1×105 cells/mL and maintained in culture for 6 days during which time cells received two treatments of TGFβ (20 ng/mL) on days 3 and 5. Human fetal brain tissue was obtained from the Fetal Tissue Repository (Albert Einstein College of Medicine, Bronx, NY). Total RNA was isolated using standard Trizol (Invitrogen) protocols and stored at −80° C. In some embodiments, a suspension of small clumps of cells are produced and used in a similar manner as the single cell suspension.
iMGL Transplantation in MITRG and Rag5xfAD Brains
All animal procedures were performed in accordance with NIH and University of California guidelines approved IAUC protocols (IAUC #2011-3004). MITRG mice were purchased from Jax (The Jackson Laboratory, #017711) and have been previously characterized (Rongvaux et al., 2014). MITRG mice allow for xenotransplantation and is designed to support human myeloid engraftment. iMGLs were harvested at day 38 and suspended in injection buffer: 1× HBSS with M-CSF (10 ng/ml), IL-34 (50 ng/ml), and TGFβ-1 (25 ng/ml). iMGLs were delivered using stereotactic surgery as previously described (Blurton-Jones, et al, 2009) using the following coordinates; AP: −0.6, ML: ±2.0, DV: −1.65. Brains were collected from mice at day 60 post-transplantation per established protocols (Blurton-Jones, et al, 2009). Rag5xfAD mice were generated in this lab and previously characterized (Marsh et al., 2016). Rag5xfAD mice display robust beta-amyloid pathology and allow for xenotransplantation of human cells. iMGLs were transplanted into the hippocampi using the following coordinates; AP: −2.06, ML: ±1.75, DV: −1.95. After transplantation mice were killed and brains collected using previously established protocol. Briefly, mice were anesthetized using sodium-barbiturate and perfused through the left-ventricle with cold 1× HBSS for 4 min. Perfused mice were decapitated and brain extracted and dropped-fixed in PFA (4% w/v) for 48 hours at 4° C. Brains were then washed 3 times with PBS and sunk in sucrose (30% w/v) solution for 48 hours before coronal sectioning (40 μm) using a microtome (Leica). Free-floating sections were stored in PBS sodium azide (0.05%) solution at 4° C. until IHC was performed.
Serial dilutions of proteins (2 μl) were blotted on a pre-wet nitrocellulose paper and allowed to dry. After drying, blots were blocked with 5% BSA in 1×Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature with slight shaking. Next, blots were incubated with primary antibodies (see below) at room temperature for 1 hour. Blots were then washed 3 times for 5 min each with TBST. Blots were then incubated with HRP conjugated secondary antibody (Santa Cruz) at 1:10,000 for 1 h at room temperature with mild shaking. After 1 h, blots were washed 3 times for 5 min each with TBST. After wash, blots were dried on filter paper and incubated with Pierce ECL Western blotting development substrate (Thermo Fisher) for 10 min in the dark. Blots were imaged on ChemiDoc XRS+ imaging system (BioRad).
AD-GWAS qPCR Primers
The following validated and available Taqman primers were used: APOE Hs00171168_m1, CR1 Hs00559342_m1, CD33 Hs01076281_m1, ABCA7 Hs01105117_m1, TREM2 Hs00219132_m1, TREML2 Hs01077557_m1, TYROBP (DAP12) Hs00182426_m1, PICALM Hs00200318_m1, CLU Hs00156548_m1, MS4A6A Hs01556747_m1, BIN1 Hs00184913_m1, CD2AP Hs00961451_m1, CASS4 Hs00220503_m1, MEF2C Hs00231149_m1, DSG2 Hs00170071_m1, MS4A4A Hs01106863_m1, ZCWPW1 Hs00215881_m1, INPP5D Hs00183290_m1, and PTK2B Hs00169444_m1.
It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present inventions herein disclosed should not be limited by the particular disclosed embodiments described above. Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. For example, actions such as “administering a population of expanded NK cells” include “instructing the administration of a population of expanded NK cells.” In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 10 nanometers” includes “10 nanometers.”
iHPC transplantation allows for studying human microglial development in a complete brain environment. Normal development and aging of human microglia in a complete CNS environment can be studied by transplanting iHPCs in the brains of mice (see
This application is a Continuation Application of U.S. application Ser. No. 16/489,338, filed Aug. 27, 2019, which is the U.S. National Phase Application under 35 U.S.C. § 371 of International Application No. PCT/US2018/019763, filed Feb. 26, 2018, designating the U.S. and published in English as WO 2018/160496 A1 on Sep. 7, 2018, which claims the benefit of U.S. Provisional Application No. 62/464,925, filed Feb. 28, 2017. The contents of the aforementioned applications are expressly incorporated herein by reference in their entirety.
This invention was made with government support under AG048099 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62464925 | Feb 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16489338 | Aug 2019 | US |
| Child | 18749358 | US |
